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https://openalex.org/W2967233312
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https://aguassubterraneas.abas.org/asubterraneas/article/download/29583/19113
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Química ambiental: monitoramento físico-químico da água de um poço artesiano na cidade de Remígio-PB
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Águas Subterrâneas
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Alerta: Os artigos publicados nesta seção não são avaliados por pares e não são indexados. A intenção da seção ECNT é prover um espaço para divulgação
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Técnicas Alerta: Os artigos publicados nesta seção não são avaliados por pares e não são indexados. A intenção da seção ECNT é p
de dados e estudos de interesse local, sem caráter científico. Sendo assim, a Revista Águas Subterrâneas não se respons
Técnicas Alerta: Os artigos publicados nesta seção não são avaliados por pares e não são indexados. A intenção da seção ECNT é prover um espaço para divulgação
de dados e estudos de interesse local, sem caráter científico. Sendo assim, a Revista Águas Subterrâneas não se responsabiliza pelo conteúdo publicado. Técnicas Disclaimer: Articles published in this section are not peer-reviewed and are not indexed. The intention of the ECNT section
dissemination of data and studies of local interest, with no scientific character. Therefore, Revista Águas Subterrâneas is not r Disclaimer: Articles published in this section are not peer-reviewed and are not indexed. The intention of the ECNT section is to provide a space for the
dissemination of data and studies of local interest, with no scientific character. Therefore, Revista Águas Subterrâneas is not responsible for this content. Environmental chemistry: physical-chemical monitoring of water from an artesian
well in the city of Remígio-PB Aldeni Barbosa da Silva1; Edmilson Dantas da Silva Filho2; John Carlos Silva Câmara1; Martha Lisboa Oliveira dos Santos1; Daniel
Rodrigues dos Santos1; Marjorye Lavigne Alves de Freitas1; Joelson Souza Izidro dos Santos2; Pedro Lucas Nunes da Silveira2 1 Instituto Federal de Educação Ciência e Tecnologia da Paraíba, Esperança, Paraíba. 2 Instituto Federal de Educação Ciência e Tecnologia da Paraíba, Campina Grande, Paraíba. aldeni.silva@ifpb.edu.br, edmilson.silva@ifpb.edu.br, john.carlos@academico.ifpb.edu.br, martha.lisboa@academico.ifpb.edu.br,
rodrigues.daniel@academico.ifpb.edu.br, marjorye.lavigne@academico.ifpb.edu.br, joelsonisidro700@gmail.com,
pedrolucasns2000@gmail.com Química ambiental: monitoramento físico-químico da água de um
poço artesiano na cidade de Remígio-PB Environmental chemistry: physical-chemical monitoring of water from an artesian
well in the city of Remígio-PB SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 1 Resumo Esse trabalho teve o objetivo de estudar a qualidade físico-química da água de um poço artesiano localizado na zona urbana da
cidade de Remígio-PB. A amostra de água foi coletada em garrafa plástica de 2 litros em um poço artesiano localizado na zona
urbana da cidade de Remígio/PB e foi encaminhada ao laboratório de Química do Instituto Federal da Paraíba, campus de
Campina Grande, para a realização das análises. Todas as análises foram realizadas em triplicata. Os parâmetros físico-
químicos da água foram determinados seguindo-se as metodologias do manual do Instituto Adolfo Lutz. Os parâmetros analisa-
dos foram os seguintes: pH, temperatura, cor aparente, alcalinidade, condutividade elétrica, sólidos totais dissolvidos, porcen-
tagem de cinzas, cloreto, turbidez, dureza total, dureza de cálcio e magnésio, cloro total e porcentagem de ferro. A amostra
apresentou um valor médio para pH de 6,52 a uma temperatura de 26,3oC, um valor médio de 368 uH para cor aparente,
106,7 uT para turbidez, 42,67 mg/L de CaCO3 para alcalinidade e 104,5 mg/L para cloreto. Com relação a dureza total, o valor
médio encontrado foi de 92,67 mg/L de CaCO3, 911,1 µS/cm para condutividade elétrica, 5.957 mg/L para sólidos totais
dissolvidos, 0,0165 % de cinzas, 0,24 e 0,94 mg/L para cloro total e ferro, respectivamente. Diante disso, conclui-se que a
água é imprópria para o consumo humano, pois os parâmetros cor aparente, turbidez, sólidos totais dissolvidos e porcentagem
de ferro estão totalmente fora do que é preconizado pela Portaria de Consolidação no 05/2017 do Ministério da Saúde e da
resolução de nº 357/2005 do Conselho Nacional do Meio Ambiente. Palavras-chave:
Água potável. Água subterrânea. Análise de água. Qualidade da água. 1. INTRODUÇÃO A água é necessidade primordial para a vida, recurso natural indispensável ao ser humano e aos demais seres vivos, além de ser suporte
essencial aos ecossistemas. Utilizada para o consumo humano e para as atividades socioeconômicas, é retirada de rios, lagos, represas
e aquíferos, tendo influência direta sobre a saúde, a qualidade de vida e o desenvolvimento das populações (SCURACCHIO, 2010; SILVA
et al, 2017; BANDEIRA et al. 2018). A água ocupa aproximadamente 75% da superfície da Terra e é o constituinte inorgânico mais abundante da matéria viva, integrando
aproximadamente dois terços do corpo humano e atingindo até́ 98% para certos animais aquáticos, assim como, legumes, frutas e verdu-
ras. Constitui-se no solvente universal da maioria das substâncias, modificando-as e modificando-se em função destas (LIBÂNIO, 2010;
SILVA et al. 2018). As águas subterrâneas são as principais fontes de água potável nas regiões com déficit de água superficial ou em localidades sem siste-
ma centralizado de abastecimento de água. Em muitos locais ela é um complemento às águas superficiais, sendo muitas vezes consumi-
da indiscriminadamente pela população, não levando em conta sua qualidade, o que acaba causando prejuízos para o bem-estar dos
consumidores. Geralmente os problemas com a qualidade da água subterrânea são maiores em regiões rurais devido à infiltração de
agrotóxicos, fertilizantes, esgotos domésticos e excrementos de animais nos aquíferos ou, diretamente, nos poços artesianos. Nestas
regiões, os efeitos são mais significativos em seus consumidores, devido à ausência de outras opções de abastecimento (MIRLEAN et al. 2005; SILVA FILHO et al. 2019). No Brasil, 80% dos esgotos são lançados em corpos d’água sem qualquer tratamento; destes 85% são esgotos domésticos e 15% esgo-
tos industriais. Em áreas urbanas a elevada densidade populacional produz alto volume de esgoto, e em cidades desprovidas de sistema
de esgotamento sanitário eficiente, as águas subterrâneas podem ser contaminadas por meio da infiltração oriunda de fossas negras e
pelo escoamento superficial da água da chuva em contato com o esgoto lançado a céu aberto (CALHEIROS; OLIVEIRA, 2006; CAPP et al.,
2012). Os parâmetros físico-químicos determinam as características de potabilidade necessárias para que a água seja propícia para o consumo
humano. Esses parâmetros são regulamentados por normas e/ou padrões definidos em portarias do Ministério da Saúde (RICHTER;
NETTO, 1999; SANTOS; MOHR, 2013). 1. INTRODUÇÃO No Brasil, as legislações vigentes que tratam de potabilidade da água para consumo humano e de águas subterrâneas são, respectiva-
mente, a Portaria de consolidação nº 05 de 28 de setembro de 2017, do Ministério da Saúde (BRASIL, 2017) e a Resolução nº 396, de 3
de abril de 2008, do Conselho Nacional do Meio Ambiente (CONAMA) (BRASIL, 2008). De acordo com a definição da portaria n.º 05/2017
do Ministério da Saúde, no Art. 5º, água potável é a água para consumo humano cujos parâmetros microbiológicos, físicos, químicos e
radioativos atendam ao padrão de potabilidade e que não ofereça riscos à saúde. Diante disso, o objetivo do presente trabalho foi o de estudar a qualidade físico-química da água de um poço artesiano localizado na zona
urbana da cidade de Remígio-PB. Abstract This work had the objective of studying the physical-chemical quality of the water of an artesian well located in the urban area of
the city of Remígio-PB. The water sample was collected in a 2 liter plastic bottle in an artesian well located in the urban area of
the city of Remígio / PB and was sent to the Chemistry laboratory of the Federal Institute of Paraíba, campus of Campina
Grande, to carry out the analyzes. All analyzes were performed in triplicate. The physico-chemical parameters of the water were
determined following the methodologies of the Adolfo Lutz Institute manual. The parameters analyzed were: pH, temperature,
apparent color, alkalinity, electrical conductivity, total dissolved solids, percentage of ash, chloride, turbidity, total hardness,
calcium and magnesium hardness, total chlorine and percentage of iron. The sample had a mean pH value of 6.52 at a temper-
ature of 26.3 °C, an average value of 368 uH for apparent color, 106.7 uT for turbidity, 42.67 mg/L CaCO3 for alkalinity and
104, 5 mg / L for chloride. In relation to total hardness, the mean value found was 92.67 mg/L CaCO3, 911.1 μS/cm for electri-
cal conductivity, 5,957 mg/L for total dissolved solids, 0.0165% ash, 0.24 and 0.94 mg / L for total chlorine and iron, respec-
tively. Therefore, it is concluded that water is unfit for human consumption, since the parameters apparent color, turbidity, total
dissolved solids and iron percentage are totally out of the one recommended by the Consolidation Ordinance no. 05/2017 of
the Ministry of Health and Resolution No. 357/2005 of the National Environmental Council. DOI: http:/dx.doi.org/10.14295/ras.v33i3.29583 DOI: http:/dx.doi.org/10.14295/ras.v33i3.29583 SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 1 2.1. Área de Estudo O estudo foi desenvolvido em Remígio/PB, cidade com área territorial de 180,897 km², altitude média de 535 metros, apresentando uma
população estimada em 19.368 habitantes, densidade demográfica de 98,77 hab/km2 (IBGE, 2016), e coordenadas geográficas de
06°53’30” S e 35°49’51” W (CIDADE BRASIL, 2017). SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 2 2.2.2. Cor aparente 2.2.2. Cor aparente
A cor aparente foi determinada pelo método de comparação óptica, utilizando-se o Colorímetro digital (Hanna Instruments HI 727 Chec-
ker HC Handheld Colorimeter, For Color of Water) (Figura 1B). 2.2.3. Condutividade Elétrica, Sólidos Totais Dissolvidos e Porcentagem de cinzas
A Condutividade Elétrica, os Sólidos Totais Dissolvidos e a Porcentagem de Cinzas foram determinadas através do condutivímetro portátil
da Tecnopon, modelo mCA–150, com resultados expressos na escala de μS/cm (Figura 1C). Figura 1 - A. Medidor de pH de bancada (Microprocessado) da marca Tecnopon, modelo mPA210. B. Colorímetro digital (Hanna Instru-
ments HI 727 Checker HC Handheld Colorimeter, For Color of Water). C. Condutivímetro portátil da Tecnopon, modelo mCA–150. 2.2.4. Turbidez
A medida da turbidez baseou-se na medida do grau de interferência à passagem de luz através da água. A turbidez foi determinada pelo
método nefelométrico, com um turbidímetro da marca DEL LAB modelo DLT-WV (Figura 2A). 2.2.6. Porcentagem de ferro A porcentagem de Ferro foi determinada pela adaptação do método EPA Fenantrolina 315 B, para águas nat
utilizando-se o Colorímetro Checker digital para medição de ferro (Hanna Instruments HI 721) (Figura 2C). 2.2.5. Cloro total O cloro total foi determinado pelo método adaptado da USEPA 330.5 (método DPD), utilizando-se o Colorímetro Checker digital para me-
dição de cloro (Hanna Instruments HI 711) (Figura 2B). 2.2.4. Turbidez A medida da turbidez baseou-se na medida do grau de interferência à passagem de luz através da água. A turbidez foi determinada pelo
método nefelométrico, com um turbidímetro da marca DEL LAB modelo DLT-WV (Figura 2A). 2.2.8. Cloreto O cloreto, por sua vez, foi verificado pelo método de Mohr, em mg/L de Cl-, no qual, após a adição, para cada 10 ml da amostra de água
com 90 ml de água destilada, de 1 ml do indicador cromato de potássio (K2CrO4), cuja cor é amarelo-esverdeada, titula-se inicialmente
com a solução padrão de nitrato de prata (AgNO3) a 0,00141 N e, em seguida, para tornar o precipitado colorido, repetiu-se o procedi-
mento (a chamada prova em branco), dessa vez com 100 ml de água destilada, onde acrescentou-se uma pitada de carbonato de cálcio
(CaCO3) para a titulação com o AgNO3. 2.2.7. Alcalinidade total A alcalinidade foi determinada pelo método volumétrico, com a adição, para cada 100 mL da amostra, de duas gotas do indicador de
fenolftaleína (permanecendo incolor), em seguida três gotas do indicador metil-orange (cor amarela), titulando-se com ácido clorídrico
(HCl) a 0,1 M, até o surgimento da coloração salmão-rósea, cujos resultados são expressos em mg/L de CaCO3. 2.2.1. pH e Temperatura p
p
O pH e a temperatura foram determinados com o medidor de pH de bancada (Microprocessado) da marca Tecnopon, modelo mPA210
(figura 1A), previamente calibrado com soluções-tampão de pH 7,0 e de pH 4,0, com resultados expressos em escala logarítmica de pH. SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 3 2.2 Amostras para análises físicas e químicas A amostra de água destinada para as análises físico-químicas foi coletada em garrafa plástica de 2 litros em um poço artesiano localiza-
dos na zona urbana da cidade de Remígio/PB e foi encaminhada ao laboratório de Química (LQ) do Instituto Federal da Paraíba, campus
de Campina Grande, para a realização das análises. Os parâmetros físico-químicos da água foram determinados seguindo-se as metodologias do manual do Instituto Adolfo Lutz, notadamen-
te os métodos Físico-Químicos para Análise de Alimentos, da 4ª versão, do Capítulo VIII – Águas (INSTITUTO ADOLFO LUTZ, 2008). Os
valores foram avaliados conforme as recomendações da Portaria de consolidação nº 05 de 28 de setembro de 2017, do Ministério da
Saúde (BRASIL, 2017). Todas as análises foram realizadas em triplicata. Os parâmetros analisados e os métodos de análises foram os seguintes: Todas as análises foram realizadas em triplicata. Os parâmetros analisados e os métodos de análises fo Todas as análises foram realizadas em triplicata. Os parâmetros analisados e os métodos de análises foram os seguintes: SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 2 pH e temperatura De acordo com a Portaria de consolidação nº 5, de 28 de setembro de 2017, do Ministério da Saúde, o pH é padrão de potabilidade,
devendo as águas para consumo humano apresentar valores entre 6,0 e 9,5 (BRASIL, 2017). A amostra de água do poço artesiano atendeu aos padrões estipulados para pH, apresentando valores que variaram de 6.40 a 6,67
(Tabela 1). O potencial hidrogeniônico (pH) consiste na concentração dos íons H+ nas águas e representa a intensidade das condições ácidas ou
alcalinas do ambiente aquático. No valor do pH, aliada a dissociação da molécula de água, incorpora-se o hidrogênio resultante da disso-
ciação de ácidos orgânicos naturais ou inorgânicos presentes presente em efluentes industriais (PIVELI; KATO, 2006). Ingerir água com pH neutro ou levemente alcalino contribui para que o corpo humano mantenha o pH nos níveis adequados para os
processos fisiológicos. No entanto, cabe destacar que o pH das águas subterrâneas pode variar entre 5,5 e 8,5 pois as concentrações
iônicas são mais elevadas que as das águas superficiais devido a interação entre água e rochas, sendo os diferentes valores relaciona-
dos às características químicas das mesmas (PEDROSA; CAETANO, 2002). Stein et al. (2012) afirmaram que as águas do aquífero Barreiras tendem a serem pouco ácidas com pH médio de 6,18. De acordo com
Rocha et al. (2005), as características químicas das águas subterrâneas refletem os meios por onde percolam, guardando uma estreita
relação com os tipos de rochas drenados e com os produtos das atividades humanas adicionados ao longo de seu trajeto. As águas sub-
terrâneas tendem a ser mais ricas em sais dissolvidos do que as águas superficiais. Silva & Araújo (2003) observaram que 82,8% de amostras apresentaram um pH ácido, abaixo de 6,0, não atendendo ao recomendado
pela legislação. Silva et al. (2017) ao estudarem os parâmetros físico-químicos da água utilizada para consumo em poços artesianos na cidade de Remí-
gio-PB, observaram que todas as amostras apresentaram um caráter ácido, com valores que variaram de 4,5 a 5,9, não atendendo,
portanto, aos padrões estipulados. A temperatura média encontrada nesse estudo foi em média de 26,3 oC (Tabela 1). De acordo com Libânio (2010), a temperatura da
água e dos fluidos em geral, indica a magnitude da energia cinética do movimento aleatório das moléculas e sintetiza o fenômeno de
transferência de calor à massa líquida. pH e temperatura Segundo o mesmo autor, a alteração da temperatura das águas naturais decorre em especial da
insolação, esta é influenciada significativamente pelo clima e pela latitude, e, quando de origem antrópica, do lançamento de despejos
industriais. 2.2.9. Dureza total e Dureza de cálcio e de magnésio A dureza total foi definida como a soma das concentrações de cálcio e magnésio, ambas expressas como carbonato de cálcio, em mili-
gramas por litro. O ácido etilenodiaminotetracético e seus sais sódicos (EDTA) formaram complexos quelados solúveis com certos cátions
metálicos. Uma solução contendo íons de cálcio e magnésio, com uma pequena quantidade do indicador negro de eriocromo T, em pH
(10,0±0,1) tornou-se purpura. Titulando-se essa solução com EDTA, cálcio e magnésio foram quelados e uma viragem de cor purpura a
azul indicou o ponto final. SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 3 Figura 2 - A Turbidímetro da marca DEL LAB modelo DLT-WV. B. Colorímetro Checker digital para medição de cloro (Hanna Instruments HI
711). C. Colorímetro Checker digital para medição de ferro (Hanna Instruments HI 721). Figura 2 - A Turbidímetro da marca DEL LAB modelo DLT-WV. B. Colorímetro Checker digital para medição de cloro (Hanna Instruments HI
711). C. Colorímetro Checker digital para medição de ferro (Hanna Instruments HI 721). SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 4 Turbidez De acordo com a análise realizada, a amostra apresentou um valor médio para turbidez de 106,7 (Tabela 1), estando totalmente em
desacordo com a portaria de consolidação No 05/2017 do Ministério da Saúde que preconiza um Valor Máximo Permitido (VMP) igual a
5. O alto nível de turbidez está intimamente relacionado à existência de matérias orgânicas e argilas suspensas na água, em que, a agrega-
ção destes componentes dá origem a coloides que interferem na penetração da luz (APHA, 1995; FERREIRA et al., 2015). Outros parâme-
tros, como por exemplo, o tamanho e a geometria das partículas que podem interferir na coloração e dispersão da luz na água, presença
de algas, plâncton, zinco, ferro, manganês e até mesmo areia, sendo que, para comprovar o alto índice de turbidez é necessário realizar
testes quantitativos para liberar a água para o consumo humano (ROBERTO et al., 2017). Pereira-Silva et al. (2011), ao realizarem a avaliação da qualidade da água em microbacias hidrográficas de uma Unidade de Conserva-
ção do Nordeste do estado de São Paulo, observaram que a turbidez foi significativamente superior ao longo de todo período para a mi-
crobacia Mb1, em relação às outras duas microbacias. Relações entre turbidez e sólidos suspensos podem ser indicativas de processos
não-naturais, como despejo de efluentes e atividades agropecuárias (VIDAL et al. 2000). Cor aparente Em relação a cor aparente, a amostra não atendeu ao padrão vigente, apresentando um valor médio de 368 uH (Tabela 1). A Portaria de
consolidação nº 5/ 2017do Ministério da Saúde estabelece para cor aparente o Valor Máximo Permitido de 15 (quinze) uH como padrão
de aceitação para consumo humano. SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 4 Segundo Richtter e Azevedo Netto (2002), a água pura é virtualmente ausente de cor. A presença de substâncias dissolvidas ou em sus-
pensão altera a cor da água, dependendo da quantidade e da natureza do material presente. A cor da água é produzida pela reflexão da luz em partículas minúsculas, denominadas coloides, finamente dispersas de origem predo-
minantemente orgânica e dimensão inferior a 10 µm, relacionando-se com a concentração de carbono orgânico presente no ambiente
aquático. Pode também ser resultado da presença de compostos de ferro e manganês ou do lançamento de diversos tipos de resíduos
industriais. Quando a cor se manifesta em águas subterrâneas, via de regra é resultado da presença destes compostos de ferro e man-
ganês (LIBÂNIO, 2010). Paludo (2010) ao estudar a qualidade da água nos poços artesianos do município de Santa Clara do Sul, observou que todas as amos-
tras apresentaram valor zero com relação a cor. Valores semelhantes foram encontrados por Moura et al (2009), ao analisarem as águas
dos poços artesianos do campus CAVG – UFPEL. Silva et al. (2017) ao estudarem os parâmetros físico-químicos da água utilizada para consumo em poços artesianos na cidade de Remí-
gio-PB, observaram que todas as amostras atenderam ao padrão vigente, com exceção da água coletada no poço 2, que apresentou um
valor de 500 uH. Alcalinidade Total A amostra de água apresentou uma alcalinidade média de 42,67 mg/L de CaCO3, devido a presença de bicarbonatos, encontrando-se
dentro do padrão de potabilidade permitido pela portaria de consolidação de nº 05/2017 (Tabela 1). A alcalinidade nas águas naturais, responsável pela capacidade de neutralização de ácidos, geralmente apresenta como principais res-
ponsáveis: bases conjugadas de ácido carbônico, carbonatos e bicarbonatos; outras bases derivadas do íon amônio e dos ácidos sulfúri-
co e fosfórico também podem contribuir para a alcalinidade (ESTEVES, 2011; PIRATOBA et al., 2017) Silva Filho et al. (2019) ao estudarem a qualidade físico-química e microbiológica da água de poço tubular situado no sitio alegre no
município de Lagoa Seca-PB, encontraram um valor médio de 73 mg/L, encontrando-se dentro do padrão de potabilidade permitido pela
portaria de consolidação de nº 05/2017, que estabelece um valor máximo permitido de 100 mg/L (BRASIL, 2017). A maioria das águas naturais apresentam valores de alcalinidade na faixa de 30 a 500 mg/L de CaCO3. Segundo (Morais, 2008), esse
parâmetro está intimamente associado ao pH e indica que tais amostras apresentam a alcalinidade de bicarbonatos (pH entre 4,5 e 8,2). SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 5 Cloreto Verificou-se que o teor de cloreto foi em média de 104,5 mg/L de Cl- (Tabela 1), estando dentro dos padrões permitidos pela portaria de
consolidação No 05/2017 do Ministério da Saúde que estabelece um teor de 250 mg/L de Cl- como valor máximo permitido para água
potável. O cloreto é outro íon que facilmente se desloca na solução, assim, este íon quando adicionado ao solo por meio da água de irrigação
facilmente pode atingir as águas subterrâneas alterando o padrão de qualidade das mesmas (SILVA et al. 2008; ANDRADE et al. 2009). Outra fonte em potencial de aporte de cloreto ao solo e as águas subterrâneas, nas regiões costeiras, são os aerossóis marinhos (MEIRE-
LES et al. 2007; LUNA et al. 2013). O cloreto é um íon importante nas águas subterrâneas e superficiais, podendo ter origem antrópica e geológica, sendo a lixiviação de
rochas, esgotos domésticos e industriais a sua principal origem (USEPA, 2015). Segundo a CETESB (2012), uma pessoa expele na urina
aproximadamente 4 g de cloreto por dia. O cloreto é um íon importante na produtividade global dos ecossistemas aquáticos, faz parte de
importantes processos fisiológicos como a troca e/o transporte de outros íons para os meios intracelular e extracelular. SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 5 SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 5 Resultados semelhantes foram encontrados por Silva et al. (2018) quando realizaram uma análise físico-química da água utilizada para
consumo nas escolas municipais da zona urbana de Esperança/PB, enquanto que Silva Filho et al. (2019) encontraram resultados con-
traditórios (323,3 mg/L), ao estudarem a qualidade físico-química e microbiológica da água de poço tubular situado no sitio alegre no
município de Lagoa Seca-PB. Dureza total, Dureza de cálcio e magnésio A dureza da água é expressa em mg/L de equivalente em carbonato de cálcio (CaCO3) e pode ser classificada em mole ou branda: < 50
mg/L de CaCO3; moderada: entre 50 mg/L e 150 mg/L de CaCO3; dura: entre 150 mg/L e 300 mg/L de CaCO3; e muito dura: >300
mg/L de CaCO3 (BRASIL, 2014). Baseando-se na classificação citada anteriormente, a amostra apresentou dureza moderada (92,67 mg/L de CaCO3) (Tabela 2). A amos-
tra apresentou uma dureza média de cálcio de 56,20 mg/L de CaCO3 e uma dureza média de magnésio de 36,47 mg/L de CaCO3, es-
tando, portanto, dentro dos padrões estipulados pelo Ministério da Saúde (Tabela 2). De acordo com essa classificação, convém ressaltar que a referida amostra está dentro dos padrões de potabilidade brasileiro, america-
no e da Organização Mundial de Saúde (OMS), que estabelecem o limite de 500 mg/L CaCO3, idêntico ao adotado no Canadá (GUIDELI-
NES FOR CANADIAN DRINKING WATER QUALITY, 2004). De acordo com essa classificação, convém ressaltar que a referida amostra está dentro dos padrões de potabilidade brasileiro, america-
no e da Organização Mundial de Saúde (OMS), que estabelecem o limite de 500 mg/L CaCO3, idêntico ao adotado no Canadá (GUIDELI-
NES FOR CANADIAN DRINKING WATER QUALITY, 2004). De acordo com Esteves (2011), geralmente ambientes com pH próximos à neutralidade apresentam uma dureza mole dado pelas baixas
concentrações de carbonatos, sendo a reação com o CO2 para a produção de bicarbonato desprezível. De acordo com Esteves (2011), geralmente ambientes com pH próximos à neutralidade apresentam uma dureza mole dado pelas baixas
concentrações de carbonatos, sendo a reação com o CO2 para a produção de bicarbonato desprezível. Piratoba et al. (2017), quando realizaram a caracterização de parâmetros de qualidade da água na área portuária de Barcarena, PA,
Brasil, observaram que os teores médios de dureza mudaram entre 16,21 e 17,52 mg L-1 de CaCO3 para o período menos chuvoso e
12,55 a 12,64 mg L-1 de CaCO3 para o período chuvoso, sendo menores para este período. Silva Filho et al. (2019) encontraram uma dureza total de 415 mg/L de CaCO3, ao estudarem a qualidade físico-química e microbiológica
da água de poço tubular situado no sitio alegre no município de Lagoa Seca-PB. Tabela 1. Valores de pH, cor aparente, temperatura, turbidez, alcalinidade total e cloreto de um poço artesiano localizado na zona urbana
da cidade de Remígio/PB. SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 6 Dureza total, Dureza de cálcio e magnésio Parâmetros
Unidades
Poço
pH
-
6,52
Cor Aparente
uH
368
Temperatura
°C
26,3
Turbidez
uT
106,7
Alcalinidade Total
mgCaCO3/L
42,67
Cloreto
mg.L
104,5 Tabela 1. Valores de pH, cor aparente, temperatura, turbidez, alcalinidade total e cloreto de um poço artesiano
da cidade de Remígio/PB. Cloro Total A amostra apresentou uma média de 0,24 mg/L de cloro total (Tabela 2). Os valores encontrados estão dentro do padrão estipulado pela
Portaria de Consolidação no 05/2017 do Ministério da Saúde (BRASIL, 2017), que estipula um valor máximo permitido de 0,5 mg/L. Trindade et al. (2015) ao avaliarem a qualidade água em três escolas públicas da cidade de Macapá, Amapá, observaram valores médios
de cloro residual para o ponto coletado na caixa d’água nas escolas A, B e C de 0,02 ± 0,01; 0,09 ± 0,01 e 0,10 ± 0,03 mg.L-1, respecti-
vamente. Na torneira da cozinha foram: 0,02 ± 0,02, 0,00 ± 0,00 e 0,20 ± 0,01 mg.L-1, respectivamente; e no bebedouro foram: 0,02 ±
0,01, 0,02 ± 0,01 e 0,21 ± 0,02 mg.L-1, respectivamente. Silva Filho et al. (2019) encontraram um valor médio de cloro total de 0,0 mg/L ao estudarem a qualidade físico-química e microbiológica
da água de poço tubular situado no sitio alegre no município de Lagoa Seca-PB. Sólidos Totais Dissolvidos Com relação aos Sólidos Totais Dissolvidos, a amostra de água apresentou um valor médio de 5.957 mg/L (Tabela 2), estando totalmen-
te fora do estipulado pelas normas vigentes. Sólidos totais dissolvidos são constituídos por partículas de diâmetro inferior a 10-3 μm e que permanecem em solução mesmo após a
filtração. A entrada de sólidos na água pode ocorrer de forma natural (processos erosivos, organismos e detritos orgânicos) ou antropo-
gênica (lançamento de lixo e esgotos). O padrão de potabilidade refere-se apenas aos sólidos totais dissolvidos (limite: 1000 mg/L), já
que esta parcela reflete a influência de lançamento de esgotos, além de afetar a qualidade organoléptica da água (BRASIL, 2014). Silva et al. (2017) encontrou resultados satisfatórios ao analisarem os parâmetros físico-químicos da água utilizada para consumo em
poços artesianos na cidade de Remígio-PB. Silva Filho et al. (2019) também encontraram resultados satisfatórios ao estudarem a quali-
dade físico-química e microbiológica da água de poço tubular situado no sitio alegre no município de Lagoa Seca-PB. Condutividade Condutividade
A condutividade elétrica da água do poço foi em média de 911,1 μS/cm a temperatura média de 26,3 oC (Tabela 2). De acordo com
Libânio (2010), águas naturais apresentam usualmente condutividade elétrica inferior a 100 μS/cm, podendo atingir 1000 μS/cm em
corpos d’água receptores de elevadas cargas de efluentes domésticos e industriais. A condutividade elétrica da água do poço foi em média de 911,1 μS/cm a temperatura média de 26,3 oC (Tabela 2). De acordo com
Libânio (2010), águas naturais apresentam usualmente condutividade elétrica inferior a 100 μS/cm, podendo atingir 1000 μS/cm em
corpos d’água receptores de elevadas cargas de efluentes domésticos e industriais. Segundo Boesch (2002) e Esteves (2011), a condutividade elétrica é um parâmetro que pode mostrar modificações na composição dos
corpos d’água, mas não especifica quantidades e componentes. É um parâmetro importante para controlar e determinar o estado e a
qualidade de água (PIÑEIRO DI BLASI et al., 2013; PIRATOBA et al., 2017). Segundo Boesch (2002) e Esteves (2011), a condutividade elétrica é um parâmetro que pode mostrar modificações na composição dos
corpos d’água, mas não especifica quantidades e componentes. É um parâmetro importante para controlar e determinar o estado e a
qualidade de água (PIÑEIRO DI BLASI et al., 2013; PIRATOBA et al., 2017). Silva et al. (2017) avaliando os parâmetros físico-químicos da água utilizada para consumo em poços artesianos na cidade de Remígio-
PB, encontraram uma condutividade que variou de 370,0 a 557,1 μS/cm. Silva et al. (2017) avaliando os parâmetros físico-químicos da água utilizada para consumo em poços artesianos na cidade de Remígio-
PB, encontraram uma condutividade que variou de 370,0 a 557,1 μS/cm. Silva Filho et al. (2019) observaram uma condutividade de 1321,3 μS/cm ao estudarem a qualidade físico-química e microbiológica da
água de poço tubular situado no sitio alegre no município de Lagoa Seca-PB. Silva Filho et al. (2019) observaram uma condutividade de 1321,3 μS/cm ao estudarem a qualidade físico-química e microbiológica da
água de poço tubular situado no sitio alegre no município de Lagoa Seca-PB. Porcentagem de cinzas O teor de cinzas para a amostra da água coletada foi em média de 0,0165 cz (Tabela 2). Silva et al. (2017) encontraram teores que vari-
aram de 0,5816 a 0,8587 cz, para a porcentagem de cinzas a 5g, e variou de 0,1936 a 0,2934 cz para porcentagem de cinzas a 18 g. Vasconcelos et al. (1999) estudando a relação entre as massas úmida, seca e de cinza em materiais biológicos, observaram que a per-
centagem média de cinzas por categoria de alimentos foi de 0,70 para os bulbos, 3,1 para os grãos, 1,5 para os vegetais folhosos e 1,05
para as carnes. Os valores encontrados na literatura para os vegetais radiculares foram de 0,72 (Phillip et al., 1993) e 0,76 (IAEA, 1989). Para os produtos de consumo animal, o valor médio para a percentagem de cinzas obtido neste trabalho foi de 2,1 e na literatura é 2,3
(Phillip et al., 1993). O valor médio da percentagem de cinzas citado na literatura para sucos de frutas é de 0,68 (Phillip et al., 1993) e de
0,61 (IAEA, 1989) sendo também concordantes com os obtidos para as amostras de laranja (0,6). Oliveira et al. (2014), observaram que as geleias diet tiveram os conteúdos de cinzas variando de 0,57% (GD1) a 0,81% (GD3), estando
superiores aos dados de Yuyama et al. (2008) e Polesi et al. (2011) ao caracterizarem geleias diet de cubiu (Solanum sessiliflorum) e
manga, respectivamente. SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 7 4. CONCLUSÕES Conclui-se que de acordo com as análises físico-químicas realizadas, a água é imprópria para o consumo humano, pois os parâmetros
cor, turbidez, sólidos totais dissolvidos e porcentagem de ferro estão totalmente fora do que é preconizado pela Portaria de Consolidação
no 05/2017 do Ministério da Saúde e da resolução de nº 357/2005 do Conselho Nacional do Meio Ambiente. Porcentagem de Ferro Origina-se da dissolução de compostos de rochas e solos. Por ser um dos elementos mais abundantes, o ferro é habitualmente encontra-
do nas águas naturais, superficiais e subterrâneas, apresentando-se na forma insolúvel (Fe+3) e dissolvida (Fe+2), como óxidos, silicatos,
carbonatos, cloretos, sulfatos e sulfitos. A segunda forma é frequente em águas subterrâneas de poços artesianos, e no fundo de lagos e
reservatórios de acumulação onde se verificam baixas concentrações de oxigênio dissolvido (LIBÂNIO, 2010). A porcentagem de ferro encontrada na amostra de água coletada foi de 0,94 mg/L (Tabela 1), estando totalmente foram dos padrões
preconizados pela Portaria de Consolidação no 05/2017 do Ministério da Saúde (BRASIL, 2017), que estipula um valor máximo permitido
de 0,3 mg/L. Este íon, apesar de não ser tóxico traz diversos problemas para o abastecimento público de água, pois confere cor e sabor à água, provo-
cando manchas em roupas e utensílios sanitários, (LIBÂNEO, 2010). Além de causar depósitos e incrustações ao longo das tubulações e
podem estar associados ao aparecimento de bactérias ferruginosas nocivas, quando sua concentração é excessiva (RICHTER; AZEVEDO
NETTO, 2002). Silva Filho et al. (2019) encontraram um valor médio de ferro de 0,1 mg/L ao estudarem a qualidade físico-química e microbiológica da
água de poço tubular situado no sitio alegre no município de Lagoa Seca-PB. SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 7 Tabela 2. Valores de dureza total, dureza de cálcio e magnésio, condutividade elétrica, sólidos totais, porcentagem de cinzas, cloro total e
porcentagem de ferro de um poço artesiano localizado na zona urbana da cidade de Remígio/PB. Tabela 2. Valores de dureza total, dureza de cálcio e magnésio, condutividade elétrica, sólidos totais, porcentagem de cinzas, cloro total e
porcentagem de ferro de um poço artesiano localizado na zona urbana da cidade de Remígio/PB. p
g
p ç
g /
Parâmetros
Unidades
Resultado
Dureza Total
mgCaCO3/L
92,67
Dureza de Cálcio
mgCaCO3/L
56,20
Dureza de Magnésio
mgCaCO3/L
36,47
Condutividade
µS/cm
911,1
Sólidos Totais Dissolvidos
mg/L
5.957
Porcentagem de cinzas
cz
0,0165%
Cloro Total
mg/L de Cl-
0,24
Porcentagem de Ferro
Mg/L
0,94 APHA, AMERICAN PUBLIC HEALTH ASSOCIATION. Standard methods for the examination of water and wastewater. 19a ed. New York:
APHA, WWA, WPCR, p. 1268, 1995. APHA, AMERICAN PUBLIC HEALTH ASSOCIATION. Standard methods for the examination of water and wastewater. 19a ed. New York:
APHA, WWA, WPCR, p. 1268, 1995. ANDRADE, E. M.; AQUINO, D. N.; CRISÓSTOMO, L. A.; RODRIGUES, J. O.; LOPES, F. B. Impacto da lixiviação de nitrato e cloreto no lençol
freático sob condições de cultivo irrigado. Ciência Rural, v. 39, n. 1, p. 88-95, 2009. ANDRADE, E. M.; AQUINO, D. N.; CRISÓSTOMO, L. A.; RODRIGUES, J. O.; LOPES, F. B. Impacto da lixiviação de nitrato e cloreto no lençol
freático sob condições de cultivo irrigado. Ciência Rural, v. 39, n. 1, p. 88-95, 2009. BANDEIRA, P. L.; MENEZES, W. S.; SILVA FILHO, E. D.; SILVA, A. B.; CORDEIRO NETO, R. S.; GONZAGA, F. S. Caracterização físico-química
da água de poços tubulares utilizada para consumo na zona rural da idade de Lagoa Seca-PB. Engenharia Ambiental, v. 15, n. 1, p. 98-
110, 2018. BOESCH, D. F. Challenges and opportunities for science in reducing nutrient over-enrichment of coastal ecosystems. Estuaries, v. 25, n. 4b, p. 886– 900, 2002. BRASIL. RESOLUÇÃO CONAMA no 396, de 3 de abril de 2008. Dispõe sobre a classificação e diretrizes ambienta
das águas subterrâneas e dá outras providências. Publicada no DOU nº 66, de 7 de abril de 2008, Seção 1, pág BRASIL. Manual de Controle da Qualidade da Água para Técnicos que Trabalham em ETAS. Ministério da Saúde, Fundação Nacional de
Saúde. Brasília: Funasa, 112 p., 2014. BRASIL. Portaria de consolidação de nº de 28 de setembro de 2017 do Ministério da Saúde. Dispõe sobre os procedimentos de controle
e de vigilância da qualidade da água para consumo humano e seu padrão de potabilidade. CALHEIROS, D. F.; OLIVEIRA, M. D. Contaminação de corpos d’água nas áreas urbanas de Corumbá e Ladário. ADM – Artigo de Divulgação
na
Mídia,
Embrapa
Pantanal,
Corumbá-MS,
n. 89,
p.1-4. nov. 2006. Disponível
em:
<http://www.riosvivos.org.br/Noticia/Contaminacao+de+corpos+d+agua+na+regiao+de+Corumba/9526>. Acessado em: 20 nov. 2009. CAPP, N.; AYACH, L. R.; SANTOS, T. M. B.; GUIMARÃES, S. T. L. Qualidade da água e fatores de contaminação de poços rasos na área ur-
bana de Anastácio (MS). Geografia Ensino & Pesquisa, v. 16. n. 3, p. 77-91, 2012. CETESB. COMPANHIA AMBIENTAL DO ESTADO DE SÃO PAULO. 2012. Website. http://www.cetesb.sp.gov.br. Acesso: 27/12/2018. CAPP, N.; AYACH, L. R.; SANTOS, T. M. B.; GUIMARÃES, S. T. L. Qualidade da água e fatores de contaminação de poços rasos na área ur-
bana de Anastácio (MS). SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 8 ESTEVES, F. Fundamentos de limnologia. 3. ed. Rio de Janeiro: Interciência, 2011. 826 p. NSTITUTO
BRASILEIRO
DE
GEOGRAFIA
E
ESTATÍSTICA.
Cidades.
2016.
Dispon bge.gov.br/v3/cidades/municipi o/2512705. Acesso: 14/01/2017. http://cidades.ibge.gov.br/v3/cidades/municipi o/2512705. Acesso: 14/01/2017. INSTITUTO ADOLFO LUTZ. Águas. 2008, p. 347-408. In: Métodos químicos e físicos para análises de alimentos. Edição IV. São Paulo: 1ª g g
INSTITUTO ADOLFO LUTZ. Águas. 2008, p. 347-408. In: Métodos químicos e físicos para análises de alimentos. Edição IV. São Paulo: 1ª Edição Digital. SES – CCD – IAL. Secretaria de Estado da Saúde – Coordenadoria de Controle de Doenças. 1020 p. 2008. LIBÂNIO, M. Fundamentos de Qualidade e Tratamento de Água. Campinas/SP. 3ª Edição, Editora Átomo, 494p., 2010. LIBÂNIO, M. Fundamentos de Qualidade e Tratamento de Água. Campinas/SP. 3ª Edição, Editora Átomo, 494p., 2010. S., ANDRADE, E. M., CRISÓSTOMO, L. A., MEIRELES, A. C. M.; AQUINO, D. N. Dinâmica do nitrato e cloreto no s LUNA, N. R. S., ANDRADE, E. M., CRISÓSTOMO, L. A., MEIRELES, A. C. M.; AQUINO, D. N. Dinâmica do nitrato e cloreto no solo e a qualida-
de das águas subterrâneas do distrito de irrigação Baixo Acaraú, CE. Revista Agro@mbiente On-line, v. 7, n. 1, p. 53-62, 2013. de das águas subterrâneas do distrito de irrigação Baixo Acaraú, CE. Revista Agro@mbiente On-line, v. 7, n. 1, p. 53-62, 2013. MEIRELES, A. C. M.; FRISCHKOM, H.; ANDRADE, E. M. Sazonalidade da qualidade das águas do açude Edson Queiroz, bacia do Acaraú, no
Semiárido cearense. Revista Ciência Agronômica, v. 38, n. 1, p. 25-31, 2007. MIRLEAN, N.; MACHADO, M. I.; OSINALDI, G. M.; DEMOLINER, A.; BAISCH, P. O impacto industrial na composição química das águas sub-
terrâneas com enfoque de consumo humano (Rio Grande, RS). Química Nova, v. 28, n. 5, p. 788-791, 2005. MORAIS, P. B. Tratamento físico-químico de efluentes líquidos. Universidade de Campinas, 14p, 2008. q
q
p
p
MOURA, M. H. G. BUENO, R. M.; MILANI, I. C. B.; COLLARES, G. L. Análise das águas dos poços artesianos do campus CAVG - UFPEL. 2ª
Mostra de Trabalhos de Tecnologia Ambiental. Rio Grande do Sul: Pelotas, 2009. OLIVEIRA, E. N. A.; ROCHA, A. P. T.; GOMES, J. P.; SANTOS D. C. Processamento e Caracterização Físico-Química de Geleias Diet de Umbu-
Cajá (Spondias spp.). Biosci. J., Uberlandia, v. 30, n. 4, p. 1007-1016, July/Aug. 2014. PALUDO, D. Qualidade da Água nos Poços Artesianos do Município de Santa Clara do Sul. CENTRO UNIVERSITÁRIO UNIVATES, Monografia,
75p., 2010. PEDROSA, Célio A.; CAETANO, Francisco A. Águas Subterrâneas. Agência Nacional das Águas-ANA, Superint
Hidrológicas
-
SIH,
Brasília,
2002. NSTITUTO
BRASILEIRO
DE
GEOGRAFIA
E
ESTATÍSTICA.
Cidades.
2016.
Dispon em<www.ana.gov.br/gestaoRecHidricos/InfoHidrologicas/aguasSubterr/EstudoAguasSubterraneasANA22-08-02.doc.>Consulta:
02/06/19. PEREIRA-SILVA, E. F. L.; PIRES, J. S. R.; HARDT, E.; SANTOS, J. E.; FERREIRA, W. A. Avaliação da qualidade da água em microbacias hidro-
gráficas de uma Unidade de Conservação do Nordeste do estado de São Paulo, Brasil. Revista Brasileira de Biociência, v. 9, n. 3, p. 371-
381, 2011. In: Environmental Measurements Laboratory Procedures Manual, EML, HASL 300. U.S. Department of Energy
Donald C. Bogen, Earlo Knutson. 29th Edition, Vol I, February 1993. PHILLIP, W. KREY, H. L. B. In: Environmental Measurements Laboratory Procedures Manual, EML, HASL 300. U.S. Department of Energy. Edited by Nancy A Chieco, Donald C. Bogen, Earlo Knutson. 29th Edition, Vol I, February 1993. PIÑEIRO DI BLASI, J. I.; MARTÍNEZ TORRES, J.; GARCÍA NIETO, P. J.; ALONSO FERNÁNDEZ, J. R.; DÍAZ MUÑIZ, C.; TABOADA, J. Analysis and
detection of outliers in water quality parameters from ‘different automated monitoring stations in the Miño river basin (NW Spain). Ecolo-
i
l E
i
i
60
60 66 2013 PIÑEIRO DI BLASI, J. I.; MARTÍNEZ TORRES, J.; GARCÍA NIETO, P. J.; ALONSO FERNÁNDEZ, J. R.; DÍAZ MUÑIZ, C.; TABOADA, J. Analysis and
detection of outliers in water quality parameters from ‘different automated monitoring stations in the Miño river basin (NW Spain). Ecolo-
gical Engineering, v. 60, p. 60–66, 2013. O, H. M. C.; MORALES, G. P.; GONÇALVES, W. G. Caracterização de parâmetros de qualidade da água na área
A, Brasil. Revista Ambiente & Água, v. 12, n. 3, p. 435-456, 2017. PIRATOBA, A. R. A.; RIBEIRO, H. M. C.; MORALES, G. P.; GONÇALVES, W. G. Caracterização de parâmetros de q
portuária de Barcarena, PA, Brasil. Revista Ambiente & Água, v. 12, n. 3, p. 435-456, 2017. PIVELI, R. P.; KATO, M. Características físicas das águas: cor, turbidez, sólidos, temperatura, sabor e odor. In: Qualidade das águas e
poluição: aspectos físico-químicos. Ed. ABES, Rio de Janeiro, 285 p., 2006. p
ç
p
q
p
POLESI, L. F.; MATTA JUNIOR, M. D.; MATSUOKA, C. R.; CEBALLOS, C. H. M.; ANJOS, C. B. P.; SPOTO, M. H. F.; SARMENTO, S.B.S. Caracteri-
zação física e química de geleia de manga de baixo valor calórico. Revista Brasileira de Produtos Agroindustriais, Campina Grande, v. 13,
n. 1, p. 85-90, 2011. A.; NETTO, J. M. Tratamento de água: tecnologia atualizada. São Paulo: Editora Edgard Blücher, 1999. HTER, C. A.; NETTO, J. M. Tratamento de água: tecnologia atualizada. São Paulo: Editora Edgard Blücher, 1999. APHA, AMERICAN PUBLIC HEALTH ASSOCIATION. Standard methods for the examination of water and wastewater. 19a ed. New York:
APHA, WWA, WPCR, p. 1268, 1995. Geografia Ensino & Pesquisa, v. 16. n. 3, p. 77-91, 2012. Ã CETESB. COMPANHIA AMBIENTAL DO ESTADO DE SÃO PAULO. 2012. Website. http://www.cetesb.sp.gov.br. Acesso: 27/12/2018. CIDADE BRASIL. 2017. Município de Remígio. Disponível em: http://www.cidade-brasil.com.br/municipio-remigio.html. Acesso:
14/06/2019. ESTEVES, F. Fundamentos de limnologia. 3. ed. Rio de Janeiro: Interciência, 2011. 826 p. SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 8 FERREIRA, A. C.; ROCHA, L. C.; FIGUEIREDO, M. do A. Análise do índice de qualidade de água na bacia do Córrego do Rio Acima, São João
DelRei/MG. Revista Nacional de Gerenciamento de Cidades, v. 03, n. 15, p. 94-105, 2015. HEALTH CANADA. Guidelines for Canadian drinking water quality. 2004. Disponível em:<www.hc-sc.gc.ca/ewh-semt/pubs/water-
eau/enteric-enterovirus/index-eng.php>. Acesso em: 05 jul. 2019. AEA, International Agency Energy Atomic. Technical Reports Series No 295. Measurements of Radionuclides
ment. A Guidebook. 1989. ment. A Guidebook. 1989. IBGE
–
INSTITUTO
BRASILEIRO
DE
GEOGRAFIA
E
ESTATÍSTICA. Cidades. 2016. Disponível
em:
htt // id d
ibg g
b / 3/ id d
/
i i i /2512705 A
14/01/2017 IBGE
–
INSTITUTO
BRASILEIRO
DE
GEOGRAFIA
E
ESTATÍSTICA. Cidades. 2016. Disponível
em:
http://cidades.ibge.gov.br/v3/cidades/municipi o/2512705. Acesso: 14/01/2017. NSTITUTO
BRASILEIRO
DE
GEOGRAFIA
E
ESTATÍSTICA.
Cidades.
2016.
Dispon HTER, C. A., AZEVEDO NETTO J. M. Tratamento de água: tecnologia atualizada. São Paulo: Edgard Blucher, 332p. RICHTER, C. A.; NETTO, J. M. Tratamento de água: tecnologia atualizada. São Paulo: Editora Edgard Blücher, 1999. RICHTER, C. A., AZEVEDO NETTO J. M. Tratamento de água: tecnologia atualizada. São Paulo: Edgard Blucher, 332p., 2002. ROBERTO, M. C.; GUIMARÃES, A. P. M.; RIBEIRO, J. L.; CARVALHO, A. V.; NERES, J. C. I.; CERQUEIRA, F. B. Avaliação do pH, turbidez e análi-
se microbiológica da água do córrego Guará Velho em Guaraí, Estado do Tocantins. Revista Desafios, v. 4, n. 4, p. 3-14, 2017. ,
;
,
g
g
g
,
R, C. A., AZEVEDO NETTO J. M. Tratamento de água: tecnologia atualizada. São Paulo: Edgard Blucher, 332p., 20 AZEVEDO NETTO J. M. Tratamento de água: tecnologia atualizada. São Paulo: Edgard Blucher, 332p., 2002. ROBERTO, M. C.; GUIMARÃES, A. P. M.; RIBEIRO, J. L.; CARVALHO, A. V.; NERES, J. C. I.; CERQUEIRA, F. B. Avaliação do pH, turbidez e anál
se microbiológica da água do córrego Guará Velho em Guaraí, Estado do Tocantins. Revista Desafios, v. 4, n. 4, p. 3-14, 2017. ROCHA, A. L.; NISHIYAMA, L.; BELLINGIERI, P. H. Qualidade da água nas áreas aterradas com entulho em Uberlândia, Minas Gerais, Brasil. Holos Environment, v.5 n.1, p.81-93. 2005. SANTOS, R. S.; MOHR, T. Saúde e qualidade da água: análises microbiológicas e físico-químicas de águas subterrâneas. Revista Contexto
& Saúde, v. 13, n. 24/25, p. 46-53, 2013. SCURACCHIO, P. A. Qualidade da Água Utilizada para Consumo em Escolas no Município de São Carlos – SP. Dissertação (Mestrado). Araraquara, 2010, 57p. SILVA, R. C. S.; ARAUJO, T. M. Qualidade da água do manancial subterrâneo em áreas urbanas de Feira de Santana (BA). Ciência & Saúde
Coletiva, v. 8, n. 4, p. 1019-1028, 2003. SILVA, M. O.; FREIRE, M. B. G. S.; MENDES, A. M. S.; FERNANDES, M. B.; OLIVEIRA, D. A. Composição do lixiviado de quatro solos do Rio
Grande do Norte irrigados com águas salinas. Revista Caatinga, v. 21, n. 1, p. 189-203, 2008. SILVA, A. B.; BRITO, J. M.; SILVA, R. A.; BRAZ, A. S.; SILVA FILHO, E. D. S. Parâmetros físico-químicos da água utilizada para consumo em
poços artesianos na cidade de Remígio-PB. Águas Subterrâneas, v. 31, n. 2.p. 109-118, 2017. SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 9 SILVA, A. B; BRITO, J. SILVA, A.B. et al.. Águas Subterrâneas - Seção Estudos de Caso e Notas Técnicas, 2019. 10 NSTITUTO
BRASILEIRO
DE
GEOGRAFIA
E
ESTATÍSTICA.
Cidades.
2016.
Dispon M.; DUARTE, J. S.; BRAZ, A. S.; SILVA, R. A.; SILVA FILHO, E. D. Análise físico-química da água utilizada para consumo
nas escolas municipais da zona urbana de Esperança/PB. Biota Amazônia, Macapá, v. 8, n. 3, p. 49-52, 2018. SILVA FILHO, E. D.; SILVA, A. B.; GONZAGA, F. A. S.; MENEZES, W. M. S.; DANTAS, G. M.; SANTOS, J. S. I.; MADUREIRA, I. A. Estudo da quali-
dade físico-química e microbiológica da água de poço tubular situado no sitio alegre no município de Lagoa Seca-PB. Águas Subterrâneas,
v. 33, n. 1, 8p., 2019. p
p
ç /
,
p ,
,
, p
,
SILVA FILHO, E. D.; SILVA, A. B.; GONZAGA, F. A. S.; MENEZES, W. M. S.; DANTAS, G. M.; SANTOS, J. S. I.; MADUREIRA, I. A. Estudo da quali-
dade físico-química e microbiológica da água de poço tubular situado no sitio alegre no município de Lagoa Seca-PB. Águas Subterrâneas,
v. 33, n. 1, 8p., 2019. STEIN, P.; DINIZ FILHO, J. B.; LUCENA, L. R. F.; CABRAL, N. M. T. Qualidade das águas do aquífero Barreiras no setor sul de Natal e norte
de Parnamirim, Rio Grande do Norte, Brasil. Revista Brasileira de Geociências, v. 42 (Suppl 1), p. 226-237, 2012. TRINDADE, G. A.; SÁ-OLIVEIRA, J. C.; SILVA, E. S. Avaliação da qualidade da água em três escolas públicas da cidade de Macapá, Amapá. Biota Amazônia, v. 5, n. 1, p. 116-122, 2015. TRINDADE, G. A.; SÁ-OLIVEIRA, J. C.; SILVA, E. S. Avaliação da qualidade da água em três escolas públicas da cidade de Macapá, Amapá. Biota Amazônia, v. 5, n. 1, p. 116-122, 2015. USEPA. UNITED STATES. Environmental Protection Agency - USEPA. Secondary maximum contaminant levels: a strategy for drinking water
quality and consumer acceptability. 2015. Disponível em: http://www.waterrf.org/PublicReportLibrary/4537.pdf. Acesso em:
27/06/2019. Biota Amazônia, v. 5, n. 1, p. 116 122, 2015. USEPA. UNITED STATES. Environmental Protection Agency - USEPA. Secondary maximum contaminant levels: a strategy for drinking water
quality and consumer acceptability. 2015. Disponível em: http://www.waterrf.org/PublicReportLibrary/4537.pdf. Acesso em:
27/06/2019. VASCONCELLOS, L. M. H.; LAURIA, D. C.; SILVA, L. H. C. Relação entre as massas úmida, seca e de cinza em materiais biológicos - uma
ferramenta para amostragens em campo e análise de amostras. Nota Técnica. Química Nova, v. 22, n. 6, p. 889-893, 1999. VIDAL, M.; LÓPEZ, A.; SANTOALLA, M.C.; VALLES, V. VASCONCELLOS, L. M. H.; LAURIA, D. C.; SILVA, L. H. C. Relação entre as massas úmida, seca e de cinza em materiais biológicos - uma
ferramenta para amostragens em campo e análise de amostras. Nota Técnica. Química Nova, v. 22, n. 6, p. 889-893, 1999.
VIDAL, M.; LÓPEZ, A.; SANTOALLA, M.C.; VALLES, V. Factor analysis for the study of water resources contamination due to the use of live-
stock slurries as fertilizer. Agricultural Water Management, v. 45, n. 1, p. 1-15, 2000.
YUYAMA, L. K. O.; PANTOJA, L.; MAEDA, R. N.; AGUIAR, J. P. L.; SILVA, S. B. Desenvolvimento e aceitabilidade de geleia dietética de cubiu
(Solanum sessiliflorum Dunal). Ciência e Tecnologia de Alimentos, Campinas, v.28, n.4, p.929-934, 2008. A, L.; MAEDA, R. N.; AGUIAR, J. P. L.; SILVA, S. B. Desenvolvimento e aceitabilidade de geleia dietética de cubiu
nal). Ciência e Tecnologia de Alimentos, Campinas, v.28, n.4, p.929-934, 2008. NSTITUTO
BRASILEIRO
DE
GEOGRAFIA
E
ESTATÍSTICA.
Cidades.
2016.
Dispon Factor analysis for the study of water resources contamination due to the use of live-
stock slurries as fertilizer. Agricultural Water Management, v. 45, n. 1, p. 1-15, 2000. YUYAMA, L. K. O.; PANTOJA, L.; MAEDA, R. N.; AGUIAR, J. P. L.; SILVA, S. B. Desenvolvimento e aceitabilidade de geleia dietética de cubiu
(Solanum sessiliflorum Dunal). Ciência e Tecnologia de Alimentos, Campinas, v.28, n.4, p.929-934, 2008.
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English
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Static reinforcement and vibration reduction of structures using topology optimization
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Mechanics & industry
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Received: 23 August 2022 / Accepted: 13 January 2023 Abstract. This paper presents a topology optimization formulation based on the Solid Isotropic Material with
Penalization (SIMP) method and solved by the Modified Optimality Criteria (MOC) algorithm. It addresses
mechanical design problems such as structural reinforcement adding elastic material or vibration reduction
using viscoelastic layers. The aim is thus to attach on a pre-existing given structure a design domain in order
to improve the behavior of this elastic structure, according to an objective function. This can be useful when
one wants to use, for example, additive manufacturing to reinforce a pre-existing structure or to maximize
structural damping. Two objective functions are tested in both linear statics and dynamics: a compliance
based objective function and a displacement based one. In the dynamic case, written in the frequency domain,
the two proposed objective functions include the viscoelastic material model (a Zener fractional derivative
one) used to fill the design domain. The displacement criteria is developed using a general formula able
to take into account as many degree of freedom as necessary. Finally, some applications based on beams
and CubeSat-like structures are shown in this article. The proposed examples show that in both statics and
dynamics, the optimization of a restrained design domain attached to an existing structure can improve its
behavior: stiffness improvement or vibration reduction. Keywords: Static Reinforcement / Vibration Reduction / Topology optimization / Viscoelasticity * e-mail: antoine.legay@lecnam.net This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. tributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0)
stricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://cre
which permits unrestricted use, distribution, and reproduction in any medium, provided the original w Static reinforcement and vibration reduction of structures using
topology optimization Laboratoire de M´ecanique des Structures et des Syst`emes Coupl´es (LMSSC), Structural Mechanics and Coupled
System Laboratory, Conservatoire National des Arts et M´etiers (Cnam), 292 rue Saint-Martin, 75003 Paris, France Mechanics
&Industry Mechanics & Industry 24, 14 (2023)
© S. Burri and A. Legay, Published by EDP Sciences, 2023
https://doi.org/10.1051/meca/2023003 Mechanics & Industry 24, 14 (2023)
© S. Burri and A. Legay, Published by EDP Sciences, 2023
https://doi.org/10.1051/meca/2023003 Mechanics & Industry 24, 14 (2023)
© S. Burri and A. Legay, Published by EDP Sciences, 2023
https://doi.org/10.1051/meca/2023003 Available online at:
www.mechanics-industry.org Available online at:
www.mechanics-industry.org Available online at:
www.mechanics-industry.org REGULAR ARTICLE REGULAR ARTICLE 1 Introduction and motivation formulation) and stiffness matrices of each substructure
are considered separately and assembled afterwards in
the global system. During the optimization process, the
discretized operators of Ω1 remain constant (stiffness
and mass matrices) while they vary for Ω2 according
to its material density distribution. In order to perform
this topology optimization, the design variables are the
elemental material density distribution in Ω2. The structural mass is often a main issue which has to
be cautiously considered during the design process of
a mechanical structure, for instance for a vehicle or an
equipment in the aerospace industry. This is a structural
optimization problem where one wants to reduce the mass
regarding to another criterion such as, non-exhaustively:
stiffness maximization usually expressed as a compliance
minimization problem, displacement lowering or vibration
reduction [1]. Such objectives can be effectively addressed
by using a topology optimization approach [2–11]. One can therefore imagine to reinforce Ω1 using an
efficient manufacturing process such as additive manu-
facturing [19] to build Ω2. Concerning the purpose of vibration reduction, it is per-
formed in the frequency domain and damping is taken into
account thanks to viscoelastic materials [20–22]. Among
the various rheological existing models of viscoelastic
behavior, the four-parameter Zener fractional-derivative
model is used in this work. It allows to efficiently repre-
sent the frequency-dependence of the damping properties
with only four parameters: correlations between numerical
and experimental tests show a good accuracy [23]. The present article uses an algorithm based on the well
known SIMP-method [12–14] in order to focus on these
objectives and tries to address the situation when one
wants to add to a pre-existing given structure Ω1, a sec-
ond domain Ω2 with the aim of either reinforcing the
pre-existing structure, lowering the displacement of a cho-
sen area, or reducing vibrations of Ω1 using the design
domain Ω2. Although few papers exist on the subject
of structural reinforcement using topology optimization
[15–18], the idea of considering separated substructures is
newly addressed here. This means that mass (for dynamic [
]
The next part of the present paper is dedicated to
the building of the discretized numerical model of the
physical problem, including a sensitivity analysis of the
solution with respect to the design variables, namely the
elemental material densities in the design domain. The S. Burri and A. 1 Introduction and motivation Legay: Mechanics & Industry 24, 14 (2023) 2 2 Ω2
Ω1
xe = 0
xe = 1
Interface I
⃗F
Fig. 1. Geometry and finite element mesh of the problem
composed of a given structure Ω1 and a design domain Ω2. third section details the topology optimization problem
formulation, introducing the two different used objective
functions: compliance and displacement based objective
functions. The fourth section presents a selection of appli-
cations corresponding to a static reinforcement and to a
vibration reduction. The numerical implementation is done using Python
and Fortran languages. Gmsh [24] is used for pre-
treatment (meshing) and post-treatment (plotting). 2.1
Geometry Fig. 1. Geometry and finite element mesh of the problem
composed of a given structure Ω1 and a design domain Ω2. The studied problem is composed of a purely elastic given
domain Ω1 and a design domain Ω2 which can be either
purely elastic or viscoelastic (Fig. 1). The whole problem
is assumed to be linear. The interface between the two
domains is denoted by I. In the static case, the aim is
to reinforce the given structure Ω1 subjected to the static
load ⃗F. In the dynamic case, considered in the frequency
domain, the aim is to dissipate energy in order to protect
the given structure Ω1 from spurious vibrations due to the
harmonic excitation ⃗F. The first case can be degenerated
from the second by taking the angular frequency ω to zero. This last expression is the one that is implemented to
describe the material contained in the sub-part Ω2. 2.3
Implementation of design variables For the purpose of topology optimization, the design
domain Ω2
contains a set of design variables x =
[x1, x2, . . . , xn]T which is the set of elemental material
densities (n is the number of elements in Ω2-domain’s
mesh). These densities are such that 0 ≤xe ≤1 where 0
is the lower bound of xe corresponding to an absence of
material in the element and 1 is the upper bound meaning
a presence of material [26] (Fig. 1). A design domain Ω2 is then attached to Ω1, in which
each element e has a material density variable xe. The aim
is to find the best material distribution in Ω2 according to
an objective function associated to constrains functions. This optimization problem is solved using a topology
optimization procedure described in Section 3.1. An efficient way to use this is the well-known penal-
ization algorithm SIMP from Bendsøe [12] and Zhou &
Rozvany [13]. However, a derivative from this law estab-
lished by Sigmund [27] and called the Modified SIMP-law
is used here. This formulation has several advantages,
including the fact that it avoids stiffness (or mass) matrix
to become singular. 2.5
Sensitivity analysis of the solution with respect to
the design variables Using the finite element method, the dynamic discretized
system of the whole problem is written in the frequency
domain as: In the following work, a topology optimization procedure
is used (Sect. 3.1). This algorithm needs to compute the
sensibility of the solution according to the design variables
xe. Since only matrix S2(ω, x) is depending on design
variables, the derivative of equation (11) with respect to
the design variable xe leads to the following equation: K −ω2M
U = F,
(6) (6) where K is the complex stiffness matrix (taking into
account viscoelastic terms), M is the mass matrix, ω
is the angular frequency and F represents the external
nodal forces. The design variable vector x, composed of
elemental material densities in design sub-domain Ω2 is
introduced in order to use a topology optimization proce-
dure. Using a sub-domain decomposition between domain
Ω1 and Ω2, the global discretized system equation (6) is
written: ∂S2(ω, x)
∂xe
U(x) + S(ω, x)∂U(x)
∂xe
= 0. (12) (12) The sensitivity of the solution with respect to the design
variable xe is then given by: ∂U(x)
∂xe
= −S−1(ω, x)∂S2(ω, x)
∂xe
U(x),
(13) (13) "K111
K11I
012
K1I1
K1II + K2II(ω, x)
K2I2(ω, x)
021
K22I(ω, x)
K222(ω, x)
#
−ω2
"M111
M11I
012
M1I1
M1II + M2II(x)
M2I2(x)
021
M22I(x)
M222(x)
#! ×
"U 1(x)
U I(x)
U 2(x)
#
=
"F 1
0I
02
#
,
(7) "K111
K11I
012
K1I1
K1II + K2II(ω, x)
K2I2(ω, x)
021
K22I(ω, x)
K222(ω, x)
#
−ω2
"M111
M11I
012
M1I1
M1II + M2II(x)
M2I2(x)
021
M22I(x)
M222(x)
#! ×
"U 1(x)
U I(x)
U 2(x)
#
=
"F 1
0I
02
#
,
(7) where U(x) is the solution of equation (11). In this last
equation, the derivative of the S2(ω, x) matrix needs to
be computed. (7) 2.2
Viscoelastic model In this work, the viscoelastic behavior of the dissipa-
tive material in Ω2 is modeled using a four-parameter
Zener fractional-derivative viscoelastic model, introduced
by Bagley and Torvik [20]. For that purpose, the Young
modulus is considered as a complex one such as: The effective Young modulus in the element e of Ω2 is
expressed as a function of the elemental material densities
xe such that: E⋆
eff.(ω, xe) =
hEmin
E0
+ xp
e
1 −Emin
E0
i
E⋆(ω),
(4) E⋆(ω) = E′(ω) + iE′′(ω),
(1) (1) (4) where E′(ω) is the storage modulus, E′′(ω) the loss mod-
ulus and i2 = −1. Both moduli can be expressed as a
function of the static stiffness E0, the dynamic stiffness
E∞, the relaxation time τ and the fractional order of the
derivative α [25], which represent the four parameters of
the model: where E⋆(ω) is given by equation (3), Emin = ϵE0 is the
imposed lower bound of the effective Young modulus (ϵ <
1) and p is a penalization factor. In the same way, the
effective volumetric mass density in the element e of Ω2 is
expressed as a function of the elemental material densities
xe such that: E′(ω) = E0 + (E0 + E∞)(ωτ)α cos( απ
2 ) + E∞(ωτ)2α
1 + 2(ωτ)α cos( απ
2 ) + (ωτ)2α
,
E′′(ω) =
(E∞−E0)(ωτ)α sin( απ
2 )
1 + 2(ωτ)α cos( απ
2 ) + (ωτ)2α . (2 ρeff.(xe) =
hρmin
ρ0
+ xm
e
1 −ρmin
ρ0
i
ρ0,
(5) (5) where ρ0 is the volumetric mass density of the material
used for the design domain Ω2, ρmin = ϵρ0 is the imposed
lower bound of the effective volumetric mass density and
m is a penalization factor. In this work, the penalization
factor p related to the Young modulus is set to 3 and the
penalization factor m related to the volumetric mass is set
to 1 [5]; the ϵ coefficient is chosen to be 10−3. (2)
h (2)
Finally, the Young modulus can be expressed using the
four above parameters: E⋆(ω) = E0 + E∞(iωτ)α
1 + (iωτ)α
. (3) E⋆(ω) = E0 + E∞(iωτ)α
1 + (iωτ)α
. (3) S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 3 3 2.6
Derivatives of the stiffness and mass matrices of
the design domain The derivative of S2(ω, x) with respect to xe involves
the derivative of the stiffness and mass matrices of Ω2,
denoted respectively by K2(ω, x) and M2(x): where 1, 2 and I denote respectively sub-domain Ω1, sub-
domain Ω2 and the interface I between Ω1 and Ω2. The
stiffness matrix K2(ω, x) is complex and frequency depen-
dent due to the viscoelastic material of sub-domain Ω2
(Eq. (3)). Moreover, this last matrix depends on the design
variable vector x. where 1, 2 and I denote respectively sub-domain Ω1, sub-
domain Ω2 and the interface I between Ω1 and Ω2. The
stiffness matrix K2(ω, x) is complex and frequency depen-
dent due to the viscoelastic material of sub-domain Ω2
(Eq. (3)). Moreover, this last matrix depends on the design
variable vector x. ∂S2(ω, x)
∂xe
= ∂K2(ω, x)
∂xe
−ω2 ∂M2(x)
∂xe
. (14) (14) The stiffness matrix K2(ω, x) can be expressed as the
assembly of the elemental stiffness matrices kj(ω, xj) over
the n elements of Ω2-domain mesh: In order to simplify the notations, the following matri-
ces are introduced: S1(ω) =
"K111
K11I
012
K1I1
K1II
0I2
021
02I
022
#
−ω2
"M111
M11I
012
M1I1
M1II
0I2
021
02I
022
#
,
(8)
S2(ω, x) =
"011
01I
012
0I1 K2II(ω, x) K2I2(ω, x)
021 K22I(ω, x) K222(ω, x)
#
−ω2
"011
01I
012
0I1 M2II(x) M2I2(x)
021 M22I(x) M222(x)
#
,
(9) S1(ω) =
"K111
K11I
012
K1I1
K1II
0I2
021
02I
022
#
−ω2
"M111
M11I
012
M1I1
M1II
0I2
021
02I
022
#
,
(8) K2(ω, x) = An
j=1kj(ω, xj). (15) K2(ω, x) = An
j=1kj(ω, xj). (15) (15) The elemental stiffness matrix kj(ω, xj) can be written as kj(ω, xj) = E⋆
eff.(ω, xj)k0
j,
(16) S2(ω, x) =
"011
01I
012
0I1 K2II(ω, x) K2I2(ω, x)
021 K22I(ω, x) K222(ω, x)
#
−ω2
"011
01I
012
0I1 M2II(x) M2I2(x)
021 M22I(x) M222(x)
#
,
(9 S2(ω, x) =
"011
01I
012
0I1 K2II(ω, x) K2I2(ω, x)
021 K22I(ω, x) K222(ω, x)
#
−ω2
"011
01I
012
0I1 M2II(x) M2I2(x)
021 M22I(x) M222(x)
#
,
(9)
kj(ω, xj) = E⋆
eff.(ω, xj)k0
j,
(16)
where k0
j is the stiffness matrix of element j computed
with a unit Young modulus. 3.2.1
Dynamic case The compliance is given in terms of the complex displace-
ment field U(x), and the stiffness and mass matrices of
the problem as [30,31] 3 Topology optimization problem
formulation c(ω, x) = |U
T (x)
S1(ω) + S2(ω, x)
U(x)|,
(28) (28) 3.2
Compliance based objective function The elemental stiffness and mass matrices obtained
with a unit Young modulus and a unit volumetric mass
density are computed once at the beginning of the com-
putation and stored in order to be used later on during
the topology optimization process. 2.6
Derivatives of the stiffness and mass matrices of
the design domain Thus, the derivative of
M2(x) with respect to xe is given by ∂M2(x)
∂xe
= An
j=1
∂ρeff.(xj)
∂xe
m0
j,
(21) (21) The iterative process is stopped until either a conver-
gence of the objective function is reached, meaning when
the difference of the elemental densities between 2 con-
secutive iterations is less than a fixed relative criterion
(noted as ζ in this work); or when the maximum number
of iterations is reached (noted as nmax in this work). with ∂ρeff.(xj)
∂xe
= mxm−1
e
1 −ρmin
ρ0
ρ0δej. (22) (22) Since m = 1 in this work, the simplification gives Since m = 1 in this work, the simplification gives ∂ρeff.(xj)
∂xe
= (ρ0 −ρmin)δej. (23) (23) 2.6
Derivatives of the stiffness and mass matrices of
the design domain Thus, the derivative of
K2(ω, x) with respect to xe is given by (16) )
where k0
j is the stiffness matrix of element j computed
with a unit Young modulus. Thus, the derivative of
K2(ω, x) with respect to xe is given by 9)
where k0
j is the stiffness matrix of element j computed
with a unit Young modulus. Thus, the derivative of
K2(ω, x) with respect to xe is given by (9) ∂K2(ω, x)
∂xe
= An
j=1
∂E⋆
eff.(ω, xj)
∂xj
k0
e,
(17) (17) where S1(ω) does not depend on x and represents the con-
tribution of Ω1, while S2(ω) depends on x and represents
the contribution of the design domain Ω2. with g
The global dynamic equation (Eq. (6)) then becomes ∂E⋆
eff.(ω, xe)
∂xj
= pxp−1
e
1 −Emin
E0
E⋆(ω)δej,
(18)
S1(ω) + S2(ω, x)
U(x) = F. (10) (18) (10) where δej is the Kronecker symbol (no implicit sum-
mation). The same development is applied to the mass
matrix. The mass matrix M2(x) is expressed as the assem-
bly of the elemental mass matrices mj(xj) over the n By denoting S(ω, x) = S1(ω) + S2(ω, x), the equation can
be written as S(ω, x)U(x) = F. (11) (11) ( )
bly of the elemental mass matrices mj(xj) over the n S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 4 4 elements of Ω2-domain mesh: elements of Ω2-domain mesh: M2(x) = An
j=1mj(xj). (19) (19) A density filtering strategy [3] is used to avoid checker-
board patterns. This minimization problem is solved using
a modified-SIMP algorithm (see Sect. 2.3) associated to a
solver called Modified Optimality Criteria (MOC) method
[16] which is an extension of the Optimality Criteria (OC)
method [14,28]. This algorithm has been chosen thanks to
its capability to be adjustable depending on the optimiza-
tion problem to solve. Also, it is much more suitable than
OC due the modified parameter introduced for dealing
with dynamic criteria, and relatively easier to implement
and analyze compare to other complex algorithms such as
MMA [29]. The elemental mass matrix mj(xj) can be written as The elemental mass matrix mj(xj) can be written as mj(xj) = ρeff.(xj)m0
j,
(20) (20) where m0
j is the mass matrix of element j computed with
a unit volumetric mass density. 3.2.2 Static case The compliance based objective function in the static
case (denoted by cs(x)) can be derived from the previous
section by taking ω = 0: This expression can be rewritten as: This expression can be rewritten as: cs(x) = c(0, x) = |U
T (x)
S1(0) + S2(0, x)
U(x)|,
(36) (x) = c(0, x) = |U
T (x)
S1(0) + S2(0, x)
U(x)|,
(36)
re
∂d(ω, x)
∂xe
= −U
T (x)∂S2
T (x)
∂xe
Y + Y
T ∂S2(x)
∂xe
U(x), (45) ∂d(ω, x)
∂xe
= −U
T (x)∂S2
T (x)
∂xe
Y + Y
T ∂S2(x)
∂xe
U(x), (45) where where Y is the solution of the following system: where Y is the solution of the following system: S1(0) = K1,
(37) (37) S(x)Y = βU(x). (46) (46) This equation has to be solved for each topology optimiza-
tion iterative step, while equation (45) gives the sensitivity
of the displacement based objective function: This equation has to be solved for each topology optimiza-
tion iterative step, while equation (45) gives the sensitivity
of the displacement based objective function: S2(0, x) = K2(0, x). (38) (38) In this case, S(0, x) = S(0, x) since S2(0, x) is a real
matrix. The derivative of g(0, x) with respect to xe
becomes: ∂d(ω, x)
∂xe
= −2ℜ
U
T (x)∂S2
T (x)
∂xe
Y
,
(47) (47) ∂g(0, x)
∂xe
= −U
T ∂K2(0, x)
∂xe
U(x),
(39) ∂g(0, x)
∂xe
= −U
T ∂K2(0, x)
∂xe
U(x),
(39)
where ℜis the real part of the imaginary number. = −U
T ∂K2(0, x)
∂xe
U(x),
(39)
where ℜis the real part of the imaginary number. (39) (39)
where ℜis the real part of the imaginary number. 3.3.1
Dynamic case For some applications, it can be relevant to use a local
criterion (e.g. when focusing on a targeted component
embedded in a the structure Ω1), such as the minimization
of the displacement of a local point (or set of points). The
following displacement based function given in terms of
the displacement field U(x) [32,33] is then introduced: ∂g(ω, x)
∂xe
= ∂U
T (x)
∂xe
S(ω, x)U(x) + U(x)T S(ω, x)∂U(x)
∂xe
+U
T (x)∂S2(ω, x)
∂xe
U(x). (32) (32) Using equation (13), this equation becomes Using equation (13), this equation becomes d(ω, x) = U
T (x)βU(x),
(41) (41) ∂g(ω, x)
∂xe
= −U
T (x)∂S2(ω, x)
∂xe
S
−1(ω, x)S(ω, x)U(x). (33 where β is a diagonal localization matrix which can
be defined by (using the Kronecker symbol δij with no
implicit summation): where β is a diagonal localization matrix which can
be defined by (using the Kronecker symbol δij with no
implicit summation): (33) It can be rewritten as: βij = biδij,
(42) (42) ∂g(ω, x)
∂xe
= −U
T ∂S2(ω, x)
∂xe
X,
(34) (34) The coefficient bi is prescribed by the user (0 or 1)
depending on whether one wants to consider the degree of
freedom (dof) i or not. The coefficient bi is prescribed by the user (0 or 1)
depending on whether one wants to consider the degree of
freedom (dof) i or not. where X is the solution of the following system: where X is the solution of the following system: (
)
The derivative of d(ω, x) with respect to xe is S(ω, x)X = S(ω, x)U(x). (35) (35) ∂d(ω, x)
∂xe
= ∂U
T (x)
∂xe
βU(x) + U
T (x)β ∂U(x)
∂xe
. (43) (43) The system of equation (35) has to be solved for each
iteration of the topology optimization loop, but not for
each design variable xe. Once the sensitivity of the g(x)
function with respect to xe is obtained, the compliance
sensitivity is given by equation (31). Using equation (13), this last equation becomes ∂d(ω, x)
∂xe
= −U
T (x)∂S2
T (ω, x)
∂xe
S
−1(ω, x)βU(x)
−U
T (x)βS−1(ω, x)∂S2(ω, x)
∂xe
U(x). (44) 3.1 Minimization problem where U
T (x) denotes the transpose-conjugate of the U(x)
vector and |.| denotes the norm of a complex number. By
introducing the following g(ω, x) complex function where U
T (x) denotes the transpose-conjugate of the U(x)
vector and |.| denotes the norm of a complex number. By
introducing the following g(ω, x) complex function In this work, the aim of the topology optimization is to
find the material density set of design variables ˜xω mini-
mizing the objective function f(ω, x) for a given angular
frequency ω according to constraints: ˜xω = Argmin
x
f(ω, x),
(24)
g(ω, x) = U
T (x)
S1(ω) + S2(ω, x)
U(x),
(29) (24)
g(ω, x) = U
T (x)
S1(ω) + S2(ω, x)
U(x),
(29) ˜xω = Argmin
x
f(ω, x),
(24) (29) (24) such that the compliance can be expressed as a function of g(ω, x): 5)
the compliance can be expressed as a function of g(ω, x): γv0 ≤0,
(25)
the compliance can be expressed as a function of g(ω, x): v(x) −γv0 ≤0,
(25)
0 ≤xe ≤1, ∀e ∈[1, n],
(26)
the compliance can be expressed as a function of g(ω, x):
c(ω, x) = (g(ω, x)g(ω, x))
1
2 . (30) v(x) −γv0 ≤0,
(25)
the compliance can be expressed as a func (25) (26)
c(ω, x) = (g(ω, x)g(ω, x))
1
2 . (30) 0 ≤xe ≤1, ∀e ∈[1, n],
(26)
c(ω, x) = (g(ω, x)g(ω, x))
1
2 . (30) (27)
The sensitivity of c(ω, x) with respect to xe is then given
by: (27)
The sensitivity of c(ω, x) with respect to xe is then given
by: S(ω, x)U(x) = F, where v(x) is the volume of material in Ω2 associated
to the distribution of material density x, v0 is the total
volume of Ω2 and γ is the final target of material volume
ratio in Ω2, chosen by the user. Two different objective
functions are considered in this work. The first function
is based on the compliance (denoted by c(ω, x)) while ∂c(ω, x)
∂xe
= 1
2(g(ω, x)g(ω, x))−1
2
∂g(ω, x)
∂xe
g(ω, x)
+g(ω, x)∂g(ω, x)
∂xe
. (31) (31) S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 5 This last expression involves the sensitivity of g(ω, x)
with respect to xe, which can be computed from equa-
tion (29): where E⋆(0) = E0 in the expression of equation (3). 4.1.1 Reinforcement of a 3D cantilever beam This example is adapted from the cantilever beam from
[34] and is composed of an elastic plate (Ω1) surrounded
by two layers which compose Ω2, as shown in Figure 2. The beam is fully clamped on its left side while a verti-
cal load of 100 N is applied on the right side of Ω1. The
structure is discretized using 8-node hexahedral elements:
2160 in Ω1 and 8640 in Ω2, leading to a total of 10,800 ele-
ments and 37,719 degrees of freedom. The chosen point for
the local displacement minimization is point A (Fig. 2), so
matrix β only has 3 unit terms corresponding to the 3 dofs
associated to the node A. The optimum solution for both
objective functions, static compliance cs(x) and displace-
ment based function ds(x), are compared. The material for
both domains Ω1 and Ω2 is aluminum whose properties
are given in Table 1. g
The considered initial structure is shown in Figure 5
and is composed of a skeleton (Ω1) standing on its 4
clamped feet. A vertical surface load is applied on the
upper surface. Design domain Ω2 is chosen to be the
volume resulting from the 6 faces of the cube times the
thickness of the skeleton in the third dimension. The struc-
ture is discretized using 8-node hexahedral elements: 2808
in Ω1 and 9504 in Ω2, leading to a total number of 12,312
elements and 49,560 degrees of freedom. The material for
both domains Ω1 and Ω2 is aluminum whose properties are
given in Table 1. The two criteria, compliance cs(x) and
displacement based objective functions ds(x), are used. The chosen point for the local displacement minimization
is one of the upper corner (point A), so matrix β only
has 3 unit terms corresponding to the 3 dofs associated to
this node A. The target for the final volume ratio is set
to γ = 25% of the total volume of Ω2, the stop criterion
ζ is empirically set to 1% and the maximum number of
iterations nmax is set to 200. In this case, the target for the final volume ratio is set
to γ = 25% of the total volume of Ω2, the stop criterion ζ
is empirically set to 2.5% and the maximum number nmax
of iterations is set to 100. 4 Applications The application addressed in this section concerns a
CubeSat-like structure which is a standard format cre-
ated by Puig-Suari and Twiggs [35,36] for the sake of
university projects, in order to send small satellites into
space. The specifications for the main configuration is
called “1U” and is a 10 × 10 × 10 cm cube whose weight
can not exceed 1.33 kg. Therefore, it seems appropriate to
use topology optimization on an initial CubeSat skeleton
in order to optimize the stiffness of the overall structure
while controlling the mass of the added material. 3.3.2
Static case and the static compliance sensitivity is then given by The displacement based function in the static case
(denoted by ds(x)) can be derived from the previous
section by taking ω = 0: ∂cs(x)
∂xe
= −U
T ∂K2(0, x)
∂xe
U(x). (40) (40) ds(x) = d(0, x) = U T (x)βU(x),
(48) ssion of equation (3). ds(x) = d(0, x) = U T (x)βU(x),
(48) where E⋆(0) = E0 in the expression of equation (3). where E⋆(0) = E0 in the expression of equation (3). where E⋆(0) = E0 in the expression of equation (3). (48) 6
S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 6
S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 6 Ω1
Ω2
⃗F
A
30 cm
4 cm
4 cm
2 cm
10 cm
Fig. 2. 3D cantilever beam (Ω1) reinforced by a design domain
(Ω2). )
)
)
Ω1
Ω2
⃗F
A
30 cm
4 cm
4 cm
2 cm
10 cm
Fig. 2. 3D cantilever beam (Ω1) reinforced by a design domain
(Ω2). where U(x) is the solution of:
(K1 + K2(0, x))U(x) = F. (49)
The sensitivity of ds(x) with respect to xe becomes:
∂d(0, x)
∂xe
= −2U T (x)∂K2
T (0, x)
∂xe
Y
(50)
where Y is the solution of the following system:
K(x)Y = βU(x). (51)
Ω1
Ω2
⃗F
A
30 cm
4 cm
4 cm
2 cm
10 cm
Fig. 2. 3D cantilever beam (Ω1) reinforced by a design domain
(Ω2). where U(x) is the solution of: where U(x) is the solution of: (K1 + K2(0, x))U(x) = F. (49) The sensitivity of ds(x) with respect to xe becomes: The sensitivity of ds(x) with respect to xe becomes: ∂d(0, x)
∂xe
= −2U T (x)∂K2
T (0, x)
∂xe
Y
(50) where Y is the solution of the following system: where Y is the solution of the following system: Fig. 2. 3D cantilever beam (Ω1) reinforced by a design domain
(Ω2). K(x)Y = βU(x). (51) (51) 4.1.1 Reinforcement of a 3D cantilever beam The optimal final shapes obtained by minimizing cs(x)
and ds(x) are denoted respectively by ˜xc and ˜xd; they are
given in Figure 3. One can see that the final shapes are
quite similar, with only few differences on a local aspect. In both cases, computations have converged towards a
physical shape. On a numerical aspect, the evolution of
both objective functions (Fig. 4) show that a convergence
is reached around 80 iterations for both criteria. In the
final shape for solution ˜xc, there are: The optimal final shapes obtained by minimizing cs(x)
and ds(x) are denoted respectively by ˜xc and ˜xd; they are
given in Figure 6. One can observe two clearly defined
reinforcement shapes depending on the objective func-
tion. The evolution of the objective functions are given
in Figure 7. It shows that the criteria ζ is reached at
around 100 iterations for the displacement objective func-
tion while there are spurious oscillations in the material
densities for the compliance objective function and the
maximum number of iteration nmax is reached. Neverthe-
less it does not affect the final shape of the design domain
since it concerns only a few elements. Indeed, in the final
shape for solution ˜xc, there are: 2140 elements such that xe > 0.99 6420 elements such that xe < 0.01 80 elements such that 0.01 < xe < 0.99 In the final shape for solution ˜xd, there are: 2140 elements such that xe > 0.99 6400 elements such that xe < 0.01 100 elements such that 0.01 < xe < 0.99 2360 elements such that xe > 0.99 The quantity of non-converged elements (material density
between 0.01 and 0.99) is about 1% in both cases. 7064 elements such that xe < 0.01 80 elements such that 0.01 < xe < 0.99 S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 7 Table 1. Aluminum material properties. Table 1. Aluminum material properties. p
p
Young Modulus E
Poisson coefficient ν
Volumetric mass density ρ
70 GPa
0.3
2700 kg m−3
(a) View of the optimal solution ˜xc
(b) View of the optimal solution ˜xd
(c) Side view of the optimal solution ˜xc
(d) Side view of the optimal solution ˜xd
Fig. 3. 4.1.1 Reinforcement of a 3D cantilever beam Optimal converged final shapes for the 3D cantilever beam: elements in Ω2 such that xe > 0.99 are in red, elements in Ω1
are in blue. (a) View of the optimal solution ˜xc (b) View of the optimal solution ˜xd (a) View of the optimal solution ˜xc
(c) Side view of the optimal solution ˜xc (b) View of the optimal solution ˜xd (a) View of the optimal solution ˜xc (b) View of the optimal solution ˜xd
(d) Side view of the optimal solution ˜xd (d) Side view of the optimal solution ˜xd (c) Side view of the optimal solution ˜xc (d) Fig. 3. Optimal converged final shapes for the 3D cantilever beam: elements in Ω2 such that xe > 0.99 are in red, elements in Ω1
are in blue Fig. 3. Optimal converged final shapes for the 3D cantilever beam: elements in Ω2 such that xe > 0.99 are in red, elements in Ω1
are in blue. Fig. 3. Optimal converged final shapes for the 3D cantilever beam: elements in Ω2 such that xe > 0
are in blue. Fig. 4. Evolution of the objective functions cs(xi)/cs(x1) and ds(xi)/ds(x1) over the optimization iterations for the 3D cantilever
beam where x1 is the design at the end of the first iteration while xi is the design at iteration i. Fig. 4. Evolution of the objective functions cs(xi)/cs(x1) and ds(xi)/ds(x1) over the optimization iterations for the 3D cantilever
beam where x1 is the design at the end of the first iteration while xi is the design at iteration i. S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 8 8 1 cm
⃗F
Ω1
Ω2
10 cm
10 cm
10 cm
1 cm
1 cm
8 cm
8 cm
7 cm
1 cm
A
Fig. 5. CubeSat-like structure (Ω1) reinforced by a design domain (Ω2). Note that half of Ω2 volume is represented with a
translation from Ω1 in order to see its shape. 1 cm
⃗F
Ω1
10 cm
10 cm
10 cm
1 cm
1 cm
A 10 cm 10 cm Ω2
8 cm
8 cm
7 cm
1 cm Ω1 Ω2 Fig. 5. CubeSat-like structure (Ω1) reinforced by a design domain (Ω2). Note that half of Ω2 volume is represented with a
translation from Ω1 in order to see its shape. Harmonic force Harmonic force Fig. 8. Optimization of an internal viscoelastic layer (Ω2) of a 2D sandwich beam composed of 2 external aluminum layers (Ω1). n internal viscoelastic layer (Ω2) of a 2D sandwich beam composed of 2 external aluminum layers (Ω Fig. 8. Optimization of an internal viscoelastic layer (Ω2) of a 2D sandwich beam composed of 2 ex In the final shape for solution ˜xd, there are: the length direction of the beam, there are 198 elements. There is a total of 1386 elements in Ω2. The target final
volume ratio is γ = 50% of the total volume of Ω2, the
stop criterion ζ is empirically set to 1% and the maximum
number of iterations nmax is set to 100. 2368 elements such that xe > 0.99
7072 elements such that xe < 0.01
64 elements such that 0.01 < xe < 0.99 2368 elements such that xe > 0.99 7072 elements such that xe < 0.01 64 elements such that 0.01 < xe < 0.99 64 elements such that 0.01 < xe < 0.99 Firstly, the viscoelastic layer is optimized using the
compliance objective function c(ω, x) at three different
harmonic excitations ω (500 Hz, 1000 Hz and 2000 Hz). The solutions in terms of material densities are denoted
respectively by ˜xc
500 Hz, ˜xc
1000 Hz and ˜xc
2000 Hz. These optimal
material densities are plotted in Figure 9. The conver-
gence of the compliance over the iterations is plotted in
Figure 10. It can be seen that for the three harmonic
excitations, there are oscillations of the objective func-
tion and the optimization process is stopped at a fixed
number of iteration (here nmax=100 iterations). How-
ever, these spurious oscillations concern a few elements
and do not affect the final solution (Tab. 3). The fre-
quency response functions of the beam are then computed
using these three material densities. The compliance func-
tions c(ω, ˜xc
500 Hz), c(ω, ˜xc
1000 Hz) as well as c(ω, ˜xc
2000 Hz) are
shown in Figure 11. The black curve is obtained using
a fully filled layer of viscoelastic material (xe = 1, ∀e). It can be seen that for a given angular frequency, a The quantity of non-converged elements (material density
between 0.01 and 0.99) is less than 1% in both cases. 4.1.1 Reinforcement of a 3D cantilever beam (a) Overall view of the optimal solution ˜xc (b) Overall view of the optimal solution ˜xd (a) Overall view of the optimal solution ˜xc
(b) Overall view of the optimal solution ˜xd
(c) Side view of the optimal solution ˜xc
(d) Side view of the optimal solution ˜xd
Fig. 6. Optimal converged final shapes for the 3D CubeSat-like structure: elements in Ω2 such that xe > 0.99 are in red, elements
in Ω1 are in blue. (a) Overall view of the optimal solution ˜xc (b) Overall view of the optimal solution ˜xd (a) Overall view of the optimal solution ˜xc
(c) Side view of the optimal solution ˜xc (b) Overall view of the optimal solution x
(d) Side view of the optimal solution ˜xd (d) Side view of the optimal solution ˜xd (c) Side view of the optimal solution ˜xc Fig. 6. Optimal converged final shapes for the 3D CubeSat-like structure: elements in Ω2 such that xe > 0.99 are in red, elements
i
Ω
i
bl Fig. 6. Optimal converged final shapes for the 3D CubeSat-like structure: elements in Ω2 such that xe > 0.99 are in red, elements
in Ω1 are in blue. Fig. 6. Optimal converged final shapes for the 3D CubeSat-like structure: elements in Ω2 such that xe > 0.99 are in red, elements
in Ω1 are in blue. S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 9 Fig. 7. Evolution of the objective functions cs(xi)/cs(x1) and ds(xi)/ds(x1) over the optimization iterations for the 3D CubeSat-like
structure where x1 is the design at the end of the first iteration while xi is the design at iteration i. Fig. 7. Evolution of the objective functions cs(xi)/cs(x1) and ds(xi)/ds(x1) over the optimization iterations for the 3D CubeSat-like
structure where x1 is the design at the end of the first iteration while xi is the design at iteration i. e = 1 mm
e = 1 mm
h = 1 mm
Ω1
Ω1
Ω2
L = 0.1 m
A
Harmonic force
Fig. 8. Optimization of an internal viscoelastic layer (Ω2) of a 2D sandwich beam composed of 2 external aluminum layers (Ω1). 4.2.1 Optimization of a visoelastic layer for a 2D sandwich
beam Optimal solutions of the visco-elastic layer of the 2D sandwich beam (black: xe > 0.99) for
bottom to top: ˜xc
500 Hz, ˜xc
1000 Hz, ˜xc
2000 Hz. g
y
(
)
bottom to top: ˜xc
500 Hz, ˜xc
1000 Hz, ˜xc
2000 Hz. Fig. 10. Dynamic compliance c(x) evolution over the optimization iterations for the 2D sandwich beam and for the three different
harmonic excitations: c(500 Hz, xi), c(1000 Hz, xi) and c(2000 Hz, xi). bottom to top: x500 Hz, x1000 Hz, x2000 Hz. Fig. 10. Dynamic compliance c(x) evolution over the optimization iterations for the 2D sandwich beam and for the three different
harmonic excitations: c(500 Hz, xi), c(1000 Hz, xi) and c(2000 Hz, xi). Fig. 10. Dynamic compliance c(x) evolution over the optimization iterations for the 2D sandwich beam and for the three different
harmonic excitations: c(500 Hz, xi), c(1000 Hz, xi) and c(2000 Hz, xi). Table 3. Number of elements in Ω2 in terms of their final
material densities for the 2D sandwich beam. The same conclusions can be done with solution ˜xc
1000 Hz
at the frequency 1000 Hz; and with solution ˜xc
2000 Hz at the
frequency 2000 Hz. xe > 0.99
xe < 0.01
0.01 < xe < 0.99
˜xc
500 Hz
623
754
9
˜xc
1000 Hz
675
625
86
˜xc
2000 Hz
676
617
93
˜xd
500 Hz
693
693
0
˜xd
1000 Hz
677
622
87
˜xd
2000 Hz
686
663
37 Secondly, the viscoelastic layer is optimized using the
displacement objective function d(ω, x) at the same three
different harmonic excitations ω (500 Hz, 1000 Hz and
2000 Hz). The solutions in terms of material densities
are denoted respectively by ˜xd
500 Hz, ˜xd
1000 Hz and ˜xd
2000 Hz. These optimal material densities are plotted in Figure 12. The convergence of the displacement objective function
over the iterations is plotted in Figure 13. As for the
compliance criteria, there are spurious oscillations for two
harmonic excitations (1000 Hz and 2000 Hz) (Table 3). The frequency response functions of the beam are then
computed using these three material densities. The dis-
placement functions d(ω, ˜xd
500 Hz), d(ω, ˜xd
1000 Hz) as well as
d(ω, ˜xd
2000 Hz) are shown in Figure 14. The black curve is
obtained using a fully filled layer of viscoelastic mate-
rial (xe = 1, ∀e). 4.2.1 Optimization of a visoelastic layer for a 2D sandwich
beam 4.2.1 Optimization of a visoelastic layer for a 2D sandwich
beam The problem is described in Figure 8. It is assumed a plane
stress state. The sandwich beam is composed of 2 exter-
nal aluminum layers (domain Ω1) and a viscoelastic layer
(domain Ω2) in between them. The material properties
are given in Table 1 (for aluminum) and 2 (for the vis-
coelastic layer). The left side of the beam is clamped while
an harmonic force (±10 N) is imposed at the right side at
the angular velocity ω. The structure is discretized using
quadrangular 4-node elements. There are 6 elements in the
thickness direction of the aluminum layers and 7 elements
in the thickness direction of the viscoelastic layer. Along 10
S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023)
Table 2. Viscoelastic material properties. E0
E∞
ν
ρ
α
τ
4.2 MPa
1.62 GPa
0.45
1460 kg m−3
0.59
0.52×10−6 s
Fig. 9. Optimal solutions of the visco-elastic layer of the 2D sandwich beam (black: xe > 0.99) for the compliance criteria, from
bottom to top: ˜xc
500 Hz, ˜xc
1000 Hz, ˜xc
2000 Hz. S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 10 Table 2. Viscoelastic material properties. E0
E∞
ν
ρ
α
τ
4.2 MPa
1.62 GPa
0.45
1460 kg m−3
0.59
0.52×10−6 s
Fig. 9. Optimal solutions of the visco-elastic layer of the 2D sandwich beam (black: xe > 0.99) for the compliance criteria, from
bottom to top: ˜xc
500 Hz, ˜xc
1000 Hz, ˜xc
2000 Hz. Fig. 10. Dynamic compliance c(x) evolution over the optimization iterations for the 2D sandwich beam and for the three different
harmonic excitations: c(500 Hz, xi), c(1000 Hz, xi) and c(2000 Hz, xi). Table 2. Viscoelastic material properties. E0
E∞
ν
ρ
α
τ
4.2 MPa
1.62 GPa
0.45
1460 kg m−3
0.59
0.52×10−6 s
Fig. 9. Optimal solutions of the visco-elastic layer of the 2D sandwich beam (black: xe > 0.99) for the compliance criteria, from
bottom to top: ˜xc
500 Hz, ˜xc
1000 Hz, ˜xc
2000 Hz. Fig. 9. Optimal solutions of the visco-elastic layer of the 2D sandwich beam (black: xe > 0.99) for the compliance criteria, from
bottom to top: ˜xc
˜xc
˜xc Fig. 9. 4.2.1 Optimization of a visoelastic layer for a 2D sandwich
beam As for the compliance based criteria, solution can be better than an other in terms of mini-
mizing the compliance. For instance, the material density
solution ˜xc
500 Hz given by the minimization of the compli-
ance at 500 Hz exhibits the minimum of compliance at
500 Hz compared to the other solutions, even the solu-
tion computed with a full layer of viscoelastic material. solution can be better than an other in terms of mini-
mizing the compliance. For instance, the material density
solution ˜xc
500 Hz given by the minimization of the compli-
ance at 500 Hz exhibits the minimum of compliance at
500 Hz compared to the other solutions, even the solu-
tion computed with a full layer of viscoelastic material. S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 11 Fig. 11. Frequency response functions of the 2D sandwich beam in terms of dynamic compliance: c(ω, ˜xc
500 Hz), c(ω, ˜xc
1000 Hz) and
c(ω, ˜xc
2000 Hz). Fig. 11. Frequency response functions of the 2D sandwich beam in terms of dynamic compliance: c(ω, ˜xc
500 Hz), c(ω, ˜xc
1000 Hz) and Fig. 11. Frequency response functions of the 2D sandwich beam in terms of dynamic compliance: c(ω, ˜xc
500 Hz), c(ω, ˜xc
1000 Hz) and
c(ω, ˜xc
2000 Hz). Fig. 11. Frequency response functions of the 2D sandwich beam in terms of dynamic compliance: c(ω, ˜xc
500 Hz), c(ω, ˜xc
1000 Hz) and
c(ω, ˜xc
2000 Hz). Fig. 12. Optimal solutions of the visco-elastic layer of the 2D sandwich beam (black: xe > 0.99) for the displacement criteria, from
bottom to top: ˜xd
500 Hz, ˜xd
1000 Hz, ˜xd
2000 Hz. bottom to top: ˜xd
500 Hz, ˜xd
1000 Hz, ˜xd
2000 Hz. Fig. 13. Displacement objective function d(x) evolution over the optimization iterations for the 2D sandwich beam and for the
three different harmonic excitations: d(500 Hz, xi), d(1000 Hz, xi) and d(2000 Hz, xi). Fig. 13. Displacement objective function d(x) evolution over the optimization iterations for the 2D sandwich beam and for the
three different harmonic excitations: d(500 Hz, xi), d(1000 Hz, xi) and d(2000 Hz, xi). S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 12 Fig. 14. Frequency response functions of the 2D sandwich beam in terms of displacement objective function: d(ω, ˜xd
500 Hz),
d(ω, ˜xd
1000 Hz) and d(ω, ˜xd
2000 Hz). Fig. 14. 4.2.1 Optimization of a visoelastic layer for a 2D sandwich
beam Optimal solutions of the viscoelastic layer of the 2D CubeSat-like structure (black: xe > 0.99) for the compliance criteria,
from left to right: ˜xc
50 Hz, ˜xc
100 Hz, ˜xc
200 Hz. 4.2.1 Optimization of a visoelastic layer for a 2D sandwich
beam Frequency response functions of the 2D sandwich beam in terms of displacement objective function: d(ω, ˜xd
500 Hz),
d(ω, ˜xd
1000 Hz) and d(ω, ˜xd
2000 Hz). Ω1
Ω2
Ω2
Ω2
⃗F
45o
A
96 cm
96 cm
19 cm
1 cm
Fig. 15. Optimization of an internal viscoelastic layer (Ω2) for a 2D CubeSat-like structure (Ω1). 1 cm Fig. 15. Optimization of an internal viscoelastic layer (Ω2) for a 2D CubeSat-like structure (Ω1). true; the topology optimization process can not give a bet-
ter solution than the one given by a fully filled layer of
viscoelastic material. Nevertheless, the solution given by
the optimization is lighter. the same conclusions can be made: the density optimized
solution obtained for a given frequency gives the minimum
of the displacement based function at this frequency. One
can notice that in this case, for 500 Hz, it is not completely S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 13 (a) Solution ˜xc
50 Hz at 50 Hz
(b) Solution ˜xc
100 Hz at 100 Hz (c) Solution ˜xc
200 Hz at 200 Hz
Fig. 16. Deformed structure for the three target frequencies and for the compliance objective function (scale factor of the deformed
mesh is 5000; black correspond to no displacement, yellow to maximum). (a) Solution ˜xc
50 Hz at 50 Hz
(b) Solution ˜xc
100 Hz at 100 Hz (c) Solution ˜xc
200 Hz at 200 Hz
Fig. 16. Deformed structure for the three target frequencies and for the compliance objective function (scale factor of the deformed
mesh is 5000; black correspond to no displacement, yellow to maximum). (b) Solution ˜xc
100 Hz at 100 Hz (c) Solution ˜xc
200 Hz at 200 Hz Fig. 16. Deformed structure for the three target frequencies and for the compliance objective function (scale factor of the deformed
mesh is 5000; black correspond to no displacement, yellow to maximum). Fig. 16. Deformed structure for the three target frequencies and for the compliance objective function (scale factor of the deformed
mesh is 5000; black correspond to no displacement, yellow to maximum). Fig. 17. Optimal solutions of the viscoelastic layer of the 2D CubeSat-like structure (black: xe > 0.99) for the compliance criteria,
from left to right: ˜xc
50 Hz, ˜xc
100 Hz, ˜xc
200 Hz. Fig. 17. 4.2.2 Optimization of a viscoelastic layer for a 2D
CubeSat-like structure Firstly, the viscoelastic layer is optimized using the
compliance objective function c(ω, x) at three different
harmonic excitations ω (50 Hz, 100 Hz and 200 Hz). The solutions in terms of material densities are denoted
respectively by ˜xc
50 Hz, ˜xc
100 Hz and ˜xc
200 Hz. These optimal
material densities are plotted in Figure 17. The conver-
gence of the compliance over the iterations is plotted
in Figure 18. As for the 2D sandwich beam, it can be
seen that for the three harmonic excitations, there are
oscillations of the objective function and the optimiza-
tion process is stopped at a fixed number of iteration
(here 100 iterations). However, these spurious oscillations
concern a few elements and do not affect the final solu-
tion (Table 4). These oscillations are more important for
˜xc
50 Hz, nevertheless if only elements with xe > 0.99 are
considered the solution is acceptable. Moreover, for this
solution, there are 369 elements with a material density
less than 0.1; and so only 128 elements between 0.1 and
0.99. The frequency response functions of the structure The problem is described in Figure 15. It is assumed a
plane stress state. The structure is composed of external
aluminum layers (domain Ω1) and internal viscoelastic
layers (domain Ω2). Its shape is the same as the 3D
CubeSat-like structure studied in the previous section. The material properties are given in Table 1 (for alu-
minum) and 2 (for the viscoelastic layers). The target
final volume ratio is γ = 50% of the total volume of Ω2,
the stop criterion ζ is empirically set to 1% and the maxi-
mum number of iterations nmax is set to 100. The bottom
side of the structure is clamped while an harmonic force ⃗F
is imposed on the upper left corner at the angular velocity
ω (∥⃗F ∥= 1 N). (∥
∥
)
The structure is discretized with a total of 3222 quad-
rangular 4-node elements, the number of elements in Ω2
is 966. The mesh can be seen in Figure 16. S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 14 Fig. 18. Dynamic compliance c(x) evolution over the optimization iterations for the 2D CubeSat-like structure and for the three
different harmonic excitations: c(50 Hz, xi), c(100 Hz, xi) and c(200 Hz, xi). Fig. 18. 4.2.2 Optimization of a viscoelastic layer for a 2D
CubeSat-like structure Dynamic compliance c(x) evolution over the optimization iterations for the 2D CubeSat-like structure and for the three
different harmonic excitations: c(50 Hz, xi), c(100 Hz, xi) and c(200 Hz, xi). Table 4. Number of elements in Ω2 in terms of their final material densities for the 2D Cu elements in Ω2 in terms of their final material densities for the 2D CubeSat-like structure. of elements in Ω2 in terms of their final material densities for the 2D CubeSat-like structu xe > 0.99
xe < 0.01
0.01 < xe < 0.99
˜xc
50 Hz
469
339
158
˜xc
100 Hz
499
411
56
˜xc
200 Hz
508
432
26
˜xd
50 Hz
450
0
516
˜xd
100 Hz
499
414
53
˜xd
200 Hz
509
435
22
Fig. 19. Frequency response functions of the 2D CubeSat-like structure in terms of dynamic compliance: c(ω, ˜xc
50 Hz), c(ω, ˜xc
100 Hz)
and c(ω, ˜xc
200 Hz). Fig. 19. Frequency response functions of the 2D CubeSat-like structure in terms of dynamic compliance: c(ω, ˜xc
50 Hz), c(ω, ˜xc
100 Hz)
and c(ω, ˜xc
200 Hz). S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 15 Fig. 20. Optimal solutions of the viscoelastic layer of the 2D CubeSat-like structure (black: xe > 0.99) for the displacement criteria,
from left to right: ˜xd
50 Hz, ˜xd
100 Hz, ˜xd
200 Hz. Fig. 20. Optimal solutions of the viscoelastic layer of the 2D CubeSat-like structure (black: xe > 0.99) for the displacement criteria,
from left to right: ˜xd
50 Hz, ˜xd
100 Hz, ˜xd
200 Hz. Fig. 20. Optimal solutions of the viscoelastic layer of the 2D CubeSat-like structure (black: xe > 0.99) for the displacement criteria,
from left to right: ˜xd
50 Hz, ˜xd
100 Hz, ˜xd
200 Hz. Fig. 21. Displacement objective function evolution over the optimization iterations for the 2D CubeSat-like structure and for the
three different harmonic excitations: d(50 Hz, xi), d(100 Hz, xi) and d(200 Hz, xi). Fig. 21. Displacement objective function evolution over the optimization iterations for the 2D CubeSat-like structure and for the
three different harmonic excitations: d(50 Hz, xi), d(100 Hz, xi) and d(200 Hz, xi). Secondly, the viscoelastic layer is optimized using the
displacement objective function d(ω, x) at the same three
different harmonic excitations ω (50 Hz, 100 Hz and
200 Hz). 4.2.2 Optimization of a viscoelastic layer for a 2D
CubeSat-like structure The solutions in terms of material densities
are denoted respectively by ˜xd
50 Hz, ˜xd
100 Hz and ˜xd
200 Hz. These optimal material densities are plotted in Figure 20. The convergence of the displacement criteria over the
iterations is plotted in Figure 21. As previously, two
of the three optimizations have spurious oscillations in
terms of objective function over the iteration process
(Table 4). These oscillations are more important for
˜xd
50 Hz: for this case, there are no elements with xe <
0.01 but there are 362 elements with a material den-
sity less than 0.1; and so only 154 elements between
0.1 and 0.99. In order to illustrate the oscillations in are then computed using these three material densities. The compliance functions c(ω, ˜xc
50 Hz), c(ω, ˜xc
100 Hz) as well
as c(ω, ˜xc
200 Hz) are shown in Figure 19. The black curve
is obtained using a fully filled layer of viscoelastic mate-
rial (xe = 1, ∀e). As for the 2D sandwich beam, it can
be seen that for a given angular frequency, a solution
can be better than another one in terms of minimizing
the compliance. For instance, the material density solu-
tion ˜xc
50 Hz given by the minimization of the compliance
at 50 Hz exhibits the minimum of compliance at 50 Hz
compared to the other solutions, even the solution com-
puted with a full layer of viscoelastic material. The same
conclusions can be done with solution ˜xc
100 Hz at the fre-
quency 100 Hz; and with solution ˜xc
200 Hz at the frequency
200 Hz. are then computed using these three material densities. The compliance functions c(ω, ˜xc
50 Hz), c(ω, ˜xc
100 Hz) as well
as c(ω, ˜xc
200 Hz) are shown in Figure 19. The black curve
is obtained using a fully filled layer of viscoelastic mate-
rial (xe = 1, ∀e). As for the 2D sandwich beam, it can
be seen that for a given angular frequency, a solution
can be better than another one in terms of minimizing
the compliance. For instance, the material density solu-
tion ˜xc
50 Hz given by the minimization of the compliance
at 50 Hz exhibits the minimum of compliance at 50 Hz
compared to the other solutions, even the solution com-
puted with a full layer of viscoelastic material. 4.2.2 Optimization of a viscoelastic layer for a 2D
CubeSat-like structure The same
conclusions can be done with solution ˜xc
100 Hz at the fre-
quency 100 Hz; and with solution ˜xc
200 Hz at the frequency
200 Hz. S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 16 Fi
22 L
t 3 it
ti
f
th
l ti
˜d
f th 2D C b S t lik
t
t
f
l ft t
i ht it
ti
98 99
d 100 (
d Fig. 22. Last 3 iterations for the solution ˜xd
50 Hz of the 2D CubeSat-like structure, from left to right: iterations 98, 99, and 100 (red:
xe = 1, blue: xe = 0). Fig. 22. Last 3 iterations for the solution ˜xd
50 Hz of the 2D CubeSat-like structure, from left to right: iterations 98, 99, and 100 (red:
xe = 1, blue: xe = 0). Fig. 22. Last 3 iterations for the solution ˜xd
50 Hz of the 2D CubeSat-like structure, from left to right: iterations 98, 99, and 100 (red:
x
1 blue: x
0) Fig. 23. Frequency response functions of the 2D CubeSat-like structure in terms of displacement amplitude: d(ω ˜xd
50 H ) d(ω ˜xd
100 H ) Fig. 23. Frequency response functions of the 2D CubeSat-like structure in terms of displacement amplitude: d(ω, ˜xd
50 Hz), d(ω, ˜xd
100 Hz)
and d(ω, ˜xd
200 Hz). Fig. 23. Frequency response functions of the 2D CubeSat-like structure in terms of displacement amplitude: d(ω, ˜xd
50 Hz), d(ω, ˜xd
100 Hz)
and d(ω, ˜xd
200 Hz). of viscoelastic material. Nevertheless, the solution given
by the optimization is lighter. the solution, the last three iterations are plotted on
Figure 22. Nevertheless if only elements with xe > 0.99
are considered the solution is acceptable. The frequency
response functions of the structure are then computed
using these three material densities. The displacement
functions d(ω, ˜xd
50 Hz), d(ω, ˜xd
100 Hz) as well as d(ω, ˜xd
200 Hz)
are shown in Figure 23. The black curve is obtained using
a fully filled layer of viscoelastic material (xe = 1, ∀e). As
for the compliance based criteria, the same conclusions
can be made: the density optimized solution obtained for
a given frequency gives the minimum of the displacement
based function at this frequency. 5 Conclusion The aim of this work is to use a topology optimiza-
tion process to design a domain Ω2 interacting through
an interface with a fixed given structure Ω1 in order to
improve the behavior of the initial structure according to
a given criteria. The problem can be static or dynamic. In
the dynamic case, the problem is treated in the frequency
domain and the used material in the design domain
has viscoelastic properties able to reduce the vibration
amplitude. This viscoelastic material is modeled using However, sometimes, for this application, the topol-
ogy optimization process does not improve so much the
solution compared to the one given by a fully filled layer S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 17 a four-parameter Zener fractional-derivative viscoelastic
model. [2] K. Suzuki, N. Kikuchi, A homogenization method for
shape and topology optimization, Comput. Methods Appl. Mech. Eng. 93, 291–318 (1991) Two objective functions are introduced in this paper:
a global compliance based one and a local displacement
based one. They are both defined for the dynamic as well
as for the static case. Results show that the optimum
solutions obtained for the two objective function are very
often quite similar. [3] O. Sigmund, A 99 line topology optimization code written
in Matlab, Struct. Multidiscipl. Optim. 21, 120–127 (2001) [4] M. Stolpe, K. Svanberg, An alternative interpolation
scheme for minimum compliance topology optimization,
Struct. Multidiscipl. Optim. 22, 116–124 (2001) [5] D. Tchierniak, Topology optimization of resonating struc-
tures using SIMP method, Int. J. Numer. Methods Eng. 54, 1605–1622 (2002) The formulation of the problems allows to have a com-
plex shape of the additional design, even a design domain
separated into several volumes, as it is the case in a few
presented applications. Moreover, the chosen material for
the design domain can be different than the one used
for the initial structure. The shape and the material of
the design domain have simply to be coherent with func-
tional surfaces of the system and with the manufacturing
process. [6] A. Takezawa, M. Daifuku, Y. Nakano, K. Nakagawa,
T. Yamamoto, M. Kitamuraa, Topology optimization of
damping material for reducing resonance response based
on complex dynamic compliance, J. Sound Vibr. 365,
230–243 (2016) [7] R.B. Haber, C.S. Jog, M.P. Bendsøe, A new approach
to variable-topology shape design using a constraint on
perimeter, Struct. Optim. 5 Conclusion 11, 1-12 (1996) The applications in the static case show that it is
possible to improve a given structure by adding mate-
rial around it using a topology optimization process. The strategy may be applied to a damaged structure
needed to be repaired. Additive manufacturing may
be used to manufacture the domain Ω2 in order to
reinforce Ω1. [8] L. Shu, M.Y. Wang, Z. Fang, Z. Ma, P. Wei, Level
set based structural topology optimization for minimiz-
ing frequency response, J. Sound Vibr. 330, 5820–5834
(2011) (
)
[9] J. Cao, K. Cai, P. Fei Wang, D. Yan, J. Shi, Multiple
materials layout optimization in a layered structure, Mech. Ind. 17, 404 (2016) The examples in the dynamic case show that the
proposed strategy enables to design additive viscoelas-
tic layers to damp the vibrations at a given frequency. The proposed examples deal with sandwich structures for
which the viscoelastic layer in between two layers of metal
since it is known to be more efficient than on the external
surface of the structure. The final shape of the viscoelastic
layer drastically depends on the target frequency. [10] O.M. Silva, M.M. Neves, A. Lenzi, A critical analysis
of using the dynamic compliance as objective func-
tion in topology optimization of one-material structures
considering steady-state forced vibration problems, J. Sound Vibr. 444, 1–20 (2019) (
)
[11] S. Burri, Contributions `a l’optimisation topologique de
liaisons amortissantes pour des applications spatiales. Th`ese de doctorat. CNAM, HESAM Universit´e (2020). https://tel.archives-ouvertes.fr/tel-03179845 Nevertheless, especially in the context of dynamics and
viscoelasticity, the topology optimization process often
reaches the maximum number of iterations, exhibiting
spurious oscillations of the material densities between
few elements. However, these oscillations concern a few
elements, and do not affect the quality of the final
solution. [12] M.P. Bendsøe, Optimal shape design as a material distri-
bution problem, Struct. Optim. 1, 193-202 (1989) [13] N. Zhou, G.I.N. Rozvany, The COC algorithm. Part II:
Topological, geometrical and generalized shape optimiza-
tion. Comput. Methods Appl. Mech. Eng. 89, 309–336
(1991) Conclusions show that it is possible to deal with two
different substructures with the aim of controlling the first
one using the second one. The way it is implemented here
allows for a great variety of configurations for future users. [14] M.P. Bendsøe, O. Sigmund, Topology Optimization –
Theory, Methods and Applications (Springer, Berlin,
Heidelberg, 2004) [15] A. Diaz, N. 5 Conclusion Kikuchi, Solutions to shape and topology
eigenvalue optimization problems using a homogenization
method. Int. J. Numer. Methods Eng. 35, 1487–1502
(1992) Funding information [16] Z.-D. Ma, N. Kikuchi, I. Hagiwara, Structural topology
and shape optimization for a frequency response problem. Comput. Mech. 13, 157–174 (1993) ArianeGroup is gratefully acknowledged for its financial
support. [17] Z.-D. Ma, H.-S. Cheng, N. Kikuchi, Structural design for
obtaining desired eigenfrequencies by using the shape and
topology optimization method. Comput. Syst. Eng. 5, 77–
89 (1994) Declaration of conflicting interests The authors declare no potential conflicts of interest with
respect to the research, authorship, and/or publication of
this article. [18] M. Bruggi, A. Talierci, Topology optimization of the fiber-
reinforcement retrofitting existing structures, Int. J. Solids
Struct. 50, 121–136 (2013) (
)
[19] J.-P. Kruth, M.C. Leu, T. Nakagawa, Progress in addi-
tive manufacturing and rapid prototyping, CIRP Ann. 47,
525–540 (1998) References [1] F. Rigaud, M. Charlotte, C. Kerdreux, P. Marechal, Multi-
objective optimization of rotary-wing aircrafts at the
predesign stage, Mech. Ind. 15, 267–277 (2014) [20] R.L Bagley, P. Torvik, Fractional calculus – a differ-
ent approach to the analysis of viscoelastically damped
structures, AIAA J. 21, 741–748 (1983) S. Burri and A. Legay: Mechanics & Industry 24, 14 (2023) 18 [21] A. Germant, XLV. On fractional differentials, Philos. Mag. Ser. 1 25, 540–549 (1938) [29] K. Svanberg, The method of moving asymptotes – a
new method for structural optimization. Int. J. Numer. Methods Eng. 24, 359–373 (1987) [22] B. Morin, A. Legay, J.-F. De¨u, Reduced order models for
dynamic behavior of elastomer damping devices, Finite
Elem. Anal. Des. 143, 66–75 (2018) [30] Z.-D. Ma, N. Kikuchi, I. Hagiwara, Structural topology
and shape optimization for a frequency response problem,
Comput. Mech. 13, 157–174 (1993) [23] L. Rouleau,
A. Legay,
J.-F. De¨u,
Mod´elisation
vibro-acoustique
de
structures
sandwich
munies
de
mat´eriaux visco´elastiques. Th`ese de Doctorat CNAM
(2013) [31] G.H. Yoon, Structural topology optimization for fre-
quency response problem using model reduction schemes,
Comput. Methods Appl. Mech. Eng. 199, 1744–1763
(2010) [24] C. Geuzaine, J.-F. Remacle, Gmsh: a 3-D finite element
mesh generator with built-in pre- and post-processing
facilities. Int. J. Numer. Methods Eng. 79, 1309–1331
(2019) [32] K.-S. Yun, S.-K. Youn, Topology optimization of viscoelas-
tic damping layers for attenuating transient response of
shell structures, Finite Element Anal. Des. 141, 154–165
(2018) [25] A.C. Galucio, J.-F. De¨u, R. Ohayon, Finite element
formulation of viscoelastic sandwich beams using frac-
tional derivative operators. Comput. Mech. 33, 282–291
(2004) [33] C.S. Jog, Topology design of structures subjected to
periodic loading, J. Sound Vibr. 253, 687–709 (2002) [34] K. Liu, A. Tovar, An efficient 3D topology optimization
code written in Matlab, Struct. Multidiscipl. Optim. 50,
1175–1196 (2014) (
)
[26] M.P. Bendsøe, N. Kikuchi, Generating optimal topolo-
gies in structural design using a homogenization method,
Comput. Methods
Appl. Mech. Eng. 71,
197–224
(1988) (
)
[35] H. Heidt, J. Puig-Suari, A.S. Moore, S. Nakasuka, R.J. Twiggs, CubeSat: a new generation of picosatellite for
education and industry low-cost space experimentation, in
14th Annual AIAA/USU Conference on Small Satellites
(2000) [27] O. Sigmund, Morphology-based black and white filters for
topology optimization. Struct. Multidiscipl. Optim. 33,
401–424 (2007) [28] G.I.N. Rozvany, Structural Design via Optimality Criteria:
The Prager Approach to Structural Optimization. (Kluwer
Academic Publishers, Springer Dordrecht, 1989) [36] R. Cite this article as: S. Burri and A. Legay, Static Reinforcement and Vibration Reduction of Structures using Topology
Optimization, Mechanics & Industry 24, 14 (2023) References Hevner, W. Holemans, J. Puig-Suari, R.J. Twiggs,
An advanced standard for CubeSats, in 25th Annual
AIAA/USU Conference on Small Satellites (2011)
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Effect of finasteride on lipid profile in individuals with androgenetic alopecia
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African journal of pharmacy and pharmacology
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hort Communication
Effect of finasteride on lipid profile in individuals with
androgenetic alopecia
Seied Reza Seied Mohammad Doulabi1, Hossein Kavoussi1*, Danial Isapour1,
Amirhossein hashemian2 and Ali Taheriniya3 1School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran. artment of Biostatistics and Epidemiology, School of Health, Kermanshah University of Medical Scienc
Kermanshah, Iran. 3Department of Emergency Medicine, Alborz University of Medical Sciences, Karaj, Iran. Kermanshah, Iran. 3Department of Emergency Medicine, Alborz University of Medical Sciences, Karaj, Iran Kermanshah, Iran. artment of Emergency Medicine, Alborz University of Medical Sciences, Karaj, Iran. Accepted 4 June, 2012 Accepted 4 June, 2012 Atherosclerosis constitutes one of the most frequent diseases and one of the important predisposing
factors for atherosclerosis is lipid profile change. Androgen changes lipid profile, mainly high density
lipoprotein (HDL), and oral finasteride are used for treating androgenetic hair loss as a risk factor for
atherosclerosis. This study was conducted in order to determine the lipid profile changes by 1 mg
finasteride tablets daily in patients with androgenetic hair loss. Twenty-five patients with androgenetic
hair loss were prescribed one 1 mg finasteride tablet daily. Fasting plasma levels of low density
lipoprotein (LDL), HDL, triglyceride, total cholesterol and HDL to LDL ratio of patients before therapy
and after 3 and 6 months of therapy were measured. The study was conducted in the form of a before-
after clinical trial. Data were analyzed using SPSS software version 16. A statistically significant rise in
triglyceride plasma level (p=0.014) and significant decreases in HDL plasma level (p<0.001) was
observed after 3 and 6 months of therapy, respectively. Plasma levels of LDL, total cholesterol and HDL
to LDL ratio were not changed significantly but changes in ratio between third and sixth month were
significant. Therefore finasteride may decrease dihydrotestosterone and increase testosterone that
could lead to complications of the lipid profile by reducing HDL and increasing total cholesterol. Key words: Atherosclerosis, finasteride, testosterone, lipid profile. *Corresponding author. E-mail: s8371916@kums.ac.ir. Tel: +98
9127016682. Fax: 00988318368013. African Journal of Pharmacy and Pharmacology Vol. 7(6), pp. 315-317, 15 February, 2013
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.353
ISSN 1996-0816 ©2013 Academic Journals African Journal of Pharmacy and Pharmacology Vol. 7(6), pp. 315-317, 15 February, 2013
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.353
ISSN 1996-0816 ©2013 Academic Journals Short Communication Short Communication RESULTS Changes in parameters were compared. Analysis of data
indicated a significant decrease in fasting plasma level of
HDL after 6 months of therapy (p<0.001) as well as a
significant increase in plasma level of triglyceride after 3
months of therapy (p=0.006). No significant change was
observed in the fasting plasma levels of LDL and total
cholesterol. Although HDL to LDL ratio did not show
significant change after 3 (p=0.073) and 6 months
(p=0.056), however, changes in ratio between third and
sixth month were significant (p=0.006) (Table 1). Experimental design 1987). Androgenetic hair loss is considered a risk factor
for atherosclerosis (Dogramaci et al., 2009). The increase
in HDL level following total testectomy and the subse-
quent decrease in testosterone have been demonstrated
in patients with prostatic cancer (Moorjani et al., 1987). Furthermore, the impact of testosterone on lowering HDL
and increasing cholesterol and triglyceride has been
established (Asscheman et al., 1994). Also, an inverse
relationship exists between the plasma level of free
testosterone and serum triglyceride (Hämäläinen et al.,
1987). HDL to LDL ratio has been confirmed to be not
only an important factor for development of cardio-
vascular disease (Reaven, 1988; Onyesom et al., 2012)
but also used to efficacy of anti-lipid drug therapy
(Kannel, 2005). Blood samples were obtained from patients at three stages (before
therapy, after three months and six months of treatment with 1 mg
finasteride tablets) and their fasting plasma levels of LDL, HDL,
triglyceride and total cholesterol were measured. Patients who did
not take finasteride regularly or had discontinued therapy were
eliminated from the study. Sample collection Twenty-two patients underwent the second phase of tests and
finally 16 patients with all three tests and regular consumption of 1
mg finasteride tablets during 6 months remained in the study. MATERIALS AND METHODS This study took place in Hajdaie Clinic of Kermanshah University of
Medical Sciences in the city of Kermanshah, west of Iran. This is a
before-after clinical trial. Subjects Twenty-five patients with androgenetic hair loss as confirmed by a
dermatologist were selected. Prior to entry, the patients were
inquired about using other drugs which may influence the lipid
profile, such as statins and patients who used these drugs were
excluded from the study. All patients were referred to one single
laboratory for tests and they were fasting before the tests. During
the study, patients were evaluated for nutritional status. Patients
had different degrees of androgenetic hair loss and this difference
did not affect our study. All patients expressed their informed
consent in writing prior to the study. The proposal of the study was
approved by the Ethics Committee of Kermanshah University of
Medical Sciences and registered in IRCT database. Statistical analysis There are a few studies that evaluated the effect of
using finasteride tablets on lipid profile with variable
results (Moorjani et al., 1987; Denti et al., 2000; Amory et
al., 2008; Duskova et al., 2010). According to predictor
factor of androgenetic alopecia for atherosclerosis and
the effect of using oral finasteride tablets on lipid profile,
we undertook the present study to determine the impact
of using 1 mg finasteride tablets on lipid profile of patients
with androgenetic hair loss. Analysis was performed using SPSS 16.0 (SPSS Inc., Chicago,
U.S.A.). The statistical analysis was performed using the dependent
t-test with an assumption that p-value ≤ 0.05 is significant. INTRODUCTION Atherosclerosis constitutes one of the most common
diseases, with an increasing frequency. It is predicted
that atherosclerosis will be the top cause of death in
2020. Atherosclerosis may involve vessels of different
regions of the body and thus induce stroke and
myocardial infarction (Fauci et al., 2008). Lowering the
low density lipoprotein (LDL) level through administration
of statins may prevent atherosclerosis. Increasing high
density lipoprotein (HDL) and lowering triglyceride are
two other major factors for preventing atherosclerosis
(McRobb et al2009) indicated that androgens may cause
vascular calcification. Smoking is considered as a risk factor associated with
elevated serum level of triglyceride, cholesterol and LDL
but HDL was almost similar in both groups of smoker and
non-smoker (Al-Ajlan, 2012). Tulbaghia violacea is a
valuable medicinal plant used in South Africa for
management of heart diseases and many human
disorders and methanolic extract of it in rat reduces
plasma level of triglyceride, cholesterol, VLDL and LDL
(Olorunnisola et al., 2012). Finasteride is used for treating benign prostatic
hyperplasia and hormonal hair loss, exerting its effect
through inhibition of 5-α reductase (Moorjani et al., 1987). Long-term therapy with 1 mg finasteride tablets may
prevent androgenetic hair loss (Barud et al., 1999). It also
suppresses
the
conversion
of
testosterone
to
dihydrotestosterone, the most potent metabolite of
testosterone (Asscheman et al., 1994; Hämäläinen et al., Afr. J. Pharm. Pharmacol. 316 Table 1. Lipid profile changes. Lipid
Before therapy
After 3 months
After 6 months
Mean ± SD
Mean ± SD
Percent change
Mean ± SD
Percent change
HDL
43.6 ± 9a
44.91 ± 10.7
3.6 ± 17.1
38.5 ± 8.1
-13.2 ± 12a
LDL
103.8 ± 27.8
99.8 ± 26.2
-1.7 ± 22.1
103.2 ± 27.2
-2.6 ± 12.2
HDL/ LDL
0.43 ± 0.1
0.48 ± 0.16
8.9 ± 22.6b
0.39 ± 0.11
-9.2 ± 19.2b
Triglyceride
93 ± 32.9c
117.2 ± 48.2c
32.8 ± 51
94.8 ± 28.3
16.5 ± 48.2
Total cholesterol
162.1 ± 36.6
166.1 ± 32.9
5.3 ± 24.6
160 ± 38.3
-0.9 ± 19.5
Values with different superscripts differ significantly (p < 0.05). Table 1. Lipid profile changes. Doulabi et al. 317 atherosclerosis. Moreover, a study by Moorjani et al. (1987) indicated that anti-androgenic medication such as
finasteride may increase HDL in patients with prostatic
cancer while leaving LDL intact. An Italian study on
patients with benign prostatic hyperplasia indicated that
using 5 mg finasteride tablets may increase HDL and
decrease LDL after 6 months of therapy, which may be
due to the direct impact of the drug on hepatic
metabolism
or
through
dihydrotestosterone
(DHT)
suppression (Denti et al., 2000), (Movérare-Skrtic et
al2006) reported that treatment with DHT may increase
HDL and decrease TG. Amory et al. (2008) indicated that
suppressing DHT with 5-α reductase inhibitors, such as
finasteride tablet, does not lead to adverse modifications
of the lipid profile. Asscheman H, Gooren LJ, Megens JA, Nauta J, Kloosterboer HJ,
Eikelboom F (1994). Serum testosterone level is the major
determinant of the male-female differences in serum levels of high-
density lipoprotein (HDL) cholesterol and HDL2 cholesterol. Metabolism, 43: 935-939. Asscheman H, Gooren LJ, Megens JA, Nauta J, Kloosterboer HJ,
Eikelboom F (1994). Serum testosterone level is the major
determinant of the male-female differences in serum levels of high-
d
i
li
i
(HDL)
h l
l
d
HDL
h l
l density lipoprotein (HDL) cholesterol and HDL2 cholesterol. Metabolism, 43: 935-939. Barud W, Wójcicka G, Palusiński R, Bilan A, Hanzlik J (1999). [Dihydrotestosterone therapy of men with coronary artery disease
and processes of peroxidation]. Pol Merkur Lekarski, 7: 248-50. Denti L, Pasolini G, Cortellini P, Sanfelici L, Benedetti R, Cecchetti A,
Ferretti S, Bruschieri L, Ablondi F, Valenti G (2000). Changes in HDL-
cholesterol and lipoprotein Lp(a) after 6-month treatment with
finasteride in males affected by benign prostatic hyperplasia (BPH). Atherosclerosis, 152: 159-166. Dogramaci AC, Balci DD, Balci A, Karazincir S, Savas N, Topaloglu C
(2009). Is androgenetic alopecia a risk for atherosclerosis? Eur. Acad. Dermatol. Venereol., 23: 673-677. Duskova M, Hill M, Starka L (2010). Changes of metabolic profile in
men treated for androgenetic alopecia with 1 mg finasteride. Endocr. Regul., 44: 3-8. p
p
In addition, Duskova et al. (2010) conducted a study on
12 patients to observe that finasteride an initial rise in
LDL, HDL and total cholesterol which became constant
with progression of the study. Doulabi et al. We assume that the
different results from previous studies may be related to
dosage or duration consumption of finasteride, age and
nutritional status of patients and different genetic ability of
drug hepatic metabolism. While HDL to LDL ratio has
been proved to be a predictor factor of cardiovascular
disease (Reaven., 1988; Onyesom et al., 2012), our
study did not show significant change after 3 and 6
months but changes ratio between third and sixth month
were significant. g
Hämäläinen E, Tikkanen H, Härkönen M, Näveri H, Adlercreutz H
(1987). Serum lipoproteins, sex hormones and sex hormone binding
globulin in middle-aged men of different physical fitness and risk of
coronary heart disease. Atherosclerosis, 67: 155-162. Kannel WB (2005). Risk stratification of dyslipedemia: Insights from the
Framingham study. Curr. Med. Chem. Cardiovasc. Hematol. Agents,
3: 187-193. Fauci AS, Libby P Braunwald E, Kasper DL, Hauser SL, Longo DL,
Jameson JL, Loscalzo J(2008). The Pathogenesis, Prevention and
Treatment of Atherosclerosis In: Harrison’s principles of internal
medicine. New York. 17th ed. McGraw-Hill, pp. 1501-1509. McRobb L, Handelsman DJ, Heather AK (2009). Androgen-induced
progression of arterial calcification in apolipoprotein E-null mice is
uncoupled from plaque growth and lipid levels. Endocrinology, 150:
841-848. Moorjani S, Dupont A, Labrie F, Lupien PJ, Brun D, Gagné C, Giguére
M, Bélanger A (1987). Increase in plasma high-density lipoprotein
concentration following complete androgen blockage in men with
prostatic carcinoma. Metabolism, 36: 244-250. The risk of stroke and myocardial infarction is higher in
young men compared to young women. This change in
lipid profile may be due to testosterone elevation by using
finasteride. As earlier mentioned, it is recommendable to
identify the risk factors for atherosclerosis in patients with
androgenetic hair loss so that 1 mg finasteride tablets
may be waived for high-risk patients. Moreover, the
adverse effect of the drug may be countered through
recommendations made to the patients regarding lifestyle
modification and abstaining from high-fat food. Further
studies aimed at the effects of finasteride exerted on
vessels through lowered DHT may corroborate our
findings. Movérare-Skrtic S, Venken K, Andersson N, Lindberg MK, Svensson J,
Swanson C (2006). Dihydrotestosterone treatment results in obesity
and altered lipid metabolism in orchidectomized mice. Obesity, 14(4):
662-672. Olorunnisola OS, Bradley G, Afolayan J (2012). Effect of methanolic
extract of Tulbaghia violacea rhizomes on antioxidant enzymes and
lipid profile in normal rats. AJPP, 6: 1026-1030 Onyesom I, Osioma E, Testimi OL, Rotu AR (2012). Doulabi et al. Biomarkers of
Metabolic Syndrome in Serum of Some Cigarette Smokers in Delta
State, Nigeria. Am. J. Biochem., 2: 7-10. Reaven GM (1988). Role of insulin resistance in human disease. Diabetes, 37: 1595-1607. ACKNOWLEDGMENT We would like to appreciate DR. Qasem Mirbahari the
pathologist for help us in laboratory issues in conducting
this clinical trial. DISCUSSION Our study is the first to indicate that 1 mg finasteride
tablets used for treatment of androgenetic hair loss
causes a significant decrease in HDL after 6 months of
therapy and a significant increase in triglyceride after 3
months of therapy also changes ratio of HDL/LDL
between third and sixth months significantly (p-
value=0.006), all of which may be increase the risk for Doulabi et al. REFERENCES Al-Ajlan
AR
(2012). Tobacco
smoking
vs. lipid
profile
and
anthropometric measures: A cross-sectional study among students in
the Riyadh College of Health Sci. AJPP, 6: 717-723. y
g
Amory JK, Anawalt BD, Matsumoto AM, Page ST, Bremner WJ, Wang
C, Swerdloff RS, Clark RV (2008). The effect of 5alpha-reductase
inhibition with dutasteride and finasteride on bone mineral density,
serum lipoproteins, hemoglobin, prostate specific antigen and sexual
function in healthy young men. J. Urol., 179: 2333-2338. Amory JK, Anawalt BD, Matsumoto AM, Page ST, Bremner WJ, Wang
C, Swerdloff RS, Clark RV (2008). The effect of 5alpha-reductase
inhibition with dutasteride and finasteride on bone mineral density,
serum lipoproteins, hemoglobin, prostate specific antigen and sexual
function in healthy young men. J. Urol., 179: 2333-2338.
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Experimental Research on Regeneration Characteristic of ED Regeneration for Lithium Bromide Desiccant Solution with High Concentration: Operating Condition and Electrode Solution
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Energies
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cc-by
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Received: 11 August 2020; Accepted: 8 September 2020; Published: 11 September 2020 Abstract: Electrodialysis is regarded as a novel liquid regeneration method, and the regenerated
solution can satisfy the dehumidification requirements even in a hot and humid environment. LiBr solution is an important choice for a liquid desiccant air conditioning system due to its great
dehumidifying ability, so it is necessary to conduct experimental exploration of the regeneration
characteristics of ED regeneration for LiBr solution. In this paper, the effects of solution concentration,
circulation flow rate, current and electrode solution on the performance of the electrodialysis
regeneration system were studied by constructing an experimental electrodialysis regeneration
system. The results show that growing the starting concentration of the LiBr solution adversely
affects the regeneration characteristics of the electrodialyzer and of the air conditioning system
dehumidified by the solution. Under test conditions, as the initial concentration of LiBr solution
increased from 45% to 55%, the performance coefficient (COP) of the system decreased from 2.12 to
1.05. When the dehumidification requirement is met, the initial concentration of the LiBr solution
should be reduced. Increasing the circulating flow rate can improve the regeneration performance of
the electrodialyzer and the capability of the air conditioning system dehumidified by the solution,
but excessively increasing the circulating flow rate will decrease the regeneration performance of the
electrodialyzer and the performance of the air conditioning system dehumidified by the solution. Increasing the current can increase the concentration of the LiBr solution in the regeneration cells in a
short time, but it will reduce the regeneration performance of the electrodialyzer and the characteristic
of the air conditioning system dehumidified by the solution. The current needs to be minimized
when meeting regeneration requirements. With the growth in the flow rate of the electrode solution,
the regeneration performance of the electrodialyser decreases continuously. Keywords: liquid desiccant; regeneration; electrodiaalysis; LiBr; electrode solution Experimental Research on Regeneration
Characteristic of ED Regeneration for Lithium
Bromide Desiccant Solution with High Concentration:
Operating Condition and Electrode Solution Qing Cheng 1,2 and Han Wang 1,2,*
1
Jiangsu Province Key Laboratory of Aerospace Power System, Nanjing 210016, China;
chengqingny@njtech.edu.cn
2
School of Energy Science and Engineering, Nanjing Tech University, Nanjing 211816, China
*
Correspondence: wangh_njt@njtech.edu.cn Qing Cheng 1,2 and Han Wang 1,2,*
1
Jiangsu Province Key Laboratory of Aerospace Power System, Nanjing 210016, China;
chengqingny@njtech.edu.cn
2
School of Energy Science and Engineering, Nanjing Tech University, Nanjing 211816, China
*
Correspondence: wangh_njt@njtech.edu.cn energies energies 1. Introduction Due to high energy loss of buildings’ air conditioning systems, improving the energy efficiency of
buildings plays a vital role in reducing global energy use. In the past few decades, in order to promote
building energy conservation, much research has been done in the application and development of
sustainable and green technologies [1,2]. Liquid desiccant dehumidification technology has received
widespread attention due to its high energy saving potential [3,4], and this technology has been
practically applied in the air dehumidification of buildings. Energies 2020, 13, 4733; doi:10.3390/en13184733 www.mdpi.com/journal/energies www.mdpi.com/journal/energies 2 of 14 Energies 2020, 13, 4733 In the air conditioning system dehumidified by a solution, regeneration of the dehumidification
solution is key to ensuring the continuous work of the air conditioning system dehumidified by the
solution [5,6]. The current thermal regeneration method is the main means of regenerating desiccant
solutions. The thermal regeneration method whose heat source comes from heat pumps, waste,
and solar energy has been extensively discussed [7,8]. However, when the outside environment is
hot and humid, the desiccant solution needs more heat energy and higher temperature in order to be
regenerated, and the hot desiccant solution after heat regeneration needs to be cooled before it can
work to meet the dehumidification requirement. This shortage limits the widespread application of
liquid desiccant air conditioning systems utilizing the thermal regeneration method. In addition to the thermal regeneration method, the electrodialysis (ED) technology for desiccant
solutions is also a reliable method. Electrodialysis technology is mainly used in chemical, food,
pharmaceutical, seawater desalination, wastewater treatment, and ion separation fields [9,10]. The application of electrodialysis for desiccant solution regeneration was first proposed by Li et al. [11]. It is found that, through theoretical analysis, the electrodialysis regeneration method is more stable than
the thermal regeneration method because the weather conditions do not affect the performance of the
electrodialyzer. Cheng et al. [12] also proposed a two-stage photovoltaic electrodialysis regeneration
system. Theoretical analysis found that the two-stage regeneration system has more energy efficiency
than the single stage under reasonable operating conditions. After this, Cheng et al. 1. Introduction [13–15] conducted
a series of experimental studies on electrodialysis regeneration for LiCl solution, and the results showed
that when a 5A current was used to regenerate the dehumidification solution, the temperature of the
dehumidification solution did not rise above 3 ◦C; at the same time, the regeneration performance of
the electrodialyzer was obviously affected by the concentration difference. Al-Jubainawi et al. [16]
carried out experiments using an electrodialyzer with 10 membrane pairs and numerically simulated
the regeneration process of the electrodialyzer using COMSOL Multiphysics. Li and Zhang [17] found
that the current intensity and solute concentration were the key factors affecting the regeneration
performance of the electrodialyzer. At present, lithium chloride is the main solution for the experimental study of electrodialysis
regeneration of desiccant solutions, and experimental exploration of the regeneration of lithium
bromide solution with high concentrations by an electrodialyzer is rarely conducted. Li and Zhang [17]
conducted a theoretical study on the performance of electrodialysis regeneration systems using different
desiccant solutions and found that the characteristic of the electrodialysis regeneration system utilizing
LiBr solution is better than those of lithium chloride (LiCl) and calcium chloride (CaCl2) at the same
current efficiency performance. Liu et al. [18] analyzed and compared the mass transfer performance
between two common hygroscopic salt solutions (LiBr solution and LiCl solution) and humid air,
and the study showed that the dehumidification characteristic of lithium bromide (LiBr) is better than
LiCl when the two solutions are used under the same treatment conditions. However, when the LiBr solution and the LiCl solution have the same dehumidifying ability,
the concentration of the LiBr solution is higher than that of LiCl. The literature data so far show that
the solution concentration is the key factor affecting the regeneration characteristic of ED. Therefore,
the characteristic of the electrodialyzer to regenerate lithium bromide and lithium chloride is also very
different, so it is necessary to conduct experimental exploration of the regeneration characteristic of ED
regeneration for LiBr solution. On the other hand, electrode solution is significant for the regeneration
characteristic of ED, so it is also important to study the effect of electrode solution on the regeneration
characteristic of ED for LiBr solution with a high concentration. 2.1. Electrodialysis Method
2.1. Electrodialysis Method In an electrodialyzer, cation-exchange membranes (CMV) and anion-exchange membranes (AMV)
are alternately placed between a cathode and an anode. An example of an electrodialyzer with one
membrane pair is shown in Figure 1. In an electrodialyzer, cation‐exchange membranes (CMV) and anion‐exchange membranes
(AMV) are alternately placed between a cathode and an anode. An example of an electrodialyzer
with one membrane pair is shown in Figure 1. Figure 1. Schematic diagram of electrodialyzer. Figure 1. Schematic diagram of electrodialyzer. Figure 1. Schematic diagram of electrodialyzer. Figure 1. Schematic diagram of electrodialyzer. Figure 1. Schematic diagram of electrodialyzer. Figure 1. Schematic diagram of electrodialyzer. Under the effect of electric field force, Br− moves to the anode through the membranes of anion
exchange, and Li+ moves to the cathode through the membranes of cation exchange. During the
migration process, the membranes of anion exchange only allow Br− to pass and retain Li+; the
membranes of cation exchange only allow Li+ to pass and retain Br−. So, the concentration of the LiBr
solution in the regenerate panels will increase and the concentration of the LiBr solution in the dilute
panels will decrease. The concentrated LiBr solution in the regenerate panels flows into the
regeneration solution tank, and the dilute LiBr solution in the dilute panel flows into the desalination
solution tank. Finally, the concentrated LiBr solution required in the air conditioning system
dehumidified by solution can be obtained in the regeneration solution tank. Under the effect of electric field force, Br−moves to the anode through the membranes of anion
exchange, and Li+ moves to the cathode through the membranes of cation exchange. During the
migration process, the membranes of anion exchange only allow Br−to pass and retain Li+;
the membranes of cation exchange only allow Li+ to pass and retain Br−. So, the concentration
of the LiBr solution in the regenerate panels will increase and the concentration of the LiBr solution
in the dilute panels will decrease. The concentrated LiBr solution in the regenerate panels flows
into the regeneration solution tank, and the dilute LiBr solution in the dilute panel flows into the
desalination solution tank. Finally, the concentrated LiBr solution required in the air conditioning
system dehumidified by solution can be obtained in the regeneration solution tank. 2. Method
2. Method 2.1. Electrodialysis Method
2.1. Electrodialysis Method 1. Introduction This paper focuses on studying the effects of LiBr solution concentration, flow rate, current,
and electrode solution on the regeneration characteristic of the electrodialyzer to regenerate lithium
bromide solution and to analyze the regeneration performance of the electrodialysis regeneration
system for LiBr solution based on the experimental data. 3 of 14
3 of 14 Energies 2020, 13, 4733
Energies 2020 13 x FOR 2.2. Test Device
2.2. Test Device A simplified schematic of the experimental setup is shown in Figure 2. The electrodialysis
regeneration system is mainly composed of an electrodialyzer and a rectifier. The system also
includes three circulation pumps, three solution tanks, and three flow meters, which are used in the
three circulation circuits of regeneration, desalination, and electrodes. The electrodialyzer is the core
device of the test. The electrodialyzer used in this test consists of 30 membrane pairs, and the effective
size of the membrane is 200 × 100 mm. In this experimental system, an AMV anion‐exchange
membrane and CMV cation‐exchange membrane (both supplied by AGC) are applied. Meanwhile,
the thickness of the regeneration panel and the desalination panel of the electrodialyzer was 1 mm. A simplified schematic of the experimental setup is shown in Figure 2. The electrodialysis
regeneration system is mainly composed of an electrodialyzer and a rectifier. The system also includes
three circulation pumps, three solution tanks, and three flow meters, which are used in the three
circulation circuits of regeneration, desalination, and electrodes. The electrodialyzer is the core device
of the test. The electrodialyzer used in this test consists of 30 membrane pairs, and the effective size of
the membrane is 200 × 100 mm. In this experimental system, an AMV anion-exchange membrane and
CMV cation-exchange membrane (both supplied by AGC) are applied. Meanwhile, the thickness of
the regeneration panel and the desalination panel of the electrodialyzer was 1 mm. 4 of 14
f Energies 2020, 13, 4733 Figure 2. Schematic diagram of electrodialyzer. Figure 2. Schematic diagram of electrodialyzer. Figure 2. Schematic diagram of electrodialyzer. Figure 2. Schematic diagram of electrodialyzer. During the experiment, in order to calculate the concentration of the LiBr solution, the density
and temperature of the lithium bromide solution need to be measured. The purity of LiBr used in the
test was over 99%. The main measuring instruments and their measurement accuracy are shown in
Table 1. In the analysis of the experiment results, the average values of testing parameters have been
employed for evaluating the regeneration characteristic of the ED regenerator. During the experiment, in order to calculate the concentration of the LiBr solution, the density
and temperature of the lithium bromide solution need to be measured. The purity of LiBr used in the
test was over 99%. The main measuring instruments and their measurement accuracy are shown in
Table 1. 2.2. Test Device
2.2. Test Device in which y
is relative uncertainty, y is the variable, xi is the independent measured variable, n is
the number of independent measured variables. According to Equation (1), the maximum composite relative uncertainty of the LiBr solution
concentration in the experiment is 0.383%. 2.2. Test Device
2.2. Test Device In the analysis of the experiment results, the average values of testing parameters have been
employed for evaluating the regeneration characteristic of the ED regenerator. Table 1. Test instrument and accuracy. Table 1. Test instrument and accuracy. Table 1. Test instrument and accuracy. Name
Model
Test Dimension
Accuracy
Rectifier
HCP‐DC
0–20 A
±0.01 A
Density meter
AU‐120L
0.0001–99.9999 g/cm3
0.0001 g/cm3
Thermocouple
TC‐TT‐T‐30‐5M
−200–350 °C
±0.5 °C
Flowmeter
LFS15
16–160 L/h
±5 L/h
Data acquisition instrument
Agilent 34970A
‐
‐
For concentration calculation the relative uncertainty can be determined by Equation (1)[19]
Table 1. Test instrument and accuracy. Name
Model
Test Dimension
Accuracy
Rectifier
HCP-DC
0–20 A
±0.01 A
Density meter
AU-120L
0.0001–99.9999 g/cm3
0.0001 g/cm3
Thermocouple
TC-TT-T-30-5M
−200–350 ◦C
±0.5 ◦C
Flowmeter
LFS15
16–160 L/h
±5 L/h
Data acquisition instrument
Agilent 34970A
-
-
For concentration calculation, the relative uncertainty can be determined by Equation (1) [19]. Table 1. Test instrument and accuracy. Table 1. Test instrument and accuracy. For concentration calculation, the relative uncertainty can be determined by Equation (1)[19]. For concentration calculation, the relative uncertainty can be determined by Equation (1) [19]. For concentration calculation, the relative uncertainty can be determined by Equation (1)[19]. For concentration calculation, the relative uncertainty can be determined by Equation (1) [19]. 2
1
δ
i
n
i
i
y
x
x
y
(1)
δy
y =
r
Pi=1
n
δy
δxi δxi
2
y
(1) (1)
(1) y
y
i
hi h δy
i
l ti
t i t
i th
i bl
i th i d
d
t
d
i bl
i
in which δy
y is relative uncertainty, y is the variable, xi is the independent measured variable, n is the
number of independent measured variables. y
y
i
hi h δy
i
l ti
t i t
i th
i bl
i th i d
d
t
d
i bl
i
in which δy
y is relative uncertainty, y is the variable, xi is the independent measured variable, n is the
number of independent measured variables. in which y
is relative uncertainty, y is the variable, xi is the independent measured variable, n is
the number of independent measured variables. According to Equation (1), the maximum composite relative uncertainty of the LiBr solution
concentration in the experiment is 0.383%. According
concentration in
2.3. Test Scheme concentration in the experiment is 0.383%. 2.3. Test Scheme
Twenty‐six tests were selected and divided into 3 groups, as shown in Table 2, to explain the
effect of solution concentration, flow rate, current, and electrode solution on the performance of the
Twenty-six tests were selected and divided into 3 groups, as shown in Table 2, to explain the
effect of solution concentration, flow rate, current, and electrode solution on the performance of
the electrodialysis regeneration system for LiBr solution regeneration. In this scheme, values of
current and flow rate of desiccant solutions are calculated in the design stage of the ED regenerator; 5 of 14 Energies 2020, 13, 4733 the initial concentration of desiccant solutions is based on the dehumidification requirement under
different environment climates. In Table 2, flow rate in panels includes the flow rates of desiccant
solution in all panels, including electrode chambers. Each test was run for 66 min, and all experiments
were performed continuously under ambient conditions. During the experiment, the density and
temperature of the inlet and outlet solutions of the regeneration panel and the desalination panel were
measured every 6 min, and the rectifier voltage was recorded. Table 2. Test conditions. Serial Number
Flow Rate in Panels
(L/h)
Current (A)
Initial Concentration of
Desiccant Solution (%)
Group 1
80
5
45, 50, 55
80, 120, 160
5
50
80
3, 5, 8
50
Group 2
80
5
27, 35, 42
80, 120, 160
5
35
80
3, 5, 8
35
Group 3
120
8
45, 50, 55
80, 120, 160
8
45
80
8
25 Table 2. Test conditions. Table 2. Test conditions. Analytical Method for Experiment 2.4. Analytical Method for Experiment The experimental operating cost of an electrodialysis
regeneration system for desiccant solution can be obtained as follows: Ps = UI
(5) Ps = UI (5) Note that U is the rectifier voltage, V, in the formula. The dehumidification capacity of the electrodialysis regeneration system for desiccant solution
can be described by the following equation: Qr =
rwmreg
Coreg −Cireg
Cireg
(6) (6) which rw is the latent heat of vaporization of water, kJ/kg. Therefore, the theoretical and experimental performance coefficient (COP) of the electrodialysis
regeneration system for desiccant solution is as follows: COPl = Qr
Pl
=
rwmreg
Coreg −Cireg
"
zFmreg(Coreg −Cireg)
ζMd
#2 ×
N
Cireg(Ram + Rcm +
Lr
Aeρreg +
Lr
Aeρdiu )
(7) (7) COPs = Qr
Ps
=
rwmreg
Coreg −Cireg
UICireg
(8) (8) 2.4. Analytical Method for Experiment The concentration variety of the LiBr solution in the regeneration panels is a significant index
for evaluating the regeneration performance of the electrodialysis and can be obtained by the
following methods: (2) Ccadd = Coreg −Cireg
(2) in which Ccadd is the concentration difference at the outlet and inlet of the regeneration panels of the
electrodialyzer, Coreg is the concentration of LiBr solution exiting the regeneration panels, Cireg is the
concentration of LiBr solution entering the regeneration panels. in which Ccadd is the concentration difference at the outlet and inlet of the regeneration panels of the
electrodialyzer, Coreg is the concentration of LiBr solution exiting the regeneration panels, Cireg is the
concentration of LiBr solution entering the regeneration panels. Current efficiency is also a crucial evaluation parameter of the electrodialysis regeneration system,
which is the ratio of the charge transferred by the ion to the total charge applied to the electrodialyzer. It can be calculated by the following equation: ζ =
zFmreg
Coreg −Cireg
INMd
(3) (3) in which z is solution valence, F is Faraday constant, C/mol, mreg is the flow rate of concentrated
solution, kg/s; I is operating current of electrodialysis, A, N is membrane logarithm of electrodialysis
regenerator; Md is molar mass of solution, kg/mol. The operating cost of an electrodialysis regeneration system for desiccant solution is mainly
dependent on the electricity costs of the electrodialysis regenerator and solution pump. The operating
cost of the solution pump is ignored in the following studies, as it is much lower than the operating
cost of the electrodialyzer. Therefore, the theoretical operating cost of an electrodialysis regeneration
system for desiccant solution can be obtained as follows: Pl =
zFmreg
Coreg −Cireg
ζMd
2
×
(Ram + Rcm +
Lr
Aeρreg +
Lr
Aeρdiu )
N
(4) (4) N in which Rcm is the resistance of the anion-exchange membrane, Ω, Ram is the resistance of the
cation-exchange membrane, Ω, Lr is the thickness of one regeneration panel, m, Ae is the effective
transfer area of the membrane, m2, ρreg is the conductivity of the regenerate solution, s/m, ρdiu is the Energies 2020, 13, 4733 6 of 14 conductivity of the desalination solution, s/m. The experimental operating cost of an electrodialysis
regeneration system for desiccant solution can be obtained as follows: conductivity of the desalination solution, s/m. 3.1. Effect of Initial Concentration 3.1. Effect of Initial Concentration The regeneration performance of the electrodialyzer with different initial concentrations is shown
in Figure 3a. When the initial lithium bromide concentration was increased from 45% to 55%, the current
efficiency of the electrodialyzer to regenerate the lithium bromide solution decreased from 14.72% to
10.73%. At the end of the experiment, the lithium bromide solution concentration in the regeneration
tank decreased from 0.408% to 0.224% with the increase of the initial lithium bromide concentration. This is due to the increase in the initial concentration, which continuously increases the viscosity
of the lithium bromide solution, which leads to a decrease in the regeneration performance of the
electrodialysis regenerator. y
g
The effects of the initial concentration of LiBr solution in the electrodialysis regeneration system
on the energy consumption and COP of the system under theoretical and experimental conditions are
respectively shown in Figure 3b,c. When the initial concentration of LiBr solution was increased from
45% to 55%, the energy consumption of the system increased slightly under theoretical conditions,
and the theoretical COP of the system decreased from 9.17 to 5.46. Under test conditions, as the initial concentration of LiBr solution was raised from 45% to 55%,
the energy consumption of the system increased rapidly from 50 to 107.5 W; the COP of the system
decreased from 2.12 to 1.05. The main reason for this is that, when the concentration of the lithium
bromide solution is between 45% and 55%, the conductivity of the lithium bromide decreases as the
concentration increases, which leads to an increase in the resistance of the electrodialyzer and an
increase in energy consumption during regeneration. 7 of 14
7 of 14 Energies 2020, 13, 4733
Energies 2020 13 x FO 2020, 13, x FOR PEER REVIEW
7
44
46
48
50
52
54
56
10
11
12
13
14
15
16
Ctadd
C (%)
(%)
(a)
0.20
0.24
0.28
0.32
0.36
0.40
Ctadd (%)
44
46
48
50
52
54
56
10.86
10.87
10.88
10.89
10.90
10.91
Pl
COPl
C (%)
Pl( W)
(b)
5
6
7
8
9
COPl
44
46
48
50
52
54
56
50
60
70
80
90
100
110
Ps
COPs
C (%)
Ps( W)
(c)
1.0
1.2
1.4
1.6
1.8
2.0
2.2
COPs
igure 3. 3.2. Effect of Flow Rate The regeneration performance of the electrodialyzer as a function of flow rate is discussed. As we
can see from Figure 4a, the current efficiency of the electrodialyzer appears to increase first and
then decrease when the flow rate is continuously increased. With the flow rate increased from 80
to 160 L/h, the current efficiency of the electrodialyzer to regenerate the lithium bromide solution
increased from 13% to 15.83% and then decreased to 12.23%. Ultimately, the concentration of the
lithium bromide solution in the regeneration tank first increased and then decreased. When the flow
rate was 80, 120, and 160 L/h, the concentration increase was 0.347%, 0.386%, and 0.294%, respectively. Properly increasing the flow rate can enhance the regeneration performance of the electrodialyzer by
improving the diffusing performance between ions on the boundary of membrane and ions in the
mainstream of the liquid desiccant, but excessively increasing the flow rate will reduce the time for the
mainstream of the liquid desiccant to stay in the electrodialyzer, and the ions will not have enough time
to pass through the membrane, which will reduce the regeneration performance of the electrodialyzer. The effects of solution flow rate in the channel on the system energy consumption and system
performance under the theoretical and experimental conditions in ED regeneration for LiBr solution
are respectively shown in Figure 4b,c. Increasing the flow rate can save energy for the system. Under theoretical conditions, when the inlet flow rate is increased from 80 to 160 L/h, the energy
consumption of the system is basically unchanged, and the system performance tends to increase first
and then decrease. The maximum system COP can reach 10.49. Under the test conditions, when the
flow rate was increased from 80 to 160 L/h, the system energy consumption decreased from 82.5 to 78 W. As the flow rate increases, the system performance tends to increase first and then decrease. When the
flow rates are 80, 120, and 160 L/h, the system COPs are 0.624, 0.780, and 0.60, respectively. This is
because the regeneration performance of the electrodialyzer increases first and then decreases owing to
the increase in the inlet flow rate, but the energy consumption decreases with the change in the flow rate. Therefore, when the electrodialyzer is used to regenerate the dehumidifying solution, an appropriate
solution flow rate should be selected so that the electrodialyzer has the desired performance. 3.1. Effect of Initial Concentration (a) Effect of initial concentration on regeneration performance of electrodialyzer; (b) effec
f initial concentration on performance of electrodialysis regeneration system under theoretica
onditions; (c) effect of initial concentration on performance of electrodialysis regeneration system
nder experimental conditions. gure 3. (a) Effect of initial concentration on regeneration performance of electrodialyzer; (b) effect
initial concentration on performance of electrodialysis regeneration system under theoretical
nditions; (c) effect of initial concentration on performance of electrodialysis regeneration system
der experimental conditions. 44
46
48
50
52
54
56
10
11
12
13
14
15
16
Ctadd
C (%)
(%)
(a)
0.20
0.24
0.28
0.32
0.36
0.40
Ctadd (%)
44
46
48
50
52
54
56
10.86
10.87
10.88
10.89
10.90
10.91
Pl
COPl
C (%)
Pl( W)
(b)
5
6
7
8
9
COPl
80
90
100
110
Ps
COPs
W)
(c)
1.6
1.8
2.0
2.2
Ps 44
46
48
50
52
54
56
10
11
12
13
14
15
16
Ctadd
C (%)
(%)
(a)
0.20
0.24
0.28
0.32
0.36
0.40
Ctadd (%) 44
46
48
50
52
54
56
10.86
10.87
10.88
10.89
10.90
10.91
Pl
COPl
C (%)
Pl( W)
(b)
5
6
7
8
9
COPl 44
46
48
50
52
54
56
50
60
70
80
90
100
110
Ps
COPs
C (%)
Ps( W)
(c)
1.0
1.2
1.4
1.6
1.8
2.0
2.2
COPs Figure 3. (a) Effect of initial concentration on regeneration performance of electrodialyzer; (b) effect
of initial concentration on performance of electrodialysis regeneration system under theoretical
conditions; (c) effect of initial concentration on performance of electrodialysis regeneration system
under experimental conditions. Figure 3. (a) Effect of initial concentration on regeneration performance of electrodialyzer; (b) effect
of initial concentration on performance of electrodialysis regeneration system under theoretical
conditions; (c) effect of initial concentration on performance of electrodialysis regeneration system
under experimental conditions. 8 of 14 Energies 2020, 13, 4733 3.3. Effect of Current Under various applied currents, the current efficiency of the electrodialyzer to regenerate the
lithium bromide solution and the incremental change in the solution concentration in the regeneration
tank are shown in Figure 5a. When the current was increased from 3 to 8A, the current efficiency
of the electrodialyzer decreased from 17.45% to 10.2%; at the end of the experiment, the solution
concentration in the regeneration tank increased from 0.259% to 0.484%. This shows that a large current
can lead to an increase in the concentration of the desiccant solution in a short time, but it will cause
the regeneration performance of the electrodialyzer to decrease. The effects of current on the performance of the system and energy consumption under the
theoretical and experimental conditions of the electrodialysis regeneration system for LiBr solution are
respectively shown in Figure 5b,c. With the increase in current, the energy consumption of the system
increases rapidly under both theoretical and experimental conditions, and the COP of the system
continues to decrease. When the current is increased from 3 to 8 A, the system energy consumption
increases from 3.915 to 27.84 W under theoretical conditions, and the system performance coefficient
decreases from 16.237 to 3.584; under the experimental conditions, the system energy consumption
increases from 30.9 to 189.6 W, and the system performance decreases from 2.073 to 0.523. 9 of 14
olution
m Energies 2020, 13, 4733
performance unde
are respectively sh pe i e y
o
i
igu e
,
I
ea i g
e
o
a e a
a e e e gy o
e y e
80
100
120
140
160
12
13
14
15
16
17
Ctadd
Qv (L/h)
(%)
(a)
0.28
0.30
0.32
0.34
0.36
0.38
0.40
Ctadd( %)
2020, 13, x FOR PEER REVIEW
9
80
100
120
140
160
9.50
9.55
9.60
9.65
9.70
Pl
COPl
Qv (L/h)
Pl( W)
8.0
8.5
9.0
9.5
10.0
10.5
11.0
COPl
(b)
80
100
120
140
160
78
79
80
81
82
83
Ps
COPs
Qv (L/h)
Ps( W)
(c)
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
COPs
gure 4. (a) Effect of flow rate on regeneration performance of electrodialyzer; (b) effect of flow rate
n performance of electrodialysis regeneration system under theoretical conditions; (c) effect of flow
te on performance of electrodialysis regeneration system under test conditions. 3.3. Effect of Current d
th
ti
l
diti
h
th i l t fl
t i i
d f
80 t 160 L/h th
gure 4. (a) Effect of flow rate on regeneration performance of electrodialyzer; (b) effect of flow rate
n performance of electrodialysis regeneration system under theoretical conditions; (c) effect of flow
te on performance of electrodialysis regeneration system under test conditions. 80
100
120
140
160
12
13
14
15
16
17
Ctadd
Q (L/h)
(%)
(a)
0.28
0.30
0.32
0.34
0.36
0.38
0.40
Ctadd( %)
13, x FOR PEER REVIEW 80
100
120
140
160
9.50
9.55
9.60
9.65
9.70
Pl
COPl
Qv (L/h)
Pl( W)
8.0
8.5
9.0
9.5
10.0
10.5
11.0
COPl
(b) (b) 80
100
120
140
160
78
79
80
81
82
83
Ps
COPs
Qv (L/h)
Ps( W)
(c)
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
COPs Figure 4. (a) Effect of flow rate on regeneration performance of electrodialyzer; (b) effect of flow rate
on performance of electrodialysis regeneration system under theoretical conditions; (c) effect of flow
rate on performance of electrodialysis regeneration system under test conditions. Figure 4. (a) Effect of flow rate on regeneration performance of electrodialyzer; (b) effect of flow rate
on performance of electrodialysis regeneration system under theoretical conditions; (c) effect of flow
rate on performance of electrodialysis regeneration system under test conditions. 10 of 14
me, but Energies 2020, 13, 4733
large current can le
it will cause the reg 3
4
5
6
7
8
10
12
14
16
18
Ctadd
I (A)
(%)
0.25
0.30
0.35
0.40
0.45
0.50
Ctadd( %)
(a)
3
4
5
6
7
8
0
5
10
15
20
25
30
Pl
COPl
I (A)
Pl( W)
(b)
2
4
6
8
10
12
14
16
18
COPl
3
4
5
6
7
8
40
80
120
160
200
Ps
COPs
I (A)
Ps( W)
0.4
0.8
1.2
1.6
2.0
COPs
(c)
Figure 5. (a) The effect of current on the performance of the electrodialyzer; (b) the effect of current
on the performance of the electrodialysis regeneration system under theoretical conditions; (c) the
effect of current on the performance of the electrodialysis regeneration system under test conditions. Figure 5. The effects of current on
3.4. Effect of Electrode Solution The effects of current on the performance of the system and energy consumption under the
theoretical and experimental conditions of the electrodialysis regeneration system for LiBr solution
are respectively shown in Figure 5b,c. With the increase in current, the energy consumption of the
system increases rapidly under both theoretical and experimental conditions, and the COP of the
system continues to decrease. When the current is increased from 3 to 8 A, the system energy
consumption increases from 3.915 to 27.84 W under theoretical conditions, and the system
performance coefficient decreases from 16.237 to 3.584; under the experimental conditions, the system
energy consumption increases from 30.9 to 189.6 W, and the system performance decreases from
2.073 to 0.523. Figure 6a shows the regeneration performance of the electrodialyzer using different electrode
solutions with different concentrations. It is shown from the figure that, at the concentration of LiBr
solution of 55%, the regeneration performance of the electrodialyzer decreases with the concentration
of the electrode solution. When the electrode solution is LiBr solution with the concentration of 55%,
50%, 45%, the concentration difference of LiBr solutions at the inlet and outlet of regenerate panels in
the electrodialysis regenerator is around 0.07%, 0.06%, 0.05%; when the electrode solution is Li2SO4
solution with a concentration of 25%, the concentration difference of LiBr solution at the inlet and
outlet of regenerate panels is around 0.04%. 2.073 to 0.523. 3.4. Effect of Electrode Solution
Figure 6a shows the regeneration performance of the electrodialyzer using different electrode
solutions with different concentrations. It is shown from the figure that, at the concentration of LiBr
solution of 55%, the regeneration performance of the electrodialyzer decreases with the concentration
of the electrode solution. When the electrode solution is LiBr solution with the concentration of 55%,
50% 45% the concentration difference of LiBr solutions at the inlet and outlet of regenerate panels in
Figure 6b shows the regeneration performance of the electrodialysis regenerator with different
circulation rates of the electrode solution. It can be seen that, with the change in flow rate of the
electrode solution, the regeneration performance of the electrodialyzer decreases continuously. At the
flow rate of the electrode solution of 80, 120, and 160 L/h, the concentration difference of LiBr solution
at the inlet and outlet of the regenerate panels is around 0.07%, 0.05%, and 0.04%. 3.3. Effect of Current (a) The effect of current on the performance of the electrodialyzer; (b) the effect of current on
the performance of the electrodialysis regeneration system under theoretical conditions; (c) the effect of
current on the performance of the electrodialysis regeneration system under test conditions. 3
4
5
6
7
8
10
12
14
16
18
Ctadd
I (A)
(%)
0.25
0.30
0.35
0.40
0.45
0.50
Ctadd( %)
(a)
3
4
5
6
7
8
0
5
10
15
20
25
30
Pl
COPl
I (A)
Pl( W)
(b)
2
4
6
8
10
12
14
16
18
COPl 3
4
5
6
7
8
10
12
14
16
18
Ctadd
I (A)
(%)
0.25
0.30
0.35
0.40
0.45
0.50
Ctadd( %)
(a) 3
4
5
6
7
8
0
5
10
15
20
25
30
Pl
COPl
I (A)
Pl( W)
(b)
2
4
6
8
10
12
14
16
18
COPl 3
4
5
6
7
8
40
80
120
160
200
Ps
COPs
I (A)
Ps( W)
0.4
0.8
1.2
1.6
2.0
COPs
(c) I (A) Figure 5. (a) The effect of current on the performance of the electrodialyzer; (b) the effect of current
on the performance of the electrodialysis regeneration system under theoretical conditions; (c) the
effect of current on the performance of the electrodialysis regeneration system under test conditions. Figure 5. (a) The effect of current on the performance of the electrodialyzer; (b) the effect of current on
the performance of the electrodialysis regeneration system under theoretical conditions; (c) the effect of
current on the performance of the electrodialysis regeneration system under test conditions. The effects of current on
3.4. Effect of Electrode Solution This is because the
increase in the circulating flow rate in the electrode panels enhances the migration process of the water
molecules in the electrode solution to the LiBr solution in the regeneration panels. 11 of 14
igration
neration Energies 2020, 13, 4733
because the increa
process of the wat panels. (a)
(b)
Figure 6. The effect of electrode solution. (a) The effect of concentration of electrode solution. (b) Th
effect of flow rate of electrode solution. Figure 6. The effect of electrode solution. (a) The effect of concentration of electrode solution. (b) Th
effect of flow rate of electrode solution. (b) Figure 6. The effect of electrode solution. (a) The effect of concentration of electrode solution. (b) The
effect of flow rate of electrode solution. Figure 6. The effect of electrode solution. (a) The effect of concentration of electrode solution. (b) The
effect of flow rate of electrode solution. 3.5. Temperature Change of the Regeneration Tank Solution 3.5. Temperature Change of the Regeneration Tank Solution Figure 7 shows the relationship between the operating current and the temperature change of the
regenerate desiccant solution. The hot liquid desiccant shows weak dehumidification performance in
the dehumidifier, so the increase of liquid desiccant temperature is harmful to the dehumidification
performance and the hot liquid desiccant should be cooled before it enters the dehumidifier. As a
result, both concentration and temperature of liquid desiccant after regeneration should be researched
to evaluate the performance of the ED regenerator. 12 of 14
tering the Energies 2020, 13, 4733
temperature rise i
dehumidifier, whi 0
6
12 18 24 30 36 42 48 54 60 66
32
33
34
35
36
3A
5A
8A
t (
oC)
(min)
Figure 7. Effect of current on temperature of lithium bromide regeneration solution. Figure 7. Effect of current on temperature of lithium bromide regeneration solution. Figure 7. Effect of current on temperature of lithium bromide regeneration solution
Figure 7. Effect of current on temperature of lithium bromide regeneration solution. Under the same current conditions, the concentration and flow rate of the desiccant liquid have
little effect on the temperature of the regeneration solution, so they are not listed here. 3.6. The effects of current on
3.4. Effect of Electrode Solution Discussion
As the research result is based on the experimental result, the results of this research can be
employed with conditions including currents varying from 3 to 8 A, flow rates of liquid desiccant
i
f
80 t
160 L/h
d
t
ti
f LiB
l ti
i
f
25% t
55% Th
It is concluded that, when the current increases, the temperature difference of the regenerate
desiccant solution increases continuously at the end of the test; when the current is 3, 5, and 8 A,
the temperature of the regeneration solution increases by 1.1, 1.6, and 3.7 ◦C, respectively. As we
can see from the figure, the temperature of the regenerate solution will keep constant after rapidly
increasing at the beginning of the test. When an electrodialyzer is used to regenerate LiBr solution,
the solution temperature rise is limited, and the desiccant solution does not need to be cooled before
entering the dehumidifier, which decreases the system energy consumption. varying from 80 to 160 L/h, and mass concentrations of LiBr solution varying from 25% to 55%. The
experimental result shows that, with the increase in the concentration of the liquid desiccant, more
electrolytic gas will be generated at the electrodes resulting in an increase in resistance which is not
Under the same current conditions, the concentration and flow rate of the desiccant liquid have
little effect on the temperature of the regeneration solution, so they are not listed here. considered in
d
i
t
l
3.6. Discussion desiccant solution, the initial concentration of the LiBr solution should be minimized to make the
electrodialysis regeneration system have ideal performance under the premise of meeting the needs
of dehumidification. Although increasing the current can improve the regeneration effect, the energy
consumption of the electrodialysis regeneration system for LiBr solution is greatly increased. Therefore, when the regeneration requirement is met, the applied current should be minimized. Meanwhile, when the concentration of LiBr solution in the regeneration panels is higher than that of
As the research result is based on the experimental result, the results of this research can be
employed with conditions including currents varying from 3 to 8 A, flow rates of liquid desiccant
varying from 80 to 160 L/h, and mass concentrations of LiBr solution varying from 25% to 55%. The experimental result shows that, with the increase in the concentration of the liquid desiccant,
more electrolytic gas will be generated at the electrodes, resulting in an increase in resistance, which is
not considered in the theoretical conditions. Therefore, when an electrodialyzer is used to regenerate
the desiccant solution, the initial concentration of the LiBr solution should be minimized to make the
electrodialysis regeneration system have ideal performance under the premise of meeting the needs of
dehumidification. Although increasing the current can improve the regeneration effect, the energy
consumption of the electrodialysis regeneration system for LiBr solution is greatly increased. Therefore,
when the regeneration requirement is met, the applied current should be minimized. Meanwhile,
when the concentration of LiBr solution in the regeneration panels is higher than that of the electrode
solution, the solution in the regeneration panels will penetrate into the electrode panel, thereby leading
to a drop in the regeneration performance of the electrodialyzer. There is a major difference between
theoretical and experimental values in this paper—this is mainly because the resistance of electrode
chambers and electrodes is neglected in the theoretical analysis, but the comparative results show
that the resistance of electrode chambers is also important for the ED regenerator, as is the huge gas
production in electrode chambers resulting from the large operational current. Consequently, it is
important to evaluate the resistance of electrode chambers in the ED regenerator in the future work. 13 of 14 Energies 2020, 13, 4733 13 of 14 4. Conclusions Funding: This research was funded by (Jiangsu Province Key Laboratory of Aerospace Power System) g
number (CEPE2018002) Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 4. Conclusions In this paper, an experimental system of electrodialysis regeneration for LiBr solution was
established, and the effects of solution concentration, flow rate, current, and electrode solution on
the regeneration performance of electrodialysis and the performance of an air conditioning system
dehumidified by solution based on ED regeneration were studied. The results are as follows: 1. Increasing the starting concentration of the LiBr solution adversely affects the regeneration
performance of the electrodialyzer and the characteristic of the air conditioning system
dehumidified by the solution. When the dehumidification requirement is met, the initial
concentration of the LiBr solution should be reduced. 2. Increasing the circulating flow rate can improve the regeneration performance of the electrodialyzer
and the characteristic of the air conditioning system dehumidified by the solution, but excessively
increasing the flow rate will reduce the regeneration performance of the electrodialyzer and the
performance of the air conditioning system dehumidified by the solution. 3. Increasing the operating current can increase the concentration of the LiBr solution in the
concentration cells in a short time, but it will reduce the regeneration performance of the
electrodialyzer and the characteristic of the air conditioning system dehumidified by the solution. The current needs to be minimized when meeting regeneration requirements. 4. Through theoretical analysis and experimental research, it is found that the theoretical performance
of the air conditioning system dehumidified by the solution is much larger than the experimental
value, which is mainly because the theoretical power consumption of the system is much smaller
than the experimental value. This shows that seeking to lower the energy consumption of the
electrodialysis system is a method by which to improve the performance of electrodialysis in
the future. 5. With the increase in the flow rate of the electrode solution, the regeneration performance of the
electrodialyzer decreases continuously. 5. With the increase in the flow rate of the electrode solution, the regeneration performance of the
electrodialyzer decreases continuously. 6. When the electrodialyzer regenerates the LiBr solution, the temperature rise of the LiBr solution is
small. Therefore, the LiBr solution does not need to be pre-cooled before entering the dehumidifier,
which decreases the system’s energy consumption. Author Contributions: Investigation, H.W.; methodology, Q.C. All authors have read and agreed to the publis
version of the manuscript. References 1. Lin, W.; Ma, Z.; Sohel, M.I.; Cooper, P. Development and evaluation of a ceiling ventilation system enhanced
by solar photovoltaic thermal collectors and phase change materials. Energy Convers. Manag. 2014, 88,
218–230. [CrossRef] 1. Lin, W.; Ma, Z.; Sohel, M.I.; Cooper, P. Development and evaluation of a ceiling ventilation system enhanced
by solar photovoltaic thermal collectors and phase change materials. Energy Convers. Manag. 2014, 88,
218–230. [CrossRef] 2. Kreiner, H.; Passer, A.; Wallbaum, H. A new systemic approach to improve the sustainability performance of
office buildings in the early design stage. Energy Build. 2015, 109, 385–396. [CrossRef] 3. Jiang, X.; He, H.; Li, X.; Mao, H. Performance analysis and mixing ratio selection of mixed liquid desiccant
for a dehumidification system. Indoor Built Environ. 2020, 29, 711–720. [CrossRef] 4. Guo, Y. Electrodialysis-Assisted Liquid Desiccant Dehumidification: Experimental Investigation and System
Development. Ph.D. Thesis, University of Wollongong, Wollongong, Australia, 2017. Available online:
https://ro.uow.edu.au/theses1/114 (accessed on 10 September 2020). 5. Duong, H.; Ansari, A.; Nghiem, L.; Cao, H.; Vu, T.; Nguyen, T. Membrane processes for the regeneration of
liquid desiccant solution for air conditioning. Curr. Pollut. Rep. 2019, 5, 308–318. [CrossRef] 5. Duong, H.; Ansari, A.; Nghiem, L.; Cao, H.; Vu, T.; Nguyen, T. Membrane processes for the regeneration of
liquid desiccant solution for air conditioning. Curr. Pollut. Rep. 2019, 5, 308–318. [CrossRef] 6. Duong, H.; Hai, F.; Al-Jubainawi, A.; Ma, Z.; He, T. Liquid desiccant lithium chloride regeneration by
membrane distillation for air conditioning. Sep. Purif. Technol. 2017, 177, 121–128. [CrossRef] 6. Duong, H.; Hai, F.; Al-Jubainawi, A.; Ma, Z.; He, T. Liquid desiccant lithium chloride regeneration by
membrane distillation for air conditioning. Sep. Purif. Technol. 2017, 177, 121–128. [CrossRef] Energies 2020, 13, 4733 14 of 14 7. Niu, X.; Xiao, F.; Ma, Z. Investigation on capacity matching in liquid desiccant and heat pump hybrid
air-conditioning systems. Int. J. Refrig. 2012, 35, 160–170. [CrossRef] 8. Elsarrag, E. Evaporation rate of a novel tilted solar liquid desiccant regeneration system. Sol. Energy 2008, 82,
663–668. [CrossRef] 9. Galama, A.H.; Saakes, M.; Bruning, H.; Rijnaarts, H.H.M.; Post, J.W. Seawater predesalination with
electrodialysis. Desalination 2014, 342, 61–69. [CrossRef] 10. Valero, D.; García-García, V.; Expósito, E.; Aldaz, A.; Montiel, V. Application of electrodialysis for the
treatment of almond industry wastewater. J. Membr. Sci. 2015, 476, 580–589. [CrossRef] 11. Li, X.; Zhang, X. Photovoltaic–electrodialysis regeneration method for liquid desiccant cooling system. Sol. Energy 2009, 83, 2195–2204. [CrossRef] 12. References Cheng, Q.; Zhang, X.; Li, X. Double-stage photovoltaic/thermal ED regeneration for liquid desiccant cooling
system. Energy Build. 2012, 51, 64–72. [CrossRef] 13. Cheng, Q.; Zhang, X.; Jiao, S. Experimental comparative research on electrodialysis regeneration for liquid
desiccant with different concentrations in air-conditioning system dehumidified by solution. Energy Build. 2017, 155, 475–483. [CrossRef] 14. Cheng, Q.; Zhang, X.; Jiao, S. Influence of concentration difference between dilute cells and regenerate cells
on the performance of electrodialysis regenerator. Energy 2017, 140, 646–655. [CrossRef] 15. Cheng, Q.; Xu, W. Performance analysis of a novel multi-function liquid desiccant regeneration system for
air-conditioning system dehumidified by solution. Energy 2017, 140, 240–252. [CrossRef] 16. Al-Jubainawi, A.; Ma, Z.; Guo, Y.; Nghiem, L.D.; Cooper, P.; Li, W. Factors governing mass transfer during
membrane electrodialysis regeneration of LiCl solution for liquid desiccant dehumidification systems. Sustain. Cities Soc. 2017, 28, 30–41. [CrossRef] 17. Li, X.; Zhang, X. Membrane air-conditioning system driven by renewable energy. Energy Convers. Manag. 2012, 53, 189–195. [CrossRef] 17. Li, X.; Zhang, X. Membrane air-conditioning system driven by renewable energy. Energy Convers. Manag. 2012, 53, 189–195. [CrossRef] 18. Liu, X.H.; Yi, X.Q.; Jiang, Y. Comparison of Mass Transfer Performance of Two Common Liquid Hygroscopic
Agents. J. Chem. Ind. Eng. 2009, 60, 567–573. 18. Liu, X.H.; Yi, X.Q.; Jiang, Y. Comparison of Mass Transfer Performance of Two Common Liquid Hygroscopic
Agents. J. Chem. Ind. Eng. 2009, 60, 567–573. 19. Guo, Y.; Ma, Z.; Al-Jubainawi, A.; Cooper, P.; Nghiem, L.D. Using electrodialysis for regeneration of aqueous
lithium chloride solution in liquid desiccant air conditioning systems. Energy Build. 2016, 116, 285–295. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Value Creation in Collaborative Networks: The Case of RFiD Implementation
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IFIP advances in information and communication technology
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cc-by
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To cite this version: Carine Dominguez, Blandine Ageron, Gilles Neubert. Value Creation in Collaborative Networks: The
Case of RFiD Implementation. 11th IFIP WG 5.5 Working Conference on Virtual Enterprises (PRO-
VE), Oct 2010, Saint-Etienne, France. pp.664-671, 10.1007/978-3-642-15961-9_79. hal-01055933 Value Creation in Collaborative Networks: The Case of
RFiD Implementation
Carine Dominguez, Blandine Ageron, Gilles Neubert Carine Dominguez, Blandine Ageron, Gilles Neubert Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01055933
https://inria.hal.science/hal-01055933v1
Submitted on 25 Aug 2014 L’archive ouverte pluridisciplinaire HAL, est
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teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Value Creation in Collaborative Networks: The Case of
RFiD Implementation Carine Dominguez1, Blandine Ageron2, Gilles Neubert3 1Carine Dominguez, University Jean Monnet, Coactis laboratory, 6 rue basse des rives
42023 Saint-Etienne, France (Corresponding author : carine.dominguez@univ-st-etienne.fr)
2Blandine Ageron, Université de Grenoble, CERAG laboratory, IUT Valence,
Rue Barthelemy de Laffemas, 26000 Valence, France
3Gilles Neubert, LIESP Laboratory, Saint-Etienne Business School,
51-53 Cours Fauriel, 42009 Saint Etienne, France Abstract. In this article, we question the way CNs are built around the
implementation of RFiD in a supply chain and what are the value creation
logics among these CNs. We address three main questions: the way and types
of CNs in a RFiD implementation project, the nature of the aggregator and
value creation of each actor during the different phases of the project and
finally the interconnections between CNs in the value creation logic. We
provide answers to theses questions based on a case study that describes the
implementation of the RFiD innovative technology in a jewellery supply chain. Keywords: collaborative networks, supply chain, RFiD, value generator, value
creation. 1.1 Inter-organizational information systems supporting collaborative networks [5] define a collaborative network (CN) is constituted by a variety of entities
(organizations and people) that are largely autonomous, geographically distributed,
and heterogeneous in terms of their: operating environment, culture, social capital and
goals. Nevertheless these entities collaborate to better achieve common or compatible
goals, and whose interactions are supported by inter-organizational information
systems. Different forms of collaborative network organizations (CNOs) can be
distinguished in the literature [6] from temporary networks that are goal-oriented
(virtual organizations and teams, dynamic supply chains, virtual government) to more
long-term strategic networks (virtual organization breeding environment, clusters,
business ecosystems, supply chains, districts) that remain relatively stable with a clear
definition of members’ roles along the value chain. Supply chains are specific CNOs defined as a network of organizations interlinking
suppliers, manufacturers and distributors in the different processes and activities that
produce value in the form of products and services delivered to end customer [7]. Given the dynamic and often temporary nature of CNs, it is important to
understand their life cycle that can be describe into five main steps [6]: creation,
operation, evolution, dissolution and metamorphosis. Traditionally, the main inter organizational information systems supporting
products in the supply chain are bar codes linked to EDI [14]. One of the key
requirements of temporary CN is to develop a generic Information System to control
the interoperability of the participating organizations, distributed, heterogeneous and
concurrent systems for the purpose of accessing and sharing the necessary data and
resources. Because temporary CNs are short terms alliances, formed to address fast-
changing opportunities, they should be connected to the IS of the long-term CN via
“plug-and-play” options [1]. RFiD technology is emerging in order to improve
process efficiency, notably by reducing daily receipts and inventories [26]. 1 Introduction There are different forms of collaborative networks. In this article, we question the
way CNs are built dynamically around the implementation of RFiD in a supply chain
and what are the value creation logics among these CNs. To do so, we observe the dynamic construction of three types of collaborative
networks (CNs) that emerge with the implementation of RFiD in jewellery products. At each phase of the RFiD project, we aim at understanding the value generator
(investment in the collaboration) and value creation (gains) of each actor. We also
question the interconnections of value creation between different CNs based on the
fact the RFiD impact studies to date largely focus on a single firm setting. This leads
to characterise the specificity of collaboration of each network and the type of value
created in this project. We finally open the discussion on the appropriate models that
could describe how to create value in RFiD supply chain projects via the different
RFiD CNs. 714 C. Dominguez, B. Ageron, G. Neubert 1.2 Collaborative networks around RFiD in a supply chain Supply chain collaboration implies that the chain members become involved in
coordinating activities that span boundaries of their organizations in order to fulfil end
users needs [2]. According to [24], the intensity of collaboration depends on the
following impact horizons: Short-term, medium-term or long-term. Collaboration between supply chain partners has been covered extensively in the
strategic management literature ([2]; [15]; [11]; [3]). Several research surveys have
shown that improvement of inter-enterprise processes is one of the major issues in
Supply Chain Management [4]. [13] have recognised that the application of
information technology in the supply chain management has become one of a major
issue of both managers and researchers, as Information technology improve the
performance of both individual firm and supply chain as a whole. Information
technology plays major role in various supply chain collaboration practices as it
enables and integrates information exchanges between supply chain partners. In this
context, EDI, Internet, standards and others technologies have been developed in
order to support increasing exchanges. By the same time, new collaborative practices 715 Value creation in collaborative networks emerge between supply chain partners, such as Vendor/Supplier management
inventory, Collaborative Planning, Forecasting and Replenishment (CPFR). And as
the volume of information exchanged and the intensity of interactions between
partners further increased, there has been an amplified requirement towards an
adequate underlying technology infrastructure. In order to cope with these new
collaboration practices, new ways of information exchanges have emerged [23] The recent industry report CGI [10] emphasizes the new collaborative processes
that will be empowered through the use of RFID. The emergence of these new
technologies revolutionize many of the supply chain operations by reducing costs,
improving service levels, etc [26]. 1.3 Value creation in RFiD collaborative networks Different value generators have been highlighted in the literature. [22] propose main
value generators in CNs that are good indicators of the metric of the collaboration:
physical and financial assets, human, organizational and relational capital. Finally, there are both tangible and intangible types of benefits that can be reached
with RFiD in a manufacturing process [16]. The main benefits are production
planning, process optimization, IT management, improving quality and customer
service, increasing reputation and improving inter organizational collaboration. RFID provides multiple benefits for the supply-chain. It can add value along the
entire supply-chain and related logistical operations and business relationships for
more effective business process design. It provides a means of tracking supplier items
from supplier through the distribution network to the point of consumption. The
possibilities of RFID let its use in the supply-chain somewhat transformational ([20];
[9]) have identified the four main processes that can be directly affected by RFID. Finally, the logic of value creation in these networks included different layers. While the layers may embrace sequential activity chains, other co-producing value
creation activities will also take place in parallel, further enhancing value creation at
the network level [1]. The notion of value creation is central to strategic management
theory and includes value not only in supply and IS flows but also on knowledge
sharing, learning and innovation [17] in what can be labelled strategic supplier
networks. Firms acting in different value constellations [21] are characterized by
social embeddedness [12], governance mechanism and the development of a sense of
trust and reciprocity. 2 The case study analysis The majority of research on RFiD projects has focused on quantitative methodologies
and there are very few case studies focusing on the description of the complexity of
RFID implementation project in a supply chain such as those developed by [18] and
[19]. We conducted a single case study with 15 semi-structured interviews, to reach
an in-depth and insight understanding into this contemporary phenomenon [27]. Our case study was performed with one of the main French retailer. The choice of
this case study is largely explained by the fact that: 716 C. Dominguez, B. Ageron, G. Neubert • The company is a pioneer in the implementation of the RFID technology and no
research has been done in France. • The company is a pioneer in the implementation of the RFID technology and no
research has been done in France. • The jewellery supply chain with RFID has never been studied before. • The jewellery supply chain with RFID has never been studied before. 2.2 The case study analysis In order to analyse the value creation logic in the RFiD project, we use the framework
of [9] that we adapt and complete to illustrate our case study. Fosso and Wamba’s
framework, specifically developed for RFID projects, is composed of a sequence of
three main phases that describe a RFID project implementation: opportunities
seeking, pilot project and validation and RFID project deployment. In the following
section, we will present only phase 1 : Opportunity seeking; phases 2 : Pilot project
and validation and phase 3: RFID project development will be discussed later during
the session. Value creation in collaborative networks How had these CNs been built over time? Different logics explain the
configuration of these networks. The PCN was the initial and historical one: Casino
was working with its LSP to supply jewellery products for many years with bar codes. It is interesting to come back to the main criteria that explain the shaping of theses
CNs. To do so, let us enter into more details in the description of the phase 1 “seeking
opportunities” step 4 (See 3.2.1) and the phase 2 “pilot project and validation” step 7
(See 3.2.2). First, the choice of the ISSP in the IS CN can be surprising as the retailer
Casino decided to choose a very small-sized firm with high expertise in IS. Among
the different criteria to select an ISSP, the fact that the company that was selected had
been working in the past, and for many years, with the LSP brought trust in the
relationship. Trust build in the PCN was spread out in the IS CN. Second, the shaping
of the RFiD CN did not follow the same logic. Casino sent an invitation to tender to
different companies positioned in the RFiD market. Two main RFiD tag suppliers
were selected in the final short list and the one that was finally chosen proposed the
lower global cost. After that, the RFiD tag supplier managed to convince Casino to
work with its own CNS, hence connecting the Label supplier and the RFiD printer
and reader supplier. We can present three main CNs in this project (Fig.2). • The Product Collaborative Network (PCN): the more stable CN that deals with
supplying Casino’s stores (POS) with jewellery products. Different actors are part
of this CN: suppliers of jewels, the logistics service provider (LSP), Casino’s 80
Stores called “Boutique Or”. • The Information System Collaborative Network (ISCN): the network that is in
charge of the evolution and maintenance of Casino’ IS linked with the jewellery
supply chain. Different actors are part of this CN: Casino’s CIO (Information
Systems Department) and the Information System Service Provider (ISSP) • The RFiD Collaborative Network (RFiD CN): it is the temporary network that
is build with the RFiD project. Different actors are part of this CN: the label
supplier, the printer pupplier: this company proposes printing machines to encode
and print the RFiD tags inside the LSP warehouse, the reader supplier and the tag
supplier. • The RFiD Collaborative Network (RFiD CN): it is the temporary network that
is build with the RFiD project. Different actors are part of this CN: the label
supplier, the printer pupplier: this company proposes printing machines to encode
and print the RFiD tags inside the LSP warehouse, the reader supplier and the tag
supplier. Jewelry
Suppliers
Delivery
Receiving
Order picking
Warehousing
Selling
Inventory
Receiving
Casino CIO
Department
Jewelry Suppliers
Logistic Service Provider
Point of sale (POS)
RFID tag
Supplier
Label Supplier
Information
Systems
Service
Provider
RFiD Supply Chain
(Temporary CN)
Product Supply Chain
(Long-term strategic CN)
IT Supply Chain (Long-
term strategic CN)
RFiD Printer supplier
RFID Reader supplier
Fig. 2. Temporary and long-term collaborative networks value creation linked with the RFiD
project. Product Supply Chain
(Long-term strategic CN) RFiD Printer supplier
RFID Reader supplier Fig. 2. Temporary and long-term collaborative networks value creation linked with the RFiD
project. 717 Value creation in collaborative networks 2.2.1 Phase 1: Opportunities seeking This initial phase deals with the opportunities to implement the RFID project in a
specific value chain. It is composed of six steps. In the table below, we describe for
each phase what actors were involved, what did they do, what type of value they
invest in the CN called value generator [22] and the value created (gains) for each
actor in the different CNs. Phase 1:
Opportunities
seeking
Actors and CNs
involved
Value generator
(investment in the
collaboration)
Value creation
(gains)
Step1: Primary
motivation (Why?)
- IS CN: IT consultancy
company came and propose
its services to Casino CIT
- IS CN (Operation): Casino
CIT sees the project as an
opportunity to develop an
innovative technology and
- IS CN (CIO): Scanning
the environment and IT
opportunities in business
(Organizational)
- CIO: IS innovation
(Organizational) Phase 1:
Opportunities
seeking
Actors and CNs
involved
Value generator
(investment in the
collaboration)
Value creation
(gains)
Step1: Primary
motivation (Why?)
- IS CN: IT consultancy
company came and propose
its services to Casino CIT
- IS CN (Operation): Casino
CIT sees the project as an
opportunity to develop an
innovative technology and
- IS CN (CIO): Scanning
the environment and IT
opportunities in business
(Organizational)
- CIO: IS innovation
(Organizational) C. Dominguez, B. Ageron, G. Neubert 718 C. Dominguez, B. Ageron, G. 2.2.2 Phase 2: Pilot project and Validation 2.2.2 Phase 2: Pilot project and Validation The objective of this second phase is to develop a pilot project in order to validate the
feasibility of the RFID implementation. The goal is to identify the business
opportunities that could come from this new technology and to propose process and
IT reconfiguration to fit with RFID. It is made of four steps. 2.2.1 Phase 1: Opportunities seeking Neubert gain visibility inside the
company
Step2: Analysis of
the product value
chain
(What
and
Why?)
- IS CN (Operation):
*CIO was searching for
products disconnected to the
main Casino’s IS to lower
risks in case of project
failure
-
IS
CN
(CIO):
minimize risks linked
to
the
main
IS
Infrastructure
(Physical)
- PCN (Casino POS)
identification
of
jewellery
products
as
best suited to test RFiD
(Organizational)
Step3:
Identification of the
critical
activities
(Which?)
- PCN (Operation): LSP in
cooperation with CIT study
the productivity and cost
gains in different processes
and finally hold receiving
and inventories
- PCN: LSP and
Casino POS (Human)
- PCN (Casino POS):
identification of SC
activity improvement
opportunities
(Organizational)
Step4: Mapping of
the
network
of
firms supporting the
PVC
(Who
and
with Whom?)
- IS CN (Evolution): CIO
Casino
- IS RFiD (Creation): CIO
-
IS
CN
(CIO):
identification of RFiD
competencies in the
market:
Information
Systems LSP, Label
Supplier, RFiD Tag
supplier (Human)
- IS CN (CIO): RFiD
market
knowledge
(Human)
Step5: Mapping of
intra-organizational
processes for the
identified
opportunities (How
within
organization?)
- IS CN (Evolution): CIO
Casino
-
PCN
(Evolution):
the
jewellery
supply
chain
foresees potential changes in
its
processes
with
the
introduction of RFiD
- IS CN (Evolution):
CIO Casino is looking
for
complementary
competencies
- PCN:LSP: process
modifications to adapt
to
Casino
POS
demand
(Organizational)
-ISCN
(CIO):
competencies
identification: make of
buy decision
- PCN (Casino POS)
identification
of
SC
process
improvement
opportunities
(Organizational)
Step6: Mapping of
inter-organizational
processes for the
identified
opportunities (How
between
organization?)
- ISCN (Evolution): CIO
Casino
-
ISCN
(CIO):
competencies
identification
(make
or buy decision)
-
ISCN
(CIO):
add
complementary
IS
resources
outside
the
core
IS
infrastructure
(Relational) 3 Discussion The main results of the case study analysis are the following: • During the project, different CNs are built or broken: the RFiD CN is built upon
the project itself, the choice of Casino to externalize the IS part of the project to an
ISSP initiated a long-term ISCN working in cooperation with the PCN. • Some CNs are stable (ISCN and PCN) whereas others are temporary [6], formed
only during the project opportunities seeking and pilot project. The project
deployment phase transforms a temporary collaboration between the RFiD CN
and the IS CN/PCNs mainly based on an innovative project to a business
relationship between the Tag Supplier and Casino. • The RFiD Temporary CN only invested during the Phases 1 and 2 of the project
(Human, Organizational, Physical) and captured value in the last Phase 3 of the
project (Brand and Relational above all). Hence, the temporary RFiD CN largely
participated to increase the value of the stable CNs • Value creation (gains) is largely oriented in a CN logic rather than in an individual
logic based on criteria such as size and power. For instance, the ISSP, which is a
small company, that is working with CIO in the ISCN reinforced its business
relationship with Casino (Relational value) and managed to build a long-term
ISCN with CIO, owing to its past business relationships with LSP. The configuration of temporary and long-term strategic CNs are based on
collaborative characteristics such as personal and historical relationships (the
information system service provider get used to work with the LSP, the RFiD tag
supplier knows personally some CIO department managers) [12], trust and partners
competencies. Moreover, the innovative capacity of all the stakeholders of the
collaborative network and their willingness to explore new opportunities are
important enablers of these configurations. The RFiD technology engenders
organizational change and process optimization ([20]; [9]). The RFiD technology also
generates intangible benefits [16] but these seem not the same as in manufacturing
processes. Knowledge and innovation capacities [17] seem to be the main gains for
stakeholders participating in a supply chain temporary CNs. This hypothesis had to be
developed in further research through a comparison case-study research design. 1.
Bolton, R., Dewey, A., Horstmann, P., Requirements for Plug and Play, Information
Infrastructure Frameworks and Architectures to Enable Virtual Enterprises. Proceedings of
SPIE '96, pp.2-19 (1996). 2.
Bowersox, D.J., The strategic benefits of logistics alliances, Harvard Business Review,
68:4, pp.36-43 (1990). pp y
g
pp
4.
Boyson, S., Corsi, T.M., Dresner, M.E., Harrington, L.H., Logistics and the Extended
Enterprise: Benchmarks and Best Practices for the Manufacturing Professional, Wiley,
New York, NY, (1999). 3.
Bowersox, D.J., Closs, D.J. and Keller, S.B., How supply chain competency leads to
business success, Supply Chain Management Review, 4:4, pp.70-78 (2000). 2.2.3 Phase 3: RFID Project Deployment The last phase concerns the validation of the pilot project and the deployments of the
technology on the whole supply chain. 719 Value creation in collaborative networks References pp y
g
pp
4. Boyson, S., Corsi, T.M., Dresner, M.E., Harrington, L.H., Logistics and the Extended
Enterprise: Benchmarks and Best Practices for the Manufacturing Professional, Wiley,
New York, NY, (1999). C. Dominguez, B. Ageron, G. Neubert 720 5. Camarinha-Matos L. M., Afsarmanesh H. Collaborative networks: a new scientific
discipline, Journal of Intelligent Manufacturing, Vol.6, pp.439-452 (2005) 6. Camarinha-Matos L., Afsarmanesh H., Galeano N., Molina A. Collaborative networked
organizations - concepts and practice in manufacturing enterprises, Computers and
Industrial Engineering, 57:1, pp.46-60 (2009). g
g
pp
(
)
7. Christopher M. L., Logistics and supply chain management, Pitman publishing, London
(1992). 8. Czajkowski, K., Fitzgerald, S., Foster, I., Kesselman, C.: Grid Information Services for
Distributed Resource Sharing. In: 10th IEEE International Symposium on High
Performance Distributed Computing, pp. 181--184. IEEE Press, New York (2001) 9. Fosso Wamba S., Lefebvre L.A., Lefebvre E., Integrating RFID Technology and EPC
Network into a B2B retail supply chain: a step toward intelligent business processes,
Journal of Technology Management and Innovation, 2:2, pp.114-124. 10. GCI, EPC: A Shared Vision for Transforming Business Processes, Global Commerce
Initiative, available at: www.gci-net.org, (2005) 1. 11. Gilmour, P., Benchmarking supply chain operations, International Journal of Physical
Distribution & Logistics Management, 29:4, pp.283-90, (1999). g
g
pp
11. Gulati R., Does familiarity breed trust? The implications of repeated ties for contractual
h i
i
lli
A
d
f M
t J
l 38 85 112 (1995) 11. Gulati R., Does familiarity breed trust? The implications of repeated ties for contractual
choice in alliances, Academy of Management Journal, 38, 85-112 (1995). choice in alliances, Academy of Management Journal, 38, 85-112 (1995). 12. Gunasekaran A., Ngai E.W.T., Information systems in supply chain integration and
management, European Journal of Operational Research, 159:2, pp.269-295 (2004). 13. Holland C.: Cooperative supply chain management: the impact of inter-organizational
information systems, 4:2, pp.117-133 (1995). 14. Hanman, S., Benchmarking your firm’s performance with best practice, International
Journal of Logistics Management, 8:2, pp.1-18, (1997). 15. Ivantysynova L., Klafft M., Ziekow H., Günther O., Kara S., RFiD in manufacturing: the
investment decision, Pacific Asia Conference on Information Systems (PACIS),
http://aisel.aisnet.org/pacis2009/41 (2009). 16. Kogut B., The Network as Knowledge: Generative Rules and the Emergence of Structure,
Strategic Management Journal, 21:3, pp. 405-425 (2000). 17. Loebbecke, C., Piloting RFID along the supply chain: a case analysis, Electronic Markets,
17:1, pp.29-37 (2007). 18. References Loebbecke, C., Use of innovative content integration information technology at the point
of sale, European Journal of Information Systems, 16, pp.228-236 (2008). 19. Niedermann F., Mathieu R.G., Morley R. and Kwon I.W., Examining RFiD in Supply
Chain Management, Communications of the ACM, 50:7, pp.92-101 (2007). 20. Norman N., Ramirez R., From value chain to value constellation: designing interactive
strategy, Harvard Business Review, 71, pp. 65-77 (1993). gy
pp
(
)
21. Parung J., Bititci U., A metric for collaborative networks, Business Process Management
Journal, 14:5, pp.654-674 (2008) 22. Pramatari K., Collaborative supply chain practices and evolving technical approaches,
Supply Chain Management:An International Journal, 12:3, pp.210-220 (2007). 23. Simatupang, T.M., Sridharan, R., The collaborative supply chain: a scheme for
information sharing and incentive alignment, International Journal of Logistics
Management, 13:2, pp.257-274 (2002). g
pp
24. Tzeng S.F., Chen W.H., Pai F.Y., Evaluating the business value of RFiD: Evidence from
five case studies, International Journal of Economics, 112, pp.601-163 (2007) 25. Veeramani D., Tang J., Gutierrez A., A framework for assessing the value of RFID
implementation by Tier-one suppliers to major retailers, Journal of Theoretical and
Applied Electronic Commerce Research, 3:1, pp.55-70 (2008) pp
pp
26. Yin R.K., Case Study Research: Design and Method, Sage Publication (2003)
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Team leadership assessment after advanced life support courses comparing real teams vs. simulated teams
|
Frontiers in psychology
| 2,022
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cc-by
| 6,396
|
TYPE Original Research
PUBLISHED 07 December 2022
DOI 10.3389/fpsyg.2022.1020124 TYPE Original Research
PUBLISHED 07 December 2022
DOI 10.3389/fpsyg.2022.1020124 TYPE Original Research
PUBLISHED 07 December 2022
DOI 10.3389/fpsyg.2022.1020124 Team leadership assessment
after advanced life support
courses comparing real teams
vs. simulated teams OPEN ACCESS
EDITED BY
Tayana Soukup,
King’s College London,
United Kingdom
REVIEWED BY
Margarete Boos,
University of Göttingen, Germany
Hadis Ashrafizadeh,
Dezful University of Medical
Sciences, Iran
*CORRESPONDENCE
Robert Greif
robert.greif@insel.ch
SPECIALTY SECTION
This article was submitted to
Quantitative Psychology and
Measurement,
a section of the journal
Frontiers in Psychology
RECEIVED 16 August 2022
ACCEPTED 31 October 2022
PUBLISHED 07 December 2022
CITATION
Nabecker S, Huwendiek S, Roten F-M
Theiler L and Greif R (2022) Team
leadership assessment after advanced
life support courses comparing real
teams vs. simulated teams. Front. Psychol. 13:1020124. doi: 10.3389/fpsyg.2022.1020124
COPYRIGHT
© 2022 Nabecker, Huwendiek, Roten Sabine Nabecker1,2,3,4, Sören Huwendiek5,
Fredy-Michel Roten2, Lorenz Theiler6 and Robert Greif2,3,7* 1Department of Anesthesiology and Pain Management, Sinai Health System, University of Toronto,
Toronto, ON, Canada, 2Department of Anaesthesiology and Pain Medicine, Bern University Hospital,
University of Bern, Bern, Switzerland, 3ERC ResearchNET, Niel, Belgium, 4Graduate School for
Health Sciences (GHS), University of Bern, Bern, Switzerland, 5Department for Assessment and
Evaluation, Institute for Medical Education, University of Bern, Bern, Switzerland, 6Department of
Anaesthesia, Cantonal Hospital Aarau, Aarau, Switzerland, 7School of Medicine, Sigmund Freud
University Vienna, Vienna, Austria Aim:
Efective
team
leadership
is
essential
during
cardiopulmonary
resuscitation
(CPR)
and
is
taught
during
international
advanced
life
support (ALS) courses. This study compared the judgement of team
leadership during summative assessments after those courses using diferent
validated
assessment
tools
while
comparing
two
diferent
summative
assessment methods. Nabecker S, Huwendiek S, Roten F-M,
Theiler L and Greif R (2022) Team
leadership assessment after advanced
life support courses comparing real
teams vs. simulated teams. Front. Psychol. 13:1020124. doi: 10.3389/fpsyg.2022.1020124 Methods: After ALS courses, twenty videos of simulated team assessments
and 20 videos of real team assessments were evaluated and compared. Simulated team assessment used an instructor miming a whole team, whereas
real team assessment used course participants as a team that acted on
the team leader’s commands. Three examiners individually evaluated each
video on four diferent validated team leadership assessment tools and on
the original European Resuscitation Council’s (ERC) scenario test assessment
form which does not assess leadership. The primary outcome was the
average performance summary score between all three examiners for each
assessment method. COPYRIGHT
© 2022 Nabecker, Huwendiek, Roten,
Theiler and Greif. This is an
open-access article distributed under
the terms of the Creative Commons
Attribution License (CC BY). Team leadership assessment
after advanced life support
courses comparing real teams
vs. simulated teams 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. Results:
The average performance summary score for each of the four
assessment tools was significantly higher for real team assessments compared
to simulated team assessments (all p-values < 0.01). The summary score
of the ERC’s scenario test assessment form was comparable between
both assessment methods (p = 0.569), meaning that participants of both
assessments performed equally. Conclusion: Team leadership performance is rated significantly higher in real
team summative assessments after ALS courses compared to simulated team
assessments by four leadership assessment tools but not by the standard 01 frontiersin.org Frontiers in Psychology Nabecker et al. 10.3389/fpsyg.2022.1020124 10.3389/fpsyg.2022.1020124 ERC’s scenario test assessment form. These results suggest that summative
assessments in ALS courses should integrate real team assessments, and a
new assessment tool including an assessment of leadership skills needs to
be developed. education, assessment, CPR, advanced life support, ERC, human factors Methods
Study design Training is essential for lay persons, first responders (Marx
et al., 2020; Nabecker et al., 2021a), and all healthcare providers
(Smith et al., 2015; Greif et al., 2021) in small groups of up to 6
participants per instructor (Nabecker et al., 2021b) spaced over
time (Yeung et al., 2020). Attendance in accredited advanced life
support (ALS) courses improves patient outcomes (Lockey et al.,
2018). The European Resuscitation Council (ERC) like other
international resuscitation councils includes training in human
factors especially team leadership (Greif et al., 2015; Kuzovlev
et al., 2021; Soar et al., 2021) in their ALS courses. Nonetheless,
the summative end-of-course assessments still largely focus on
adherence to guidelines but do not assess team leadership and
team members’ success in task management. The videos of the summative assessments at the end of ALS
courses were recorded between December 2017 and March 2019
and analyzed between March 2021 and September 2021. All
course participants and instructors provided written informed
consent for the study participation and the video recording. Frontiers in Psychology Nabecker et al. frontiersin.org Introduction and the official ERC scenario test assessment form for the
comparison of the two assessment methods. The study results might trigger changes in the assessment
approach of international resuscitation councils or provide
evidence that a new assessment tool for ALS courses needs to
be developed. In Europe, the average survival rate at hospital discharge
after out-of-hospital cardiac arrest ranges from 0 to 18% and
in hospitals from 15 to 34% (Grasner et al., 2021; Perkins et al.,
2021). This underlines how important proper cardiopulmonary
resuscitation (CPR) education is to enable rescuers to improve
patient outcomes. Setting In the study year of 2017/2018, all fifth-year medical
students at the University of Bern, Switzerland, participated
in a mandatory 8-h immediate life support (ILS) course and
all sixth-year medical students in a 16-h ALS course. All
these courses were accredited ERC courses based on the 2015
ERC’s resuscitation guidelines (Greif et al., 2015; Monsieurs
et al., 2015). All instructors in these courses were ERC-
certified ILS/ALS instructors. The study was registered at
clinicaltrials.gov (NCT03412032) and was performed at the
University of Bern, Switzerland. Competency assessment practices of learners are different
in various international ALS organizations. The ERC uses an
assessment method where course participants are assessed as
team leaders, while one instructor simulates a whole team. In
courses run by the American Heart Association (AHA), a group
of course participants acts as the team and the assessed course
participant leads this team through a cardiac arrest scenario test. Recently, we assessed course participants’ and instructors’
perceptions of how these two different assessment methods
can test human factors. In short, real team assessment was
preferred over simulated team assessment (Nabecker et al.,
2022). This has been the first study comparing simulated vs. real team assessments. To the best of our knowledge, there
are currently no other publications available on this topic. As different assessment methods are used during summative
assessments in international CPR councils, it is important
to establish which assessment method is better to test team
leadership as an important human factor during cardiac
arrest treatment. At the end of each course, a mandatory summative
assessment was held, which used the validated ERC cardiac
arrest scenario tests (Ringsted et al., 2007). The courses
were randomized with the “Research Randomizer” (https://
www.randomizer.org) software to one of the two different
assessment methods: (1) Simulated team assessment: Only the assessed course
participant and two instructors were in the assessment
room. One instructor mimes a whole team. The miming
instructor performs different team roles at once. The
instructor does only act on the team leader’s commands. The instructor does not introduce any standardized The aim of this study was, therefore, to evaluate how well
these two assessment methods allow instructors to test team
leadership. We used four different validated assessment tools 02 frontiersin.org Nabecker et al. 10.3389/fpsyg.2022.1020124 (1) The checklist developed by Andersen et al. Study measurements The primary outcome parameter was defined as the average
summary performance score for each assessment tool compared
between both assessment methods. The null hypothesis of
this study was that the summary performance scores of the
included rating tools would be comparable between the real
team and the simulated team assessments. The detailed results
of each assessment tool used in this analysis are presented as
Supplementary material. (4) (4) The “leadership and behavior dimensions” are derived
from a systematic review by Rosenman et al. (2015). We
used the leadership dimension table as a yes/no checklist. The first item on this checklist was rated as one point
if the response was yes either to leadership, defined in
terms of clinical expertise, or to leadership, defined in
terms of having a leader. There were additional three
different areas with 37 items, namely, transition process,
action process, and interpersonal skills. Each yes response
counted as one point. This gives a maximum score of 38
points for this tool. (4) The “leadership and behavior dimensions” are derived
from a systematic review by Rosenman et al. (2015). We
used the leadership dimension table as a yes/no checklist. The first item on this checklist was rated as one point
if the response was yes either to leadership, defined in
terms of clinical expertise, or to leadership, defined in
terms of having a leader. There were additional three
different areas with 37 items, namely, transition process,
action process, and interpersonal skills. Each yes response
counted as one point. This gives a maximum score of 38
points for this tool. Setting The last item is a global assessment
of the team leader on a scale of below expected for level/as
expected for level/above expectations for level/top 5%. Again, we dichotomized the responses. Each response for
“As expected for level”, “above expectations for level”,
or “top 5%” counted as 1 point, and “Below expected
for level” as 0 points. This instrument, therefore, has a
maximum score of 17 points. (2) The
Concise
Assessment
of
Leader
Management
instrument by Nadkarni et al. (2018): It asks the
question whether the role of a team leader has been
announced or not (yes = 1/no = 0), followed by 15 items
assessing on a scale of rarely/sometimes/mostly/always
the following areas: leadership, communication, team
management, and medical management. For this analysis,
we dichotomized the answers and counted a mostly or
always response as 1 point, and a rarely or sometimes
response as 0 points. Medical knowledge is assessed by
free-text entries; therefore, we did not include this item in
the current analysis. The last item is a global assessment
of the team leader on a scale of below expected for level/as
expected for level/above expectations for level/top 5%. Again, we dichotomized the responses. Each response for
“As expected for level”, “above expectations for level”,
or “top 5%” counted as 1 point, and “Below expected
for level” as 0 points. This instrument, therefore, has a
maximum score of 17 points. All assessments were videotaped; videos were saved on a
protected research server at the University of Bern. Study participants Three certified ERC instructors (FMR, CS, and AH) watched
a total of 40 randomly chosen videos (using the random selection
function in Excel), 20 from the simulated team assessment and
20 from the real team assessment. (3) (3) The Team Emergency Assessment Measure (TEAM)
rating scale by Cooper et al. (2010): This scale consists
of 11 items rated on a scale of 0 = never/hardly ever, 1
= seldom, 2 = about as often as not, 3 = often, and 4 =
always/nearly always. Three different areas are covered,
namely, leadership, teamwork, and task management. Again, we dichotomized the responses, and each response
for “often” and “always/nearly always” was counted as 1
point and all others as 0 points. The last item on this scale
is an overall rating on a Numeric Rating Scale from 1 to
10. Each response from 6 to 10 was counted as 1 point,
and 5 and below as 0. Therefore, the maximum score for
this rating scale was 12 points. Setting (2010): The
checklist consists of 22 yes/no items targeting seven
main topics, namely, initial therapy, continuous loops,
information and supplementary therapy, spontaneous
circulation, correction, maintenance algorithm, and
technology. Each yes response was counted as 1 point,
giving a maximum score of 22 points. reactions. The assessed course participant acted as team
leader and led this “Pro-forma” team through a cardiac
arrest scenario test. This assessment method is currently
the ERC standard test format. reactions. The assessed course participant acted as team
leader and led this “Pro-forma” team through a cardiac
arrest scenario test. This assessment method is currently
the ERC standard test format. (2) Real
team
assessment:
Three
additional
course
participants are with the assessed course participants in
the assessment room. Only the team leader is assessed
and the team members act as resuscitation team on the
team leader’s commands but are not allowed to advise the
team leader on medical decisions. The assessed course
participant acts as the team leader and leads this team
through a cardiac arrest scenario test. The other course
participants do not receive any further instructions,
and they behave naturally. This assessment method
approximates the current method used by the AHA. (2) Real
team
assessment:
Three
additional
course
participants are with the assessed course participants in
the assessment room. Only the team leader is assessed
and the team members act as resuscitation team on the
team leader’s commands but are not allowed to advise the
team leader on medical decisions. The assessed course
participant acts as the team leader and leads this team
through a cardiac arrest scenario test. The other course
participants do not receive any further instructions,
and they behave naturally. This assessment method
approximates the current method used by the AHA. (2) The
Concise
Assessment
of
Leader
Management
instrument by Nadkarni et al. (2018): It asks the
question whether the role of a team leader has been
announced or not (yes = 1/no = 0), followed by 15 items
assessing on a scale of rarely/sometimes/mostly/always
the following areas: leadership, communication, team
management, and medical management. For this analysis,
we dichotomized the answers and counted a mostly or
always response as 1 point, and a rarely or sometimes
response as 0 points. Medical knowledge is assessed by
free-text entries; therefore, we did not include this item in
the current analysis. Frontiers in Psychology Study process The three examiners rated team leadership and performance
individually using four different validated team leadership
and/or team performance checklists/tools as well as the original
ERC ALS scenario test assessment forms. The intraclass
correlation coefficient (ICC) was calculated for each rating tool. By using four different validated tools, we aimed to show the
comparability of the assessment methods or the superiority of
one assessment method. We did not provide cross-validation of
the included rating tools. This was not the purpose of this study. (5) The ERC ALS scenario test assessment forms are the
official test forms used by the ERC. Items on this
assessment form are derived from the ALS algorithms and (5) The ERC ALS scenario test assessment forms are the
official test forms used by the ERC. Items on this
assessment form are derived from the ALS algorithms and The four rating tools were: Frontiers in Psychology 03 frontiersin.org 10.3389/fpsyg.2022.1020124 Nabecker et al. to the simulated team assessments (p < 0.01). The summary
score of the official ERC ALS scenario test assessment forms
was comparable between both assessment methods (p = 0.569),
meaning that participants of both assessments performed
equally. do not include human factors or leadership items. The
assessment form has 24 items rated on a scale from 1 to
4 (1 = outstanding, 2 = adequate, 3 = marginal, and 4 =
insufficient). Each outstanding or adequate response was
counted as 1 point, and all others as 0 points. A passing
score was counted as 1 point, and not passing or retesting
as 0 points. Therefore, the maximum score for the ERC
ALS scenario test assessment form was 25 points. In the Supplementary material to this report, we provide the
detailed scores for each item and each checklist/tool included in
this study. Hereafter, we showcase the most interesting findings. y
g
g
In the checklist by Andersen et al. (2010), which was
developed as a formative assessment tool for the measurement
of performance of resuscitation teams with a maximum score
of 22 points, the simulated team assessment received a mean of
12.2 points and the real team assessment received 14.0 points
(p < 0.01, Table 1). It was possible to rate most items on this
checklist with one or both assessment methods; however, there
were 4 items that showed to be difficult to be rated with either
assessment method. Study process Difficulty in rating was discussed between
the three examiners, and it was defined that some of the items
simply could not be assessed with the rating tool used. Those
were the use of cognitive aids or supplementary information
and the correction of hyperventilation or defibrillation (compare
Supplementary Table 1). For example, during an assessment, it is
not allowed to use cognitive aids; therefore, this item could not
be rated. Study analysis We did not perform a formal sample size calculation for
this analysis as no data were available to base such a calculation
(an extensive literature search in PubMed and Medline with the
following search terms was performed (education, assessment,
CPR, ALS, ERC, leadership, team, simulation, and human
factor). Not a single publication resulted from that search
comparing the two different assessment methods. However, we
decided to include 20 videos from each of the two assessments as
we assumed that more valuable information will not be derived
by including more additional videos because the research
question was whether the different assessment tools were able to
assess team leadership but not to evaluate the individual course
participant’s performance. In
the
Concise
Assessment
of
Leader
Management
instrument by Nadkarni et al. (2018) for formative assessment
of team leader performance during pediatric resuscitations,
a maximum score of 17 was achievable. The simulated team
assessment reached a mean of 7.7 points, and the real team
assessment reached a mean of 10.5 points (p < 0.01, Table 1). Most items were able to be scored; however, 3 items were
difficult to be scored by either assessment method. Those
were if a team leader was announced or not, reinforcement of
closed-loop communication, and engagement of team members
in decision-making (compare Supplementary Table 2). Statistical analysis was performed using the STATA version
16.0 (StataCorp LT, Texas, USA) and IBM SPSS Statistics
version 28.0.1.1 (IBM, New York, USA) software. The primary
outcome was calculated by averaging each examiner’s summary
performance score for each assessment tool. The Mann–
Whitney U-test was used to evaluate summary performance
scores between assessment groups. Fisher’s exact test was used
to evaluate the detailed results presented in the supplemental
material to this report. The ICC was calculated to compare inter-
rater reliability between the 3 examiners. Data are presented
as mean ± standard deviation (95% confidence interval) or
value (percentage). A probability of <5% was considered
statistically significant. The TEAM rating scale by Cooper et al. (2010), developed
to rate medical emergency teamwork performance, had a
maximum score of 12 points. The simulated team assessment
scored a mean of 5.6 points, and the real team assessment 9.5
points (p < 0.01, Table 1). All items were able to be scored with
both assessment methods (compare Supplementary Table 3). Ethical considerations The checklist derived from the leadership and behavior
dimensions by Rosenman et al. (2015) allowed a maximum
score of 38 points. The simulated team assessment scored a
mean of 15.4 points and real team assessment scored 22.0
points (p < 0.01, Table 1). On this checklist, multiple items
were difficult to be scored by either assessment, and those
include team leader incorporates team member’s suggestions,
team leader briefs the team, team leader plans/decides
how to do things, team leader debriefs the team/provides
feedback/identifies areas for team improvement, team leader
asks for help when needed, team leader notices changes
in system/team environment, team leader identifies errors, The Cantonal Ethics Committee of Bern, Switzerland (Req-
2017-00578, 7 August 2017) reviewed the study and judged
that it does not fall under the Swiss Human Research Act of
biomedical studies. Frontiers in Psychology Results Table 1 shows the summary score for each checklist/tool
used in this study and the ICC. All summary scores were
significantly higher for the real team assessments compared Frontiers in Psychology 04 frontiersin.org 10.3389/fpsyg.2022.1020124 Nabecker et al. TABLE 1 Primary outcome parameter: summary score in each used checklist/tool between simulated team assessment and real team assessment. TABLE 1 Primary outcome parameter: summary score in each used checklist/tool between simulated team assessment and real team assessment. Simulated team
Real team
p-valueA
(n = 20)
(n = 20)
Checklist by Andersen et al. (2010)
12.2 ± 2.1
(11.2–13.2)
14.0 ± 2.0
(13.1–14.9)
<0.01
% of max. score of 22
55.5
63.6
Intraclass correlation coefficient (ICC)
0.932
0.849
Concise assessment of leader management by
Nadkarni et al. (2018)
7.7 ± 2.9
(6.3–9.0)
10.5 ± 3.4
(8.9–12.1)
<0.01
% of max. score of 17
45.3
61.8
Intraclass correlation coefficient (ICC)
0.819
0.801
Team Emergency Assessment Measure (TEAM)
by Cooper et al. (2010)
5.6 ± 1.7
(4.8–6.3)
9.5 ± 2.4
(8.4–10.6)
<0.01
% of max. score of 12
46.7
79.2
Intraclass correlation coefficient (ICC)
0.444
0.611
Leadership competencies by Rosenman et al. (2015)
15.4 ± 3.0
(14.0–16.8)
22.0 ± 4.6
(19.8–24.2)
<0.01
% of max. score of 38
40.5
57.9
Intraclass correlation coefficient (ICC)
0.85
0.765
Cardiac arrest simulation Test
17.9 ± 4.3
17.2 ± 3.8
0.569
(15.9–19.8)
(15.4–18.9)
% of max. score of 25
71.6
68.8
Intraclass correlation coefficient (ICC)
0.877
0.896
AMann–Whitney U-test. Scores are presented as mean ± standard deviation (95% confidence interval) and % of the total score. Mann–Whitney U-test. Scores are presented as mean ± standard deviation (95% confidence interval) and % of the total score. U test. ed as mean ± standard deviation (95% confidence interval) and % of the total scor leadership and performance of resuscitation teams if that is
intended in the end-of-course assessment. Participants scored
comparably in both assessment methods on the official ERC ALS
scenario test assessment forms. Not surprising, this assessment
tool does not include human factors. team leader coaches/provides supervision as needed, team
leader assists with conflict management/resolution, and team
leader motivates and empowers team members (compare
Supplementary Table 4). On the official ERC ALS scenario test assessment forms,
a maximum score of 25 points was achievable, the simulated
team assessment received a mean of 17.9 points, and the real
team assessment received 17.2 points (p = 0.569, Table 1). Results All
items were able to be scored with both assessment methods. Supplementary Table 5 shows the detailed results of the scenario
test assessment forms. In the current literature, there is only one study available
comparing simulated vs. real team assessments. This study was
performed by our research group and showed that instructors
and participants favor real team assessments and judged the
real team assessments mainly to be superior in assessing human
factors. There is no other study available objectively evaluating
real team vs. simulated team assessments. Some items on the checklist by Andersen et al. (2010),
developed as a formative assessment tool for team performance,
were difficult to be scored for both assessment types, e.g., the
use of cognitive aids and supplementary information, as well
as the correction of hyperventilation and/or defibrillation. All
those items are important during real cardiac arrests. The use
of cognitive aids and supplementary information is, however,
prohibited during summative assessments. The occurrence of
hyperventilation and defibrillation errors during an assessment
is highly circumstantial. It is not easy to standardize “poor”
team member performance during an assessment that needs
to be equal for all assessed participants. This checklist focuses Discussion Assessed team leaders of real team assessments of ALS
courses score significantly higher on four different validated
assessment tools for team leadership performance than assessed
team leaders of simulated team assessments. Therefore, the
assessment method impacts how well-human factors can be
assessed; simulated team assessments do not allow course
participants to show leadership skills appropriately. This adds
further evidence that a change in assessment practice to
real team assessments might be beneficial to measure team Frontiers in Psychology frontiersin.org 05 Nabecker et al. 10.3389/fpsyg.2022.1020124 mainly on team performance. Teamwork issues were identified
as being most challenging by another study (Walsh et al., 2017). Training team membership is an integral part of all CPR courses. However, during the current final course assessments, only
the team leader is assessed. Thoughts should be given as to
whether the team should also be assessed during the final course
assessment, which would be possible if real team assessments
were used. There is currently no evidence available if formative
assessments throughout the course could be used to replace the
summative end-of-course assessment or at least the aspect of
team membership (Greif et al., 2020). Therefore, future studies
are necessary to establish if formative assessments can replace
summative assessments for the assessment of leadership and
other human factors. real cardiac arrests but are less relevant for assessments after
ALS courses. Therefore, in a newly developed assessment tool,
multiple items from this checklist can be omitted. The official ERC ALS scenario test assessment form only asks
for adherence to guidelines and does not test leadership skills. It
is, therefore, not surprising that both assessment methods scored
equally with it. As participants of simulated team assessments scored
significantly lower in human factor aspects than participants
of real team assessments with the included checklist, but equal
performance was found with the current ERC ALS scenario
test assessment forms, we assume that this assessment method
does not allow participants to show their full skill set in
human factors. Therefore, future summative assessments in CPR
courses should use real team assessments to account for that. None of the included assessment tools in this study seems to be
perfect in scoring human factors targeted for team leadership. Therefore, future projects should consider creating a new and
properly designed assessment tool specifically targeted for ALS
course assessments that allow assessing team leadership as well
as adherence to current resuscitation guidelines. Conclusion Participants of real team assessments of ALS courses score
significantly higher on assessment tools evaluating human
factors in comparison with simulated team assessments. The
simulated team assessment prevents participants to show their
learnt skills. These results support that real team assessment
should be considered to be implemented in ALS courses. A new
assessment tool should be created to incorporate human factor
assessment as well as adherence to guidelines. The checklist derived from leadership and behavioral
dimensions by Rosenman et al. (2015) assesses team leadership
comprehensively; however, there have been multiple items
that none of the checklists were able to capture. Some are
irrelevant or even prohibited during an assessment, e.g., the team
leader incorporates team members’ suggestions, or motivates
or empowers team members. Other items are again highly
dependent on circumstantial factors if they even occur, e.g.,
the team leader assists with conflict management/resolution. Those items are important to address during the debriefing of Discussion On
the
Concise
Assessment
of
Leader
Management
instrument by Nadkarni et al. (2018), again, some items
were difficult to be scored as this tool was developed for
formative assessments, not for summative assessments. The
team leader was defined a priori as the person being assessed. The engagement of team members in decision-making was
prohibited. Closed-loop communication seems difficult to be
assessed, even though it should be possible to be judged at
least by the real team assessment. Communication is a key
component of human factors during cardiac arrests, and it
has to be clear, brief, and empathetic and should provide a
feedback loop (Jones et al., 2018; Ulmer et al., 2021). Literature
shows that ongoing training on leadership principles improves
communication (Hunziker et al., 2010, 2011; Lee et al., 2021). These results suggest a lack of competency of course participants
in this specific human factor. However, if this is really the case,
the implications for education and training need to be evaluated
in further studies. Limitations of this study are the single-center study
design and the inclusion of only medical students as course
participants, which might limit the generalizability of the
results. All examiners were instructors at the same center. It is, therefore, possible that results would differ if different
instructors from different areas or cultures had rated the
participants’ performance. We included three examiners, two
men and one woman, with a similar extended experience
as instructors to perform the video analysis. By using three
examiners and adding the total scores together, differences in
the rating of human factors were minimized. Another strength
of this study is the execution of a randomized controlled trial. All items on the TEAM rating scale (Cooper et al., 2010)
were able to be scored by either one of the assessment methods. This scale focuses on team leadership, teamwork, and team
membership, and is widely used in ERC training courses
without having established its scientific value in comparison
with other scoring systems. There are certain aspects missing
that other scores can map, e.g., situational awareness (Jones
et al., 2018). Therefore, in the next step, it is important to
develop a new assessment tool, which allows judging all major
human factor aspects in conjunction with adherence to the
current guidelines. Frontiers in Psychology Publisher’s note This project was funded by an institutional research
grant awarded to Dr. Sabine Nabecker by the Department of
Anaesthesiology and Pain Medicine, Bern University Hospital,
Inselspital, University of Bern, Bern, Switzerland. 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. Cooper, S., Cant, R., Porter, J., Sellick, K., Somers, G., Kinsman, L., et al.
(2010). Rating medical emergency teamwork performance: development of
the team emergency assessment measure (TEAM). Resuscitation 81, 446–452.
doi: 10.1016/j.resuscitation.2009.11.027 Data availability statement The datasets presented in this article are not readily available
because the original contributions presented in this study are
included in the article/Supplementary material, further inquiries
can be directed to the corresponding author with a dedicated Frontiers in Psychology frontiersin.org 06 10.3389/fpsyg.2022.1020124 Nabecker et al. research question and local ethics committee approval. Requests
to access the datasets should be directed to robert.greif@insel.ch. like to thank Ferdinand Habermann and Thilo Schweizer (both
doctoral thesis students, University of Bern) for their assistance
in data control and evaluation. Furthermore, the authors thank
all life support instructors of the Bern Simulation- and CPR-
Centre at the Bern University Hospital who performed the
courses as well as the assessments. In addition, we would
like to thank Anisa Hana (AH) and Christian Seidl (CS) who
performed the assessment of the video clips for this study. Finally, we thank all medical students at the University of
Bern who agreed to participate in this study. This report was
previously presented, in part, at the AMEE conference 2018 in
Basel, Switzerland. Ethics statement Ethical approval was not required for the study on human
participants in accordance with the local legislation and
institutional requirements. The Cantonal Ethics Committee of
Bern, Switzerland (Req-2017-00578, 07.08.2017) reviewed the
study and judged that it does not fall under the Swiss Human
Research Act of biomedical studies. All course participants and
instructors provided written informed consent for the study
participation and the video recording. Acknowledgments We thank Fabian Schneeberg, Florian Ueltschi, Bettina
Eberle (all doctoral thesis students, University of Bern), Lisa
Lüthi, Raffaela Flury, Ayla Frischknecht, Nadine Mathieu,
Marius Bigler, Irina Zürrer, Jeremy Glasner, Julia Rosser, Fabian
Lütolf, Barbara Keller, and Nino Räschle for their support in
the performance of the study. Furthermore, the authors would Supplementary material The Supplementary Material for this article can be
found online at: https://www.frontiersin.org/articles/10.3389/
fpsyg.2022.1020124/full#supplementary-material Author contributions SN is currently a committee member in the ERC’s
Science and Education Committee – Instructor Educator
Support (SEC-IES). She is also a committee member of the
Canadian Anesthesiologists Society’s Continuing Education and
Professional Development (CEPD) committee. RG is the Board
Director of Guideline and ILCOR of the ERC and the Chair
of the ILCOR Task Force on Education, Implementation, and
Team. SN, F-MR, and RG are currently life support instructors
with the ERC. SN had the idea of the study, performed data acquisition,
data
evaluation,
first
manuscript,
and
approved
final
manuscript. SH and LT contributed to data evaluation,
significant contribution to manuscript, and approved final
manuscript. F-MR contributed to data acquisition, significant
contribution to manuscript, and approved final manuscript. RG performed data acquisition, data evaluation, significant
contribution to manuscript, and approved final manuscript. All authors contributed to the article and approved the
submitted version. The remaining authors declare that the research was
conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict
of interest. Frontiers in Psychology frontiersin.org References Andersen, P. O., Jensen, M. K., Lippert, A., Østergaard, D., and Klausen, T. W. (2010). Development of a formative assessment tool for measurement of
performance in multi-professional resuscitation teams. Resuscitation 81, 703–711. doi: 10.1016/j.resuscitation.2010.01.034 Frontiers in Psychology 07 frontiersin.org frontiersin.org Nabecker et al. 10.3389/fpsyg.2022.1020124 simulated team and real team assessment: a randomised controlled cohort trial. Front. Cardiovasc. Med. 9, 840114. doi: 10.3389/fcvm2022840114 Grasner, J. T., Herlitz, J., Tjelmeland, I. B. M., Wnent, J., Masterson,
S.,
Lilja,
G.,
et
al. (2021). European
resuscitation
council
guidelines
2021: epidemiology of cardiac arrest in Europe. Resuscitation 161, 61–79. doi: 10.1016/j.resuscitation.2021.02.007 Nabecker, S., Huwendiek, S., Theiler, L., Huber, M., Petrowski, K., Greif, R.,
et al. (2021b). The effective group size for teaching cardiopulmonary resuscitation
skills – A randomized controlled simulation trial. Resuscitation 165, 77–82. doi: 10.1016/j.resuscitation.2021.05.034 Greif, R., Bhanji, F., Bigham, B. L., Bray, J., Breckwoldt, J., Cheng,
A., et al. (2020). Education, implementation, and teams: 2020 international
consensus on cardiopulmonary resuscitation and emergency cardiovascular
care science with treatment recommendations. Resuscitation 156, A188–A239. doi: 10.1161/CIR.0000000000000896 Nabecker, S., Theodorou, M., Huwendiek, S., Kasper, N., and Greif, R. (2021a). Out-of-hospital cardiac arrest: comparing organised groups to individual first
responders: a qualitative focus group study. Eur. J. Anaesthesiol. 38, 1096–1104. doi: 10.1097/EJA.0000000000001335 Greif, R., Lockey, A., Breckwoldt, J., Carmona, F., Conaghan, P., Kuzovlev,
A., et al. (2021). European resuscitation council guidelines 2021: education for
resuscitation. Resuscitation 161, 388–407. doi: 10.1016/j.resuscitation.2021.02.016 Nadkarni, L. D., Roskind, C. G., Auerbach, M. A., Calhoun, A. W., Adler,
M. D., Kessler, D. O., et al. (2018). The development and validation of a
concise instrument for formative assessment of team leader performance
during
simulated
pediatric
resuscitations. Simul. Healthc. 13,
77–82. doi: 10.1097/SIH.0000000000000267 Greif, R., Lockey, A. S., Conaghan, P., Lippert, A., De Vries, W., Monsieurs,
K. G., et al. (2015). European resuscitation council guidelines for resuscitation
2015: section 10. education and implementation of resuscitation. Resuscitation 95,
288–301. doi: 10.1016/j.resuscitation.2015.07.032 Perkins, G. D., Graesner, J. T., Semeraro, F., Olasveengen, T., Soar,
J., Lott, C., et al. (2021). European resuscitation council guidelines 2021:
executive summary. Resuscitation 161, 1–60. doi: 10.1016/j.resuscitation.2021. 02.003 Hunziker, S., Bühlmann, C., Tschan, F., Balestra, G., Legeret, C., Schumacher, C.,
et al. (2010). Brief leadership instructions improve cardiopulmonary resuscitation
in a high-fidelity simulation: a randomized controlled trial. Crit. Care Med. 38,
1086–1091. doi: 10.1097/CCM.0b013e3181cf7383 Ringsted, C., Lippert, F., Hesselfeldt, R., Rasmussen, M. B., Mogensen, S. S., Frost, T., et al. (2007). References Assessment of advanced life support competence
when combining different test methods–reliability and validity. Resuscitation 75,
153–160. doi: 10.1016/j.resuscitation.2007.03.003 Hunziker, S., Johansson, A. C., Tschan, F., Semmer, N. K., Rock, L., Howell, M. D., et al. (2011). Teamwork and leadership in cardiopulmonary resuscitation. J. Am. Coll Cardiol. 57, 2381–2388. doi: 10.1016/j.jacc.2011.03.017 Rosenman, E. D., Ilgen, J. S., Shandro, J. R., Harper, A. L., and Fernandez, R. A. (2015). Systematic review of tools used to assess team leadership in health
care action teams. Acad. Med. 90, 1408–1422. doi: 10.1097/ACM.00000000000
00848 Jones, C. P., Fawker-Corbett, J., Groom, P., Morton, B., Lister, C., Mercer, S. J. (2018). Human factors in preventing complications in anaesthesia: a systematic
review. Anaesthesia 73, 12–24. doi: 10.1111/anae.14136 Kuzovlev, A., Monsieurs, K. G., Gilfoyle, E., Finn, J., Greif, R., Bigham, B. L.,
et al. (2021). The effect of team and leadership training of advanced life support
providers on patient outcomes: a systematic review. Resuscitation 160, 126–139. doi: 10.1016/j.resuscitation.2021.01.020 Smith, G. B., Welch, J., DeVita, M. A., Hillman, K. M., and Jones, D. (2015). Education for cardiac arrest–Treatment or prevention? Resuscitation 92, 59–62. doi: 10.1016/j.resuscitation.2015.04.018 Soar, J., Maconochie, I., Wyckoff, M. H., Olasveengen, T. M., Singletary, E. M.,
Greif, R., et al. (2021). International consensus on cardiopulmonary resuscitation
and emergency cardiovascular care science with treatment recommendations:
summary from the basic life support; advanced life support; neonatal life support;
education, implementation, and teams; first aid task forces. Resuscitation 140,
E826–E880. doi: 10.1161/CIR.0000000000000734 Lee, S. H., Khanuja, H. S., Blanding, R. J., Sedgwick, J., Pressimone,
K., Ficke, J. R., et al. (2021). Sustaining teamwork behaviors through
reinforcement
of
TeamSTEPPS
principles. J
Patient
Saf. 17:e582–6. doi: 10.1097/PTS.0000000000000414 Lockey, A., Lin, Y., and Cheng, A. (2018). Impact of adult advanced cardiac
life support course participation on patient outcomes – A systematic review and
meta-analysis. Resuscitation 129, 48–54. doi: 10.1016/j.resuscitation.2018.05.034 Ulmer, F. F., Lutz, A. M., Müller, F., Riva, T., Bütikofer, L., Greif, R., et al. (2021). Communication patterns during routine patient care in a pediatric intensive care
unit: the behavioral impact of in situ simulation. J. Patient Saf. 18, e573–e579. doi: 10.1097/PTS.0000000000000872 Marx, D., Greif, R., Egloff, M., Balmer, Y., Nabecker, S., et al. (2020). Recruiting medical students for a first responder project in the social age:
direct contact still outperforms social media. Emerg. Med. Int. 2020, 9438560. doi: 10.1155/2020/9438560 Walsh, O., Lydon, S., and O’Connor, P. (2017). References A mixed methods evaluation of
paediatric trainee preparedness to manage cardiopulmonary arrests. Eur. J. Pediatr. 176, 1653–1662. doi: 10.1007/s00431-017-3017-6 Monsieurs, K. G., Nolan, J. P., Bossaert, L. L., Greif, R., Maconochie, I. K., Nikolaou, N. I., et al. (2015). European resuscitation council guidelines
for resuscitation 2015: section 1. Executive summary. Resuscitation 95, 1–80. doi: 10.1016/j.resuscitation.2015.07.038 Yeung, J., Djarv, T., Hsieh, M. J., Sawyer, T., Lockey, A., Finn, J.,
et al. (2020). Spaced learning versus massed learning in resuscitation – A
systematic review. Resuscitation 156, 61–71. doi: 10.1016/j.resuscitation.2020. 08.132 Nabecker, S., Huwendiek, S., Seidl, C., Hana, A., Theiler, L., Greif, R., et al. (2022). Assessment of human factors after advanced life support courses comparing 08 Frontiers in Psychology frontiersin.org 08
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Research on the Art Design College's Computer Network Entrepreneurship Plat From
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Keywords: Students; network business platform; art and design; innovative education Abstract. At present, in the artistic design education in Colleges of China appeared in the realm of"
enterprise education" as the core content of the reform, obtained better result also gained experience. But there are also problems. Based on the innovative education of art design and professional
characteristics analysis, proves the reality and necessity of the university construction of art design
professional business platform for the network (hereinafter referred to as the “business platform for
the network." ), the effectiveness in the promotion of university art design education, and it improve
students' innovation ability. And the actual operation of the platform design to provide a preliminary
exploration. In 2011 April Academic Degrees Committee of the State Council, the Ministry of
education to add “art” for the thirteenth disciplines, design upgrades for the first grade discipline. New courses to adapt to the development of China's economic, social, science and technology and
higher education, also indicated that the promote of art and design disciplines’ academic status. Focus
on scientific and technological advances in today's world, the horizon of knowledge economy,
national competitiveness and education in the comprehensive national strength in the important role
of art and design college design education is the most fundamental task is to nurture students to
become innovative, professional skills intensive, with good employment entrepreneurship modern
design professionals. This article is an open access article under the terms and conditions of the Creative Commons Attribution (CC BY) license
(https://creativecommons.org/licenses/by/4.0) Online: 2012-06-04 Online: 2012-06-04 Advanced Engineering Forum
ISSN: 2234-991X, Vol. 4, pp 163-167
doi:10.4028/www.scientific.net/AEF.4.163
© 2012 The Author(s). Published by Trans Tech Publications Ltd, Switzerland. Research on the Art Design College's Computer Network
Entrepreneurship Plat From
Wang Ming1,a, ShanTan2,b
1 College of Furniture and Art Design, Central south university of forestry and technology, ChangSha
Hunan, P.R.China
2 Art design departments of ZengCheng School, South China Normal University, Guangzhou
Guangdong, P.R.China
awangming68610@163.com, b24794065@qq.com Smart Technologies for Communication 164 3. Entrepreneurship class too passive and routine. Heavy theory of light practice; Heavy
short-term subject conclusion, light up long-term projects; Heavy personal behavior, light the team;
many copy foreign mode, ignored China's situation and the universities, the market situation. 3. Entrepreneurship class too passive and routine. Heavy theory of light practice; Heavy
short-term subject conclusion, light up long-term projects; Heavy personal behavior, light the team;
many copy foreign mode, ignored China's situation and the universities, the market situation. 3. Entrepreneurship class too passive and routine. Heavy theory of light practice; Heavy
short-term subject conclusion, light up long-term projects; Heavy personal behavior, light the team;
many copy foreign mode, ignored China's situation and the universities, the market situation. 4. Lack of entrepreneurial scheme for the correct practice test evaluation system. College design
education internal often only to be content as evaluation standard, neglecting social meaning and
practical value, cause some practical innovation design only stays in the design on the drawing
board, hard into productivity. 4. Lack of entrepreneurial scheme for the correct practice test evaluation system. College design
education internal often only to be content as evaluation standard, neglecting social meaning and
practical value, cause some practical innovation design only stays in the design on the drawing
board, hard into productivity. 5. Off many of the resources advantage didn't get the integration and reflect lack of social
linkage. Universities can provide the opportunity and practice are limited to meet the students
entrepreneurship practice needs, students need to equal participation opportunity of business, but
the internal lack of public resources sharing platform. Especially for the innovation of the life of
professional design, need more close to the actual combat training platform of the market. 5. Off many of the resources advantage didn't get the integration and reflect lack of social
linkage. Universities can provide the opportunity and practice are limited to meet the students
entrepreneurship practice needs, students need to equal participation opportunity of business, but
the internal lack of public resources sharing platform. Especially for the innovation of the life of
professional design, need more close to the actual combat training platform of the market. p
g ,
g p
According to the survey, about 60% of the art design professional students tend to start their own
businesses. Smart Technologies for Communication But because the university student's pioneering work widespread "3 without" problem,
only a few students can design company, founded entity studio, undertake market project. Finally to
withstand test market and obtain the stable development is even less. [2]In general, there is a
general lack of awareness of college students, entrepreneurship ability lacking, own pioneering
atmosphere is far still did not form. At the same time university student's poineering work need
adjust entrepreneurial mindset, establishing market view. And the lack of professional guidance, not
a long-term stable development is the key problem affecting the university students' business. In the
market rapidly changing today, college students' learning in school during practice becomes more
and more important, need to colleges can build a wide coverage, long-term stability of the
marketization of business practice platform. Network poineering advantage and disadvantage analysis Today, the network economy has increasingly become the core of the new economy. By the end of
June, 2011, the number of users has reached 485 million, the network shopping users and more than
130 million. Starting a business network to become a reality of employment of the new choice. Starting a business network with "initial capital investment, low operating cost is low, the small
risk, market range, flexible and profits high return time" and so on characteristics and advantages,
so most of the independent business student chose through the network platform venture as the first
choice of the first business. Most art design professional students first venture third party market network platform to start a
business. Although the third party venture platform operation marketization, coverage, trading
convenience, low cost, for art design professional students in business, also have platform
environment, lack of mixed and disorderly support and guidance, trading owe safety improvement,
intellectual property is easy to attack, design evaluation and pricing standard chaos, businesses
identical and professional characteristics and market combined with not close, the same level
competition is fierce. But the network platform of the large span, informationization, low cost,
multi-level and resource sharing, easy communication network pioneering characteristic is the root
cause of the great heat. Higher school in considering entity venture platform at the same time, also
need innovation, make full use of modern network technology advantages to carry out business
network platform construction, enhances strong points and avoid weaknesses, complementary
advantages, in order to achieve optimization university innovation talents training mode of purpose. [3] [3] The current situation of Chinese enterprise education for college students and the Business
status. UNESCO published in 1999, higher education in the 21st century: vision and action for the World
Declaration, proposed the entrepreneurial skills and entrepreneurial spirit must be used as the basic
goals of higher education; On deepening the education reform in China, promoting quality
education decision also emphasizes: "stressing the cultivation of innovation ability of college
students in higher education, practice skills and entrepreneurial spirit, a general increase in the
humanistic quality of college students and science literacy."[1]In the field of higher education in
China "career education" as the core content of boom, student venture employability is undoubtedly
has become an important standard in evaluation of school success. Current internal practices in
entrepreneurship education in universities and achieved certain results at the same time, still
appeared the following questions: 1 the prevalence of entrepreneurship education of students work under the category of
misunderstanding. Lack of link between teaching and practice of enterprise education; 2. Lack of clear entrepreneurial goal of entrepreneurship education guide and complete
cultivation plan, diversity of student individuality development have not been fully discovered, lack
of professionalism, Omni-directional guidance left majority. University student's pioneering work
with no capital, no existing site, 3 without the experience of the common problems, only a few of
the students will be able to realize the establishment and organization for market entity gleam of
project; Smart Technologies for Communication Advanced Engineering Forum Vol. 4 165 1: using the network venture platform advantage, to make full use of various resources
universities to support the university student's pioneering work. Use of human resources in colleges
and universities, research projects, research achievements on the advantages, and the university
project team organization ability, and innovative thinking, professional complementary business
development and support such as the uniqueness and set up a new university business education
training mode and practice base. At the same time, the research and relying on the multiple network
technology advantage, drawing a market advantages of third party platform, strengthen and large
network venture platform, professional network technology platform, enterprise, institution,
professional personnel's communication, provide more close to the market demand of training,
trade, investment, consultation and so on various aspects of the help and services, gradually realize
the normality of the business thinking of, so as to improve the success rate of business; Will the
design results converted into productive forces, for university student's pioneering work from
concept to provide practical production management research assessment of the overall support and
service. 2: through this platform, provided a self development innovation, mutual communication,
support mutually, mutual reciprocity and mutual benefit network space for college students. Will
venture ability training and curriculum, and the combination of ascension of the university students'
of entrepreneurship and innovation spirit, cultivate the spirit of innovation and independent thinking
ability, team cooperation ability, improve overall quality, according to the actual situation of self as
early as possible location, and know the market tendency, development and orientation for the
future lay a practice base. Based on this, the formation of the daily operation of the market and
professional management team and the full for university art design speciality various talents
training services. g
3: through the platform professional advantage and design show research results, make with art
design professional feature unique network space. Will art design targeted, and cover range, close to
people living demand, more easily to the business professional features a full play. From business to
support business training, to really improve the students' autonomous learning enthusiasm, clear the
direction of study. Through to the university student's pioneering work on the reasonable guide,
according to the individual team advantage, build up the core competitive power. The significance and function about the construction of the platform of network pioneering According to the analysis of the status of colleges and universities should give full play to its own
advantages, combining the characteristics of the network platform, can create outstanding
professional research characteristics, and combining with the market the new network venture
platform for college students to provide various business support. Construction of Internet and
venture platform has the following the significance and role of: Advanced Engineering Forum Vol. 4 (1) Registration of designer (1) Registration of designer (1) Registration of designer The name, age, ID card, the user name, password (all need validation) fill out verification code,
and then submit. To work of status register: can obtain the following functions, the work can upload
their work, also can delete their works, still can change their work status, after landing also can see
the work of others, but can't operate. (2) Registration of business (2) Registration of business Name, age, ID card, the user name, password (all need validation) and fill in the name of the
company, set up a time, address, fill in verification code, and then submit. To merchants identity
register: can obtain the function, can choose all the work, change can choose to work, the selected
works, will be marked by the businessman has options, then can see after contact, if companies in
three days not to work or to the site links, will be cancelled automatically state has chosen to let
other merchants choice. (Works can also be choose cancel state) 3. Database 3. Database Picture database with oracle database handling Suggestions in large picture storage and delivery
is very useful. Will the database classification, the school subject’s professional classification, a few
major points of a few. Upload press kind of storage after work. So convenient search, also facilitate
maintenance. Works sent to the front page in add a watermark processing, this can prevent illegal
things happen. Advanced Engineering Forum Vol. 4 In strive to
improve employment can also bring more innovation employment mode, the better the
school-enterprise cooperation way, for students' learning and development offers multiple choice
and chance. 4: create a practice drive research, to promote the practice teaching of the platform. Through the
prompt understanding market trends and needs, the targeted professional curriculum. The actual
combat training into the classroom, classroom knowledge will be used in practice. Research by
practice to explore how to improve the teaching quality, reform the teaching mode, and will
continue to various teaching achievements through the platform displayed, through the market for
inspection. Realize the market and the talent cultivation and seamless connection between. 4: create a practice drive research, to promote the practice teaching of the platform. Through the
prompt understanding market trends and needs, the targeted professional curriculum. The actual
combat training into the classroom, classroom knowledge will be used in practice. Research by
practice to explore how to improve the teaching quality, reform the teaching mode, and will
continue to various teaching achievements through the platform displayed, through the market for
inspection. Realize the market and the talent cultivation and seamless connection between. p
5: through the network business platform, enterprise, strengthen the school students and the
connection between communication. Use the college each professional direction, all kinds of
expertise of interaction between the classmate, independent form innovation team, set of all of
optimization team work, for enterprise better services at the same time, form good teaching
atmosphere and entrepreneurial environment of campus. 6: through this platform, and summarize the experience and lesson of entrepreneurs, sorting and
study the characteristics of successful business, for daily teaching and new entrepreneurs to provide
practice and theory on. 6: through this platform, and summarize the experience and lesson of entrepreneurs, sorting and
study the characteristics of successful business, for daily teaching and new entrepreneurs to provide
practice and theory on. 7: starting a business network platform in the continuous practice operation process, gradually
full platform database, summarize the experience, improving the operation and management,
perfect the venture platform function, attract more colleges and students participate in them, so as to
form the stronger resource intensive advantages; Attract designers, enterprise and the independent
brand into, enhance the market operation platform independent ability. Advanced Engineering Forum Vol. 4 Gradually enhance the
teaching platform training function, at the basic teaching primarily university, on the basis of Smart Technologies for Communication 166 various resources through encourage social platform provides space for entrepreneurial,
employment, professional knowledge training in the teaching training or guidance. [4] make venture
platform not only have the business reference significance to study, more be a help to improve the
teaching quality, improve college students' integrated qualities and ability of the network sharing
space. various resources through encourage social platform provides space for entrepreneurial,
employment, professional knowledge training in the teaching training or guidance. [4] make venture
platform not only have the business reference significance to study, more be a help to improve the
teaching quality, improve college students' integrated qualities and ability of the network sharing
space. The basic principles and methods about how to establishing a business network pl 1. This platform to provide each other only choice, not engaged in cash transactions The user classification: works, businessmen,, the network administrator; 2. How to Making a
page er classification: works, businessmen,, the network administrator; 2. How to Making The user classification: works, businessmen,, the network administrator; 2. How to
age p g
(1) Ordinary HTML page is suitable for, in the web page design can use DREAMWEAVER
design software. Or choose the school most of the students are familiar with, also play the role of
design practice. p g
(1) Ordinary HTML page is suitable for, in the web page design can use DREAMWEAVER
design software. Or choose the school most of the students are familiar with, also play the role of
design practice. g p
(2) Home page to determine the style, can according to the school of the original page subject to
design, also highlights the use function of change website, and then choose some fine works in the
home page. Must be in the home page set on function, the users can see after landing site in the
design works more relevant content. design works more relevant content. g
2. Registration Summary Through the network of research and construction business platform, using higher school plan as a
whole the advantage of its resources, creating a platform of Internet and the theory research and
practice, on the one hand, to adapt to the art design speciality of the university student's pioneering
work request, for building the innovative country training high quality, creative ability, can the
various positions in for entrepreneurial talent play its role; On the other hand, the study of Internet
and venture platform in promoting college education teaching mode in the reform of the effect,
make the talent training to keep pace with The Times, and the market closely, and give full play to
the university and the main function of the higher education. This is also our university art design
education keep up with higher education to reform the world needs. Advanced Engineering Forum Vol. 4 167 [4] Liu Y J. College students starting a business network problems and countermeasures study [J].The
electronic commerce, 2011, (6):79-81. References [1] Zhang YQ, Xu J. College students' business education research review [J].China power education,
2011, (16):36-38. [1] Zhang YQ, Xu J. College students' business education research review [J].China power education,
2011, (16):36-38. [1] Zhang YQ, Xu J. College students' business education research review [J].China power education,
2011, (16):36-38. [2] Zhang M. College students' career consciousness research by university students in Zhejiang for
example [J]. Herald of education, 2011, (05):117-119. [3] Hu G L. College students to analyze the current situation and the countermeasures of starting a
business network[J]. Modern education science, 2010, (3):114-121. [3] Hu G L. College students to analyze the current situation and the countermeasures of starting a
business network[J]. Modern education science, 2010, (3):114-121. [4] Liu Y J. College students starting a business network problems and countermeasures study [J].The
electronic commerce, 2011, (6):79-81. [4] Liu Y J. College students starting a business network problems and countermeasures study [J].The
electronic commerce, 2011, (6):79-81. [4] Liu Y J. College students starting a business network problems and countermeasures study [J].The
electronic commerce, 2011, (6):79-81.
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On-Line Data-Driven Control for Uncertain Systems Based on Greedy Algorithm
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Research Square (Research Square)
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Jiahui Shen ( 22232020@zju.edu.cn ) Zhejiang University https://orcid.org/0000-0002-0948-1942 Research Article Posted Date: May 31st, 2023 Jia-Hui Shen · Xing-Gao Liu
School of Control Science and Engineering, Zhejiang University, Hangzhou, 310058, China
E-mail: lxg@zju.edu.cn (Xing-Gao Liu) DOI: https://doi.org/10.21203/rs.3.rs-2969931/v1 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 Noname manuscript No. (will be inserted by the editor) Noname manuscript No. (will be inserted by the editor) On-Line Data-Driven Control for Uncertain Systems
Based on Greedy Algorithm Jia-Hui Shen · * Xing-Gao Liu Received: date / Accepted: date Received: date / Accepted: date Abstract Considering a result that persistently exciting data can used to
replace the linear system model, this paper is devoted to applying this result in
the field of data-driven control of nonlinear systems. An on-line iteration based
on greedy algorithm to stabilize uncertain discrete-time systems is proposed. The method tends to obtain the approximate optimal control through solving
a series of programming problems. Every programming problem is linear for
the convenience of solving. Besides, in particular, the methods requires few
prior conditions, as long as the system is controllable and observable and the
equilibrium state of the system is known. First, we prove that under certain
circumstances, the solution of our linear matrix inequality can stabilize the
system. Next, a multi-objective programming problem is proposed to dealing
with situations where the required conditions are unknown. Finally, an on-
line iteration is used to enhance robustness as well as real-time evaluation. The method is illustrated to be effective through a simulation on a discretized
inverted pendulum system under repeated experiments. Keywords data-driven control · on-line iteration · greedy algorithm · matrix
inequality · uncertain discrete-time system 1 Introduction Learning from model and learning from data are different ways to analyze the
system. In 1960, Kalman proposed the parametric state-space model, laying
the the foundations for the progress of the modern control theory [1]. Model-
based control (MBC) has been developing including the methods of optimal
control [2], adaptive control [3], model-predictive control [4], and so on. With Jia-Hui Shen, * Xing-Gao Liu 2 the development of MBC, there were some shortcomings exposed. System iden-
tification [5] is always based on first principles and data calibration, which is
too difficult for increasingly complex processes nowadays [6]. Besides, MBC is
always thought to be too accurate to be robust enough in dealing with nosies
[7]. Due to the high dependence of MBC on the model, an inaccurate model
with little error may cause big problem [8]. Considering the disadvantages of MBC, data-driven control (DDC) is be-
coming more prevalent in Automation. DDC means that the controller de-
sign is based on using on-line or off-line I/O data instead of using the the
explicit information from the model [9]. How to achieve this goal, designing
control systems directly from process data is the main problem. Nowadays,
with the efforts of many researchers, there are some different DDC meth-
ods like proportional-integral-derivative (PID) [10], iterative feedback tuning
(IFT) [11], unfalsified control (UC) [12], iterative learning control (ILC) [13],
model-free adaptive control (MFAC) [14], and so on. Compared to MBC, DDC
has obvious advantages in dealing with the twinborn problem of unmodeled
dynamics and robustness [15]. The researchers also consider other needs be-
sides stability control, such as optimal control [16,17], and output tracking
[18,19], and have achieved some results. Linear systems and nonlinear systems are two categories of systems, and
generally, the former is easier to analyze. Considering the abundant theoreti-
cal foundation in addressing linear systems, linearization becomes a common
and useful method, such as Taylor’s linearization [20], piecewise linearization
[21], feedback linearization [22]. As for linear systems, Willems proposed a
fundamental result that a linear system can be represented by a finite set of
system trajectories under a persistently exciting input sequence [23]. It shows
that I/O date is equivalent to system models in certain cases, which is used in
data-driven controller design [24–26]. In this paper, we follow the previous research, proposing a method suitable
for more situations. 1 Introduction In Section 2, we transform a common controllable and
observable system into a system with fully measurable states (see Lemma 1). Next, we use Lyapunov stability condition to achieve DDC (see Theorem 2
and 3) in Section 3. Then in Section 4, to handling the situation that more
prior conditions are unknown, we propose an on-line iteration. The simulation
and conclusion are given in Section 5 and 6, respectively. 2 Problem Statement In this paper, we will study to control a deterministic discrete-time system. In
order to simplify the problem, we just consider a controllable and observable
discrete-time system xk+1 = f(xk, uk)
yk = g(xk, uk)
zk = yk + wk
(1) (1) On-Line Data-Driven Control for Uncertain Systems Based on Greedy Algorithm 3 where xk ∈Rn is the state vector, uk ∈Rm is the control vector, yk ∈Rp is
the output vector, wk ∈Rp is the error vector in measurement, and zk ∈Rp
is the measurement value. Besides, we consider the system function f(xk, uk)
is continuous for ∀xk, uk and f(0, 0) = 0, which shows that under the control
ue = 0, xe = 0 is an equilibrium state of the system (1). The output function
g(xk, uk) is also continuous for ∀xk, uk and g(0, 0) = 0, so ye = 0 is satisfied
under the equilibrium state (xe, ue). where xk ∈Rn is the state vector, uk ∈Rm is the control vector, yk ∈Rp is
the output vector, wk ∈Rp is the error vector in measurement, and zk ∈Rp
is the measurement value. Besides, we consider the system function f(xk, uk)
is continuous for ∀xk, uk and f(0, 0) = 0, which shows that under the control
ue = 0, xe = 0 is an equilibrium state of the system (1). The output function
g(xk, uk) is also continuous for ∀xk, uk and g(0, 0) = 0, so ye = 0 is satisfied
under the equilibrium state (xe, ue). If we simplify the system (1) again, such as assuming that g(xk, uk) = xk,
the system (1) will become xk+1 = f(xk, uk)
zk = xk + wk
(2) (2) with fully measurable states xk. However, it may not be possible for a common
system. To generalize this situation, consider the following conjecture. Conjecture 1 The discrete-time system (1) is observable. More precisely, the
state vector xk can be accurately inferred through the next N control vectors
and output vectors, and the required step N is known. Conjecture 1 expresses that we can solve xk if we know the control sequence
uN
k = (uk, uk+1, . . . , uk+N−1) and the output sequence yN
k = (yk, yk+1, . . . 2 Problem Statement , yk+N−1),
which means the equations
yk = g(xk, uk) = F1(xk, u1
k)
yk+1 = g(f(xk, uk), uk+1) = F2(xk, u2
k)
yk+2 = g(f(f(xk, uk), uk+1), uk+2) = F3(xk, u3
k)
... yk+N−1 = FN(xk, uN
k )
(3) (3)
. yk+N−1 = FN(xk, uN
k ) have a unique solution xk. According to this, we define a new function between
xk, and (uN
k , yN
k )
N
N q
xk, and (uN
k , yN
k ) xk = O(uN
k , yN
k ). (4) (4) We can also find yk+N = FN+1(xk, uN+1
k
) = FN+1(O(uN
k , yN
k ), (uN
k , uk+N))
which is not related to the state vector xk. In this case, we can easily analyze
the system (1) by the output measurement instead of the state measurement. Let tk = col(uN
k , yN
k ), we can obtain the state-space system shown below
uk+1
uk+2
... uk+N
yk+1
yk+2
... yk+N
=
uk+1
uk+2
... uk+N
yk+1
yk+2
... FN+1(O(uN
k , yN
k ), (uN
k , uk+N))
. (5) (5) 4 Jia-Hui Shen, * Xing-Gao Liu The system (5) is just about tk, tk+1 and uk+N, so the system (5) can
also be described by tk+1 = T(tk, uk+N). Obviously, tk = col(uN
k , yN
k ) is all
known in the system (1). Let pk = col(zN
k , uN
k ) and rk = col(wN
k , 0N
k ), then
the system will become tk+1 = T(tk, uk+N)
pk = tk + rk
(6) (6) with the fully measurable states tk. We now state a result which is key for the
developments of the article. Lemma 1 Consider the system (6). The system (6) is equivalent to the system
(2) to some extent. Proof Firstly, we consider the controllability of the system (6). For any feasible
tk, there must be a unique sequence (xN+1
k
, uN
k ) corresponding to it. Given
that the system (1) is controllable, for any xk+N, a control sequence uM
k+N can
make xk+N+M = 0. Besides, xN
k+N+M = 0 and yN
k+N+M is satisfied under the
control sequence uN
k+N+M = 0. 2 Problem Statement Hence, for the tk, a control sequence uM+N
k+N =
(uM
k+N, uN
k+N+M) can make tk+N+M = 0, that is to say the system (6) is
controllable. Then we consider the equilibrium state of the system (6). If tk = 0, ob-
viously, xk = 0 is the unique solution of the equations (3), so xN+1
k
= 0. yk+N = 0, and tk+1 = 0 as long as uk+N = 0, which shows that te = 0 is
an equilibrium state of the system (6) under the control ue = 0. Hence, the
system (6) is similar to the system (2) with some properties we need. ⊓⊔ Based on Lemma 1, we can just consider the system (2). Of course, there
are some differences, especially in dimensionality. Note that we turn the system
of order n into the system of order N(m+p), and the equations (3) show that
Np > n. In order to analyzing the system more easily, we’d better make N as
small as possible, even though the bigger N can meet Conjecture 1 better. 3 System Approximation And Controller Design In the previous section, we have changed the form of the system (1) to the the
form of the system (2). Because of the simplicity of the linear control systems,
we try to rewrite the nonlinear system (2) as xk+1 = Gxk + Huk + Lk
zk = xk + wk
(7) (7) where G and H are the linear matrix coefficients: where G and H are the linear matrix coefficients: G = ∂f
∂x
(x,u)=(xe,ue)
, H = ∂f
∂u
(x,u)=(xe,ue)
. (8) (8) On-Line Data-Driven Control for Uncertain Systems Based on Greedy Algorithm 5 5 Besides, L accounts for higher-order terms, and if (x, u) goes to (0, 0), L
will go faster, that is to say L = l1(x, u)
x
u
,
lim
(x,u)→(0,0) l1(x, u) = 0. (9) (9) More specially, if f(x, u) has continuous second-order derivatives, we can
also have
T More specially, if f(x, u) has continuous second-order derivatives, we can
also have
T L = 1
2
x
u
T
l2(x, u)
x
u
. (10) (10) Since what we can get is zk rather than xk, we rewrite the linearized system
(7) again as Since what we can get is zk rather than xk, we rewrite the linearized system
(7) again as (7) again as zk+1 = Gzk + Huk + Lk + Rk
(11) (11) where Rk = wk+1 −Gwk. It is known that if the linearized system (11) de-
fined by (G, H) can be stabilizable under the controller K, namely the system
xk+1 = (G −HK)xk is stable, K can also stabilize the original nonlinear sys-
tem (2). Hence, if we aim to stabilize our system, we can just estimate (G, H)
to describe the system instead of estimating the nonlinear function f. In order to approximate f with (G, H) better, we consider the following
conjecture. Conjecture 2 Whether the remainder Lk or the noise Rk has much less influ-
ence on the system (11) than the pair (G, H), namely ||Lk + Rk|| < ϵ and ϵ
is small enough to have little impact on analysis in the system approximation
and controller design. Conjecture 2 describes a system with low degree of nonlinearity and small
measurement error. 3 System Approximation And Controller Design If Conjecture 2 is satisfied, we approximate the system zk+1 = ˆGzk + ˆHuk
(12) (12) where ˆG and ˆH is the solution of the following least-square problem ( ˆG, ˆH) = arg min
(G,H) ||Zk+1 −GZk −HUk||F
(13) (13) where (Zk+1, Zk, Uk) represents some corresponding state vectors and control
vector (zk+1, zk, uk) in the system (11). Considering the least-square problem (13), the following result is given
naturally. Theorem 1 If the control sequence uT
k can make the system (11) with the
initial state xk meet the following condition: rank
zT
k
uT
k
= n + m
(14) (14) where zT
k is the state sequence, the least-square problem (13) has a unique
solution ( ˆG, ˆH). 6 Jia-Hui Shen, * Xing-Gao Liu Proof Under the condition that Theorem 1 describes, ( ˆG, ˆH) is the least-
squares solution of the equation ˆG ˆH
zT
k
uT
k
= zT
k+1. (15) (15) Considering the condition of equivalence of the equation (15), ( ˆG, ˆH) is
also the solution of the equation ˆG ˆH
zT
k
uT
k
zT
k
uT
k
⊤
= zT
k+1
zT
k
uT
k
⊤
. (16) (16) If the condition in Theorem 1 is satisfied, we can know the matrix
zT
k
uT
k
zT
k
uT
k
⊤
is invertible, and the solution ( ˆG, ˆH) is unique: ˆG ˆH
= zT
k+1
zT
k
uT
k
⊤
zT
k
uT
k
zT
k
uT
k
⊤!−1
. (17) (17) Hence, in order to ensure the uniqueness of the results, the rank condition
in Theorem 1 is always satisfied throughout our analysis. ⊓⊔ Hence, in order to ensure the uniqueness of the results, the rank condition
in Theorem 1 is always satisfied throughout our analysis. ⊓⊔ Having identified an approximate linear model of the linearized system
(11), we design a controller K based on the parameter ( ˆG, ˆH), that is to say
we make the system (11) a stable closed-loop system under state-feedback
law u = −Kz. Hence, we can design the controller K by letting the system
matrix ˆG −ˆHK satisfy the Lyapunov stability condition, which is proved to
be equivalent to the matrix inequality in the following result. 3 System Approximation And Controller Design Theorem 2 If the matrices Q and P > 0 satisfy
σ2P
ˆGP −ˆHQ
P ˆG⊤−Q⊤ˆH⊤
P
> 0
(18) matrices Q and P > 0 satisfy
σ2P
ˆGP −ˆHQ
P ˆG⊤−Q⊤ˆH⊤
P
> 0
(18) (18) where the parameter σ ∈(0, 1], K = QP −1 is a controller that can stabilize
the system (11). Proof Considering the Lyapunov stability condition, there are two equivalent
conditions for the same linear system xk+1 = Gxk: ∀s ∈λ(G), |s| < 1 ⇐⇒∃P > 0, s.t. G⊤PG −P < 0
(19) (19) where λ(·) represents taking the eigenvalues of a matrix. Therefore, if we want
the system to stabilize more quickly, here is the way: ∀s ∈λ(G), |s| < σ ⇐⇒∃P > 0, s.t. GPG⊤−σ2P < 0
(20) (20) On-Line Data-Driven Control for Uncertain Systems Based on Greedy Algorithm 7 where σ ∈(0, 1], and the theorem λ(G) = λ(G⊤) is used to prove it. We can
easily know that the smaller σ is, the faster the system stabilize. The way to
get K becomes to solve the following inequality due to the previous theorems: ∃P > 0, s.t. ( ˆG −ˆHK)P( ˆG −ˆHK)⊤−σ2P < 0. (21) (21) Let Q = KP, then the inequality (21) becomes the existence of one pa-
rameter σ ∈(0, 1], two matrices Q and P > 0 such that Let Q = KP, then the inequality (21) becomes the existence of one pa-
rameter σ ∈(0, 1], two matrices Q and P > 0 such that ( ˆGP −ˆHQ)P −1(P ˆG⊤−Q⊤ˆH⊤) −σ2P < 0
(22) (22) which is equivalent to the matrix inequality (18). Hence, we have turned the
inequality (21) on (σ, P, K) to the inequality (18) on (σ, P, Q) to eliminate
the multiplication term of K and P aiming to simplify the calculation, and
we can also just solve the inequality (18) to get K by calculating a posteriori
K = QP −1. ⊓⊔ ⊓⊔ However, the inequality (18) still has a nonlinear term σ2P, causing com-
plexity in solving the problem. In order to avoid this condition, we determine
the value of σ at first, then the nonlinear matrix inequality turns to a linear
matrix inequality. Besides, for a linear system with known ( ˆG, ˆH), we can also
choose other ways to design the controller K, such as pole placement. 3 System Approximation And Controller Design We deter-
mine the needed pole λK firstly, then solve K which satisfies λ( ˆG−ˆHK) = λK. Obviously, if the system under the pair ( ˆG, ˆH) is controllable, the existence of
K can be guaranteed. All the previous proof is base on Conjecture 2. However, the requirement in
Conjecture 2 is so strict that many systems can not meet it. For the most part,
the remainder Lk and the noise Rk have an influence on system approximation
and controller design. Hence, in order to take it into account, we consider a
new conjecture. Conjecture 3 The system holds that Conjecture 3 The system holds that LT
k (LT
k )⊤≤d1xT
k+1(xT
k+1)⊤
(23a)
wT
k+1(wT
k+1)⊤≤d2I, RT
k (RT
k )⊤≤d3I
(23b) for some d1, d2, d3 > 0. for some d1, d2, d3 > 0. In fact, the requirement in Conjecture 3 is much looser than that of Conjec-
ture 2. It express some natural requirements. The requirement (23a) is about
the remainder Lk. It shows that in a certain neighborhood of the equilibrium
state (xe, ue), the effect of the part Lk on the whole xk+1 is limited. The
requirement (23b) is about the noise wk+1 and Rk. It shows that the mea-
surement error has a bounded absolute error. Hence, for a common system,
Conjecture 3 can be always satisfied if d1, d2, d3 are large enough. Yet, xk+1
is not known, so we propose the following lemma. Jia-Hui Shen, * Xing-Gao Liu 8 Lemma 2 If Conjecture 3 is satisfied, it holds that Lemma 2 If Conjecture 3 is satisfied, it holds that LT
k (LT
k )⊤≤d4zT
k+1(zT
k+1)⊤+ d5I
(24) LT
k (LT
k )⊤≤d4zT
k+1(zT
k+1)⊤+ d5I (24) for some d4, d5 > 0. for some d4, d5 > 0. Proof To complete the proof, we consider a matrix inequality that ϵXFX⊤+ ϵ−1Y FY ⊤≥XFY ⊤+ Y FX⊤
(25) (25) where ϵ > 0 and F ≥0. In reality, the matrix inequality is equivalent to (√ϵX −√ϵ
−1Y )F(√ϵX⊤−√ϵ
−1Y ⊤) ≥0
(26) (26) which is clearly right. Then we consider zk = xk + wk in the constraint of Lk: )⊤= (zT
k+1 −wT
k+1)(zT
k+1 −wT
k+1)⊤
= zT
k+1(zT
k+1)⊤+ wT
k+1(wT
k+1)⊤−zT
k+1(wT
k+1)⊤−wT
k+1(zT
k+1)⊤. where the parameter σ ∈(0, 1], and C1, C2 are large enough, K = QP −1 is a
controller that can stabilize the system (11). 3 System Approximation And Controller Design (27) 1)
(27) By applying the previous matrix inequality with F = I, X = zT
k+1 and
Y = −wT
k+1, we can obtain the following relationship: xT
k+1(xT
k+1)⊤≤(1 + ϵ)zT
k+1(zT
k+1)⊤+ (1 + ϵ−1)wT
k+1(wT
k+1)⊤. (28)
Now we can easily finalize the proof through letting d4 = (1 + ϵ)d1 and
d5 = (1 + ϵ−1)d1d2. ⊓⊔ xT
k+1(xT
k+1)⊤≤(1 + ϵ)zT
k+1(zT
k+1)⊤+ (1 + ϵ−1)wT
k+1(wT
k+1)⊤. (28)
il
fi
li
th
f th
h l tti
d
(1 + )d
d xT
k+1(xT
k+1)⊤≤(1 + ϵ)zT
k+1(zT
k+1)⊤+ (1 + ϵ−1)wT
k+1(wT
k+1)⊤. (28)
ow we can easily finalize the proof through letting d4 = (1 + ϵ)d1 and
(1 + ϵ−1)d1d2. ⊓⊔ (28) xk+1(xk+1)
≤(1 + ϵ)zk+1(zk+1)
+ (1 + ϵ
)wk+1(wk+1) . (28)
Now we can easily finalize the proof through letting d4 = (1 + ϵ)d1 and
d5 = (1 + ϵ−1)d1d2. ⊓⊔ Now we can easily finalize the proof through letting d4 = (1 + ϵ)d1 and
d5 = (1 + ϵ−1)d1d2. ⊓⊔ These results show that all the nonlinear term in the system (11), the
remainder Lk and the noise Rk, can be constrained by some parameters and
the state vectors. This provides us an precondition for control the system (11)
by just using the limited information on the state vectors. Now we give new
matrix inequalities. Theorem 3 If the matrices Q and P > 0 satisfy Theorem 3 If the matrices Q and P > 0 satisfy Theorem 3 If the matrices Q and P > 0 satisfy σ2P −C1zT
k+1(zT
k+1)⊤−C2I ˆGP −ˆHQ
P ˆG⊤−Q⊤ˆH⊤
P
> 0
(29a)
I
M
P
−Q
P −Q⊤
M ⊤
P
> 0
(29b)
M =
zT
k
uT
k
⊤
zT
k
uT
k
zT
k
uT
k
⊤!−1
(29c) (29a) (29b) (29c) where the parameter σ ∈(0, 1], and C1, C2 are large enough, K = QP −1 is a
controller that can stabilize the system (11). where the parameter σ ∈(0, 1], and C1, C2 are large enough, K = QP −1 is a
controller that can stabilize the system (11). 3 System Approximation And Controller Design On-Line Data-Driven Control for Uncertain Systems Based on Greedy Algorithm 9 Proof As is similar to the proof in Theorem 1, we can obtain G H
= (zT
k+1 −RT
k −LT
k )M
(30) (30) with the equation (29c).To get the controller K through the Lyapunov stability
condition, we know G −HK can stabilize the system (11) if and only if (zT
k+1 −RT
k −LT
k )A(zT
k+1 −RT
k −LT
k )⊤−σ2P < 0
(31a)
A = M
P
−Q
P −1
P −Q⊤
M ⊤
(31b) (31b) where P > 0, Q = KP. We can easily prove that A ≥0. Therefore, we use
the inequality (25) to deal with the matrix inequality (31a): where P > 0, Q = KP. We can easily prove that A ≥0. Therefore, we use
the inequality (25) to deal with the matrix inequality (31a): (zT
k+1 −RT
k −LT
k )A(zT
k+1 −RT
k −LT
k )⊤
=zT
k+1A(zT
k+1)⊤−zT
k+1A(RT
k )⊤−zT
k+1A(LT
k )⊤−RT
k A(zT
k+1)⊤+ RT
k A(RT
k )⊤
+ RT
k A(LT
k )⊤−LT
k A(zT
k+1)⊤+ LT
k A(RT
k )⊤+ LT
k A(LT
k )⊤
≤(1 + ϵ1 + ϵ2)zT
k+1A(zT
k+1)⊤+ (1 + ϵ−1
1
+ ϵ3)RT
k A(RT
k )⊤
+ (1 + ϵ−1
2
+ ϵ−1
3 )LT
k A(LT
k )⊤
(32) (32) (32) where ϵ1, ϵ2, ϵ3 > 0. Next, we set a new constraint that A < I. Then we obtain where ϵ1, ϵ2, ϵ3 > 0. Next, we set a new constraint that A < I. Then we obtain (zT
k+1 −RT
k −LT
k )A(zT
k+1 −RT
k −LT
k )⊤
≤zT
k+1A(zT
k+1)⊤+ (ϵ1 + ϵ2)zT
k+1(zT
k+1)⊤+ (1 + ϵ−1
1
+ ϵ3)RT
k (RT
k )⊤
+ (1 + ϵ−1
2
+ ϵ−1
3 )LT
k (LT
k )⊤
≤zT
k+1A(zT
k+1)⊤+ [ϵ1 + ϵ2 + (1 + ϵ−1
2
+ ϵ−1
3 )d4]zT
k+1(zT
k+1)⊤
+ [(1 + ϵ−1
1
+ ϵ3)d3 + (1 + ϵ−1
2
+ ϵ−1
3 )d5]I. 3 System Approximation And Controller Design (33) (33) If C1, C2 are large enough to let C1 ≥ϵ1 + ϵ2 + (1 + ϵ−1
2
+ ϵ−1
3 )d4 and
C2 ≥(1 + ϵ−1
1
+ ϵ3)d3 + (1 + ϵ−1
2
+ ϵ−1
3 )d5, we can obtain (zT
k+1 −RT
k −
LT
k )A(zT
k+1 −RT
k −LT
k )⊤≤zT
k+1A(zT
k+1)⊤+ C1zT
k+1(zT
k+1)⊤+ C2I. Hence, a sufficient condition for the matrix inequality (31a) to be satisfied
is to satisfy zT
k+1A(zT
k+1)⊤+ C1zT
k+1(zT
k+1)⊤+ C2I −σ2P < 0
(34)
where A < I. Since we have let [ ˆG, ˆH] = zT
k+1M, the above inequality becomes
( ˆGP −ˆHQ)P −1(P ˆG⊤−Q⊤ˆH⊤) + C1zT
k+1(zT
k+1)⊤+ C2I −σ2P < 0
(35) zT
k+1A(zT
k+1)⊤+ C1zT
k+1(zT
k+1)⊤+ C2I −σ2P < 0
(34) (34) (35) which is equivalent to the matrix inequalities (29a) and A < I is equivalent
to the matrix inequalities (29b). The above proof implies that Theorem 3
provides a stricter condition to design K namely if the matrix inequalities
(29) is satisfied, K is a stable controller but the opposition is not necessarily
the case. ⊓⊔ Jia-Hui Shen, * Xing-Gao Liu 10 4 On-line Iteration Algorithm In the previous section, we have considered how to design a stable controller
K based on the limited data by solving the matrix inequality (18) or the ma-
trix inequalities (29). Yet, the precondition for these ways to be effective is
that Conjecture 2 or 3 is satisfied. As for Conjecture 2, as is mentioned above,
the system is needed to have low degree of nonlinearity and small measure-
ment error. The requirement is strict for a common system. In contrast, the
requirement in Conjecture 3 is much looser, which is natural for a common
system. However, it require prior knowledge of the upper bound, d1, d2, d3,
on the remainder Lk and the noise Rk. If this information is known, we can
design the controller K through Theorem 3. However, as for a discrete-time
system (1) which we just know is controllable and observable, this information
is often unknown to us. Although Theorem 3 can be always satisfied as long
as d1, d2, d3 are large enough, it can also cause that C1, C2 which satisfy the
matrix inequalities (29) are too small to satisfy C1 ≥ϵ1+ϵ2+(1+ϵ−1
2 +ϵ−1
3 )d4
and C2 ≥(1 + ϵ−1
1
+ ϵ3)d3 + (1 + ϵ−1
2
+ ϵ−1
3 )d5. All the situation mentioned
above shows that our method to design K has limitations especially in the
face of a discrete-time system with completely unknown system function and
measurement error. Hence, in order to alleviate the problem mentioned above, we now propose
a new problem without prior knowledge such as d1, d2, d3. Considering the
matrix inequalities σ2P −C1zT
k+1(zT
k+1)⊤−C2I ˆGP −ˆHQ
P ˆG⊤−Q⊤ˆH⊤
P
> 0
I
M
P
−Q
P −Q⊤
M ⊤
P
> 0
P > 0
(36) (36) on (σ, P, Q, C1, C2), we can find different parameters have different have dif-
ferent impact on the control effect of the controller K. σ is about the control
speed of the system. Smaller σ can make the system stabilize more quickly. C1, C2 is about the degree of nonlinearity and measurement error of the sys-
tem. The larger C1, C2 are, the more complex system the controller K can
stabilize, that is to say larger C1, C2 have better robustness. However, the two
requirements conflict with each other. Smaller σ will lead to smaller C1, C2. 4 On-line Iteration Algorithm Hence, we use multi-objective programming to solve this problem, through
setting different weights for different parameters. Besides, the nonlinear term
σ2P can also make problem more complex. Hence, we determine σ within an
acceptable range at first to let the problem become linear. To ensure the exis-
tence of the maximum value, we attempt to use ≥instead of >. Now here is on (σ, P, Q, C1, C2), we can find different parameters have different have dif-
ferent impact on the control effect of the controller K. σ is about the control
speed of the system. Smaller σ can make the system stabilize more quickly. C1, C2 is about the degree of nonlinearity and measurement error of the sys-
tem. The larger C1, C2 are, the more complex system the controller K can
stabilize, that is to say larger C1, C2 have better robustness. However, the two
requirements conflict with each other. Smaller σ will lead to smaller C1, C2. Hence, we use multi-objective programming to solve this problem, through
setting different weights for different parameters. Besides, the nonlinear term
σ2P can also make problem more complex. Hence, we determine σ within an
acceptable range at first to let the problem become linear. To ensure the exis-
tence of the maximum value, we attempt to use ≥instead of >. Now here is On-Line Data-Driven Control for Uncertain Systems Based on Greedy Algorithm 11 the new programming: the new programming: max
ω1C1 + ω2C2
s.t. σ2P −C1zT
k+1(zT
k+1)⊤−C2I ˆGP −ˆHQ
P ˆG⊤−Q⊤ˆH⊤
P
≥0
(37a)
I
M
P
−Q
P −Q⊤
M ⊤
P
≥0
(37b)
P ≥0, C1, C2 ≥0
(37c) (37a) (37b) (37c) where ω1, ω2 > 0 and σ ∈(0, 1]. The following result is about the solvability
condition of the programming (37). where ω1, ω2 > 0 and σ ∈(0, 1]. The following result is about the solvability
condition of the programming (37). Theorem 4 No matter what σ is, the programming (37) has feasible solutions
as long as the system under the pair ( ˆG, ˆH) is controllable. Proof Assuming C1 = C2 = 0, the programming becomes the matrix inequal-
ity (18) with an an additional constraint (37b). If the pair ( ˆG, ˆH) is control-
lable, the inequality (18) has solutions whatever σ is. 4 On-line Iteration Algorithm Let us say ( ¯P, ¯Q) satisfies
the inequality (18), then (α ¯P, α ¯Q) can also satisfy it because of the linearity
for (P, Q). Hence, there exists α which can let (α ¯P, α ¯Q) meet the constraint
(37b). It is proved that (P, Q, C1, C2) = (α ¯P, α ¯Q, 0, 0) is a feasible solution of
the programming (37), namely the programming (37) has feasible solutions. ⊓⊔ ⊓⊔ Because of the complexity of the system itself and the unknowns of some
parameters, the optimal solution of the programming (37), K = QP −1, may
not stabilize the system (11). The optimal solution has not enough robustness
in dealing with the noise and nonlinearity. Besides, we do not have other way
to evaluate this feedback controller K expect for directly using it on the system
(11) by reason of the unknown system model. Now we use the method of the
on-line iteration based on greedy algorithm to address the problem. We set a step size kn, which represents the number of steps taken by the
system in each iteration. In each iteration, we use the greedy algorithm, just
considering finding the optimal solution based on the current data. In our algo-
rithm, it means that we solve the programming (37). In reality, the optimal so-
lution K is a function of (zT
k , zT
k+1, uT
k ), for which we set K = K∗(zT
k , zT
k+1, uT
k ). Then, we use the controller K on the system (11) for kn steps. Through the out-
put of the system, we can get new sequence (zT
k+kn, zT
k+kn+1, uT
k+kn). Now we
start a new iteration, designing a new controller Knew = K∗(zT
k+kn, zT
k+kn+1, uT
k+kn
We replace K with Knew on the system (11) for the next kn steps...... We it-
erate like this until the system (11) stabilizes. Here is a new problem about the iteration—— when to end it. In reality,
we can iterate all the time, but it will increase the computational cost. Owing
to the existence of the noise Rk, the state vector zk is unable to converge to
0, and the controller K often can not converge as well. We can not obtain Jia-Hui Shen, * Xing-Gao Liu 12 a relatively easy convergence criterion. Hence, we choose a method similar
to event-triggering. 4 On-line Iteration Algorithm Set an acceptable boundary δ. If ||zk|| < δ, we stop the
iteration, and once ||zk|| ≥δ, we restart the iteration. Although it may not
help us make sure the system ultimately reaches a stable state, it is a easy way
to reduce the computational cost, and let the system be expect to be near the
equilibrium state. Algorithm 1: On-line Iteration Based on Greedy Algorithm
Input: initial output z0, sequence length T, step size kn, observable
step N, acceptable boundary δ
1 Generate a random input sequence uN
0 , t0 ←col(uN
0 , yN
0 );
2 Generate a random input sequence uT
N, obtain tT +1
0
;
3 Solve the programming (37) to obtain K, i ←0;
4 while TURE do
5
Apply u = −Kx to the system for kn step;
6
Obtain ukn
N+T +i and tkn
T +1+i;
7
if average(||tkn
T +1+i||) ≥δ then
8
Use uT
N+kn+i and tT
k−n+1+i to solve the programming (37);
9
Update K;
10
end
11
i ←i + kn;
12 end Algorithm 1: On-line Iteration Based on Greedy Algorithm 5 Simulation To evaluate whether or not our greedy algorithm is effective, here is a nonlinear
system as an example to be controlled. The example is an inverted pendulum
which has been discretized [27]. The dynamics of the pendulum is expressed
as x1(k+1) = x1(k) + ∆x2(k)
x2(k+1) = ∆g
l sin x1(k) + (1 −∆κl)x2(k) + ∆1
ml2 uk
(38) (38) where ∆is the sampling time, m is the mass of the pendulum bar, l is the
length of the pendulum bar, κ is the frictional factor and g is the gravitational
acceleration. The states x1 and x2 are the angular position and velocity, re-
spectively, u is the applied torque. It can be found that the system has an
unstable equilibrium state (xe, ue) = (0, 0). A little disturbance can cause a
huge impact. In this experiment, we set the parameters as follows: ∆= 0.1s, m = 1/2kg,
l = 1/3m, κ = 0.2, g = 9.8m/s2. The initial state is set to x0 = [−1, 10]⊤,
and the measurement error is taken as a random sequence within [−0.01, 0.01]. Our goal is to let the system stabilize. 13 On-Line Data-Driven Control for Uncertain Systems Based on Greedy Algorithm Fig. 1 The trajectories of measured system states zk
Fig. 2 The trajectories of control uk Fig. 1 The trajectories of measured system states zk Fig. 1 The trajectories of measured system states zk Fig. 2 The trajectories of control uk Fig. 2 The trajectories of control uk Fig. 2 The trajectories of control uk Fig. 2 The trajectories of control uk Example 1 We assume that the state vector xk of the system is fully measur-
able, and the measurement value zk which we get is Example 1 We assume that the state vector xk of the system is fully measur-
able, and the measurement value zk which we get is zk = xk + wk
(39) (39) where wk represents measurement error. In the first iteration, we generate a
random input sequence uT
0 of length T = 10. Let the step size kn = 1 for the
next iteration. In each iteration, we solve the programming (37), with the prior
parameters ω1 = 1, ω2 = 1, σ2 = 0.5. And the trigger event is ||zk||1 < 0.25. where wk represents measurement error. 5 Simulation In the first iteration, we generate a
random input sequence uT
0 of length T = 10. Let the step size kn = 1 for the
next iteration. In each iteration, we solve the programming (37), with the prior
parameters ω1 = 1, ω2 = 1, σ2 = 0.5. And the trigger event is ||zk||1 < 0.25. Jia-Hui Shen, * Xing-Gao Liu 14 Fig. 3 The trajectories of measured output zk Fig. 3 The trajectories of measured output zk Fig. 3 The trajectories of measured output zk The trajectories of measured system states and control are shown in Figs. 1 and 2, respectively. Finally, the system stabilizes near the equilibrium state
(xe, ue). In reality, the algorithm has been ended after 15 iterations. Example 2 We assume that the system is just observable. Only x1(k) is mea-
surable, and the measurement value zk which we get is zk = x(1)k + wk
(40) (40) where wk represents measurement error. Let N = 2, and tk = col(uk, uk+1, x1(k)
To obtain t0, we generate a random input sequence u2
0 and use it on the sys-
tem. Similar to Experience 1, in the first iteration, we generate a random input
sequence uT
3 of length T = 10. Let the step size kn = 1 for the next iteration. In each iteration, we solve the programming (37), with the prior parameters
ω1 = 1, ω2 = 1, σ2 = 0.5. And the trigger event is |zk| < 0.25. The trajectories of system output and control are shown in Figs. 3 and
4, respectively. Finally, the system also stabilizes near the equilibrium state
(xe, ue). In reality, the algorithm has been ended after 40 iterations. Yet, state trajectories obtained from repeated experiments can be very
different. We can only say that the system is expected to stabilize as a result. 6 Conclusion In this paper, the controller design problem of uncertain discrete-time systems
is considered. We propose an on-line iteration to deal with this problem. One
simulation example is given to show the performance of the present method. Compared with other controller design methods, this method requires fewer is considered. We propose an on-line iteration to deal with this problem. One
simulation example is given to show the performance of the present method. Compared with other controller design methods, this method requires fewer On-Line Data-Driven Control for Uncertain Systems Based on Greedy Algorithm 15 Fig. 4 The trajectories of control uk Fig. 4 The trajectories of control uk prior conditions, such as the error bound of noise, and so on. It can be used
on a controllable and observable system that is almost completely unknown. However, because of the random input sequence to initialize and the prior
parameters to set, it is unsuitable for systems with constraints on the state. Thus, a safer method for the systems mentioned above is under study. References 1. R. E. Kalman, A new approach to linear filtering and prediction problems, Journal of
Basic Engineering, 82(1), 34–45 (1960). 2. D. J. Bender, A. J. Laub, The linear-quadratic optimal regulator for descriptor systems:
Discrete-time case, Automatica, 23(1), 71–85 (1987). Discrete-time case, Automatica, 23(1), 71–85 (1987). ( )
(
)
3. G. C. Goodwin and K. S. Sin, Adaptive Filtering Prediction and Control. Englewood
Cliffs, NJ, USA: Prentice-Hall (1984). 4. S. Vazquez et al., Model predictive control: A review of its applications in power elec-
tronics, IEEE Industrial Electronics Magazine, 8(1), 16–31 (2014). 5. L. Ljung, System Identification: Theory for the User. Englewood Cliffs, NJ, USA:
Prentice-Hall (1987). 6. Z. S. Hou, R. H. Chi, HJ Gao. An overview of dynamic-linearization-based data-driven
control and applications, IEEE Transactions on Industrial Electronics, 64(5): 4076-4090
(2016). (
)
7. B. D. O. Anderson, A. Dehghani, Challenges of adaptive control-past, permanent and
future, Annual Reviews in Control, 32(2), 123–135 (2008). 8. R. E. Skelton, Model error concepts in control design, International Journal of Control,
49(5), 1725–1753 (1989). 9. J. Van Helvoort, Unfalsified Control: Data-Driven Control Design for Performance Im-
provement, PhD dissertation, Technische Universiteit Eindhoven, Eindhoven, Netherlands
(2007). 10. J. C. Jeng, G. P. Ge, Disturbance-rejection-based tuning of proportional-integral-
derivative controllers by exploiting closed-loop plant data, ISA transactions, 62, 312–324
(2016). Jia-Hui Shen, * Xing-Gao Liu 16 11. H. Hjalmarsson, M. Gevers, S. Gunnarson, Iterative feedback tuning—Theory and ap-
plications, IEEE Control Systems Magazine, 18(4), 26–41 (1998). 12. M. G. Safonov, T. C. Tsao, The unfalsified control concept: A direct path from exper-
iment to controller, Feedback Control, Nonlinear Systems and Complexity, Berlin, Ger-
many: Springer, 196–214 (1995). (
)
13. M. Uchiyama, Formulation of high-speed motion pattern of a mechanical arm by trial,
Transactions of the Society of Instrument and Control Engineers, 14(6), 706–712 (1978). ( )
(
)
14. Z. S. Hou, The parameter identification, adaptive control and model free learning adap-
tive control for nonlinear systems, PhD. dissertation, North-eastern University, Shenyang,
China (1994). (
)
15. Z. S. Hou, Z. Wang. From model-based control to data-driven control: Survey, classifi-
cation and perspective, Information Sciences, 235, 3-35 (2013). 16. I. Markovsky, P. Rapisarda, On the linear quadratic data-driven control, European
Control Conference(ECC), IEEE, 5313–5318 (2007). 17. B. Pang, T. Bian, Z. P. Acknowledgements This work is supported by National Key R&D Program of China
(Grant No. 2021YFC2101100), National Natural Science Foundation of China (62073288,
12075212, 12105246, 11975207) and Zhejiang University NGICS Platform, and their supports
are thereby acknowledged. The authors declare that they have no conflict of interest. References Jiang, Data-driven finite-horizon optimal control for linear time-
varying discrete-time systems, IEEE Conference on Decision and Control (CDC), IEEE,
861–866 (2018). 18. S. Formentin, A. Karimi, and S. M. Savaresi, Optimal input design for direct data-driven
tuning of model-reference controllers, Automatica, 49(6), 1874–1882 (2013). 19. K. Van Heusden, A. Karimi, D. Bonvin, Data-driven model reference control with
asymptotically guaranteed stability, International Journal of Adaptive Control and Signal
Processing, 25(4), 331–351 (2011). 20. L. Chen, K. S. Narendra, Identification and control of a nonlinear discrete-time system
based on its linearization: A unified framework, IEEE Transactions on Neural Networks,
15(3), 663–673 (2004). ( )
(
)
21. Y. G. Xi, F. Wang, Nonlinear multi-model predictive control, IFAC Proceedings Vol-
umes, 29(1), 2359-2364 (1996). 22. H. Deng, H. X. Li, Y. H. Wu, Feedback-linearization-based neural adaptive control
for unknown nonaffine nonlinear discrete-timen systems, IEEE Transactions on Neural
Networks, 19(9), 1615–1625 (2008). ( )
(
)
23. J. C. Willems, P. Rapisarda, I. Markovsky, B. L. De Moor, A note on persistency of
excitation, Systems & Control Letters, 54(4), 325–329 (2005). 24. J. Coulson, J. Lygeros, F. D¨orfler, Data-enabled predictive control: In the shallows of
the DeePC, European Control Conference (ECC), IEEE, 307–312 (2019). he DeePC, European Control Conference (ECC), IEEE, 3 25. J. Berberich, J. K¨ohler, M. A. M¨uller, F. Allg¨ower, Data-driven model predictive control
with stability and robustness guarantees, IEEE Transactions on Automatic Control, 66(4),
1702-1717 (2020). (
)
26. C. De Persis, P. Tesi, Formulas for data-driven control: Stabilization, optimality, and
robustness, IEEE Transactions on Automatic Control, 65(3), 909-924 (2019). 27. R. Beard, Improving the closed-loop performance of nonlinear systems, PhD. disserta-
tion, Rensselaer Polytechnic Institute, Troy, NY, USA (1995). Acknowledgements This work is supported by National Key R&D Program of China
(Grant No. 2021YFC2101100), National Natural Science Foundation of China (62073288,
12075212, 12105246, 11975207) and Zhejiang University NGICS Platform, and their supports
are thereby acknowledged. The authors declare that they have no conflict of interest. This paper has no associated data.
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English
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Comparative phylotranscriptomics reveals ancestral and derived root nodule symbiosis programmes
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Nature Plants
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cc-by
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Cyril Libourel, Jean Keller, Lukas Brichet, Anne-Claire Cazalé, Sébastien
Carrère, Tatiana Vernié, Jean-Malo Couzigou, Caroline Callot, Isabelle Dufau,
Stéphane Cauet, et al. To cite this version: Cyril Libourel, Jean Keller, Lukas Brichet, Anne-Claire Cazalé, Sébastien Carrère, et al.. Comparative
phylotranscriptomics reveals ancestral and derived root nodule symbiosis programmes. Nature Plants,
2023, 9 (7), pp.1067-1080. 10.1038/s41477-023-01441-w. hal-04187925 Distributed under a Creative Commons Attribution 4.0 International License Article https://doi.org/10.1038/s41477-023-01441-w HAL Id: hal-04187925
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teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1Laboratoire de Recherche en Sciences Végétales (LRSV), Université de Toulouse, CNRS, UPS, Toulouse INP, Castanet-Tolosan, France. 2LIPME, Université
de Toulouse, INRAE, CNRS, Castanet-Tolosan, France. 3INRAE, CNRGV French Plant Genomic Resource Center, Castanet-Tolosan, France. 4INRAE, US1426,
GeT-PlaGe, Genotoul, Castanet-Tolosan, France. 5These authors contributed equally: Cyril Libourel, Jean Keller.
e-mail: philippe.remigi@inrae.fr;
pierre-marc.delaux@cnrs.fr; delphine.capela@inrae.fr Comparative phylotranscriptomics reveals
ancestral and derived root nodule
symbiosis programmes Cyril Libourel
1,5, Jean Keller
1,5, Lukas Brichet2, Anne-Claire Cazalé
2,
Sébastien Carrère
2, Tatiana Vernié
1, Jean-Malo Couzigou
1,
Caroline Callot3, Isabelle Dufau
3, Stéphane Cauet
3, William Marande3,
Tabatha Bulach4, Amandine Suin4, Catherine Masson-Boivin2,
Philippe Remigi
2
, Pierre-Marc Delaux
1
& Delphine Capela
2 Symbiotic interactions such as the nitrogen-fixing root nodule symbiosis
(RNS) have structured ecosystems during the evolution of life. Here we
aimed at reconstructing ancestral and intermediate steps that shaped
RNS observed in extant flowering plants. We compared the symbiotic
transcriptomic responses of nine host plants, including the mimosoid
legume Mimosa pudica for which we assembled a chromosome-level
genome. We reconstructed the ancestral RNS transcriptome composed of
most known symbiotic genes together with hundreds of novel candidates. Cross-referencing with transcriptomic data in response to experimentally
evolved bacterial strains with gradual symbiotic proficiencies, we found the
response to bacterial signals, nodule infection, nodule organogenesis and
nitrogen fixation to be ancestral. By contrast, the release of symbiosomes
was associated with recently evolved genes encoding small proteins in each
lineage. We demonstrate that the symbiotic response was mostly in place in
the most recent common ancestor of the RNS-forming species more than
90 million years ago. symbiotic associations can be distinguished: intracellular and extracel-
lular symbioses. Extracellular symbioses include for instance plant–
cyanobacteria interactions where the bacterial symbiont is hosted in
dedicated canals and glands5,6, ectomycorrhizal symbioses between
plant roots and ascomycete or basidiomycete fungi7, or the very specific
association between Dioscorea sansibarensis and its bacterial symbiont
restricted to leaf drip tips8. Intracellular symbioses in plants are mainly
established with fungal symbionts9. The nitrogen-fixing root nodule
symbiosis (RNS) is a rare example of intracellular accommodation of
bacteria9. However, intracellular accommodation of symbionts during symbiotic associations can be distinguished: intracellular and extracel-
lular symbioses. Extracellular symbioses include for instance plant–
cyanobacteria interactions where the bacterial symbiont is hosted in
dedicated canals and glands5,6, ectomycorrhizal symbioses between
plant roots and ascomycete or basidiomycete fungi7, or the very specific
association between Dioscorea sansibarensis and its bacterial symbiont
restricted to leaf drip tips8. Intracellular symbioses in plants are mainly
established with fungal symbionts9. The nitrogen-fixing root nodule
symbiosis (RNS) is a rare example of intracellular accommodation of
bacteria9. Results and discussion Identification of an ancestral RNS transcriptomic signature
The two largest groups of RNS-forming species, the Papilionoideae
subfamily and the Mimosoid clade which is nested in the largely
non-nodulating Caesalpinioideae subfamily17,22, belong to the Fabales
order. While transcriptomic data have been obtained in response to RNS
in a number of Papilionoideae23–28, the Mimosoids have been ignored. To fill this gap, we conducted a time-course experiment with Mimosa
pudica inoculated with its bacterial symbiont Cupriavidus taiwanensis
(Supplementary Table 1). As a preliminary to gene expression studies,
we de novo sequenced the genome of M. pudica. This Mimosa species is
tetraploid (2n = 4x = 52)29 and its genome size was previously estimated
to be around 900 Mb (ref. 16). To generate a high-quality genomic
sequence, we used a combination of long-read sequencing and optical
mapping (see Methods) leading to a near-chromosome-level assembly
(Supplementary Table 2). This method produced 74 hybrid scaffolds
(from 128 kbp to 25.5 Mbp with N50 = 16.1 Mbp) for 52 expected chro-
mosomes and a total genome size of 797.25 Mb. Automated structural
annotation of the genome yielded 73,541 protein-coding genes and
5,134 non-coding RNAs. Finally, the high completeness of the annotated
genome was evidenced by a 97% (2,255 genes) Busco recovery score on
eudicots_odb10 (C:97.0% (S:10.2%, D:86.8%), F:1.1%, M:1.9%, n: 2,326). As expected for a tetraploid genome, most of the genes are duplicated. The expression of 51,214 Mimosa genes was detected in our complete
transcriptomic dataset, 43% of which were differentially expressed
(9,034 genes up/16,470 genes downregulated) during the symbiotic
interaction with C. taiwanensis in at least one time point compared to
non-inoculated roots (Supplementary Tables 3 and 4). Using a simplified phylogeny composed of the nine species
analysed here, we then mapped the presence/absence of each
DEG-containing orthogroup (hereafter called DEOG for differentially
expressed orthogroups) on each tip of the tree, allowing us to deter-
mine at which evolutionary node the genes have been recruited for
RNS, using an ancestral state reconstruction of a discrete trait with
a fixed-rates continuous-time Markov model. Using this method, we
determined at which phylogenetic node the genes became differen-
tially regulated during symbiosis (see Methods and Extended Data
Fig. 1 for node label and position on the tree). Article https://doi.org/10.1038/s41477-023-01441-w RNS occurs in different physical structures depending on the plant spe-
cies, including transcellular tubular structures that retain bacteria10 or
organelle-like structures called symbiosomes11 that completely release
bacteria in host cells. RNS occurs in different physical structures depending on the plant spe-
cies, including transcellular tubular structures that retain bacteria10 or
organelle-like structures called symbiosomes11 that completely release
bacteria in host cells. Bradyrhizobium sp. 1AE200 strain (Ledermann and Couzigou, unpub-
lished). L. albus forms peculiar lupinoid nodules30 and its genome has
been recently sequenced31. In brief, we identified 3,976/4,944 (up/
down) differentially regulated genes in mature nodules in response to
Bradyrhizobium sp. 1AE200 compared to non-inoculated roots (Sup-
plementary Tables 1, 3 and 4). These differentially expressed genes
(DEGs) represent around 33% of the 26,204 L. albus expressed genes. In extant species, RNS is found in ~17,500 species from four orders
of flowering plants12: ~17,300 species from the Fabales and 230 species
from the Fagales, Cucurbitales and Rosales, which together form the
nitrogen-fixing nodulation (NFN) clade13. Comparative phylogenomic
studies coupled with previous phylogenetic and physiological work
provided insights into the evolutionary history of RNS. Although the
original phylogenetic work14 and recent follow-up15 support convergent
gains of RNS, the most likely scenario proposes that RNS was gained
only once, before the diversification of the NFN clade. Following that
single gain, RNS diversified in each lineage and was lost subsequently
multiple times, leading to the scattered distribution observed in extant
species13,16,17. The rate of RNS loss differs between lineages, with some
displaying an evolutionarily stabilized association while others seem to
have experienced massive losses10. However, the nature of the ancestral
RNS, its functioning and how it diversified over 92–110 million years of
evolution12 remain elusive. To obtain comparable datasets, raw RNA-seq reads obtained in the
presence or absence of their respective bacterial symbionts from seven
other nodulating species (Supplementary Table 1) were remapped on
their respective genomes, and differentially expressed genes were
computed following the same approach as for M. pudica and L. albus. Due to sampling and sequencing depth heterogeneity among spe-
cies, we used different fold-change thresholds to obtain comparable
numbers of differentially expressed genes (see Methods). For each
species, we also concatenated all differentially expressed genes at
any time point to estimate the whole symbiotic response for up- and
downregulated genes. Between 2,275 (Hippophae rhamnoides) and 9,034 (M. pudica)
differentially upregulated and 1,906 (H. rhamnoides) and 16,470
(M. Article pudica) downregulated genes were detected in the nine sampled
species at any time of the symbiotic interaction (Supplementary
Tables 3, 4 and 5). As expected, species for which transcriptomic
responses were only analysed in mature nodules, such as H. rham-
noides, Datisca glomerata and L. albus, exhibited a lower proportion
of differentially regulated genes (Supplementary Tables 3 and 5) as
the earliest responses to the symbiont were probably not captured. The comparison of transcriptomic patterns across species in each
context, whether developmental or in response to the environment,
allows reconstruction of ancestral and derived responses to that con-
text. For instance, this approach has been used in plants to reconstruct
the flooding response in angiosperms18, to study the evolution of the
shoot meristem19, organs and gametes20, and to infer the ancestral
AMS transcriptome4. The observed massive symbiotic transcriptomic responses in each
species reflect either a conserved response, species-specific responses
or a mix of both patterns. To determine the evolutionary origin of these
responses, we computed orthogroups32 for the nine studied species,
together with 16 additional species from the NFN clade and Arabidopsis
thaliana as outgroup (Supplementary Table 6). The additional species
were chosen on the basis of genome quality and to cover RNS- and
non-RNS-forming clades. The list of DEGs for each species was then
cross-referenced with the list of genes present in each orthogroup. Using this approach, we were able to identify, for each species, the list
of orthogroups containing at least one up- or downregulated gene. Here we combine transcriptomics in multiple species and phylog-
enomics to reconstruct the ancestral RNS transcriptome. We further
dissect the transcriptional shifts associated with each symbiotic step
by exploiting experimentally evolved bacterial strains21, which progres-
sively recapitulate the full symbiotic interaction. We use this combina-
tion of transcriptomics, phylogenomics and experimental evolution to
reconstruct the evolution of the plant symbiotic programme. Nature Plants | Volume 9 | July 2023 | 1067–1080 Comparative phylotranscriptomics reveals
ancestral and derived root nodule
symbiosis programmes However, intracellular accommodation of symbionts during Interactions between organisms form a continuum of associations,
from parasitism to mutually beneficial symbioses1, which have contrib-
uted to the evolution and diversification of the plant lineage for billions
of years2. The mutualistic symbioses formed with fungal or bacterial
symbionts are associated with key ecological and evolutionary transi-
tions, such as the colonization of land by plants 450 million years ago,
which was enabled by the evolution of the arbuscular mycorrhizal sym-
biosis (AMS)3,4. Following this initial event, plants and their symbiotic
partners have diversified, leading to the emergence of multiple types
of mutualistic symbioses with microorganisms2. Two main groups of Interactions between organisms form a continuum of associations,
from parasitism to mutually beneficial symbioses1, which have contrib-
uted to the evolution and diversification of the plant lineage for billions
of years2. The mutualistic symbioses formed with fungal or bacterial
symbionts are associated with key ecological and evolutionary transi-
tions, such as the colonization of land by plants 450 million years ago,
which was enabled by the evolution of the arbuscular mycorrhizal sym-
biosis (AMS)3,4. Following this initial event, plants and their symbiotic
partners have diversified, leading to the emergence of multiple types
of mutualistic symbioses with microorganisms2. Two main groups of Nature Plants | Volume 9 | July 2023 | 1067–1080 1067 Article Results and discussion Blue and gold dots indicate significant downregulated
and upregulated genes, respectively. Fig. 1 | Conservation of the RNS transcriptomic response across NFN species. The tree depicts the orthofinder NFN phylogeny with A. thaliana as outgroup. Species used to compare symbiotic transcriptomes are indicated in black;
species used to compute orthogroups are indicated in grey. The volcano plots on both transcriptomic responses (Fig. 2 and Supplementary Table 7). Reversely, 54%/67% were specific to the RNS response, indicating that
RNS evolved by combining the co-option of the older lateral-root and
AMS programmes, but also via the recruitment of a substantial number
of additional pathways. members of the downstream signalling pathway SYMRK/DMI2
(refs. 35,36), CCaMK/DMI3 (ref. 37) and CYCLOPS/IPD3 (ref. 38), the mas-
ter symbiotic regulator NIN39 or the infection-associated gene RPG40,41. The phylogenetic distribution of three of them (LjNFR5/MtNFP, NIN and
RPG) has been recently linked with the ability to form RNS in the NFN
clade. Indeed, all three genes have been lost independently in multi-
ple lineages no longer able to form RNS16,17. In addition to the known
genes, 593 orthogroups with undescribed functions were detected,
including 110 that are absent from at least one non-RNS-forming spe-
cies and present in all RNS-forming species, and thus represent top
candidates for subsequent investigation by reverse genetics (Fig. 1 and
Supplementary Table 7). Results and discussion Besides expression itself,
the number of predicted DEOGs at a given node depends on several fac-
tors such as the maximum number of orthogroups present at that node
or the accuracy of the orthogroup reconstruction method. To consider
these biases, we assessed whether the experimentally determined
values for each node significantly deviated from random expectation
(see Methods). For each species, most of the genes were found to be
differentially regulated in a species-specific manner (Fig. 1 and Sup-
plementary Table 5). However, the observed numbers were either not
significantly different from the null expectation, or were lower than
expected (Supplementary Table 5). By contrast, a number of internal
nodes displayed significantly more DEOGs than expected (Supple-
mentary Table 5). In particular, 759/1,493 (up/down) orthogroups
(211%/268% increase compared to the mean null expectation, Supple-
mentary Table 5) were inferred to have been already up/downregulated
in the most recent common ancestor of all RNS-forming species. Among
the 759 ancestrally upregulated orthogroups, 157 contain genes with
a known function, such as the Nod-factor receptor LjNFR5/MtNFP33,34, In addition, we generated the transcriptome of the Papilio-
noideae Lupinus albus, from the Genisteae tribe, inoculated with Nature Plants | Volume 9 | July 2023 | 1067–1080 1068 Article https://doi.org/10.1038/s41477-023-01441-w Trema orientalis
Parasponia andersonii
Morus notabilis
Discaria trinervis
Hippophae rhamnoides
Fragaria vesca
Dryas drummondii
Betula pendula
Alnus glutinosa
Casuarina glauca
Juglans regia
Lagenaria siceraria
Cucurbita maxima
Begonia fuchsioides
Datisca glomerata
Arachis hypogaea
Aeschynomene evenia
Nissolia schottii
Glycine max
Lotus japonicus
Medicago truncatula
Lupinus albus
Castanospermum australe
Mimosa pudica
Arabidopsis thaliana
Carpinus fangiana
Fabales
Cucurbitales
Fagales
Rosales
DEOG
Down
Up
Species
specific
1,000
2,000
3,000
Indeterminate
Determinate
Rhizobia
Peripheral vasculature
Central vasculature
Frankia
RNS origin
585
323
1,493 759
47
28
159 213
0
Fig. 1 | Conservation of the RNS transcriptomic response across NFN species. The tree depicts the orthofinder NFN phylogeny with A. thaliana as outgroup. Species used to compare symbiotic transcriptomes are indicated in black;
species used to compute orthogroups are indicated in grey. The volcano plots on
the right represent the logFC (x axis) by FDR P values (y axis) for the nine species
at the latest time point. Blue and gold dots indicate significant downregulated
and upregulated genes, respectively. Species
specific the right represent the logFC (x axis) by FDR P values (y axis) for the nine species
at the latest time point. Transcriptomic rewiring by experimentally evolved
symbionts RNS is a complex interaction involving multiple physiological and
developmental processes that are often coupled and overlapping. In the case of most Fabales, these processes include the perception
and response to the symbiotic signal produced by the symbionts (the
so-called Nod-factors, NF), nodule organogenesis and the concomitant
penetration of bacteria within root and nodule tissues, symbiosome
release and persistence, and nitrogen fixation. The evolutionary tran-
sition from a non-RNS-forming state to a fully functional RNS state
probably occurred over millions of years through a number of inter-
mediate stages that cannot be captured in extant species. To define
the transcriptional modules (and their evolutionary origin) associated
with each process, we exploited a collection of bacterial mutants that
gradually induce the full symbiotic programme. Most of these bacterial
mutants originate from an evolution experiment that was developed to
replay the evolution of symbiotic abilities in a legume symbiont46–48. In
this evolution experiment, we first introduced the symbiotic plasmid
pRalta from the rhizobium C. taiwanensis LMG19424 (refs. 49–51),
one of many natural symbionts of M. pudica, into the non-symbiotic,
soil-borne bacterium Ralstonia solanacearum GMI1000. Then, we
propagated these chimaeric bacteria for 400 generations along suc-
cessive nodulation cycles on M. pudica. Throughout the experiment,
clones gradually gained symbiotic abilities46,48,52 and adaptive muta-
tions responsible for the main phenotypic changes observed in the Specifically, the combination of transcriptomic responses
and phylogenomics allowed us to identify a set of 759 orthogroups
considered as ancestrally upregulated during RNS. We also identi-
fied orthogroups and genes recruited during the diversification of
RNS in independent lineages, a pattern reminiscent of the potentia-
tion–actualization–refinement model proposed for the evolution of
novel traits42,43. Transcriptomic recruitment from pre-existing processes a
406
324
2,855
75
222
146
56
Lateral-root
response
Ancestral
RNS response
AMS
response
EDN1-3
ENODL8
EXP7
GASA
HK5-7
IPT3
LBD18
PIN4
PUB2
ROP9
bHLH1
LYKs
NOOT1,NOOT2
GASAs
KCS2
MtFER2
NPF7.8/7.9
Prx07
STYL1/2
SULTR2.2
EFD/EFD2
NIN
LjRING
MYB14
RSD
SULTR3.5
SWEET11/12
SymRK
SYN
TML1/2
CHR
ERN1/2/3
CYCLOPS
KinG1
NF-YA1/2
NSP1/2
PUB1
RPG/RPG-like
VAPYRIN
SYFO1/1L/3
RAM1
RAM2
STR
FATM
KinF
KinG2
CASTOR/POLLUX
CCD7/CCD8
BRI
AHA1/3-5/7-9
Ann1/2
ENOD93
FLOT1-6
JKD
LBD11
LOX1/5
PR5
Peroxidases
SEN1-like
SymCRK
AOX
CCD
LAX2/4/5
LjBRUSH
IPT1
MtCNGC15a/b/c
PIN6
STR/STR2
SWEET3a/b/c
ZIP6
Fig. 2 | Non-proportional Venn diagrams presenting the shared and specific
DEOGs among the ancestral RNS, AMS and lateral-root transcriptomic
responses. Numbers in the Venn represent the number of DEOGs belonging to
ancestral RNS, AMS and/or lateral-root response. a, Upregulated DEOGs. 1,271
58
388
33
43
b
HOG
NS
Up
Down
CCD7
KinG2
LYK10/EPR3
LBD1/4
FLOT1-6
CYCLOPS
PIN9
RLP1/SUNN
Tubb1
VAPYRIN
CCD8
LBD11
NRT2.1/2.2
LjRINRK1
EPP1
EXO70H4
RPK
SEN1-like
Peroxidases
ZIP2
Ancestral
RNS response
AMS
response
1,004
85
Lateral-root
response
CKX2/3
NPL,NPL-like
Peroxidases
NRT2.3
LjAMT1.1
MtPEN3-like
RALF11/12
SULTR2.1
VPE
ZPT2.1/2.2
GmbHLHm1
MtCNGC15a-c
PIN2/4/5
NRAMP1-3
LAX2/4
LYKs
PLT1/2
Peroxidases
PUB2
REM1.2
PIN1/3
SYT2
ZIP6
NF-YA1,NF-YA2
MtFER3
EIN2
EFD/EFD2
MAPK6
CHK1/CRE1
BRI
CHR
AtLOX1-like,LOX2,LOX3
FNSII,GmIFS1/GmIFS2
HK5-7
LYR3
MATE7/8
VAMP721a-e
Peroxidases
SPK1
SymCRK
bHLH1
ENOD8.1/8.2/8.3
HK1/2
DnaJ
CHK2/3
CHI-2
PHO2-like
LjGLB1
LjSST1
TSAR2
b, Downregulated DEOGs. NS, not significantly up/downregulated DEOGs. Heat
maps represent a chosen list of DEOGs based on their role in RNS, AMS or lateral-
root development. a
406
324
2,855
75
222
146
56
Lateral-root
response
Ancestral
RNS response
AMS
response
EDN1-3
ENODL8
EXP7
GASA
HK5-7
IPT3
LBD18
PIN4
PUB2
ROP9
bHLH1
LYKs
NOOT1,NOOT2
GASAs
KCS2
MtFER2
NPF7.8/7.9
Prx07
STYL1/2
SULTR2.2
EFD/EFD2
NIN
LjRING
MYB14
RSD
SULTR3.5
SWEET11/12
SymRK
SYN
TML1/2
CHR
ERN1/2/3
CYCLOPS
KinG1
NF-YA1/2
NSP1/2
PUB1
RPG/RPG-like
VAPYRIN
SYFO1/1L/3
RAM1
RAM2
STR
FATM
KinF
KinG2
CASTOR/POLLUX
CCD7/CCD8
BRI
AHA1/3-5/7-9
Ann1/2
ENOD93
FLOT1-6
JKD
LBD11
LOX1/5
PR5
Peroxidases
SEN1-like
SymCRK
AOX
CCD
LAX2/4/5
LjBRUSH
IPT1
MtCNGC15a/b/c
PIN6
STR/STR2
SWEET3a/b/c
ZIP6 b a b, Downregulated DEOGs. NS, not significantly up/downregulated DEOGs. Heat
maps represent a chosen list of DEOGs based on their role in RNS, AMS or lateral-
root development. Fig. 2 | Non-proportional Venn diagrams presenting the shared and specific
DEOGs among the ancestral RNS, AMS and lateral-root transcriptomic
responses. Numbers in the Venn represent the number of DEOGs belonging to
ancestral RNS, AMS and/or lateral-root response. a, Upregulated DEOGs. Transcriptomic recruitment from pre-existing processes The ancestral transcriptome supported multiple symbiotic
traits
Next we sought to trace the evolutionary history of transcriptomic
1,271
58
388
33
43
b
HOG
NS
Up
Down
CCD7
KinG2
LYK10/EPR3
LBD1/4
FLOT1-6
CYCLOPS
PIN9
RLP1/SUNN
Tubb1
VAPYRIN
CCD8
LBD11
NRT2.1/2.2
LjRINRK1
EPP1
EXO70H4
RPK
SEN1-like
Peroxidases
ZIP2
Ancestral
RNS response
AMS
response
a
1,004
85
406
324
2,855
75
222
146
56
Lateral-root
response
Ancestral
RNS response
AMS
response
EDN1-3
ENODL8
EXP7
GASA
HK5-7
IPT3
LBD18
PIN4
PUB2
ROP9
bHLH1
LYKs
NOOT1,NOOT2
GASAs
KCS2
MtFER2
NPF7.8/7.9
Prx07
STYL1/2
SULTR2.2
EFD/EFD2
NIN
LjRING
MYB14
RSD
SULTR3.5
SWEET11/12
SymRK
SYN
TML1/2
CHR
ERN1/2/3
CYCLOPS
KinG1
NF-YA1/2
NSP1/2
PUB1
RPG/RPG-like
VAPYRIN
SYFO1/1L/3
RAM1
RAM2
STR
FATM
KinF
KinG2
CASTOR/POLLUX
CCD7/CCD8
BRI
AHA1/3-5/7-9
Ann1/2
ENOD93
FLOT1-6
JKD
LBD11
LOX1/5
PR5
Peroxidases
SEN1-like
SymCRK
AOX
CCD
LAX2/4/5
LjBRUSH
IPT1
MtCNGC15a/b/c
PIN6
STR/STR2
SWEET3a/b/c
ZIP6
Lateral-root
response
CKX2/3
NPL,NPL-like
Peroxidases
NRT2.3
LjAMT1.1
MtPEN3-like
RALF11/12
SULTR2.1
VPE
ZPT2.1/2.2
GmbHLHm1
MtCNGC15a-c
PIN2/4/5
NRAMP1-3
LAX2/4
LYKs
PLT1/2
Peroxidases
PUB2
REM1.2
PIN1/3
SYT2
ZIP6
NF-YA1,NF-YA2
MtFER3
EIN2
EFD/EFD2
MAPK6
CHK1/CRE1
BRI
CHR
AtLOX1-like,LOX2,LOX3
FNSII,GmIFS1/GmIFS2
HK5-7
LYR3
MATE7/8
VAMP721a-e
Peroxidases
SPK1
SymCRK
bHLH1
ENOD8.1/8.2/8.3
HK1/2
DnaJ
CHK2/3
CHI-2
PHO2-like
LjGLB1
LjSST1
TSAR2
Fig. 2 | Non-proportional Venn diagrams presenting the shared and specific
DEOGs among the ancestral RNS, AMS and lateral-root transcriptomic
responses. Numbers in the Venn represent the number of DEOGs belonging to
ancestral RNS, AMS and/or lateral-root response. a, Upregulated DEOGs. b, Downregulated DEOGs. NS, not significantly up/downregulated DEOGs. Heat
maps represent a chosen list of DEOGs based on their role in RNS, AMS or lateral-
root development. Transcriptomic recruitment from pre-existing processes RNS was obtained following muta-
tions inactivating the Type Three Secretion System of R. solanacearum. A stop mutation in hrcV, a gene encoding a Type Three Secretion Sys-
tem structural protein, conferred to bacteria the capacity to nodulate
M. pudica but nodules were only extracellularly invaded (Fig. 3a). By
contrast, a stop mutation in hrpG, a gene encoding a global regulator
of hundreds of genes including Type Three Secretion System genes,
enabled bacteria to form nodules and invade them intracellularly
through the formation of symbiosomes, which are released in the
cytoplasm of nodule cells46. However, hrpG mutants degenerate very
rapidly following symbiosome release (Fig. 3a). Cumulating an hrpG
mutation with a mutation in the regulator efpR enhanced symbio-
some persistence of bacteria although to a level not yet equivalent to
a wild-type or a non-fixing mutant of C. taiwanensis48 and was not yet
sufficient to enable nitrogen fixation in interaction with M. pudica. We
reconstructed the adaptive mutations hrcV, hrpG and hrpG-efpR in the
non-symbiotic original GMI1000+pRalta strain to generate a collection
of nearly isogenic strains with increased symbiotic abilities (Fig. 3a). We analysed the transcriptome of M. pudica in response to inoculation
with each of these three mutants, as well as with the non-nodulating
parental strains GMI1000, GMI1000+pRalta and an nifH mutant of
C. taiwanensis, which is only affected in its ability to fix nitrogen53. We
harvested tissue samples, either roots, nodule primordia or nodules,
at different time points between 1 and 21 d after inoculation to capture
the most advanced symbiotic response (that is, most advanced stage/
organ development) induced by each mutant (Supplementary Table 1). Th
l
i
fi
d
bi
i
bili i
i R l
i
i Transcriptomic recruitment from pre-existing processes 1,271
58
388
33
43
b
HOG
NS
Up
Down
CCD7
KinG2
LYK10/EPR3
LBD1/4
FLOT1-6
CYCLOPS
PIN9
RLP1/SUNN
Tubb1
VAPYRIN
CCD8
LBD11
NRT2.1/2.2
LjRINRK1
EPP1
EXO70H4
RPK
SEN1-like
Peroxidases
ZIP2
Ancestral
RNS response
AMS
response
a
1,004
85
406
324
2,855
75
222
146
56
Lateral-root
response
Ancestral
RNS response
AMS
response
EDN1-3
ENODL8
EXP7
GASA
HK5-7
IPT3
LBD18
PIN4
PUB2
ROP9
bHLH1
LYKs
NOOT1,NOOT2
GASAs
KCS2
MtFER2
NPF7.8/7.9
Prx07
STYL1/2
SULTR2.2
EFD/EFD2
NIN
LjRING
MYB14
RSD
SULTR3.5
SWEET11/12
SymRK
SYN
TML1/2
CHR
ERN1/2/3
CYCLOPS
KinG1
NF-YA1/2
NSP1/2
PUB1
RPG/RPG-like
VAPYRIN
SYFO1/1L/3
RAM1
RAM2
STR
FATM
KinF
KinG2
CASTOR/POLLUX
CCD7/CCD8
BRI
AHA1/3-5/7-9
Ann1/2
ENOD93
FLOT1-6
JKD
LBD11
LOX1/5
PR5
Peroxidases
SEN1-like
SymCRK
AOX
CCD
LAX2/4/5
LjBRUSH
IPT1
MtCNGC15a/b/c
PIN6
STR/STR2
SWEET3a/b/c
ZIP6
Lateral-root
response
CKX2/3
NPL,NPL-like
Peroxidases
NRT2.3
LjAMT1.1
MtPEN3-like
RALF11/12
SULTR2.1
VPE
ZPT2.1/2.2
GmbHLHm1
MtCNGC15a-c
PIN2/4/5
NRAMP1-3
LAX2/4
LYKs
PLT1/2
Peroxidases
PUB2
REM1.2
PIN1/3
SYT2
ZIP6
NF-YA1,NF-YA2
MtFER3
EIN2
EFD/EFD2
MAPK6
CHK1/CRE1
BRI
CHR
AtLOX1-like,LOX2,LOX3
FNSII,GmIFS1/GmIFS2
HK5-7
LYR3
MATE7/8
VAMP721a-e
Peroxidases
SPK1
SymCRK
bHLH1
ENOD8.1/8.2/8.3
HK1/2
DnaJ
CHK2/3
CHI-2
PHO2-like
LjGLB1
LjSST1
TSAR2
Fig. 2 | Non-proportional Venn diagrams presenting the shared and specific
DEOGs among the ancestral RNS, AMS and lateral-root transcriptomic
responses. Numbers in the Venn represent the number of DEOGs belonging to
ancestral RNS, AMS and/or lateral-root response. a, Upregulated DEOGs. b, Downregulated DEOGs. NS, not significantly up/downregulated DEOGs. Heat
maps represent a chosen list of DEOGs based on their role in RNS, AMS or lateral-
root development. 1,271
58
388
33
43
b
HOG
NS
Up
Down
CCD7
KinG2
LYK10/EPR3
LBD1/4
FLOT1-6
CYCLOPS
PIN9
RLP1/SUNN
Tubb1
VAPYRIN
CCD8
LBD11
NRT2.1/2.2
LjRINRK1
EPP1
EXO70H4
RPK
SEN1-like
Peroxidases
ZIP2
Ancestral
RNS response
AMS
response
1,004
85
Lateral-root
response
CKX2/3
NPL,NPL-like
Peroxidases
NRT2.3
LjAMT1.1
MtPEN3-like
RALF11/12
SULTR2.1
VPE
ZPT2.1/2.2
GmbHLHm1
MtCNGC15a-c
PIN2/4/5
NRAMP1-3
LAX2/4
LYKs
PLT1/2
Peroxidases
PUB2
REM1.2
PIN1/3
SYT2
ZIP6
NF-YA1,NF-YA2
MtFER3
EIN2
EFD/EFD2
MAPK6
CHK1/CRE1
BRI
CHR
AtLOX1-like,LOX2,LOX3
FNSII,GmIFS1/GmIFS2
HK5-7
LYR3
MATE7/8
VAMP721a-e
Peroxidases
SPK1
SymCRK
bHLH1
ENOD8.1/8.2/8.3
HK1/2
DnaJ
CHK2/3
CHI-2
PHO2-like
LjGLB1
LjSST1
TSAR2
c
b, Downregulated DEOGs. NS, not significantly up/downregulated DEOGs. Heat
maps represent a chosen list of DEOGs based on their role in RNS, AMS or lateral-
root development. Transcriptomic recruitment from pre-existing processes GO terms ‘oxido-reduction’, ‘cell wall organization’, ‘terpene synthase
activity’, ‘diterpenoid biosynthetic process’, ‘gibberellin dioxyge-
nase activity’ and ‘response to oxidative stress’, some of which may
be involved in plant responses to microbial attack (Supplementary
Table 8). This indicates that the horizontal gain of a symbiotic plasmid,
a phenomenon widely observed within rhizobial populations54, may be
sufficient to limit the activation of plant immunity. In the Ralstonia hrcV
mutant forming extracellularly infected nodules, the transcriptomic
response shared with the wild-type symbiont increased up to 60%/42%
(up/down), while these proportions reached 66%/50% (up/down) with
the hrpG-efpR strain. This pattern confirms phenotypic observations
indicating that evolved Ralstonia strains are arrested at different stages
along the progression towards a fully functional mutualistic state. evolved clones were identified. RNS was obtained following muta-
tions inactivating the Type Three Secretion System of R. solanacearum. A stop mutation in hrcV, a gene encoding a Type Three Secretion Sys-
tem structural protein, conferred to bacteria the capacity to nodulate
M. pudica but nodules were only extracellularly invaded (Fig. 3a). By
contrast, a stop mutation in hrpG, a gene encoding a global regulator
of hundreds of genes including Type Three Secretion System genes,
enabled bacteria to form nodules and invade them intracellularly
through the formation of symbiosomes, which are released in the
cytoplasm of nodule cells46. However, hrpG mutants degenerate very
rapidly following symbiosome release (Fig. 3a). Cumulating an hrpG
mutation with a mutation in the regulator efpR enhanced symbio-
some persistence of bacteria although to a level not yet equivalent to
a wild-type or a non-fixing mutant of C. taiwanensis48 and was not yet
sufficient to enable nitrogen fixation in interaction with M. pudica. We
reconstructed the adaptive mutations hrcV, hrpG and hrpG-efpR in the
non-symbiotic original GMI1000+pRalta strain to generate a collection
of nearly isogenic strains with increased symbiotic abilities (Fig. 3a). We analysed the transcriptome of M. pudica in response to inoculation
with each of these three mutants, as well as with the non-nodulating
parental strains GMI1000, GMI1000+pRalta and an nifH mutant of
C. taiwanensis, which is only affected in its ability to fix nitrogen53. We
harvested tissue samples, either roots, nodule primordia or nodules,
at different time points between 1 and 21 d after inoculation to capture
the most advanced symbiotic response (that is, most advanced stage/
organ development) induced by each mutant (Supplementary Table 1). TheevolutionofimprovedsymbioticabilitiesinRalstoniastrains evolved clones were identified. Nature Plants | Volume 9 | July 2023 | 1067–1080 Transcriptomic recruitment from pre-existing processes Genetic dissection of RNS in model species proposed that its evolution
relied on the co-option of genes regulating the more ancient AMS and
lateral-root development9,27. To determine the contribution of these
two programmes to the ancestral RNS transcriptomic response, we
cross-referenced RNA-seq data obtained in the model legume Med-
icago truncatula for lateral-root development27 and AMS differentially
regulated genes shared by Lotus japonicus44 and M. truncatula45 with the
inferred ancestral transcriptome (Fig. 2 and Supplementary Table 7). Among the 759/1,493 orthogroups up/downregulated in the ancestral
RNS transcriptome, 46%/33% behave like the lateral root, the AMS or Nature Plants | Volume 9 | July 2023 | 1067–1080 1069 https://doi.org/10.1038/s41477-023-01441-w Article evolved clones were identified. RNS was obtained following muta-
tions inactivating the Type Three Secretion System of R. solanacearum. A stop mutation in hrcV, a gene encoding a Type Three Secretion Sys-
tem structural protein, conferred to bacteria the capacity to nodulate
M. pudica but nodules were only extracellularly invaded (Fig. 3a). By
contrast, a stop mutation in hrpG, a gene encoding a global regulator
of hundreds of genes including Type Three Secretion System genes,
enabled bacteria to form nodules and invade them intracellularly
through the formation of symbiosomes, which are released in the
cytoplasm of nodule cells46. However, hrpG mutants degenerate very
rapidly following symbiosome release (Fig. 3a). Cumulating an hrpG
mutation with a mutation in the regulator efpR enhanced symbio-
some persistence of bacteria although to a level not yet equivalent to
a wild-type or a non-fixing mutant of C. taiwanensis48 and was not yet
sufficient to enable nitrogen fixation in interaction with M. pudica. We
reconstructed the adaptive mutations hrcV, hrpG and hrpG-efpR in the
non symbioticoriginalGMI1000+pRaltastraintogenerateacollection
GO terms ‘oxido-reduction’, ‘cell wall organization’, ‘terpene synthase
activity’, ‘diterpenoid biosynthetic process’, ‘gibberellin dioxyge-
nase activity’ and ‘response to oxidative stress’, some of which may
be involved in plant responses to microbial attack (Supplementary
Table 8). This indicates that the horizontal gain of a symbiotic plasmid,
a phenomenon widely observed within rhizobial populations54, may be
sufficient to limit the activation of plant immunity. In the Ralstonia hrcV
mutant forming extracellularly infected nodules, the transcriptomic
response shared with the wild-type symbiont increased up to 60%/42%
(up/down), while these proportions reached 66%/50% (up/down) with
the hrpG-efpR strain. This pattern confirms phenotypic observations
indicating that evolved Ralstonia strains are arrested at different stages
along the progression towards a fully functional mutualistic state. The ancestral transcriptome supported multiple symbiotic
traits taiwanensis nifH mutants
and at 14 dpi for C. taiwanensis wild-type (WT) strain. Roots and nodule sections
were stained with X-gal. The C. taiwanensis WT picture is from ref. 83. Nod,
nodule formation; Hac, root hair curling; Extra-cell-inf, extracellular infection
of nodules; Intra-cell-inf, intracellular infection of nodules; Intra-cell-pers, ig. 3 | Genes recruited along the experimental evolution of RN Fig. 3 | Genes recruited along the experimental evolution of RNS. a, Symbiotic
phenotypes of R. solanacearum (GMI1000)-derived strains and C. taiwanensis
symbionts. M. pudica plants were inoculated with lacZ-tagged strains and
nodules were harvested at 10 dpi for Ralstonia and C. taiwanensis nifH mutants
and at 14 dpi for C. taiwanensis wild-type (WT) strain. Roots and nodule sections
were stained with X-gal. The C. taiwanensis WT picture is from ref. 83. Nod,
nodule formation; Hac, root hair curling; Extra-cell-inf, extracellular infection
of nodules; Intra-cell-inf, intracellular infection of nodules; Intra-cell-pers, An enrichment in ancestral genes was observed in the transcrip-
tional responses associated with the perception of symbiont-produced
Nod-factors in M. truncatula and L. japonicus as well as with nodule
infection and organogenesis. In addition, both processes were linked
with an impoverishment in species-specific DEOGs (Fig. 4b,c and
Supplementary Table 9). Taken together, these results suggest that
Nod-factor perception recruited ancestral DEOGs, followed by a large
species-specific diversification facilitating recognition between symbi-
otic partners. Among the ancestral DEOGs in response to Nod-factors,
we detected well-characterized NF-signalling components such as
the transcription factors NIN58,59, NF-YA1 (ref. 60), NF-YA2 and ERN1/2
(ref. 61), the infection genes RPG40,41, VAPYRIN62,63, SYFO64 and the
LysM-RLK EPR3/LYK10 (ref. 65), the LRR-RLK RINRK1 (ref. 66) or the
cytosolic kinase SymCRK67 (Fig. 4b, and Supplementary Tables 4 and 7). response of M. truncatula. As we have done above, all the genes linked
with symbiotic traits were cross-referenced with orthogroups to infer
when (that is, at which phylogenetic node) they were recruited for
symbiosis during evolution. The distributions of the gene sets for the different traits at the
different evolutionary nodes were compared to the whole symbiotic
transcriptomic response (Fig. 3). To do so, we used Fisher’s exact test
to compare the whole symbiotic transcriptomic response and each
trait node by node (Fig. 4 and Supplementary Table 9) to estimate
over/under representation of genes in the different nodes. The ancestral transcriptome supported multiple symbiotic
traits Next, we sought to trace the evolutionary history of transcriptomic
recruitment or innovation associated with each of the following sym-
biotic traits: response to NF, nodule organogenesis and infection, sym-
biosome release, symbiosome persistence and nitrogen fixation. To
do so, we exploited the gradual improvement in symbiotic abilities of
Ralstonia strains and compared the responses of M. pudica to couples
of strains that are able or unable to realize the different symbiotic traits
(Fig. 3a and Supplementary Table 3). Transcriptomic responses to direct
NF treatments were also available for two other Fabales, M. truncatula
and L. japonicus55,56. Another dataset available for M. truncatula was
obtained from laser-capture microdissection associated with nodule
tissue differentiation, corresponding to symbiosome release (FIId)
and symbiosome persistence (FIIp), plant and bacteroid cell differen-
tiation (FIIp, IZ) and nitrogen fixation (ZIII) (Supplementary Tables 3
and 4)57. To consider genes related to the different traits, we focused
on genes differentially regulated in the trait of interest in the same
way as in the whole symbiotic transcriptomic response (Fig. 3, and
Supplementary Tables 3 and 4). For example, genes upregulated for
symbiosome release in M. truncatula have to be identified as upregu-
lated in ‘FIId’ and in the whole symbiotic response RNS transcriptomic The evolution of improved symbiotic abilities in Ralstonia strains
correlated with a gradual increase in the number of M. pudica DEGs that
are also DEGs during the interaction with the wild-type C. taiwanensis
strain (Fig. 3b). The gain of the symbiotic plasmid was sufficient on its
own to activate 19%/10% (up/down) of the whole symbiotic response
(Fig. 3b and Supplementary Table 4). Accompanying this gain of sym-
biotic response, the GMI1000+pRalta strain also did not activate the
expression of 586 M. pudica genes specifically induced by the wild-type
GMI1000 R. solanacearum strain (Supplementary Table 4 and Extended
Data Fig. 2). A notable number of these genes are associated with the Nature Plants | Volume 9 | July 2023 | 1067–1080 1070 Article https://doi.org/10.1038/s41477-023-01441-w GMI1000 pRalta
hrpG-efpR
GMI1000 pRalta
GMI1000 pRalta
hrcV
GMI1000 pRalta
hrpG
C. taiwanensis
nifH
C. taiwanensis
wild-type
Strain
Nod
–, Hac
+
Nod
+, Extra-cell-inf
+
Intra-cell-inf
+
Intra-cell-pers
+/–
Intra-cell-pers
+, fix
–
Fix
+
Symbiotic
phenotype
Nodule infection and
organogenesis
Intracellular release
of bacteria
Intracellular persistence of bacteria
Nitrogen fxation
Symbiotic stage
acquired/improved
a
b
Number of genes shared with C.tai. hrcV
pRalta
GMI1000
GMI1000
hrpG
C.tai. C.tai. GMI1000
C.tai. C.tai. The ancestral transcriptome supported multiple symbiotic
traits hrcV
pRalta
GMI1000
Strains
Strain
background
Nodes
NFN node
Fabales node
M. pudica DEOGs
M. pudica specifc
Downregulated genes
Upregulated genes
hrpG
hrpG-efpR nifH
hrpG-efpR nifH
173
1,702
5,418
5,558
387
1,742
6,051
7,568
9,034
6,956
7,363
8,348
13,306
16,470
765
1,895
7,506
7,437
7,672
9,087
789
1,915
7,639
7,917
8,945
14,404
0
5,000
10,000
15,000
10 µm
100 µm
100 µm
100 µm
100 µm
100 µm
Fig. 3 | Genes recruited along the experimental evolution of RNS. a, Symbiotic
phenotypes of R. solanacearum (GMI1000)-derived strains and C. taiwanensis
symbionts. M. pudica plants were inoculated with lacZ-tagged strains and
nodules were harvested at 10 dpi for Ralstonia and C. taiwanensis nifH mutants
and at 14 dpi for C. taiwanensis wild-type (WT) strain. Roots and nodule sections
were stained with X-gal. The C. taiwanensis WT picture is from ref. 83. Nod,
nodule formation; Hac, root hair curling; Extra-cell-inf, extracellular infection
of nodules; Intra-cell-inf, intracellular infection of nodules; Intra-cell-pers,
intracellular persistence; Fix, nitrogen fixation. b, Number of genes up- and
downregulated in nodules formed by the different R. solanacearum and
C. taiwanensis mutants and shared with the symbiotic response obtained wi
C. taiwanensis WT strain. The distribution of these genes in the NFN and Fab
nodes and in the M. pudica specific gene set is indicated. Pink dots and numb
above the bars indicate the total number of DEGs in each condition. C.tai.,
C. taiwanensis. a b Number of genes shared with
hrcV
pRalta
GMI1000
GMI1000
hrpG
C.tai. C.tai. GMI1000
C.tai. C.tai. hrcV
pRalta
GMI1000
Strains
Strain
background
hrpG
hrpG-efpR nifH
hrpG-efpR nifH
765
1,895
7,506
7,437
7,672
9,087
789
1,915
7,639
7,917
8,945
0
5,000
10,000 intracellular persistence; Fix, nitrogen fixation. b, Number of genes up- and
downregulated in nodules formed by the different R. solanacearum and
C. taiwanensis mutants and shared with the symbiotic response obtained with the
C. taiwanensis WT strain. The distribution of these genes in the NFN and Fabales
nodes and in the M. pudica specific gene set is indicated. Pink dots and numbers
above the bars indicate the total number of DEGs in each condition. C.tai.,
C. taiwanensis. Fig. 3 | Genes recruited along the experimental evolution of RNS. a, Symbiotic
phenotypes of R. solanacearum (GMI1000)-derived strains and C. taiwanensis
symbionts. M. pudica plants were inoculated with lacZ-tagged strains and
nodules were harvested at 10 dpi for Ralstonia and C. Nature Plants | Volume 9 | July 2023 | 1067–1080 The ancestral transcriptome supported multiple symbiotic
traits a, Simplified example tree. ‘Species-specific
DEOGs’ represent HOGs in which only genes of a given species are present, while
‘Species DEOGs’ represent HOGs in which other species genes are present but
differentially regulated in a species-specific manner or convergently acquired
during symbiosis in this species. b, ‘Nod factor’ DEOGs oddsRatio for L. japonicus Fig. 4 | Evolutionary symbiotic-stage responses. Simplified phylogenies
highlighting oddsRatios comparing the proportion of genes that are upregulated
in the different symbiotic traits compared to the whole symbiotic response in
the different evolutionary nodes. a, Simplified example tree. ‘Species-specific
DEOGs’ represent HOGs in which only genes of a given species are present, while
‘Species DEOGs’ represent HOGs in which other species genes are present but
differentially regulated in a species-specific manner or convergently acquired
during symbiosis in this species. b, ‘Nod factor’ DEOGs oddsRatio for L. japonicus and M. truncatula. c, Nodule infection and organogenesis DEOGs oddsRatio for
M. pudica. d, ‘Symbiosome release’ DEOGs oddsRatio for M. truncatula and
M. pudica. e, ‘Symbiosome persistence’ DEOGs oddsRatio for M. truncatula
and M. pudica. f, ‘N2 fixation’ DEOGs oddsRatio for M. truncatula and M. pudica. Asterisks indicate oddsRatios significantly different from ‘1’ using a two-sided
Fisher’s exact test: *0.05 > P > 0.01, **0.01 > P > 0.001, ***P < 0.001; absence of
symbols indicates non-significance. OddsRatios and corresponding P values are
given in Supplementary Table 9. direct or indirect targets RPG, NF-YA1, NF-YA2 and ERN1/2, while other
NIN targets, such as the transcription factors of the NF-YB family, LBD11
or STY1/2 involved in the production of auxin maxima required for nod-
ule primordium emergence27,70, specifically belong to the organogen-
esis and infection module (Fig. 4c). Another well-known transcription
factor, KNOX3, regulating nodule development through activation of
cytokinin biosynthesis but acting upstream of NIN, was found as part
of this ancestral ‘organogenesis and infection’ programme71. Finally,
NOOT1 and NOOT2, which are known to maintain nodule identity in
diverse legumes, were also detected72. Besides the known genes, 31 The chitinase CHIT5 known to play a role in NF turnover in the Fabales L. japonicus68 was also found as part of this shared NF response, indicating
that modulating NF levels was part of the ancestral RNS. Organogenesis has been scrutinized in model legumes, revealing
genes, in particular transcription factors, essential for the formation
and maintenance of nodule identity69. The ancestral transcriptome supported multiple symbiotic
traits truncatula and M. Asterisks indicate oddsRatios significantly different from ‘1’ using a two
Fisher’s exact test: *0.05 > P > 0.01, **0.01 > P > 0.001, ***P < 0.001; abse
symbols indicates non-significance. OddsRatios and corresponding P v
given in Supplementary Table 9. https://doi.org/10.1038/s41477-023-01441-w Article https://doi.org/10.1038/s41477-023-01441-w
oddsRatio
0
0.5
1.0
1.5
2.0
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Lotjap - NF
Medtru - NF
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
Nod-factor response
b
***
**
***
***
**
***
oddsRatio
0
0.5
1.0
1.5
2.0
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Medtru - FIId
Mimpud - Release
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
Symbiosome release
d
***
***
***
***
***
*
oddsRatio
0
0.5
1.0
1.5
2.0
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Medtru - ZIII
Mimpud - Nfix
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
N2 fixation
f
***
***
***
***
***
***
and M. truncatula. c, Nodule infection and organogenesis DEOGs oddsRatio for
M. pudica. d, ‘Symbiosome release’ DEOGs oddsRatio for M. truncatula and
M. pudica. e, ‘Symbiosome persistence’ DEOGs oddsRatio for M. truncatula
and M. pudica. f, ‘N2 fixation’ DEOGs oddsRatio for M. truncatula and M. pudica. Asterisks indicate oddsRatios significantly different from ‘1’ using a two-sided
Fisher’s exact test: *0.05 > P > 0.01, **0.01 > P > 0.001, ***P < 0.001; absence of
symbols indicates non-significance. OddsRatios and corresponding P values are
given in Supplementary Table 9. The ancestral transcriptome supported multiple symbiotic
traits This analy-
sis indicates that all stages of RNS involve genes that were already
expressed in the most recent common ancestor of all RNS-forming spe-
cies, although in different proportions, followed by different degrees
of species-specific refinement. Nature Plants | Volume 9 | July 2023 | 1067–1080 1071 Article
https://doi.org/10.1038/s41477-023
oddsRatio
0
0.5
1.0
1.5
2.0
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Mimpud - Nodule infection and organogenesis
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
Nodule infection and organogenesis
c
***
*
***
oddsRatio
0
0.5
1.0
1.5
2.0
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Lotjap - NF
Medtru - NF
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
Nod-factor response
b
***
**
***
***
**
***
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
Simplified example tree
N1
N2
Species-specific DEOGs
N7
N12
N21
a
oddsRatio
0
0.5
1.0
1.5
2.0
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Medtru - FIId
Mimpud - Release
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
Symbiosome release
d
***
***
***
***
***
*
oddsRatio
0
0.5
1.0
1.5
2.0
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Medtru - ZIII
Mimpud - Nfix
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
N2 fixation
f
***
***
***
***
***
***
oddsRatio
0
0.5
1.0
1.5
2.0
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Medtru - FIIp
Mimpud - Persist
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
Symbiosome persistence
e
**
**
***
***
***
***
Species DEOGs
Species DEOGs
Species DEOGs
Fig. 4 | Evolutionary symbiotic-stage responses. Simplified phylogenies
highlighting oddsRatios comparing the proportion of genes that are upregulated
in the different symbiotic traits compared to the whole symbiotic response in
the different evolutionary nodes. a, Simplified example tree. ‘Species-specific
DEOGs’ represent HOGs in which only genes of a given species are present, while
‘Species DEOGs’ represent HOGs in which other species genes are present but
differentially regulated in a species-specific manner or convergently acquired
during symbiosis in this species. b, ‘Nod factor’ DEOGs oddsRatio for L. japonicus
and M. truncatula. c, Nodule infection and organogenesis DEOGs oddsR
M. pudica. d, ‘Symbiosome release’ DEOGs oddsRatio for M. truncatula
M. pudica. e, ‘Symbiosome persistence’ DEOGs oddsRatio for M. trunca
and M. pudica. f, ‘N2 fixation’ DEOGs oddsRatio for M. The ancestral transcriptome supported multiple symbiotic
traits Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
Simplified example tree
N1
N2
Species-specific DEOGs
N7
N12
N21
a
Species DEOGs
Species DEOGs
Species DEOGs oddsRatio
0
0.5
1.0
1.5
2.0
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Lotjap - NF
Medtru - NF
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
Nod-factor response
b
***
**
***
***
**
*** b a oddsRatio
0
0.5
1.0
1.5
2.0
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Medtru - FIId
Mimpud - Release
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
Symbiosome release
d
***
***
***
***
***
* oddsRatio
0
0.5
1.0
1.5
2.0
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Mimpud - Nodule infection and organogenesis
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
Nodule infection and organogenesis
c
***
*
*** d c oddsRatio
0
0.5
1.0
1.5
2.0
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Medtru - ZIII
Mimpud - Nfix
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
N2 fixation
f
***
***
***
***
***
*** oddsRatio
0
0.5
1.0
1.5
2.0
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Medtru - ZIII
Mimpud - Nfix
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
N2 fixation
f
***
***
***
***
***
***
and M. truncatula. c, Nodule infection and organogenesis DEOGs oddsRatio for
M. pudica. d, ‘Symbiosome release’ DEOGs oddsRatio for M. truncatula and
M. pudica. e, ‘Symbiosome persistence’ DEOGs oddsRatio for M. truncatula
and M. pudica. f, ‘N2 fixation’ DEOGs oddsRatio for M. truncatula and M. pudica. Asterisks indicate oddsRatios significantly different from ‘1’ using a two-sided
Fisher’s exact test: *0.05 > P > 0.01, **0.01 > P > 0.001, ***P < 0.001; absence of
symbols indicates non-significance. OddsRatios and corresponding P values are
given in Supplementary Table 9. oddsRatio
0
0.5
1.0
1.5
2.0
Arachis hypogaea
Aeschynomene evenia
Lotus japonicus
Medicago truncatula
Medtru - FIIp
Mimpud - Persist
Lupinus albus
Mimosa pudica
Arabidopsis thaliana
Cucurbitales
Fagales
Rosales
Symbiosome persistence
e
**
**
***
***
***
*** f e Fig. 4 | Evolutionary symbiotic-stage responses. Simplified phylogenies Fig. 4 | Evolutionary symbiotic-stage responses. Simplified phylogenies
highlighting oddsRatios comparing the proportion of genes that are upregulated
in the different symbiotic traits compared to the whole symbiotic response in
the different evolutionary nodes. Article (‘Release’ and ‘FIId’) compared with the ‘whole symbiotic response’
(without the genes tagged as related to ‘symbiosome release’) in both
M. pudica (oddsRatioMimpud_signalP = 3.9; Supplementary Table 9) and
M. truncatula (oddsRatioMedtru_signalP = 4.2; Supplementary Table 9). Second, we wondered whether the proteins associated with ‘symbio-
some release’ were different in size and/or amino acid composition
from the proteins expressed during the whole symbiotic response. We observed that proteins associated with ‘symbiosome release’
were significantly shorter for both M. pudica (meanRelease = 156.9 vs
meanMimpud = 401.3, t-test P = 2.78 × 10−61; Fig. 5 and Supplementary
Table 9) and M. truncatula (meanFIId = 55.4 vs meanMedtru = 306.6, t-test
P = 1.8 × 10−141; Fig. 5 and Supplementary Table 9). In addition, we
found that these proteins showed more proline residues in M. pudica
(ratiomean_Pro_prop = 2, t-test P = 1.67 × 10−19) and more cysteine residues in
M. truncatula (ratiomean_Cys_prop = 1.9, t-test P = 1.0 × 10−39; Fig. 5a,b, and
Supplementary Tables 9 and 10). Following these trends, we observed an
enrichment in species-specific orthogroups (that is, only the sequence
of the given species is present in the orthogroup) for cysteine-rich and
small proteins in M. truncatula, but not for M. pudica (Fig. 5a and Sup-
plementary Table 9). In M. truncatula, ‘symbiosome release’ is partly
mediated by small proteins with a signal peptide and containing a high
proportion of cysteine known as ‘nodule cysteine-rich’ (NCR) peptides
(Fig. 5a). In inverted repeat-lacking clade (IRLC) and some dalbergioid
legumes, these small secreted peptides have been shown to trigger
the terminal differentiation of the nitrogen-fixing symbionts via anti-
microbial activities preventing bacteroid proliferation outside the
plant79,80. These NCRs correspond to the species-specific genes iden-
tified here for M. truncatula. Reversely, we observed an enrichment
in species-specific orthogroups for proline-rich and small proteins
in M. pudica, but not for M. truncatula (Fig. 5b and Supplementary
Table 9). Proline-rich peptides have been found in insects, mammals
and plants where they play a role as antimicrobial compounds81,82. Although M. pudica symbionts are not terminally differentiated, the
revivability of C. taiwanensis bacteroids outside the plant is limited
to 20%, indicating a possible intermediary state of differentiation83. The actual function of these proline-rich short proteins remains to be
determined. Additionally, we looked at the evolutionary pattern of
the Arachis hypogaea cysteine-rich secretory protein, antigen 5, and
pathogenesis-related 1 proteins (AhCAPs) identified recently24. Convergent evolution for symbiosome formation in legumes By contrast with symbiont perception, nodule ‘infection and organo-
genesis’, and ‘nitrogen fixation’, the evolutionary pattern of ‘symbio-
some release and persistence’ of rhizobia showed a decreased link
with ancestral genes and an enrichment in species-specific DEOGs in
both M. pudica (Caesalpinioideae) and M. truncatula (Papilionoideae,
Fig. 4d,e and Supplementary Table 9). Compared to other orders of
the NFN clade, RNS is evolutionarily stable in the Papilionoideae and
the Mimosoid clade which is nested in the largely non-nodulating
Caesalpinioideae subfamily10. It has been hypothesized that this sta-
bility is linked with the occurrence of symbiosome formation, which
is almost exclusively found in these two clades10 and some species of
the non-Mimosoid Caesalpinioideae genus Chamaecrista. Such a trait
distribution might either reflect an ancestral gain in the Fabales and
multiple subsequent losses or be the result of convergent evolution. The fact that the transcriptomic signature associated with that stage
depends much more on genes regulated in a species-specific manner in
both M. truncatula and M. pudica than the other ancestral traits strongly
supports the hypothesis of convergent gains of symbiosome formation
in the two lineages (Fig. 4d,e). This species-specific transcriptomic
change may be the result of either the recruitment of existing genes
into the symbiotic transcriptomic response or the de novo evolution
of new genes in each lineage. To address this question, we analysed the
nature of the upregulated genes associated with ‘symbiosome release’
and ‘FIId’ in a species-specific manner in M. pudica and M. truncatula. First, we identified an over-representation of proteins with
a predicted signal peptide in the ‘symbiosome release’ gene set Article pudica, but not for M. truncatula (Fig. 5b and Supplementary
Table 9). Proline-rich peptides have been found in insects, mammals
and plants where they play a role as antimicrobial compounds81,82. Alth
gh M
di a
bi
t
t t
i
ll diff
ti t d th Nitrogen fixation is a unifying feature of RNS. However, it has been
predicted to be a trait that experienced important refinement during
the diversification of the NFN. Indeed, mechanisms providing condi-
tions for nitrogen fixation by the diverse symbionts in the nodules
(Frankia, alpha- and beta-proteobacteria) vary notably13. Despite this
diversification, our analysis revealed an over-representation of DEOGs
associated with N2 fixation and linked with the ancestral RNS gene
set for M. pudica and less species-specific DEOGs identified in both
M. pudica ‘N2 fixation’ and the nodule ‘ZIII’ of M. truncatula (Fig. 4f and
Supplementary Table 9). Most of these genes encode enzymes that have
not been characterized yet (Supplementary Table 4). Although symbiosome release, inferred from the M. truncatula
(‘FIId’) and M. pudica (‘Release’) datasets, displays a peculiar evolution-
ary pattern (see below and Fig. 4d), this symbiotic stage also involved
genes that are part of the ancestral transcriptomic response. Sup-
pressors of plant defence in nodules, SymCRK67 and RSD73, as well as
the transcription regulator EFD required for both plant and bacte-
roid differentiation in M. truncatula74,75 participate in this ancestral
response. Looking specifically at the M. pudica data, we found VAPYRIN,
RPG, some flotillin and remorin genes and the syntaxin SYN, which are
well-known infection-associated genes41,76–78. We thus hypothesize that
a proportion of genes linked with the ancestral RNS transcriptome
and associated with ‘symbiosome release’ reflects infection (Fig. 4d). As mentioned for the response to ‘Nod-factors’ and ‘infection and
organogenesis’, DEOGs identified for the different traits often overlap,
suggesting that genes such as transcription factors may act at different
symbiotic stages69. We identified the gene modules associated with ancient symbiotic
processes including genes whose position in the symbiotic pathway
remains to be characterized. Altogether, this indicates that the core
mechanisms governing the response to ‘Nod factors’, nodule ‘infection
and organogenesis’ and ‘nitrogen fixation’ in extant RNS-forming spe-
cies have probably been conserved since their most recent common
ancestor. Nature Plants | Volume 9 | July 2023 | 1067–1080 The ancestral transcriptome supported multiple symbiotic
traits Many of these transcription fac-
tors were recovered in the inferred ancestral transcriptomic signature
of ‘organogenesis and infection’ (Fig. 4c, and Supplementary Tables 4
and 7). Expectedly, genes involved in this module partially overlap with
the NF-responsive genes, including the master regulator NIN and its Nature Plants | Volume 9 | July 2023 | 1067–1080 1072 https://doi.org/10.1038/s41477-023-01441-w Article Article Of a
total of 48 identified AhCAPs, 44 belong to A. hypogaea species-specific
HOGs, suggesting another convergent evolution of secreted peptides
linked to symbiosomes. Altogether, the presented data support the
idea that convergence in the symbiosome release of symbionts evolved
by at least two independent but analogous molecular processes: the
de novo evolution of nodule-induced small proteins already proposed
in dalbergioid24 and IRLC80. orthogroups annotated as transcription factors and so far not analysed
in the context of RNS were detected. Their function during nodule
organogenesis and infection remains to be determined. (‘Release’ and ‘FIId’) compared with the ‘whole symbiotic response’
(without the genes tagged as related to ‘symbiosome release’) in both
M. pudica (oddsRatioMimpud_signalP = 3.9; Supplementary Table 9) and
M. truncatula (oddsRatioMedtru_signalP = 4.2; Supplementary Table 9). Second, we wondered whether the proteins associated with ‘symbio-
some release’ were different in size and/or amino acid composition
from the proteins expressed during the whole symbiotic response. We observed that proteins associated with ‘symbiosome release’
were significantly shorter for both M. pudica (meanRelease = 156.9 vs
meanMimpud = 401.3, t-test P = 2.78 × 10−61; Fig. 5 and Supplementary
Table 9) and M. truncatula (meanFIId = 55.4 vs meanMedtru = 306.6, t-test
P = 1.8 × 10−141; Fig. 5 and Supplementary Table 9). In addition, we
found that these proteins showed more proline residues in M. pudica
(ratiomean_Pro_prop = 2, t-test P = 1.67 × 10−19) and more cysteine residues in
M. truncatula (ratiomean_Cys_prop = 1.9, t-test P = 1.0 × 10−39; Fig. 5a,b, and
Supplementary Tables 9 and 10). Following these trends, we observed an
enrichment in species-specific orthogroups (that is, only the sequence
of the given species is present in the orthogroup) for cysteine-rich and
small proteins in M. truncatula, but not for M. pudica (Fig. 5a and Sup-
plementary Table 9). In M. truncatula, ‘symbiosome release’ is partly
mediated by small proteins with a signal peptide and containing a high
proportion of cysteine known as ‘nodule cysteine-rich’ (NCR) peptides
(Fig. 5a). In inverted repeat-lacking clade (IRLC) and some dalbergioid
legumes, these small secreted peptides have been shown to trigger
the terminal differentiation of the nitrogen-fixing symbionts via anti-
microbial activities preventing bacteroid proliferation outside the
plant79,80. These NCRs correspond to the species-specific genes iden-
tified here for M. truncatula. Reversely, we observed an enrichment
in species-specific orthogroups for proline-rich and small proteins
in M. Conclusion From the distribution of the trait and phylogenomic analyses, the lead-
ing hypothesis for the origin of RNS is that it was gained before the
radiation of the NFN clade more than 90 million years ago13,16. Here we
propose that RNS in the most recent common ancestor looked very
similar to RNS in extant species. With the ancestral RNS transcriptomic
signature now defined, future studies will have to decipher how this
state evolved from a non-RNS-forming state. A role for the common
symbiosis pathway in that process can be anticipated given its phylo-
genetic link with all the intracellular plant symbioses84 and the reverse
genetic data obtained in diverse RNS-forming species9. The gain of
a regulatory link between the common symbiosis pathway and the
central RNS-regulator NIN at the base of the NFN clade represents
one of the events that have played a role in the transition from the
non-NFN-forming to the NFN-forming state85. For millions of years,
RNS has been maintained in diverse lineages of the Fagales, Fabales,
Cucurbitales and Rosales, with presumably very high rates of symbiosis
loss. RNS has become evolutionarily stable in only two lineages: the First, we identified an over-representation of proteins with
a predicted signal peptide in the ‘symbiosome release’ gene set 1073 https://doi.org/10.1038/s41477-023-01441-w Article Cysteine proportion
Cysteine proportion
Cysteine proportion
Proline proportion
Proline proportion
Medtru
FIId
Mimpud
Release
Symbiosome release
WSR with SignalP without symbiosome release
Proline proportion
a
b
20
Density
2,000
20
Length of SignalP containing proteins
Length of SignalP containing proteins
Length of SignalP containing proteins
Length of SignalP containing proteins
20
Density
2,000
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200
100
200
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40
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40
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1,500
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0
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0.2
0.3
0.4
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0
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0.0075
Density
Length
distribution
of SignalP
containing
proteins
of M. Conclusion pudica
Length
distribution
of SignalP
containing
proteins
of M. truncatula
Fig. 5 | Characteristics of proteins with signal peptide from species-specific
DEOGs related to symbiosome release in M. truncatula and M. pudica. a, Scatterplot representing proportion of cysteine (x axis) and protein length
(y axis) for upregulated genes in the ‘whole symbiotic response’ (WSR) (grey) and
‘symbiosome release’ (gold). b, Scatterplot representing proportion of proline
(x axis) and protein length (y axis) for upregulated genes in the ‘whole symbiotic
response’ (grey) and ‘symbiosome release’ (gold). Proline proportion
b
40
Density
0
0.05
0.10
0.15
0.20
0
10
20
30 Cysteine proportion
20
Density
0
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15 Cysteine proportion
Cysteine proportion
Medtru
FIId
a
20
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2,000
20
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6
100
100
200
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500
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0
500
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0.01
0.02
0.03
0.04
0
5
10
15
0
5
10
15
Length
distribution
of SignalP
containing
proteins
of M. truncatula b b Proline proportion
Proline proportion
b
Length of SignalP containing proteins
200
40
Density
0
500
0
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0.10
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1,000
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40
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30 a Proline proportion Density Density Cysteine proportion
Length of SignalP containing proteins
20
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of M. pudica 100
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distribution
of SignalP
containing
proteins
of M. pudica Density WSR with SignalP without symbiosome release Symbiosome release Fig. 5 | Characteristics of proteins with signal peptide from species-specific
DEOGs related to symbiosome release in M. truncatula and M. pudica. a, Scatterplot representing proportion of cysteine (x axis) and protein length
(y axis) for upregulated genes in the ‘whole symbiotic response’ (WSR) (grey) and ‘symbiosome release’ (gold). b, Scatterplot representing proportion of proline
(x axis) and protein length (y axis) for upregulated genes in the ‘whole symbiotic
response’ (grey) and ‘symbiosome release’ (gold). ‘symbiosome release’ (gold). b, Scatterplot representing proportion of proline
(x axis) and protein length (y axis) for upregulated genes in the ‘whole symbiotic
response’ (grey) and ‘symbiosome release’ (gold). Fig. M. pudica genome structural annotation The M. pudica gene models were predicted by the eukaryotic genome
annotation pipeline egn-ep (http://eugene.toulouse.inra.fr/Down-
loads/egnep-Linux-x86_64.1.5.1.tar.gz) using trained statistical mod-
els adapted for plants (http://eugene.toulouse.inra.fr/Downloads/
WAM_plant.20180615.tar.gz). This pipeline manages automatically
probabilistic sequence model training, genome masking, transcript
and protein alignments computation, alternative splice sites detec-
tion and integrative gene modelling by the EuGene software (release
4.2a91; http://eugene.toulouse.inra.fr/Downloads/eugene-4.2a.tar.gz). Four protein databases were used to detect translated regions: (1)
the proteome of M. truncatula A17 (v5 annotation release 1.6; https://
medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/), (2) the proteome
of the previous M. pudica Illumina genome16, (3) Swiss-Prot, October
2016 and (4) the proteome of A. thaliana TAIR10 version. Proteins
similar to REPBASE were removed from the three datasets (to avoid
the integration of transposable element related proteins in the train-
ing steps). Chained alignments spanning less than 50% of the length
of the database protein were removed. The proteome of M. truncatula
(release 1.6) was used as a training proteome by EuGene. The M. pudica gene models were predicted by the eukaryotic genome
annotation pipeline egn-ep (http://eugene.toulouse.inra.fr/Down-
loads/egnep-Linux-x86_64.1.5.1.tar.gz) using trained statistical mod-
els adapted for plants (http://eugene.toulouse.inra.fr/Downloads/
WAM_plant.20180615.tar.gz). This pipeline manages automatically
probabilistic sequence model training, genome masking, transcript
and protein alignments computation, alternative splice sites detec-
tion and integrative gene modelling by the EuGene software (release
4.2a91; http://eugene.toulouse.inra.fr/Downloads/eugene-4.2a.tar.gz). Article https://doi.org/10.1038/s41477-023-01441-w 100 -minReadScore 0.65. The final polished assembly produced had
a total of 1,343 contigs with a total length of 842,189,795 bp, a largest
contig of 24,170,175 bp and an N50 contig of 13,257,064 bp. Papillionideae subfamily and the Mimosoid clade in the Fabales order10. Our results support the hypothesis that evolutionary stability was
acquired through the convergent evolution of symbiosome release and
enhanced control of the bacterial symbiont gained by the expansion
of putative antimicrobial peptide gene families. Besides providing
an evolutionary perspective linked to multiple symbiotic traits and
reconstructing a shared RNS transcriptomic response, our comparative
transcriptomic approach has determined a list of conserved ortho-
groups that can be considered as targets for further spatial, temporal
and biological characterization in multiple species. We believe that our
dataset will help the community to integrate evolutionary perspectives
in future studies. Candidate gene validation in multiple species would
help in prioritizing key regulator genes for engineering nitrogen-fixing
symbiosis in crops86–88. Preparation of M. pudica ultra-high molecular weight
Bionano optical mapping was then used to further improve this assem-
bly. Nuclei were purified from 0.5 g of dark treated young leaves accord-
ing to the Bionano plant tissue DNA isolation base protocol (30068,
Bionano Genomics), followed by ultra-high molecular weight (uHMW)
DNA extraction based on the Bionano prep SP kit (80030, Bionano
Genomics) adapted by our laboratory for plant samples. Briefly,
plant leaves were flash frozen in liquid nitrogen and disrupted with a
rotor-stator homogenizer (Qiagen). Nuclei were pelleted, washed and
digested with proteinase K in lysis buffer. After phenylmethylsulfonyl
fluoride treatment, a centrifugation step was added to eliminate cell
wall debris. The supernatant was precipitated with isopropanol and
captured with magnetic disk (Nanobind disk). After several washes,
the uHMW DNA was eluted in elution buffer. Labelling and staining of
the uHMW DNA were performed according to the Bionano prep direct
label and stain protocol (30206, Bionano Genomics). Briefly, label-
ling was performed by incubating 750 ng of genomic DNA with DLE-1
enzyme (Bionano Genomics) for 2 h in the presence of DL-Green dye
(Bionano Genomics). The DLE-1 enzyme recognizes the motif CTTAAG. Following proteinase K (Qiagen) digestion and non-fixed dye clean-up
by membrane adsorption, the DNA backbone was stained with DNA
Stain solution (Bionano Genomics) and incubated overnight at room
temperature. The labelled DNA concentration was measured using
Qubit dsDNA HS assay (Invitrogen). Conclusion 5 | Characteristics of proteins with signal peptide from species-specific
DEOGs related to symbiosome release in M. truncatula and M. pudica. a, Scatterplot representing proportion of cysteine (x axis) and protein length
(y axis) for upregulated genes in the ‘whole symbiotic response’ (WSR) (grey) and Nature Plants | Volume 9 | July 2023 | 1067–1080 1074 Article Methods M. pudica high molecular weight DNA extraction p
g
g
High molecular weight (HMW) DNA was isolated from frozen young
leaves using QIAGEN genomic-tips 100/G kit (10243) following the tis-
sue extraction protocol. Briefly, 1 g of young leaf material was ground in
liquid nitrogen with a mortar and pestle. After 3 h of lysis at 50 °C with
proteinase K and one centrifugation step, the DNA was immobilized
on the column. After several washing steps, DNA was eluted from the
column, desalted and then concentrated by alcohol precipitation. The
DNA was resuspended in TE buffer. Data collection, optical mapping and genome scaffolding Data collection, optical mapping and genome scaffolding
Labelled DNA was loaded on a Saphyr G1 chip according to the Saphyr
System user guide (30247, Bionano Genomics). Data processing was
performed using the Bionano Genomics Access software (https://
bionanogenomics.com/support-page/bionano-access-software/). Molecules (480 Gb) larger than 150 Kb with an N50 of 199 kbp were
produced and represented 533X. This corresponded to 533X cover-
age of the 900 Mb estimated size of the M. pudica genome. These
molecules were assembled using RefAligner with default parameters,
producing 110 genome maps with an N50 of 16.1 Mbp for a total genome
map length of 833 Mbp. Finally, hybrid scaffolding was performed
between the polished PacBio assembly and the optical genome maps
using hybridScaffold pipeline with default parameters. We obtained
74 hybrid scaffolds ranging from 128 kbp to 25.5 Mbp (total length
797 Mbp with N50 = 16.1 Mbp). HMW DNA was sheared using a Megaruptor 2 system (Diagenode)
to obtain a 40 Kb average size. Following an enzymatic treatment on
7.5 µg of sheared DNA sample for DNA damage repair, ligation with
hairpin adapters to both ends of the targeted double-stranded DNA
(dsDNA) molecule was performed to create a closed single-stranded
circular DNA. A nuclease treatment was performed using a SMRT-
bell enzyme clean-up kit (Pacific Biosciences). Size selection with
Blue-Pippin system (Sage Science) to remove fragments less than 15 Kb
was done on purified sample with 0.45X AMPure PB beads (Pacific
Biosciences). The size and concentration of the final library were
assessed using the Fragment Analyzer system (Agilent) and the Qubit
fluorometer and Qubit dsDNA HS reagents assay kit (Thermo Fisher),
respectively. Sequencing primer v3 and Sequel DNA Polymerase 3.0 were
respectively annealed and bound to the SMRTbell library. The library
was loaded on 8 SMRTcells 1M and sequencing was performed on the
Sequel I system with Sequel sequencing kit 3.0, a run movie time of
600 min and Software v6.0 (PacBio). Nature Plants | Volume 9 | July 2023 | 1067–1080 M. pudica genome PacBio library preparation A standard PacBio SMRTbell library was constructed from HMW DNA
samples using the SMRTbell template prep kit 1.0 (Pacific Biosciences)
according to PacBio recommendations (PN 100-938-400-03). Data collection, optical mapping and genome scaffolding Article Three input transcripts for EuGene were used. One transcrip-
tome was predicted on the basis of the mapping of reads from the 136
RNA-seq samples generated in this study (Supplementary Table 1). and 0.1% arabinose (w/v) and grown in 100 ml Erlenmeyer flasks for
5 d at 28 °C under agitation (220 r.p.m.). Liquid culture was washed
twice with 0.9% NaCl sterile solution (w/v) after centrifugation (10 min,
4,000 g). Bacterial suspension (1 ml, optical density (OD)600 = 0.05) was
used to inoculate each seed. To obtain these transcripts, the raw fastq paired-end reads were
cleaned by removing the adapters and the low-quality sequences using cut-
adapt92 (v2.1) and TrimGalore (v0.6.5, https://github.com/FelixKrueger/
TrimGalore) with the -q 30–length 20 options. The cleaned reads were
mapped against the M. pudica genome assembly using HISAT2 (ref. 93)
(v2.1.0) with the –score-min L,-0.6,-0.6–max-intronlen 10000–dta–
rna-strandness RF options. Duplicated reads were removed using the
SAMtools94,95 (v1.9) markdup command. Transcripts were predicted using
Stringtie96 (v2.1.4) with –fr -f 0.8 on each sample. All 80 gtf sample files
were merged using stringtie–merge with standard options. Transcript
fasta files were generated using gffread96,97 (v0.11.6) with the -w option. L. albus cv. amiga seeds were sterilized using 4X diluted commer-
cial bleach (9° Chl) for 2 min. Seeds were washed five times using sterile
deionized water and spotted on 12-cm-diameter round petri dishes
(nine seeds per dish) containing soft water agar medium (4.5 g l−1 bacto
agar, DifcoTM, Becton Dickinson Bioscience) for 3 d at 28 °C. Seeds with
approximately 1-cm-long rootlets were planted in 200 ml glass jars
containing sterile vermiculite and 100 ml of modified Jensen medium104
(in which no CaHPO4 was added and K2HPO4 was raised to 381 mg l−1). Seedlings were inoculated with Bradyrhizobium sp. 1AE200 or mock
solution right after planting. We also de novo predicted two transcriptomes from two batches
of ten samples (one sample per condition) of our same RNA-seq data
using DRAP pipeline98 (v1.92, http://www.sigenae.org/drap). runDrap
was used on the 20 samples, applying the Oases RNA-seq assembly
software99. runMeta was used to merge assemblies without redundancy
on the basis of predicted transcripts with fpkm 1. These transcriptomes
were employed as a training transcriptome by EuGene. Finally, 73,541
protein-coding genes, 1,107 transfer RNAs, 114 ribosomal RNAs and
3,913 ncRNAs were annotated. Differential gene expression analysis g
p
y
All RNA-seq libraries were mapped against their representative genome
(Supplementary Table 1) using nextflow105 (v20.11.0-edge) and run on
nf-core/rnaseq106 (v3.0, 10.5281/zenodo.1400710) using the ‘-profile
debug,genotoul–skip_qc–aligner star_salmon’ options. The workflow
used bedtools107 (v2.29.2), bioconductor-summarizedexperiment
(v1.20.0), bioconductor-tximeta (v1.8.0), gffread97 (v0.12.1), picard
(v2.23.9), salmon108 (v1.4.0), samtools94 (v1.10), star109 (v2.6.1d), string-
tie96 (v2.1.4), Trimgalore (v0.6.6) and ucsc (v377). DEGs for the different
species and experiments were estimated using ‘edgeR’110 in R111 (v4.1.2). Template script to estimate and identify DEGs is stored in GitHub at
https://github.com/CyrilLibourel/Universal_nodulation_transcrip-
tomic_response. Briefly, low-expressed genes with less than ten reads
across each class of samples were removed. Then, gene counts were
normalized by library size and using the trimmed mean of M-values
normalization method112. We estimated DEGs by comparing symbiotic
states to non-inoculated roots for the different species. M. pudica sym-
biotic traits (NF response, nodule organogenesis, symbiosome release
and persistence and nitrogen fixation) DEGs were analysed with the
DicoExpress tool113 that relies on the R packages ‘FactoMineR’114 and
‘edgeR’110 to identify genes that are differentially expressed between
experimental conditions using generalized linear models. The lists
of DEGs responsive genes as well as genes associated with the dif-
ferent symbiotic traits were determined by combining lists of DEGs
originating from multiple comparisons between two samples as indi-
cated in Supplementary Table 3. Genes were considered differentially
expressed when the false discovery rate (FDR) was below 0.05
(Benjamini–Hochberg correction) and at a specific log fold-change (FC)
(see Supplementary Table 3). Article Plants were grown in a walk-in growth chamber illuminated with
high-pressure sodium lamps (16 h photoperiod, 80% humidity, 26 °C
and 22 °C day and night temperatures). For each biological replicate, at
least eight independent root systems were used for collecting nodules
or mock-inoculated root systems at 21 d after planting. L. albus isolated nodules and root samples were harvested at
21 dpi for non-inoculated plants and inoculated plants with Bradyrhizo-
bium sp. 1AE200 strain. Three biological replicates of inoculated and
non-inoculated Lupinus root samples were used for RNA sequencing. Samples were ground using a pestle and mortar. RNA extraction and
DNase treatment were performed respectively using E.Z.N.A. RNA
extraction kit (Omega-Biotek) and TURBO DNA-free kit (Invitrogen)
according to manufacturers’ instructions. Quality of RNAs was assessed
using the Agilent 2100 Bioanalyzer system. RNA sequencing was per-
formed by the Eurofins genomics facility. Polyadenylated mRNA and
RNA-seq libraries were prepared according to Illumina’s protocols
using the Illumina TruSeq Stranded mRNA sample prep kit to analyse
mRNA. RNA-seq experiments were performed on an Illumina NovaSeq
6000 using a paired-end read length of 2 × 150 bp with the Illumina
NovaSeq 6000 sequencing kits. Genome assembly, annotation file and gene models are publicly
available through myGenomeBrowser100 and through NCBI under
BioProject PRJNA787464. M. pudica genome assembly The genome was assembled in three steps from PacBio reads. In
Step 1, Canu v1.8 (ref. 89) was used to trim, correct and assemble the
5,815,198 subreads for a total size of 85 Gb, that is, an estimated cover-
age of 94X. Programme parameters were corOutCoverage=40, min-
ReadLength=1000 and input genome size estimate=900 Mb. In Step
2, the raw data from PacBio Sequel bam files were aligned on the draft
assembly. For this step, we used the wrapper of minimap2 (ref. 90),
pbmm2, included in the SMRT Analysis Software v7.0.0 (https://www. pacb.com/products-and-services/analytical-software/smrt-analysis/). Programme parameters were « pbmm2 align–preset ‘SUBREAD’ -c 70
-l 500 ». In the final step (Step 3), we used this mapping result to polish
the draft assembly and generate a high-quality final assembly. For this
step, we used the variantCaller command included in the SMRT Analysis
Software v7.0.0, with the arrow algorithm. Programme parameters
were –algorithm arrow -minConfidence 40 -minCoverage 70 -coverage Four protein databases were used to detect translated regions: (1)
the proteome of M. truncatula A17 (v5 annotation release 1.6; https://
medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/), (2) the proteome
of the previous M. pudica Illumina genome16, (3) Swiss-Prot, October
2016 and (4) the proteome of A. thaliana TAIR10 version. Proteins
similar to REPBASE were removed from the three datasets (to avoid
the integration of transposable element related proteins in the train-
ing steps). Chained alignments spanning less than 50% of the length
of the database protein were removed. The proteome of M. truncatula
(release 1.6) was used as a training proteome by EuGene. Four protein databases were used to detect translated regions: (1)
the proteome of M. truncatula A17 (v5 annotation release 1.6; https://
medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/), (2) the proteome
of the previous M. pudica Illumina genome16, (3) Swiss-Prot, October
2016 and (4) the proteome of A. thaliana TAIR10 version. Proteins
similar to REPBASE were removed from the three datasets (to avoid
the integration of transposable element related proteins in the train-
ing steps). Chained alignments spanning less than 50% of the length
of the database protein were removed. The proteome of M. truncatula
(release 1.6) was used as a training proteome by EuGene. Nature Plants | Volume 9 | July 2023 | 1067–1080 1075 https://doi.org/10.1038/s41477-023-01441-w Article M. pudica RNA isolation and sequencing M. pudica seedlings of Australian origin (B&T World Seeds, France) were
grown in Gibson tubes containing nitrogen-free synthetic medium
composed of a Fahraeus slant agar101 and liquid Jensen 1/4th medium102
at 28 °C and under a 16 h photoperiod as described previously47. M. pudica tissue samples were harvested at 1, 3 and 5 d post-inoculation
(dpi) for non-inoculated plants, at 1 and 3 dpi for plants inoculated
with non-nodulating R. solanacearum strains, at 1, 3, 5, 7 and 10 dpi for
plants inoculated with R. solanacearum strains and at 1, 3, 5, 7, 14 and
21 dpi for plants inoculated with C. taiwanensis strains (Supplementary
Table 1). Samples from four independent biological replicates were
harvested at each time point. Samples from roots, nodule primor-
dia and nodules were ground using a pestle and mortar before RNA
extraction. Total RNA was isolated using the NucleoSpin RNA Plus kit
(Macherey-Nagel) according to manufacturer’s instructions, treated
with rDNase (Macherey-Nagel) for 10 min at 37 °C and then cleaned up
with the NucleoSpin RNA clean-up kit (Macherey-Nagel). RNA quality
was verified on a 2100 Bioanalyzer instrument (Agilent) and quantified
on a QubitTM fluorometer (Thermo Fisher). RNA sequencing was per-
formed at the GeT-PlaGe core facility, INRAE Toulouse. Polyadenylated
messenger RNA and RNA-seq libraries were prepared according to
Illumina’s protocols using the Illumina TruSeq Stranded mRNA sample
prep kit to analyse mRNA. Briefly, mRNAs were selected using poly-T
beads. Then, RNAs were fragmented to generate double-stranded
complementary DNA and adaptors were ligated for sequencing. Eleven
cycles of PCR were applied to amplify libraries. Library quality was
assessed using a Fragment Analyser and libraries were quantified by
qPCR using the Kapa library quantification kit. RNA-seq experiments
were performed on an Illumina NovaSeq 6000 using a paired-end read
length of 2 × 150 bp with the Illumina NovaSeq 6000 sequencing kits. Nature Plants | Volume 9 | July 2023 | 1067–1080 Statistical analyses 1. Drew, G. C., Stevens, E. J. & King, K. C. Microbial evolution and
transitions along the parasite–mutualist continuum. Nat. Rev. Microbiol. 19, 623–638 (2021). The different scripts used to cross-reference orthogroups and DEGs
to estimate differentially expressed orthogroups (DEOGs), recon-
struct the ancestral transcriptomic symbiotic response, identify
symbiosome accommodation proteins and all statistical related
analyses are freely available at the dedicated GitHub repository
‘Universal_nodulation_transcriptomic_response’. 2. Delaux, P.-M. & Schornack, S. Plant evolution driven by
interactions with symbiotic and pathogenic microbes. Science
371, eaba6605 (2021). Briefly, to determine at which evolutionary node each orthogroup
was probably transcriptionally recruited for RNS, we used the process
described below: 3. Beerling, D. The Emerald Planet: How Plants Changed Earth’s
History (Oxford Univ. Press, 2008). 4. Rich, M. K. et al. Lipid exchanges drove the evolution of
mutualism during plant terrestrialization. Science 372, 864–868
(2021). (1) DEGs to DEOGs: if at least one gene was up/downregulated in an
orthogroup (HOG), the orthogroup was coded as 1 for the spe-
cies in which the gene was differentially regulated. This process
was done for the nine species in the 56,508 orthogroups.i 5. Rai, A. N., Söderbäck, E. & Bergman, B. Cyanobacterium-plant
symbioses. New Phytol. 147, 449–481 (2000). 6. Adams, D. G. & Duggan, P. S. Cyanobacteria-bryophyte
symbioses. J. Exp. Bot. 59, 1047–1058 (2008). (2) On the basis of a simplified tree with only the nine species, we
used the ‘asr_mk_model’ function from ‘castor’ package in R
with the following options: ‘Nstates = 2, include_ancestral_likeli-
hoods = TRUE, rate_model = ‘ER’’, giving a likelihood between
0 and 1 at each node. 7. Martin, F., Kohler, A., Murat, C., Veneault-Fourrey, C. & Hibbett, D. S. Unearthing the roots of ectomycorrhizal symbioses. Nat. Rev. Microbiol. 14, 760–773 (2016). 8. Pinto-Carbó, M., Gademann, K., Eberl, L. & Carlier, A. Leaf nodule
symbiosis: function and transmission of obligate bacterial
endophytes. Curr. Opin. Plant Biol. 44, 23–31 (2018). (3) To consider a DEOG to be recruited for RNS at a specific node,
the likelihood had to be >0.6 to increase robustness for ances-
tral recruitment. 9. 9. Parniske, M. Uptake of bacteria into living plant cells, the unifying
and distinct feature of the nitrogen-fixing root nodule symbiosis. Curr. Opin. Plant Biol. 44, 164–174 (2018). (4) For each node ‘recruited’, we kept only the most ancient paren-
tal node. Orthogroups reconstruction All the repositories of RNA-seq used in this study are detailed in
Supplementary Table 1. All the repositories of RNA-seq used in this study are detailed in
Supplementary Table 1. To cross-reference expression data among species, we reconstructed
orthogroups including the nine species for which RNA-seq data dur-
ing symbiosis are available, as well as 16 representative species of
each NFN clade and A. thaliana as outgroup (Supplementary Table 6). Orthogroups reconstruction was performed with OrthoFinder v2.5.2
(ref. 115) using the ultra-sensitive Diamond mode (-S diamond_ultra_
sens option). The tree inferred by OrthoFinder was then checked for
consistent reconstruction with known species trees and OrthoFinder
reran on the basis of the species tree with the alignment and phylog-
eny methods using mafft v7.313 (ref. 116) and fasttree v2.1.10 (ref. 117),
respectively, to infer hierarchical orthologous groups (HOG). The orthogroups file generated is this study and raw gene count for
the RNA-seq listed in Supplementary Table 2 are stored in figshare The orthogroups file generated is this study and raw gene count for
the RNA-seq listed in Supplementary Table 2 are stored in figshare
at https://figshare.com/projects/Comparative_phylotranscriptom-
ics_reveals_ancestral_and_derived_root_nodule_symbiosis_pro-
grams/166196. Reporting summary 16. Griesmann, M. et al. Phylogenomics reveals multiple losses of
nitrogen-fixing root nodule symbiosis. Science 361, eaat1743
(2018). Further information on research design is available in the Nature Port-
folio Reporting Summary linked to this article. f
g p
(5) This process was conducted on all HOGs. f
(5) This process was conducted on all HOGs. 12. Ardley, J. & Sprent, J. Evolution and biogeography of actinorhizal
plants and legumes: a comparison. J. Ecol. 109, 1098–1121
(2021). To estimate the null distribution of the number of DEOGs for each
node, we first determined to which evolutionary node each orthogroup
belongs using a maximum parsimony ancestral state reconstruction for
discrete traits118 (that is, presence/absence of species genes in orthogroup). For each species, we randomly selected genes according to (1) the number
of DEGs estimated in the species and (2) the node to which the orthogroup
containing DEGs belongs. We repeated this process for the nine species
and determined to which evolutionary node each orthogroup belongs
using the same maximum parsimony ancestral state reconstruction (that
is, presence/absence of species DEGs in orthogroup). We repeated this
process 1,000 times to get the null expectation (Supplementary Table 5). 13. van Velzen, R., Doyle, J. J. & Geurts, R. A resurrected scenario:
single gain and massive loss of nitrogen-fixing nodulation. Trends
Plant Sci. 24, 49–57 (2019). 14. Soltis, D. E. et al. Chloroplast gene sequence data suggest a
single origin of the predisposition for symbiotic nitrogen
fixation in angiosperms. Proc. Natl Acad. Sci. USA 92, 2647–2651
(1995). 15. Kates, H. R. et al. Two shifts in evolutionary lability underlie
independent gains and losses of root-nodule symbiosis
in a single clade of plants. Preprint at bioRxiv https://doi. org/10.1101/2022.07.31.502231 (2022). L. albus RNA isolation and sequencing For each biological replicate, Bradyrhizobium sp. 1AE200 (Ledermann
and Couzigou, unpublished) strain was grown for 8 d on PSY medium
agar plates (15 g l−1 bacto agar, DifcoTM, Becton Dickinson Bioscience)
supplemented with erythromycin (200 µg ml−1) and 0.1% arabinose
(w/v). Several independent colonies were used to inoculate a 20 ml
PSY103 liquid culture supplemented with erythromycin (200 µg ml−1) Nature Plants | Volume 9 | July 2023 | 1067–1080 1076 Article https://doi.org/10.1038/s41477-023-01441-w Code availability Scripts used to cross-reference orthogroups and RNA-seq data are
publicly available in GitHub at https://github.com/CyrilLibourel/
Universal_nodulation_transcriptomic_response Statistical analyses For example, if all nodes were ‘recruited’, the DEOG
was considered as recruited in the common ancestor of NFN
species (that is, NFN node). This method allowed us to accept
ancestrally recruited HOGs that (1) were subsequently lost, (2)
lost the deregulation during symbiosis or (3) for which informa-
tion was missing because of heterogeneity of the sampling
effort among species. 10. de Faria, S. M. et al. The innovation of the symbiosome has
enhanced the evolutionary stability of nitrogen fixation in
legumes. New Phytol. 235, 2365–2377 (2022). 11. de La Peña, T. C., Fedorova, E., Pueyo, J. J. & Lucas, M. M. The
symbiosome: legume and rhizobia co-evolution toward a
nitrogen-fixing organelle? Front. Plant Sci. 8, 2229 (2018). Data availability 17. van Velzen, R. et al. Comparative genomics of the nonlegume
Parasponia reveals insights into evolution of nitrogen-fixing
rhizobium symbioses. Proc. Natl Acad. Sci. USA 115, E4700–E4709
(2018). Mimosa pudica annotated genome assembly is available in myGenom-
eBrowser and raw sequenced data are available in NCBI under BioPro-
ject PRJNA787464 and SRP349803. Nature Plants | Volume 9 | July 2023 | 1067–1080 1077 Article https://doi.org/10.1038/s41477-023-01441-w 38. Yano, K. et al. CYCLOPS, a mediator of symbiotic intracellular
accommodation. Proc. Natl Acad. Sci. USA 105, 20540–20545
(2008). 18. Reynoso, M. A. et al. Evolutionary flexibility in flooding response
circuitry in angiosperms. Science 365, 1291–1295 (2019). 19. Frank, M. H. & Scanlon, M. J. Transcriptomic evidence for the
evolution of shoot meristem function in sporophyte-dominant
land plants through concerted selection of ancestral
gametophytic and sporophytic genetic programs. Mol. Biol. Evol. 32, 355–367 (2015). 39. Schauser, L., Roussis, A., Stiller, J. & Stougaard, J. A plant
regulator controlling development of symbiotic root nodules. Nature 402, 191–195 (1999). 40. Arrighi, J. F. et al. The RPG gene of Medicago truncatula controls
Rhizobium-directed polar growth during infection. Proc. Natl
Acad. Sci. USA 105, 9817–9822 (2008). 20. Julca, I. et al. Comparative transcriptomic analysis reveals
conserved programmes underpinning organogenesis and
reproduction in land plants. Nat. Plants 7, 1143–1159 (2021). 41. Lace, B. et al. RPG acts as a central determinant for infectosome
formation and cellular polarization during intracellular rhizobial
infections. eLife 12, e80741 (2023). 21. de Moura, G. G. D., Remigi, P., Masson-Boivin, C. & Capela, D. Experimental evolution of legume symbionts: what have we
learnt? Genes 11, 339 (2020). 42. Blount, Z. D., Barrick, J. E., Davidson, C. J. & Lenski, R. E. Genomic
analysis of a key innovation in an experimental Escherichia coli
population. Nature 489, 513–518 (2012). 22. LPWG (Legume Phylogeny Working Group). A new subfamily
classification of the Leguminosae based on a taxonomically
comprehensive phylogeny. Taxon 66, 44–77 (2017). 43. Quandt, E. M., Deatherage, D. E., Ellington, A. D., Georgiou,
G. & Barrick, J. E. Recursive genomewide recombination and
sequencing reveals a key refinement step in the evolution of a
metabolic innovation in Escherichia coli. Proc. Natl Acad. Sci. USA
111, 2217–2222 (2014). 23. Mergaert, P., Kereszt, A. & Kondorosi, E. Gene expression in
nitrogen-fixing symbiotic nodule cells in Medicago truncatula and
other nodulating plants. Plant Cell 32, 42–68 (2020). 24. Raul, B. et al. Data availability Microscopic and transcriptomic analyses of
dalbergoid legume peanut reveal a divergent evolution leading
to nod-factor-dependent epidermal crack-entry and terminal
bacteroid differentiation. Mol. Plant Microbe Interact. 35, 131–145
(2022). 44. Handa, Y. et al. RNA-seq transcriptional profiling of an arbuscular
mycorrhiza provides insights into regulated and coordinated
gene expression in Lotus japonicus and Rhizophagus irregularis. Plant Cell Physiol. 56, 1490–1511 (2015). 45. Luginbuehl, L. H. et al. Fatty acids in arbuscular mycorrhizal fungi
are synthesized by the host plant. Science 356, 1175–1178 (2017). 25. Mun, T., Bachmann, A., Gupta, V., Stougaard, J. & Andersen, S. U. Lotus Base: an integrated information portal for the model
legume Lotus japonicus. Sci. Rep. 6, 39447 (2016). 46. Marchetti, M. et al. Experimental evolution of a plant pathogen
into a legume symbiont. PLoS Biol. 8, e1000280 (2010). 26. Quilbé, J. et al. Genetics of nodulation in Aeschynomene evenia
uncovers mechanisms of the rhizobium–legume symbiosis. Nat. Commun. 12, 829 (2021). 47. Marchetti, M. et al. Shaping bacterial symbiosis with legumes by
experimental evolution. Mol. Plant Microbe Interact. 27, 956–964
(2014). 27. Schiessl, K. et al. NODULE INCEPTION recruits the lateral root
developmental program for symbiotic nodule organogenesis in
Medicago truncatula. Curr. Biol. 29, 3657–3668.e5 (2019). 48. Capela, D. et al. Recruitment of a lineage-specific virulence
regulatory pathway promotes intracellular infection by a plant
pathogen experimentally evolved into a legume symbiont. Mol. Biol. Evol. 34, 2503–2521 (2017). 28. Battenberg, K., Potter, D., Tabuloc, C. A., Chiu, J. C. & Berry, A. M. Comparative transcriptomic analysis of two actinorhizal plants
and the legume Medicago truncatula supports the homology of
root nodule symbioses and is congruent with a two-step process
of evolution in the nitrogen-fixing clade of angiosperms. Front. Plant Sci. 9, 1256 (2018). 49. Amadou, C. et al. Genome sequence of the β-rhizobium
Cupriavidus taiwanensis and comparative genomics of rhizobia. Genome Res. 18, 1472–1483 (2008). 50. Chen, W. M. et al. Ralstonia taiwanensis sp. nov., isolated from
root nodules of Mimosa species and sputum of a cystic fibrosis
patient. Int. J. Syst. Evol. Microbiol. 51, 1729–1735 (2001). 29. Dahmer, N. et al. Chromosome numbers in the genus Mimosa L.:
cytotaxonomic and evolutionary implications. Plant Syst. Evol. 291, 211–220 (2011). 51. Chen, W.-M., James, E. K., Prescott, A. R., Kierans, M. & Sprent, J. I. Nodulation of Mimosa spp. by the β-proteobacterium Ralstonia
taiwanensis. Mol. Plant Microbe Interact. 16, 1051–1061 (2003). 30. González-Sama, A., Lucas, M. Data availability 58. Marsh, J. F. et al. Medicago truncatula NIN is essential for
rhizobial-independent nodule organogenesis induced by
autoactive calcium/calmodulin-dependent protein kinase. Plant
Physiol. 144, 324–335 (2007). 78. Yuan, S. et al. A ubiquitin ligase of symbiosis receptor kinase
involved in nodule organogenesis. Plant Physiol. 160, 106–117
(2012). 59. Vernié, T. et al. The NIN transcription factor coordinates diverse
nodulation programs in different tissues of the Medicago
truncatula root. Plant Cell 27, 3410–3424 (2015). 79. van de Velde, W. et al. Plant peptides govern terminal
differentiation of bacteria in symbiosis. Science 327, 1122–1126
(2010). 60. Soyano, T., Kouchi, H., Hirota, A. & Hayashi, M. NODULE
INCEPTION directly targets NF-Y subunit genes to regulate
essential processes of root nodule development in Lotus
japonicus. PLoS Genet. 9, e1003352 (2013). 80. Czernic, P. et al. Convergent evolution of endosymbiont
differentiation in dalbergioid and inverted repeat-lacking clade
legumes mediated by nodule-specific cysteine-rich peptides. Plant Physiol. 169, 1254–1265 (2015). 61. Cerri, M. R. et al. Medicago truncatula ERN transcription factors:
regulatory interplay with NSP1/NSP2 GRAS factors and expression
dynamics throughout rhizobial infection. Plant Physiol. 160,
2155–2172 (2012). 81. Scocchi, M., Tossi, A. & Gennaro, R. Proline-rich antimicrobial
peptides: converging to a non-lytic mechanism of action. Cell. Mol. Life Sci. 68, 2317–2330 (2011). 62. Bapaume, L. et al. VAPYRIN marks an endosomal trafficking
compartment involved in arbuscular mycorrhizal symbiosis. Front. Plant Sci. 10, 666 (2019). 82. Cao, H. et al. Identification of a novel proline-rich antimicrobial
peptide from Brassica napus. PLoS ONE 10, e0137414 (2015). 83. Marchetti, M., Catrice, O., Batut, J. & Masson-Boivin, C. Cupriavidus taiwanensis bacteroids in Mimosa pudica
indeterminate nodules are not terminally differentiated. Appl. Environ. Microbiol. 77, 2161–2164 (2011). 63. Murray, J. D. et al. Vapyrin, a gene essential for intracellular
progression of arbuscular mycorrhizal symbiosis, is also essential
for infection by rhizobia in the nodule symbiosis of Medicago
truncatula. Plant J. 65, 244–252 (2011). 84. Radhakrishnan, G. V. et al. An ancestral signalling pathway is
conserved in intracellular symbioses-forming plant lineages. Nat. Plants 6, 280–289 (2020). 64. Liang, P. et al. Formin-mediated bridging of cell wall, plasma
membrane, and cytoskeleton in symbiotic infections of Medicago
truncatula. Curr. Biol. 31, 2712–2719.e5 (2021). 85. Cathebras, C. et al. A novel cis-element enabled bacterial
uptake by plant cells. Preprint at bioRxiv https://doi.org/10.1101/
2022.03.28.486070 (2022). 65. Kawaharada, Y. et al. Receptor-mediated exopolysaccharide
perception controls bacterial infection. Nature 523, 308–312
(2015). 86. Bloch, S. E., Ryu, M. Data availability M., de Felipe, M. R. & Pueyo, J. J. An unusual infection mechanism and nodule morphogenesis in
white lupin (Lupinus albus). New Phytol. 163, 371–380 (2004). 52. Tang, M., Bouchez, O., Cruveiller, S., Masson-Boivin, C. & Capela, D. Modulation of quorum sensing as an adaptation to nodule cell
infection during experimental evolution of legume symbionts. mBio 11, e03129-19 (2020). 31. Hufnagel, B. et al. High-quality genome sequence of white
lupin provides insight into soil exploration and seed quality. Nat. Commun. 11, 492 (2020). 32. Emms, D. M. & Kelly, S. OrthoFinder: phylogenetic orthology
inference for comparative genomics. Genome Biol. 20, 238 (2019). 53. Daubech, B. et al. Spatio-temporal control of mutualism in legumes
helps spread symbiotic nitrogen fixation. eLife 6, e28683 (2017). 33. Madsen, E. B. et al. A receptor kinase gene of the LysM type is
involved in legume perception of rhizobial signals. Nature 425,
637–640 (2003). 54. Andrews, M. et al. Horizontal transfer of symbiosis genes within
and between rhizobial genera: occurrence and importance. Genes 9, 321 (2018). 34. Amor, B. B. et al. The NFP locus of Medicago truncatula controls
an early step of Nod factor signal transduction upstream of a
rapid calcium flux and root hair deformation. Plant J. 34, 495–506
(2003). 55. Jardinaud, M. F. et al. A laser dissection-RNAseq analysis
highlights the activation of cytokinin pathways by nod factors
in the Medicago truncatula root epidermis. Plant Physiol. 171,
2256–2276 (2016). 35. Stracke, S. et al. A plant receptor-like kinase required for both
bacterial and fungal symbiosis. Nature 417, 959–962 (2002). 56. Kelly, S., Mun, T., Stougaard, J., Ben, C. & Andersen, S. U. Distinct
Lotus japonicus transcriptomic responses to a spectrum of
bacteria ranging from symbiotic to pathogenic. Front. Plant Sci. 9,
1218 (2018). 36. Catoira, R. et al. Four genes of Medicago truncatula controlling
components of a Nod factor transduction pathway. Plant Cell 12,
1647–1666 (2000). 57. Roux, B. et al. An integrated analysis of plant and bacterial
gene expression in symbiotic root nodules using laser-capture
microdissection coupled to RNA sequencing. Plant J. 77, 817–837
(2014). 37. Lévy, J. et al. A putative Ca2+ and calmodulin-dependent protein
kinase required for bacterial and fungal symbioses. Science 303,
1361–1364 (2004). Nature Plants | Volume 9 | July 2023 | 1067–1080 1078 Article https://doi.org/10.1038/s41477-023-01441-w 77. Liu, C. W. et al. A protein complex required for polar growth of
rhizobial infection threads. Nat. Commun. 10, 2848 (2019). Data availability H., Ozaydin, B. & Broglie, R. Harnessing
atmospheric nitrogen for cereal crop production. Curr. Opin. Biotechnol. 62, 181–188 (2020). 66. Li, X. et al. Atypical receptor kinase RINRK1 required for rhizobial
infection but not nodule development in lotus japonicus. Plant
Physiol. 181, 804–816 (2019). 87. Huisman, R. & Geurts, R. A roadmap toward engineered
nitrogen-fixing nodule symbiosis. Plant Commun. 1, 100019
(2020). 67. Berrabah, F. et al. A nonRD receptor‐like kinase prevents nodule
early senescence and defense‐like reactions during symbiosis. New Phytol. 203, 1305–1314 (2014). 88. Haskett, T. L. et al. Engineered plant control of associative
nitrogen fixation. Proc. Natl Acad. Sci. USA 119, e2117465119
(2022). 68. Malolepszy, A. et al. A plant chitinase controls cortical infection
thread progression and nitrogen-fixing symbiosis. eLife 7, e38874
(2018). 89. Koren, S. et al. Canu: scalable and accurate long-read assembly
via adaptive k-mer weighting and repeat separation. Genome Res. 27, 722–736 (2017). 69. Chakraborty, S., Valdés-López, O., Stonoha-Arther, C. &
Ané, J.-M. Transcription factors controlling the rhizobium–legume
symbiosis: integrating infection, organogenesis and
the abiotic environment. Plant Cell Physiol. 63, 1326–1343
(2022). 90. Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094–3100 (2018). 91. Foissac, S. et al. Genome annotation in plants and fungi: EuGene
as a model platform. Curr. Bioinform. 3, 87–97 (2008). 70. Shrestha, A. et al. Lotus japonicus Nuclear Factor YA1, a nodule
emergence stage-specific regulator of auxin signalling. New
Phytol. 229, 1535–1552 (2020). 92. Martin, M. Cutadapt removes adapter sequences from
high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011). 71. Azarakhsh, M. et al. KNOTTED1-LIKE HOMEOBOX 3: a new
regulator of symbiotic nodule development. J. Exp. Bot. 66,
7181–7195 (2015). 93. Kim, D., Paggi, J. M., Park, C., Bennett, C. & Salzberg, S. L. Graph-based genome alignment and genotyping with HISAT2
and HISAT-genotype. Nat. Biotechnol. 37, 907–915 (2019). 72. Magne, K. et al. MtNODULE ROOT1 and MtNODULE ROOT2 are
essential for indeterminate nodule identity. Plant Physiol. 178,
295–316 (2018). 94. Li, H. et al. The Sequence Alignment/Map (SAM) format and
SAMtools. Bioinformatics 25, 2078–2079 (2009). 95. Li, H. A statistical framework for SNP calling, mutation discovery,
association mapping and population genetical parameter
estimation from sequencing data. Bioinformatics 27, 2987–2993
(2011). 73. Sinharoy, S. et al. The C2H2 transcription factor REGULATOR OF
SYMBIOSOME DIFFERENTIATION represses transcription of the
secretory pathway gene VAMP721a and promotes symbiosome
development in Medicago truncatula. Plant Cell 25, 3584–3601
(2013). 96. Pertea, M. et al. Data availability StringTie enables improved reconstruction of a
transcriptome from RNA-seq reads. Nat. Biotechnol. 33, 290–295
(2015).ff 74. Vernié, T. et al. EFD is an ERF transcription factor involved in
the control of nodule number and differentiation in Medicago
truncatula. Plant Cell 20, 2696–2713 (2008). 97. Pertea, G. & Pertea, M. GFF Utilities: GffRead and GffCompare. F1000Research 9, 304 (2020). 75. Jardinaud, M.-F. et al. MtEFD and MtEFD2: two transcription
factors with distinct neofunctionalization in symbiotic nodule
development. Plant Physiol. 189, 1587–1607 (2022). 98. Cabau, C. et al. Compacting and correcting Trinity and Oases
RNA-seq de novo assemblies. PeerJ 5, e2988 (2017). 99. Schulz, M. H., Zerbino, D. R., Vingron, M. & Birney, E. Oases:
robust de novo RNA-seq assembly across the dynamic
range of expression levels. Bioinformatics 28, 1086–1092
(2012). 76. Haney, C. H. & Long, S. R. Plant flotillins are required for infection
by nitrogen-fixing bacteria. Proc. Natl Acad. Sci. USA 107, 478–483
(2010). Nature Plants | Volume 9 | July 2023 | 1067–1080 1079 Article https://doi.org/10.1038/s41477-023-01441-w 100. Carrere, S. & Gouzy, J. myGenomeBrowser: building and sharing
your own genome browser. Bioinformatics 33, 1255–1257 (2016). performed at LRSV and LIPME was also supported by the ‘Laboratoires
d’Excellence (LABEX)’ TULIP (ANR-10-LABX-41) and the ‘École
Universitaire de Recherche (EUR)’ TULIP-GS (ANR-18-EURE-0019). J.K.,
C.L. and P-M.D were supported by the project Engineering Nitrogen
Symbiosis for Africa (ENSA) currently funded through a grant to the
University of Cambridge by the Bill and Melinda Gates Foundation
(OPP1172165) and the UK Foreign, Commonwealth and Development
Office as Engineering Nitrogen Symbiosis for Africa (OPP1172165). This
project received funding from the European Research Council (ERC)
under the European Union’s Horizon 2020 research and innovation
programme (grant agreement no. 101001675 - ORIGINS) to P.-M.D. P.R. received funding from the EU in the framework of the Marie-Curie
FP7 COFUND People Program, through the award of an AgreenSkills+
fellowship (under grant agreement no. 609398) and from the
European Union’s Horizon 2020 research and innovation programme
under the Marie Skłodowska-Curie grant agreement no. 845838. 101. FÅHRAEUS, G. The infection of clover root hairs by nodule
bacteria studied by a simple glass slide technique. Microbiology
16, 374–381 (1957). 102. Jensen, H. L. Nitrogen fixation in leguminous plants. I. General
characters of root-nodule bacteria isolated from species of
Medicago and Trifolium in Australia. Proc. Linn. Soc. NSW 66,
98–108 (1942). 103. Mesa, S. et al. Reprints and permissions information is available at 116. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment
software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013). Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. 117. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree: computing large
minimum evolution trees with profiles instead of a distance
matrix. Mol. Biol. Evol. 26, 1641–1650 (2009).f 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/. 118. Louca, S. & Doebeli, M. Efficient comparative phylogenetics on
large trees. Bioinformatics 34, 1053–1055 (2018). Author contributions 106. Ewels, P. A. et al. The nf-core framework for community-curated
bioinformatics pipelines. Nat. Biotechnol. 38, 276–278 (2020). C.L., J.K., C.M.-B., P.R., P.-M.D. and D.C. designed the project. C.L., J.K.,
L.B., A.-C.C., S. Carrere, T.V., J.-M.C., C.C., I.D., S. Cauet, W.M., T.B., A.S.,
P.R., P.-M.D. and D.C. conducted experiments. C.L., J.K., S. Cauet, P.R.,
P.-M.D. and D.C. analysed data. C.L., J.K., P.R., P.-M.D. and D.C. wrote the
manuscript with input from all authors. 107. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for
comparing genomic features. Bioinformatics 26, 841–842 (2010). 108. Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript
expression. Nat. Methods 14, 417–419 (2017). Competing interests
The authors declare no competing interests. 109. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013). 110. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a
Bioconductor package for differential expression analysis of
digital gene expression data. Bioinformatics 26, 139–140 (2009). Data availability Comprehensive assessment of the regulons
controlled by the FixLJ-FixK2-FixK1 cascade in Bradyrhizobium
japonicum. J. Bacteriol. 190, 6568–6579 (2008). 104. Vincent, J. M. A Manual for the Practical Study of Root-Nodule
Bacteria (IBP Handbook No. 15) (Oxford and Edinburgh: Blackwell
Scientific Publications, 1972). 105. di Tommaso, P. et al. Nextflow enables reproducible
computational workflows. Nat. Biotechnol. 35, 316–319 (2017). Additional information Extended data is available for this paper at
https://doi.org/10.1038/s41477-023-01441-w. 111. R Core Team. R: A Language and Environment for Statistical
Computing (R Foundation for Statistical Computing, 2020). Supplementary information The online version
contains supplementary material available at
https://doi.org/10.1038/s41477-023-01441-w. 112. Robinson, M. D. & Oshlack, A. A scaling normalization method for
differential expression analysis of RNA-seq data. Genome Biol. 11,
3 (2010). 113. Lambert, I., Paysant-Le Roux, C., Colella, S. & Martin-Magniette,
M. L. DiCoExpress: a tool to process multifactorial RNAseq
experiments from quality controls to co-expression analysis
through differential analysis based on contrasts inside GLM
models. Plant Methods 16, 68 (2020). Correspondence and requests for materials should be addressed to
Philippe Remigi, Pierre-Marc Delaux or Delphine Capela. Peer review information Nature Plants thanks Euan James, Martin
Parniske and the other, anonymous, reviewer(s) for their contribution
to the peer review of this work. 114. Lê, S., Josse, J. & Husson, F. FactoMineR: an R package for
multivariate analysis. J. Stat. Softw. 25, 1–18 (2008). 115. Emms, D. M. & Kelly, S. OrthoFinder: phylogenetic orthology
inference for comparative genomics. Genome Biol. 20,
238 (2019). Acknowledgements We thank O. Valdes-Lopez, P. Gamas and the ENSA consortium for
discussions and feedback on the manuscript, B. Perret for the Lupinus
albus seeds and the genotoul bioinformatics platform Toulouse
Occitanie (Bioinfo Genotoul, https://doi.org/10.15454/1.557236932896
1167E12) for providing computing resources. This study was supported
by grants from the Fédération de Recherche Agrobiosciences,
Interactions et Biodiversité to P.-M.D. and P.R., and from the French
National Research Institute for Agriculture, Food and Environment
(INRAE, Plant Health and Environment Division) to D.C. Research © The Author(s) 2023 © The Author(s) 2023 Nature Plants | Volume 9 | July 2023 | 1067–1080 1080 https://doi.org/10.1038/s41477-023-01441-w Article Trema orientalis
Parasponia andersonii
N1: NFN clade
N2: Fabales clade
N3: Rosales+Cucurbitales clade
N7: Papilionoideae clade
N12: Dalbergioid+Hologalegina clade
N15: Rosales clade
N21: Hologalegina clade
N22: Dalbergioid clade
Morus notabilis
Discaria trinervis
Hippophae rhamnoides
Fragaria vesca
Dryas drummondii
Betula pendula
Alnus glutinosa
Casuarina glauca
Juglans regia
Lagenaria siceraria
Cucurbita maxima
Begonia fuchsioides
Datisca glomerata
Arachis hypogaea
Aeschynomene evenia
Nissolia schottii
Glycine max
Lotus japonicus
Medicago truncatula
Lupinus albus
Castanospermum australe
Mimosa pudica
Arabidopsis thaliana
Carpinus fangiana
Fabales
Cucurbitales
Fagales
Rosales
N3
N1
N2
N7 N21
N12
N22
N15
Extended Data Fig. 1 | Simplified phylogeny to highlight node labels and species clades used in this study. Extended Data Fig. 1 | Simplified phylogeny to highlight node labels and species clades used in this study. Nature Plants Article https://doi.org/10.1038/s41477-023-01441-w 7178
586
187
154
1683
6
19
C.tai Up
GMI1000 Up
GMI1000-pRalta Up
Extended Data Fig. 2 | Venn diagram comparing the lists of up-regulated genes in response to C. taiwanensis, GMI1000 pRalta and GMI1000. Extended Data Fig. 2 | Venn diagram comparing the lists of up-regulated genes in response to C. taiwanensis, GMI1000 pRa Nature Plants
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Disruptive effects of repeated stress on basolateral amygdala neurons and fear behavior across the estrous cycle in rats
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Scientific reports
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Shannon R. Blume1,4, Mallika Padival1,3, Janice H. Urban2,3 & J. Amiel Rosenkranz1,3 Stress is a precipitating factor in depression and anxiety disorders. Patients with these disorders often
show amygdala abnormalities. The basolateral amygdala (BLA) is integral in mood and emotion, and is
sensitive to stress. While much is known about effects of stress on BLA neuron activity and morphology
in males, less is known in females. We tested whether repeated stress exerts distinct effects on BLA in
vivo neuronal activity and morphology of Golgi-stained BLA neurons [lateral (LAT) and basal (BA) nuclei]
in adult female rats. Repeated restraint stress increased BLA neuronal firing and caused hypertrophy of
BLA neurons in males, while it decreased LAT and BA neuronal firing and caused hypotrophy of neurons
in the LAT of females. BLA neuronal activity and function, such as fear conditioning, shifts across
the estrous cycle. Repeated stress disrupted this pattern of BLA activity and fear expression over the
estrous cycle. The disruptive effects of stress on the pattern of BLA function across estrous may produce
behavior that is non-optimal for a specific phase of the estrous cycle. The contrasting effects of stress
may contribute to sex differences in the effects of stress on mood and psychiatric disorders. Psychiatric disorders such as generalized anxiety disorder, major depressive disorder, and post-traumatic stress
disorder (PTSD) afflict a huge number of people, the majority of which are female1–6. Patients suffering from these
disorders often show functional abnormalities in limbic structures, particularly the prefrontal cortex, hippocam-
pus and amygdala7–10. Stress not only can recapitulate these abnormalities observed in patients11, but can trigger
the emergence of psychiatric disorders. However, the impact of stress on the development of psychiatric disor-
ders may affect the sexes differently; for example, males typically experience more traumatic events, yet there is a
higher incidence of PTSD in females12. Furthermore, limbic structures in men and women may be differentially
sensitive to the effects of stress1,3,6,12–15.f f
Similarly in rodents, stress can exert contrasting effects on emotion-related behaviors of males and females16. Stress enhances fear conditioning performance in males17–20, whereas there are varied effects on fear learning in
females, with reports of impaired19, no effect21, or enhanced fear learning in females22,23 that may vary depending
on stressor, fear conditioning protocol or estrogen status.h The basolateral amygdala (BLA) plays a critical role in the expression and modulation of emotion, most nota-
bly measured as conditioned fear24–28. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 24 April 2019
Accepted: 8 August 2019
Published: xx xx xxxx OPEN Received: 24 April 2019
Accepted: 8 August 2019
Published: xx xx xxxx Shannon R. Blume1,4, Mallika Padival1,3, Janice H. Urban2,3 & J. Amiel Rosenkranz1,3 Materials and Methods Estradiol levels were
quantified using an estradiol radioimmunoassay kit (sensitivity to 8 pg/mL; Coat-A-Count Estradiol Kit, Siemens
Medical Solutions USA, Malvern, PA). Repeated restraint stress protocol. Male and female rats were randomly assigned to control or stress
groups. Cohorts of control and stress rats were run in parallel whenever possible. Restraint stress was applied
using a protocol that has been effective at inducing a change of BLA function20,39, as follows: Control handled
animals were weighed and placed in a Plexiglas transfer cage with bedding for 20 minutes, on 7 out of 9 days. All
control handling occurred in the animal housing room. Animals in the repeated stress group were weighed and
placed in a transfer cage with no bedding and transported to a procedure room (80–90 lux overhead lighting). In the procedure room, animals were placed into a hemi-cylinder restraint tube for 20 minutes per day, for 7 out
of 9 days. The hemi-cylinder restraint tubes were adjusted such that all animals had a similar degree of restricted
trunk movement. One day following the final control or stress handling, animals were used for experimental
measures. In a subset of rats, at autopsy, the adrenal gland was removed and wet weights were recorded. In vivo extracellular recording. Animals were anesthetized with urethane (1.5 g/kg i.p., Sigma Aldrich, St. Louis, MO) and placed in a stereotaxic device (David Kopf Instruments, Tujunga, CA). The level of anesthesia
was monitored by periodic testing of the hind limb pedal reflex. Body temperature was monitored with a rectal
temperature probe and maintained at ~37 °C with a heating pad (Model TC-1000, CWE Inc, Ardmore, PA). Prior
to surgery, a local anesthetic was injected subcutaneous above the skull (0.3 mL, 1% lidocaine, Webster Veterinary
Supply Inc., Devens, MA). A concentric bipolar stimulation electrode (David Kopf Instruments) was lowered into
the medial prefrontal cortex (mPFC; +2.7 mm rostral (R), −0.7 mm L and −3.7 mm ventral (V) from bregma)
to monitor the depth of anesthesia by recording ongoing rhythmic local field potentials. Single barrel electrodes
(World Precision Instruments, Sarasota, FL) were made using a vertical microelectrode puller (PE-2; Narishige,
Tokyo, Japan), and were broken under a microscope to a tip diameter of 1–2 μm and filled with a 2% Pontamine
Sky Blue solution in 2M NaCl. Shannon R. Blume1,4, Mallika Padival1,3, Janice H. Urban2,3 & J. Amiel Rosenkranz1,3 Differential effects of stress on fear conditioning may reflect different effects
of stress on BLA function in females. Indeed, markers of cellular activation, such as cFos or other early genes and
protein phosphorylation, demonstrate that acute stressors recruit the BLA29–34, and also hint at sex differences
in the response of the BLA to stress35–37. More direct cellular measures have demonstrated that repeated stress
increases firing of BLA neurons and produces hypertrophy of BLA dendrites in males38–42. However, very little
is known about the cellular effects of repeated stress on BLA neurons in females. This first goal of this study was
to test whether repeated restraint stress has different effects on BLA neuronal activity and morphology between
male and female rats. 1Discipline of Cellular and Molecular Pharmacology, The Chicago Medical School, Rosalind Franklin University, North
Chicago, IL, 60064, USA. 2Discipline of Physiology and Biophysics, The Chicago Medical School, Rosalind Franklin
University, North Chicago, IL, 60064, USA. 3Center for Neurobiology of Stress Resilience and Psychiatric Disorders,
The Chicago Medical School, Rosalind Franklin University, North Chicago, IL, 60064, USA. 4Present address: AbbVie,
Inc., 1 North Waukegan Road, North Chicago, IL, 60064, USA. Correspondence and requests for materials should be
addressed to J.A.R. (email: amiel.rosenkranz@rosalindfranklin.edu) Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 www.nature.com/scientificreports/ The BLA is comprised of several nuclei, including the lateral nucleus (LAT) and the basal nucleus (BA). These
nuclei contribute to overlapping but different aspects of BLA-dependent behaviors, display differences in neu-
ronal activity between males and females43 and may be differentially sensitive to stress23,35–37. Therefore, both of
these nuclei will be separately examined in this study.l The BLA is comprised of several nuclei, including the lateral nucleus (LAT) and the basal nucleus (BA). These
nuclei contribute to overlapping but different aspects of BLA-dependent behaviors, display differences in neu-
ronal activity between males and females43 and may be differentially sensitive to stress23,35–37. Therefore, both of
these nuclei will be separately examined in this study.l p
y
y
Mammalian biological rhythms, each with their own periodicities, influence a wide range of physiological
processes in parallel with behaviors to produce adaptive benefits. Female mammals in the reproductive stages
of life display ovarian cycles. Materials and Methods All experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory
Animals50 and were approved by the Institutional Animal Care and Use Committee at Rosalind Franklin
University of Medicine and Science. All appropriate efforts were made to minimize animal suffering, reduce the
number of animals used, and when possible, to use alternatives to in vivo approaches. Animals and determination of estrous cyclicity. Male and female adult Sprague Dawley rats (average
age on day of experimental measurement: female PND 77, male PND 76) (Charles River, Portage, MI) were group
housed (3 rats/cage) with food and water available ad libitum. The animal room was maintained on a 12 hour
reversed light-dark cycle at a limited range of temperature (68–79°F) and humidity (30–70%). Rats were allowed
one week to habituate to the animal facility. Following 1 week of habituation, vaginal lavage was performed in
female rats to monitor their estrous cycle. Cell cytology from lavage samples was examined microscopically as
previously described43,51. Male rats were handled in a similar manner and for a similar amount of time to approx-
imate handling of the females. Estrous cyclicity was monitored daily and followed a 4-day cycle pattern (2 days
of diestrus, 1 day of proestrus, 1 day of estrus). Females with inconsistent estrous cycles were not included for
these experiments. After 2 weeks of a consistent estrous cycle pattern (3 consecutive estrous cycles), females were
initially selected based on estrous cycle phase such that they would be in either diestrus (low estrogen) or proes-
trus (high estrogen) in 10 days from this selection. This 10 day period bridges the time from initiation of control
handling or repeated stress to the day at which experimental measures were taken (e.g. estrous females would be
diestrus 1 in 10 days; diestrus 2 females would be proestrus in 10 days). Assessment of uterine horns. After decapitation, visual inspection of the uterine horns was performed on all
females as a secondary measure to confirm estrous cycle phase determined by vaginal lavage. During proestrus
the uterus had a large, swollen, hyperemic appearance compared with those at diestrus51. To further validate
estrous cycle phase, trunk blood was collected from a subset of females at the time of decapitation. Blood samples
were immediately centrifuged, the serum collected and stored at −80 °C until quantification. Shannon R. Blume1,4, Mallika Padival1,3, Janice H. Urban2,3 & J. Amiel Rosenkranz1,3 This cycle consists of physiological changes in reproductive organs along with a
coordinated modulation of sexual and affiliative behavior in a manner that optimizes reproductive success; a fol-
licular phase (proestrus and estrus) in which behaviors are aimed at reproduction, and a luteal phase (metestrus
and diestrus44). This includes several behaviors that rely on the amygdala, which can support or suppress sexual
behaviors, such as anxiety and emotional memory45–49. Recent evidence demonstrates that BLA physiology and
function shifts across the estrous cycle in rats such that LAT activity and LAT-dependent cued conditioned fear
is greatest during diestrus while BA activity and BA-dependent contextual fear is greatest during proestrus43. The second goal of this study was to determine if repeated stress disrupts the pattern of BLA changes that usually
occurs across the estrous cycle. www.nature.com/scientificreports/ Slices were mounted on gelati-
nized slides and allowed to dry (25 minutes–1 hour), and rinsed in double distilled H2O (2 times, 4 minutes each
rinse). Slides were dehydrated in 50%, 75% and 95% ethanol for 4 minutes each, then in 100% ethanol 4 times for
3 minutes each. Slides were cleared with xylene 3 times for 4 minutes each; coverslips were applied with permount
and then allowed to dry for 2 weeks. Neuronal reconstruction. Golgi-stained LAT and BA neurons were reconstructed using Neurolucida software
(MBF Bioscience, Williston, VT) under bright field illumination with a 100x objective (Eclipse E400 microscope,
Nikon, Melville, NY). Rat brain atlases were used to determine LAT and BA borders53,54. Only neurons that
appeared to be fully impregnated were utilized. Neurons with breaks (>5 μm) in the dendrites were not included. Neurons were selected based on previously established properties of BLA neuron morphology (e.g. large cell
bodies with obvious primary dendrites and spines55). Fear conditioning. Behavioral measures were obtained in a separate group of rats that were not used for
electrophysiology or Golgi-Cox staining. Cued fear conditioning was performed as described previously20. Conditioning and extinction testing were performed four days apart for both males and females, such that the
female rats were in the same estrous phase during both sessions43. Conditioning was conducted in chambers
enclosed in sound-attenuating cabinets (Ugo Basile, Varese, Italy), with an audio speaker (Ugo Basile), a dim
house light, an infrared LED light and a ceiling mounted infrared-sensitive digital camera (Fire-i, Unibrain, San
Ramon, CA) in the cabinets. Conditioning consisted of 2 min habituation followed by 5 pairings of a neutral tone
(10 s, 1500 Hz, 85 dB) with a co-terminating foot shock (1 s, 0.3–0.5 mA). Conditioning trials were presented at
60 s inter-trial intervals. Rats remained in the chamber for 1 min after the end of the last conditioning trial, and
then were returned to their home cage. Four days later, conditioned freezing and within session acquisition of
extinction were tested in a contextually distinct chamber (different wall pattern and color, odors, and flooring) to
minimize contextual freezing. Testing consisted of a 2 min habituation followed by 15 trials of tone presentation
(20 s, 1500 Hz, 85 dB) at a 60 s intertrial interval. No foot shock was presented during testing trials. After testing,
animals were returned to their home cage. www.nature.com/scientificreports/ of surgery and electrode placements. The BLA includes the lateral nucleus (LAT) and the basal nucleus (BA). To ensure similar sampling of LAT and BA across animals, electrode penetrations through the BLA followed
a predetermined grid of coordinates. During electrophysiological recordings a digital oscilloscope (2530 B&K
Precision, Yorba Linda, CA) and audio monitor (AM10 Grass, West Warwick, RI) were used to monitor signals. Signals from the recording electrode were amplified (A-M Systems, Sequim, WA) and filtered at 0.1 Hz (low
frequency) and 5 kHz (high frequency). Signals were digitized through an interface (ITC-18, HEKA Elektronik,
Holliston, NY) to Axograph X software (Sydney, Australia) on a Mac Pro computer (Apple Inc, Cupertino, CA). The anesthesia level was monitored throughout the recording and rats were considered to be deeply anesthetized
when the primary component of local field potential oscillations in the mPFC were between 0.5 and 1.0 Hz. Data
were stored on an external hard drive for off-line analysis. Histology. Recording and electrode sites were verified histologically. At the end of an electrophysiological
recording, Pontamine Sky Blue was iontophoretically ejected from the recording electrode to mark the recording
site (at least 20 min, −30.0 μA, constant current source, Fintronics, Orange, CT). Stimulation electrode place-
ment was marked by iron deposits from applying 3 stimulation pulses (1 mA, 10 second duration) as previously
described52. Immediately following the marking, rats were decapitated and their brains removed. Brains were
stored in 4% paraformaldehyde with 0.05% potassium ferrocyanide in 0.1 M phosphate buffer for 12 hours and
then stored in 25% sucrose in 0.1 M phosphate buffer until sectioning. Brains were cut into 60 μm sections using
a freezing microtome (Leica Microsystems Inc., Wetzlar, Germany) and then Nissl stained. Recording and stimu-
lation sites were verified by light microscopy and reconstructed using a rat brain atlas53. Golgi-Cox staining of BLA neurons. Female and male brains were rapidly removed, blocked coronally to
contain all of the BLA and processed for Golgi-Cox staining according to manufacturer recommended protocol
(FD Rapid GolgiStain Kit, FD Neuro Technologies, Columbia, MD). Throughout the processing, brains were
stored in containers protected from light. Brain slices were collected into 20% sucrose in 0.1 M phosphate buffer
(pH 7.4) at room temperature (100 μm thickness, SM 2000 R microtome, Leica). Materials and Methods The recording electrode was lowered into the BLA (−4.8 to −5.6 mm lateral (L),
−2.8 to −3.8 mm caudal © from bregma and 6.5 to 9.0 mm V) using a hydraulic micromanipulator (model 640,
David Kopf Instruments). Electrophysiological recordings started no earlier than 1 hour after the completion Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 www.nature.com/scientificreports/ Results
Eff
t Effects of stress on estrous cyclicity. Repeated restraint stress increased adrenal gland weight in male
and female rats (main effect of stress, F(1,140) = 5.948, p = 0.0160, two-way ANOVA; p < 0.05 Holm-Sidak’s
multiple comparisons test, n = 65 control female, n = 31 stress female, n = 26 control male, n = 22 stress male),
indicating that this was an effective stressor in both sexes. Repeated restraint stress did not disrupt the regular
estrous cyclicity of the female rats. Control and stress rats showed consistent 4 day cycles as assessed by vaginal
lavage, and uterine size that matched diestrus and proestrus states. Estradiol levels were higher during proestrus
(Fig. 1; main effect of phase, F(1,17) = 9.627, p = 0.0065, two-way ANOVA, n = 4–7 rats/group), and repeated
stress increased estradiol levels (main effect of stress, F(1,17) = 31.87, p < 0.0001), but the change in estradiol
levels across estrous was similar in control and stress groups (Fig. 1; [average estradiol]proestrus − [estradiol]diestrus;
p = 0.252, t = 1.224, df = 9, two-tailed t-test). In vivo extracellular recordings. Firing rate of neurons. Recordings from LAT and BA nuclei of control
rats (Fig. 1; 39 female rats and 14 male rats) were compared with recordings from rats exposed to repeated stress
(Fig. 2A; 24 female rats and 12 male rats). Repeated stress increased the activity of BLA neurons in male rats
(Table 1; data not Gaussian, D’Agostino & Pearson omnibus normality test, p < 0.0001; significant difference
between medians, p = 0.0154, H(3) = 10.41, Kruskal-Wallis test; p < 0.05 Dunn’s multiple comparison post hoc
test). In contrast and contrary to our expectation, repeated stress decreased the firing of BLA neurons in female
rats (Table 1; p < 0.05 Dunn’s multiple comparison post hoc test). Because this data is a key finding, we normal-
ized data into a Gaussian distribution (Log10 transformation, D’Agostino & Pearson omnibus normality test,
p > 0.05) and compared groups with more powerful parametric tests to decrease the risk of an erroneous false
positive. This analysis uncovered a significant interaction between stress and sex on BLA neuron firing rate (sex
× stress interaction, p = 0.0112, F(1,255) = 6.527, two-way ANOVA, 1 outlier removed from LAT control male
group), confirming that repeated stress had different effects on BLA neuron firing rate in females and males. Relative number of active neurons. www.nature.com/scientificreports/ Data was collected by a computer (Dell E6500, Austin, TX) running
video-tracking software (ANY-maze software, Stoelting Co, Wood Dale, IL) that detects and records freezing
behavior. Data analysis. When possible, data was collected or analyzed blind to sex and/or treatment. Male and female
control and stress groups were run in parallel. Spontaneous neuronal activity. Recorded neurons were included in analyses if they were located within the BLA
and if the measured action potentials were readily discernable from noise (signal to noise ratio of at least 3:1). The firing rate of BLA neurons was determined as the number of action potentials per second (Hz). The number
of spontaneously active cells recorded per electrode track was measured and is reflective of the overall relative
neuronal firing activity of a structure (West and Grace, 2000). Cells recorded when the primary mPFC local field
potential oscillation frequency was outside a range of 0.5–1.0 Hz (possibly indicating fluctuation in depth of anes-
thesia) were not included in the data analysis. Neuronal morphology. Neurons that had large somata with bipolar, aspiny dendrites or small somata with few,
aspiny dendrites were not included in this analysis. Sholl analysis was used to measure dendritic length and
number of spines within concentric circles at increasing diameters of 10 μm steps from reconstructed neurons Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 www.nature.com/scientificreports/ (Sholl, 1953). Photographs of each individual LAT or BA neuron were collected using 10, 20 and 100x objectives
at similar light conditions. (Sholl, 1953). Photographs of each individual LAT or BA neuron were collected using 10, 20 and 100x objectives
at similar light conditions. Calculation of changes across estrous cycle. To measure the direction and magnitude of changes across the
estrous cycle, measures of neuronal activity, morphology and rodent behavior obtained during proestrus were
normalized to measures obtained during diestrus. This was accomplished by obtaining the average value of the
group in diestrus and subtracting this from each proestrus value [Individual Valueproestrus] – [Averagediestrus]. A
value close to zero indicates little change across the estrous cycle, >0 indicates a measure that peaks in proestrus,
<0 indicates a measure that peaks in diestrus. Statistical analysis. Statistical analysis was performed using Prism 5 software (GraphPad, La Jolla, CA). Data were compared between males and females (diestrus and proestrus combined), and compared between
diestrus and proestrus females. A p < 0.05 was considered statistically significant. www.nature.com/scientificreports/ Data were tested for normality
using the D’Agostino and Pearson omnibus normality test. If data failed the test for normality, two groups were
compared by Mann-Whitney U test while more than 2 groups were compared by non-parametric Kruskal-Wallis
test, followed by a Dunn’s multiple comparison test for significant differences between groups. If data were
normally distributed, they were screened for outliers (ROUT, Q = 0.5%; removal of any outlier is listed in the
Results). Two groups were compared with two-tailed unpaired t-test, while more than two groups were compared
with one-way Analysis of Variance (ANOVA). When comparing across more than one factor, two-way ANOVA
or two-way repeated measures ANOVA was used as appropriate. Significant results in ANOVA comparisons were
followed by post hoc Holm-Sidak’s multiple comparisons test to minimize Type I errors. Results
Eff
t In addition to impacting neuronal firing, repeated stress may impact the
number of active neurons in the BLA. To examine this possibility, we measured the number of spontaneously
active neurons encountered and recorded in the first electrode penetration of each animal. The number of active
neurons per electrode track in females (control: 3.00 ± 0.25, 39 rats; stress: 3.15 ± 0.31, 24 rats) was similar to
males (control: 2.63 ± 0.30, 12 rats; stress: 3.14 ± 0.42, 14 rats; main effect of sex, p = 0.557, two-way ANOVA,
F(1,90) = 0.347; data not depicted), and repeated stress did not significantly impact the number of active neu-
rons (main effect of stress, p = 0.316, F(1,90) = 1.018; stress × sex interaction p = 0.588, F(1,90) = 0.295, two-way
ANOVA; data not depicted). Firing rate of LAT and BA neurons. The LAT and BA nuclei of the BLA are functionally distinct in the afferents
they receive and their roles in behavior. We therefore examined whether restraint stress had a different impact on
neuronal firing rate in the LAT and BA between female and male groups. Stress increased the firing rate of LAT
neurons in males (Fig. 2B, Table 1; sex × stress interaction, p = 0.0322, F(1,122) = 4.694; main effect of stress,
p = 0.7621, F(1,122) = 0.09205, two-way ANOVA on transformed data, 1 outlier removed from LAT control male
group; males, p < 0.05 Holm-Sidak’s multiple comparisons test) but significantly decreased the firing of LAT
neurons in female rats (females, p < 0.05, Holm-Sidak’s multiple comparisons test). In the BA, repeated stress
significantly increased the firing of neurons in the BA of male rats (Fig. 3B, Table 1; sex × treatment interaction,
p = 0.045, F(1,129) = 4.111, two-way ANOVA on transformed data; males, p < 0.05, Holm-Sidak’s multiple com-
parisons test) but significantly decreased the firing of BA neurons in female rats (females, p < 0.05, Holm-Sidak’s
multiple comparisons test). Thus, repeated stress had opposite effects in males and females on both LAT and BA
neuron firing. Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 www.nature.com/scientificreports/ Figure 1. Effects of stress on endocrine measures. (A) Repeated stress caused an increase in adrenal gland
weight. (B) Estradiol levels were measured from trunk blood samples and were greater in proestrus compared
to diestrus. Repeated stress caused an increase of estradiol, but there was still higher estradiol in proestrus
compared to diestrus. Results
Eff
t (C) To quantitatively compare the change that occurs between diestrus and proestrus
(cyclicity), proestrus values were subtracted from the diestrus value. When presented graphically, as in this
schematic example, values above 0 signify that the value was greater during proestrus, while values below 0
signify that the value was greater during diestrus. (D) Estradiol levels were greater during proestrus (value > 0),
and the degree of cyclicity was not different between control and stress. (E) A representation of nucleus borders
(left; modified from97), and borders overlaid on a Nissl stained section of tissue (right). All values mean ± s.e.m. *p < 0.05 Holm-Sidak’s multiple comparisons test after 2-way ANOVA. Figure 1. Effects of stress on endocrine measures. (A) Repeated stress caused an increase in adrenal gland
weight. (B) Estradiol levels were measured from trunk blood samples and were greater in proestrus compared
to diestrus. Repeated stress caused an increase of estradiol, but there was still higher estradiol in proestrus
compared to diestrus. (C) To quantitatively compare the change that occurs between diestrus and proestrus
(cyclicity), proestrus values were subtracted from the diestrus value. When presented graphically, as in this
schematic example, values above 0 signify that the value was greater during proestrus, while values below 0
signify that the value was greater during diestrus. (D) Estradiol levels were greater during proestrus (value > 0),
and the degree of cyclicity was not different between control and stress. (E) A representation of nucleus borders
(left; modified from97), and borders overlaid on a Nissl stained section of tissue (right). All values mean ± s.e.m. *p < 0.05 Holm-Sidak’s multiple comparisons test after 2-way ANOVA. Firing rate across estrous. We previously found that the firing of BLA neurons in females fluctuates across the
estrous cycle, whereby the firing of LAT neurons is higher in diestrus, and the firing of BA neurons is higher
during proestrus43. That pattern was replicated here (Figs 2C and 3C; cycle × nucleus interaction, p = 0.026,
F(1,109) = 5.098, two-way ANOVA on transformed data). To test whether stress impacted this pattern that shifts
across estrous, a single measure of change across estrous was quantified (Methods; [firing rate]proestrus – [firing
rate]diestrus). In females exposed to stress, the pattern was disrupted such that there was no change in LAT activity
across estrous (Fig. Results
Eff
t 2D; main effect of stress, p = 0.049, F(1,65) = 4.025, two-way ANOVA on transformed data;
not significantly different than 0 after stress, p > 0.05, one sample t-test), but the pattern of change of BA activity
across estrous was even greater in rats exposed to stress (Fig. 3D; p < 0.05, Holm-Sidak’s multiple comparisons
test). Firing pattern. The firing of BLA neurons is driven by synaptic inputs. Variability of neuronal firing pattern can
reflect the excitatory-inhibitory balance of inputs and network state of that region56–60. One way to quantify this is
by coefficient of variation (CV) of the interspike-interval of firing for each neuron. Stress did not impact the over-
all CV of LAT neuron firing (Fig. 4A; stress x sex interaction, p = 0.767, F(1,122) = 0.0880; main effect of stress,
p = 0.89, F(1,122) = 0.021) or BA neuron firing BA (Fig. 4B; stress × sex interaction, p = 0.942, F(1,129) = 0.005;
main effect of stress, p = 0.81, F(1,129) = 0.057, two-way ANOVA). f
p
y
While stress did not impact overall CV, it may impact the pattern of CV changes across the estrous cycle. In the LAT, CVs were not different between the cycle stages (Fig. 4C; phase × stress interaction, p = 0.591,
F(1,82) = 0.292; main effect of phase, p = 0.547, F(1,82) = 0.376, two-way ANOVA). In the BA, while there was
no difference of CV across the estrous cycle in controls, a difference of CV between cycle stages emerged after
stress (Fig. 4C; main effect of phase, p = 0.029, F(1,81) = 4.957, two-way ANOVA; phase × stress interaction,
p = 0.1622, F(1,81) = 1.981, two-way ANOVA) with increased CV during proestrus (p < 0.05, Holm-Sidak’s mul-
tiple comparisons test). The change of CV across estrous ([CV]proestrus − [CV]diestrus) was significantly increased Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 www.nature.com/scientificreports/ Figure 2. Repeated stress decreased firing rate and cyclicity of LAT neurons in females. (A) Single LAT neurons
were recorded from male and female rats. (B) The firing rate of LAT neurons was decreased after repeated stress
in female rats, but was increased by repeated stress in male rats (left, mean + 95% confidence interval (C.I.);
right, log10 transformed data mean ± s.e.m.). (C) The firing rate of LAT neurons was lower during proestrus in
control rats. Table 1. The mean firing rate of neurons recorded in vivo. The mean ± s.e.m. is presented along with the
number of neurons. Because multiple neurons were usually recorded, the number of rats is also shown here. Table 1. The mean firing rate of neurons recorded in vivo. The mean ± s.e.m. is presented along with the
number of neurons. Because multiple neurons were usually recorded, the number of rats is also shown here. The mean firing rate of neurons recorded in vivo. The mean ± s.e.m. is presented along with the
f neurons. Because multiple neurons were usually recorded, the number of rats is also shown here. Table 1. The mean firing rate of neurons recorded in vivo. The mean ± s.e.m. is presented along with the
b
f
B
l i l
ll
d d h
b
f
i
l
h
h Results
Eff
t But there was no difference between diestrus and proestrus after repeated stress (left, mean + 95%
C.I.; right, log10 transformed data mean ± s.e.m.). (D) The cyclicity index was decreased after repeated stress. *p < 0.05 Holm-Sidak’s multiple comparisons test after 2-way ANOVA. Figure 2. Repeated stress decreased firing rate and cyclicity of LAT neurons in females. (A) Single LAT neurons
were recorded from male and female rats. (B) The firing rate of LAT neurons was decreased after repeated stress
in female rats, but was increased by repeated stress in male rats (left, mean + 95% confidence interval (C.I.);
right, log10 transformed data mean ± s.e.m.). (C) The firing rate of LAT neurons was lower during proestrus in
control rats. But there was no difference between diestrus and proestrus after repeated stress (left, mean + 95%
C.I.; right, log10 transformed data mean ± s.e.m.). (D) The cyclicity index was decreased after repeated stress. *p < 0.05 Holm-Sidak’s multiple comparisons test after 2-way ANOVA. Sex
Tx
BLA
LAT
BA
mean
n
mean
n
mean
n
Rats
Male
control
0.67 ± 0.25
42
14
stress
1.72 ± 0.46
34
12
Female
control
2.26 ± 0.37
113
1.87 ± 0.54
57
2.66 ± 0.54
56
39
stress
1.23 ± 0.22
70
0.81 ± 0.30
29
1.53 ± 0.29
41
24
diestrus
control
2.31 ± 0.77
38
2.26 ± 0.63
32
23
stress
0.40 ± 0.09
15
1.19 ± 0.34
29
15
proestrus
control
0.99 ± 0.42
19
3.19 ± 0.96
24
16
stress
1.26 ± 0.61
14
2.35 ± 0.54
12
9hih Table 1. The mean firing rate of neurons recorded in vivo. The mean ± s.e.m. is presented along with the
number of neurons. Because multiple neurons were usually recorded, the number of rats is also shown here. Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 www.nature.com/scientificreports/ Figure 3. Repeated stress decreased firing rate and increased cyclicity of BA neurons in females. (A) Single
BA neurons were recorded from male and female rats. (B) The firing rate of BA neurons was decreased after
repeated stress in female rats, but was increased by repeated stress in male rats (left, mean + 95% C.I.; right,
log10 transformed data mean ± s.e.m.). (C) The firing rate of BA neurons was greater in proestrus compared
to diestrus in control rats and after repeated stress (left, mean + 95% C.I.; right, log10 transformed data mean
± s.e.m.). Results
Eff
t (D) This sensitivity to estrous was increased after repeated stress, reflected as increase in the cyclicity
index after repeated stress (mean ± s.e.m.). *p < 0.05 Holm-Sidak’s multiple comparisons test after 2-way
ANOVA. Figure 3. Repeated stress decreased firing rate and increased cyclicity of BA neurons in females. (A) Single
BA neurons were recorded from male and female rats. (B) The firing rate of BA neurons was decreased after
repeated stress in female rats, but was increased by repeated stress in male rats (left, mean + 95% C.I.; right,
log10 transformed data mean ± s.e.m.). (C) The firing rate of BA neurons was greater in proestrus compared
to diestrus in control rats and after repeated stress (left, mean + 95% C.I.; right, log10 transformed data mean
± s.e.m.). (D) This sensitivity to estrous was increased after repeated stress, reflected as increase in the cyclicity
index after repeated stress (mean ± s.e.m.). *p < 0.05 Holm-Sidak’s multiple comparisons test after 2-way
ANOVA. in BA by stress (stress × nucleus interaction, p = 0.0105, F(1,100) = 6.799, two-way ANOVA; BA, p < 0.05,
Holm-Sidak’s multiple comparisons test), but not in LAT (LAT, p > 0.05, Holm-Sidak’s multiple comparisons
test). These results indicate that, while overall CV of LAT or BA neuron firing was not sensitive to stress, the
fluctuation of BA neuron CV across estrous was sensitive to stress. Because CV can reflect synaptic input effects
on firing, this sensitivity suggests that stress impacts whether inputs to BA neurons shift over the estrous cycle. Golgi-Cox staining of BLA neurons. Excitatory inputs drive BLA neuronal firing and are sensitive to
repeated stress in males61–63. The dendrites are a key site for synaptic inputs. As the majority of excitatory inputs
into the BLA synapse on the spines of pyramidal neurons64–66, spine number is used as an index for excitatory
input and is sensitive to stress in males (Vyas et al., 2002, #5370; Padival et al., 2013a, 2013b). Dendritic length is
sensitive to stress in males, and can play a functional role in neuronal responsiveness. To investigate changes in
dendritic spines and morphology, Golgi-impregnated BLA pyramidal neurons were reconstructed for morpho-
logical analysis (Fig. 5A,B).if g
y
( g
)
Dendritic length. In agreement with our previous studies, stress had no significant effect on dendritic length
of LAT neurons in male rats (Fig. Results
Eff
t 5C; p = 0.6903, t = 0.4030, df = 26; N = 14 control, N = 14 stress, two-tailed Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 www.nature.com/scientificreports/ www.nature.com/scientificreports
cientificreports/
Figure 4. The variability of neuronal firing across the cycle was altered by stress. The coefficient of neuronal
firing (CV) was measured as an index of firing variability. (A) The overall CV of LAT neuron firing was not
impacted by repeated stress (left). The CV was not significantly different between diestrus and proestrus in
control rats or after repeated stress (right). (B) The cyclicity index for CV of LAT neurons was similar between
control and stress groups. (C) The overall CV of BA neuron firing was not impacted by repeated stress (left). While CV of BA neurons was similar between proestrus and diestrus in control groups, after repeated stress
the CV was significantly higher in proestrus compared to diestrus (right). (D) The cyclicity index for CV of BA
neurons was increased by repeated stress. All data mean ± s.e.m. *p < 0.05 Holm-Sidak’s multiple comparisons
test after 2-way ANOVA. unpaired t-test), and increased the dendritic length of BA neurons in male rats (Fig. 5F; p = 0.0183, t = 2.505
df = 28; N = 16 control, N = 14 stress, two-tailed unpaired t-test). Very different effects of stress were found in females. Repeated stress decreased dendritic length of LAT neu
Figure 4. The variability of neuronal firing across the cycle was altered by stress. The coefficient of neuronal
firing (CV) was measured as an index of firing variability. (A) The overall CV of LAT neuron firing was not
impacted by repeated stress (left). The CV was not significantly different between diestrus and proestrus in
control rats or after repeated stress (right). (B) The cyclicity index for CV of LAT neurons was similar between
control and stress groups. (C) The overall CV of BA neuron firing was not impacted by repeated stress (left). While CV of BA neurons was similar between proestrus and diestrus in control groups, after repeated stress
the CV was significantly higher in proestrus compared to diestrus (right). (D) The cyclicity index for CV of BA
neurons was increased by repeated stress. All data mean ± s.e.m. *p < 0.05 Holm-Sidak’s multiple comparisons
test after 2-way ANOVA. Figure 4. The variability of neuronal firing across the cycle was altered by stress. Results
Eff
t The coefficient of neuronal
firing (CV) was measured as an index of firing variability. (A) The overall CV of LAT neuron firing was not
impacted by repeated stress (left). The CV was not significantly different between diestrus and proestrus in
control rats or after repeated stress (right). (B) The cyclicity index for CV of LAT neurons was similar between
control and stress groups. (C) The overall CV of BA neuron firing was not impacted by repeated stress (left). While CV of BA neurons was similar between proestrus and diestrus in control groups, after repeated stress
the CV was significantly higher in proestrus compared to diestrus (right). (D) The cyclicity index for CV of BA
neurons was increased by repeated stress. All data mean ± s.e.m. *p < 0.05 Holm-Sidak’s multiple comparisons
test after 2-way ANOVA. unpaired t-test), and increased the dendritic length of BA neurons in male rats (Fig. 5F; p = 0.0183, t = 2.505,
df = 28; N = 16 control, N = 14 stress, two-tailed unpaired t-test).ff p
Very different effects of stress were found in females. Repeated stress decreased dendritic length of LAT neu-
rons in females (Fig. 5C,D; stress × distance interaction, p < 0.0001, F(29,1769) = 5.031; main effect of stress,
p = 0.0023, F(1,61) = 10.09; stress × phase interaction, p = 0.9853, F(1,59) = 0.000, two-way RM-ANOVA). The
dendritic length was not significantly different between diestrus and proestrus (main effect of phase, p = 0.1086,
F(1,59) = 2.654, two-way RM-ANOVA). Stress did not impact the pattern of dendritic length across estrous ([Le
ngthproestrus] − [Lengthdiestrus]; Fig. 5E; p = 0.9806, t = 0.02458, df = 30, two-tailed unpaired t-test).f g
proestrus]
[
g
diestrus];
g
; p
,
,
,
p
)
In contrast, while stress had minimal effect on dendritic length of BA neurons in females (Fig. 5F,G; stress ×
distance interaction, p = 0.9150, F(29,1798) = 0.6619; main effect of stress, p = 0.3873, F(1,62) = 0.7582, two-way
RM-ANOVA), the sensitivity of dendritic length to the estrous cycle was impacted by stress (Fig. 5H; stress
× phase interaction p = 0.0149, F(1,60) = 6.287; main effect of stress p = 0.3706, F(1,60) = 0.8137, two-way
ANOVA). BA neuron dendritic length in controls was higher during diestrus, but this was flipped after stress,
such that BA dendritic length was higher during proestrus (Fig. Results
Eff
t 5H; p = 0.0076, t = 2.860, df = 30, two-tailed
unpaired t-test). These results point to unique effects of stress on LAT and BA neurons from female rats, and con-
sistently demonstrate an effect of stress on the sensitivity of LAT and BA neurons to the estrous cycle. Dendritic spines. In LAT neurons, stress did not significantly change the number of spines (Fig. 6A), but
caused a proximal shift in the distribution of dendritic spines in females (Fig. 6B; stress × distance interaction,
p < 0.0001, F(29,1769) = 2.689; main effect of stress, p = 0.4267, F(1,61) = 0.6404, two-way RM-ANOVA, N = 32 Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 www.nature.com/scientificreports/ Figure 5. Unique effects of repeated stress on dendritic length of BLA neurons in females. BLA neurons were
Golgi-stained and reconstructed. (A) Examples of reconstructed LAT neurons after control (left) or stress
(right). (B) Examples of reconstructed BA neurons after control (left) or stress (right). (C) Repeated stress
decreased the dendritic length of LAT neurons in females, but had no significant effect in males. (D) Repeated
stress significantly decreased dendritic length, particularly at intermediate distances from the soma, as indicated
by Sholl analysis. (E) Dendritic length of LAT neurons was similar between diestrus and proestrus in control
and stress groups (left). Repeated stress did not significantly impact the sensitivity of LAT neurons to estrous
(right). (F) Repeated stress did not significantly impact the overall dendritic length of BA neurons in females,
but increased it in males. (G) The distribution of the dendritic length across the dendritic tree was similar in
control and stress groups, as indicated by Sholl analysis. (H) Dendritic length was shorter during proestrus
compared to diestrus in control rats. But after repeated stress, dendritic length was shorter during diestrus, and
the cyclicity index of BA neurons was flipped (right). All data mean ± s.e.m. *p < 0.05 Holm-Sidak’s multiple
comparisons test after 2-way ANOVA. Figure 5. Unique effects of repeated stress on dendritic length of BLA neurons in females. BLA neurons were
Golgi-stained and reconstructed. (A) Examples of reconstructed LAT neurons after control (left) or stress
(right). (B) Examples of reconstructed BA neurons after control (left) or stress (right). (C) Repeated stress
decreased the dendritic length of LAT neurons in females, but had no significant effect in males. Results
Eff
t (D) Repeated
stress significantly decreased dendritic length, particularly at intermediate distances from the soma, as indicated
by Sholl analysis. (E) Dendritic length of LAT neurons was similar between diestrus and proestrus in control
and stress groups (left). Repeated stress did not significantly impact the sensitivity of LAT neurons to estrous
(right). (F) Repeated stress did not significantly impact the overall dendritic length of BA neurons in females,
but increased it in males. (G) The distribution of the dendritic length across the dendritic tree was similar in
control and stress groups, as indicated by Sholl analysis. (H) Dendritic length was shorter during proestrus
compared to diestrus in control rats. But after repeated stress, dendritic length was shorter during diestrus, and
the cyclicity index of BA neurons was flipped (right). All data mean ± s.e.m. *p < 0.05 Holm-Sidak’s multiple
comparisons test after 2-way ANOVA. neurons control, 31 neurons stress). The number of spines on LAT neurons was higher on proestrus compared
to diestrus (Fig. 6C; main effect of phase, p = 0.0129, F(1,59) = 6.575, two-way RM-ANOVA), a pattern that was
not significantly impacted by stress (main effect of stress p = 0.3750, F(1,59) = 0.7990, stress × phase interaction
p = 0.3033, F(1,59) = 1.078, two-way ANOVA). Similarly, the index for change of LAT spine number in proes-
trus and diestrus (Spinesproestrus] − [Spinesdiestrus]) was not significantly impacted by stress (p = 0.1958, t = 1.323,
df = 30, two-tailed unpaired t-test).if In BA neurons, there was no significant effect of stress on spine number in females (Fig. 6D; stress × dis-
tance interaction, p = 0.7754, F(29,1798) = 0.7931; main effect of stress, p = 0.3147, F(1,62) = 1.027, two-way
RM-ANOVA, N = 32 neurons control, 32 neurons stress). The number of BA spines changed across the estrous
cycle and was higher in diestrus (main effect of phase, p = 0.0359, F(1,30) = 4.824, two-way repeated measures
ANOVA). However, stress flipped the effects of estrous cycle on spine number in the BA so that it was higher
in proestrus after stress (Fig. 6E,F; stress × phase interaction, p = 0.0237, F(1,60) = 5.387, main effect of phase,
p = 0.7716, F(1,60) = 0.085, two-way ANOVA; p < 0.05, Holm-Sidak’s multiple comparisons test). Results
Eff
t When meas-
ured directly, the index of change across the estrous cycle was significantly different, and opposite direction, 9 Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 www.nature.com/scientificreports/ Figure 6. Repeated stress altered the estrous cyclicity of dendritic spines in BA neurons. The number of spines
was quantified from reconstructed neurons. (A) Repeated stress did not significantly change the number of
spines counted in LAT neurons. (B) There was a small but significant shift in the distribution of dendritic spines
after repeated stress, toward more proximal loci, as indicated by Sholl analysis. (C) The number of LAT neuron
spines was greater in proestrus compared to diestrus, in both control and stress groups (left). Repeated stress did
not significantly impact the sensitivity of LAT neurons to the estrous cycle (right). (D) Repeated stress did not
significantly impact the number or distribution of spines in BA neurons from females, but increased the number
of spines in BA neurons from males. (E) There was a trend towards an increase in the number of spines in BA
neurons from females at proximal sites, as indicated by Sholl analysis. (F) The number of BA neuron spines was
greater during diestrus compared to proestrus in control rats, however, the number of spines was greater during
proestrus in the stress group (left). Repeated stress flipped the estrous sensitivity of BA neurons and flipped the
cyclicity index of BA neurons (right). All data mean ± s.e.m. *p < 0.05 Holm-Sidak’s multiple comparisons test
after 2-way ANOVA. Figure 6. Repeated stress altered the estrous cyclicity of dendritic spines in BA neurons. The number of spines
was quantified from reconstructed neurons. (A) Repeated stress did not significantly change the number of
spines counted in LAT neurons. (B) There was a small but significant shift in the distribution of dendritic spines
after repeated stress, toward more proximal loci, as indicated by Sholl analysis. (C) The number of LAT neuron
spines was greater in proestrus compared to diestrus, in both control and stress groups (left). Repeated stress did
not significantly impact the sensitivity of LAT neurons to the estrous cycle (right). (D) Repeated stress did not
significantly impact the number or distribution of spines in BA neurons from females, but increased the number
of spines in BA neurons from males. Results
Eff
t (E) There was a trend towards an increase in the number of spines in BA
neurons from females at proximal sites, as indicated by Sholl analysis. (F) The number of BA neuron spines was
greater during diestrus compared to proestrus in control rats, however, the number of spines was greater during
proestrus in the stress group (left). Repeated stress flipped the estrous sensitivity of BA neurons and flipped the
cyclicity index of BA neurons (right). All data mean ± s.e.m. *p < 0.05 Holm-Sidak’s multiple comparisons test
after 2-way ANOVA. between control and stress groups (p = 0.0078, t = 2.853, df = 30, two-tailed unpaired t-test; control peak in
diestrus, stress peak in proestrus). In stark contrast to females, stress increased the number of spines in BA neurons from males (stress × distance
interaction, p < 0.0001, F(29,812) = 2.520, two-way RM-ANOVA, N = 16 control, N = 14 stress), similar to previ-
ous findings38,42,62. Stress did not significantly increase the number of spines in LAT neurons from males, though
there was a strong trend (stress × distance interaction, p = 0.0696, F(29,754) = 1.425, two-way RM-ANOVA,
N = 14 control, N = 14 stress). Effects of stress on fear conditioning. Fear conditioning is strongly dependent on the BLA, and has
been shown to be sensitive to stress. Three aspects of fear conditioning were examined, acquisition of condi-
tioned freezing, initial expression of conditioned freezing, and acquisition of extinction of conditioned freezing. Acquisition of conditioned freezing was measured on the conditioning day. During tests of conditioned fear
after 4 days, the initial conditioned fear response and eventual acquisition of extinction of the conditioned fear
response were quantified. To measure the conditioned fear response, the first three conditioned freezing tone tri-
als on the testing day (before significant extinction of response is observed) were collapsed and compared across
groups. Acquisition of extinction was measured as the last 10 trials (trials 5–15). Fear conditioning. In males, there was no effect of repeated stress on acquisition of conditioned fear (Fig. 7A;
main effect of stress, p = 0.335, F(1,15) = 0.992; stress × trial interaction, p = 0.408, F(5,75) = 1.027, two-way
repeated measures ANOVA; control n = 8 rats, stress n = 9 rats). Repeated stress increased conditioned freezing
on the test day (Fig. 7B; trials 1–3, main effect of stress, p = 0.0310, F(1,16) = 5.593, two-way repeated measures
ANOVA). Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 Results
Eff
t (A) Repeated stress did not significantly impact
freezing during fear conditioning in males. (B) The initial expression of conditioned fear (trials 1–3) and the
acquisition of extinction (trials 5–15) were both measured. Repeated stress increased the initial expression of
conditioned fear in male rats (trials 1–3), but not the acquisition of extinction (trials 5–15). (C) Repeated stress
did not significantly impact freezing during fear conditioning in females. (D) Repeated stress did increase the
initial expression of conditioned fear in female rats (trials 1–3), but decreased the acquisition of extinction
(trials 5–15). All data mean ± s.e.m. *p < 0.05 Holm-Sidak’s multiple comparisons test after 2-way ANOVA. (Fig. 7D; trials 5–15, main effect of stress, p = 0.130, F(1,56) = 2.358; stress × trial interaction, p = 0.0259,
F(10,560) = 2.060, two-way repeated measures ANOVA). This points to unique effects of stress on conditioned
fear in females.f We then tested if conditioned freezing differs between diestrus and proestrus, and if this is disrupted by
repeated stress. There was no significant difference in acquisition of conditioned fear between diestrus and proes-
trus females, measured as freezing on the day of conditioning in either control rats (Fig. 8A; main effect of phase,
p = 0.2497, F(1,28) = 1.382; phase × trial interaction, p = 0.7390, F(5,140) = 0.5488, two-way RM-ANOVA,
control N = 15 proestrus, N = 15 diestrus) or after repeated stress (Fig. 8C; main effect of phase, p = 0.4804,
F(1,26) = 0.5126; phase × trial interaction, p = 0.9225, F(5,130) = 0.2816, two-way RM-ANOVA, repeated
stress N = 14 proestrus, N = 14 diestrus). Previous work demonstrates that the conditioned fear response shifts
across estrous43. This was replicated here, with greater conditioned freezing during diestrus compared to proes-
trus (Fig. 8B; phase × trial interaction, p = 0.0023, F(15,420) = 2.409, two-way RM-ANOVA, control N = 15
proestrus, N = 15 diestrus). The initial expression of conditioned freezing (trials 1–3) was higher during diestrus
compared to proestrus (Fig. 8E; main effect of phase, p = 0.0335, F(1,28) = 5.001, two-way RM-ANOVA, con-
trol N = 14 proestrus, N = 14 diestrus). However, repeated stress disrupted this, and there was no significant
difference between diestrus and proestrus (Fig. 8E; main effect of phase, p = 0.6881, F(1,26) = 0.1648; phase ×
trial interaction, p = 0.5876, F(2,52) = 0.5372, two-way RM-ANOVA, control N = 15 proestrus, N = 15 diestrus). Results
Eff
t But there was no effect of repeated stress on the acquisition of extinction (trials 5–15, main effect
of stress, p = 0.4672, F(1,16) = 0.555; stress × trial interaction, p = 0.981, F(10,160) = 0.299, two-way repeated
measures ANOVA).ff In females, there was no effect of repeated stress on acquisition of conditioned fear (Fig. 7C; main effect of
stress, p = 0.550, F(1,56) = 0.361; stress × trial interaction, p = 0.860, F(5,280) = 0.383, two-way repeated meas-
ures ANOVA; control n = 8 rats, stress n = 9 rats). There was a strong trend towards an effect of repeated stress
on the conditioned freezing on the test day (Fig. 7D; trials 1–3, main effect of stress, p = 0.0577, F(1,56) = 3.755,
two-way repeated measures ANOVA). However, repeated stress decreased the acquisition of extinction Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 10 www.nature.com/scientificreports/ Figure 7. Effects of repeated stress on conditioned fear. (A) Repeated stress did not significantly impact
freezing during fear conditioning in males. (B) The initial expression of conditioned fear (trials 1–3) and the
acquisition of extinction (trials 5–15) were both measured. Repeated stress increased the initial expression of
conditioned fear in male rats (trials 1–3), but not the acquisition of extinction (trials 5–15). (C) Repeated stress
did not significantly impact freezing during fear conditioning in females. (D) Repeated stress did increase the
initial expression of conditioned fear in female rats (trials 1–3), but decreased the acquisition of extinction
(trials 5–15). All data mean ± s.e.m. *p < 0.05 Holm-Sidak’s multiple comparisons test after 2-way ANOVA. Figure 7. Effects of repeated stress on conditioned fear. (A) Repeated stress did not significantly impact h Figure 7. Effects of repeated stress on conditioned fear. (A) Repeated stress did not significantly impact
freezing during fear conditioning in males. (B) The initial expression of conditioned fear (trials 1–3) and the
acquisition of extinction (trials 5–15) were both measured. Repeated stress increased the initial expression of
conditioned fear in male rats (trials 1–3), but not the acquisition of extinction (trials 5–15). (C) Repeated stress
did not significantly impact freezing during fear conditioning in females. (D) Repeated stress did increase the
initial expression of conditioned fear in female rats (trials 1–3), but decreased the acquisition of extinction
(trials 5–15). All data mean ± s.e.m. *p < 0.05 Holm-Sidak’s multiple comparisons test after 2-way ANOVA. Figure 7. Effects of repeated stress on conditioned fear. Results
Eff
t This was directly compared with similar results, repeated stress dampened the shift of conditioned freezing that
usually occurs between diestrus and proestrus (Fig. 8E, right; [Freezingproestrus] − [Freezingdiestrus]; p = 0.0063,
t = 2.970, df = 27, two-tailed unpaired t-test); instead, after repeated stress, conditioned freezing (trials 1–3) was
high in both diestrus and proestrus. Fear extinction. Previous studies found that fear extinction also changes across the estrous cycle. We repli-
cated previous findings that acquisition of fear extinction differs between diestrus and proestrus in control rats
(Fig. 8F; phase × trial interaction, p = 0.0305, F(10,280) = 2.03, two-way RM-ANOVA), measured as the average
within session freezing across extinction trials (trials 5–15). After repeated stress, there was still a significant
difference of acquisition of extinction between diestrus and proestrus (Fig. 8F; main effect of phase, p = 0.0338,
F(1,26) = 5.023; phase × trial interaction, p = 0.3837, F(10,260) = 1.073, two-way RM-ANOVA; stress × phase
interaction, p = 0.0436, F(1,54) = 4.268, two-way ANOVA). However, repeated stress flipped the sensitivity of Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 www.nature.com/scientificreports/ Fi
8 R
t d t
di
t th
t
li it
f
diti Figure 8. Repeated stress disrupts the estrous cyclicity of conditioned fear and extinction. (A) In controls,
there was no significant difference in freezing during fear conditioning between diestrus and proestrus. (B). A
significant difference in conditioned freezing during testing was observed between diestrus and proestrus in
control rats, with greater freezing during diestrus. (C) After repeated stress, there was no significant difference
in freezing during fear conditioning across the estrous cycle. (D) A significant difference in conditioned
freezing during testing was observed across the estrous cycle after repeated stress, with greater freezing during
proestrus. (E) The initial conditioned freezing during testing (trials 1–3) was compared. Initial conditioned
freezing was sensitive to the estrous cycle in controls, with greater freezing during diestrus. After repeated
stress, this sensitivity was absent and freezing was high during diestrus and proestrus. The cyclicity of the initial
conditioned response (trials 1–3) was decreased after repeated stress (right). (F) The acquisition of with-in
session extinction (Trials 5–15) was compared. Freezing during extinction trials was sensitive to the estrous
cycle in control groups, with greater freezing during diestrus. Freezing during extinction trials was also sensitive Figure 8. Discussionh However, those studies usually lack the resolution to separate nuclei of the amygdala and seldom
track ovarian phase. In fact, there are recent studies that suggest decreased amygdala activation can be observed
in some female patients with depression70,71.i p
p
Biological rhythms are ubiquitous in mammals, and exert significant adaptive functions on feeding, drinking,
sleeping and reproduction. The cascade of neural and humoral activity across the estrous cycle, particularly dur-
ing proestrus/estrus, is important for ovulation and preparing the uterus for potential impregnation; as well as
generating reproductive and social behaviors geared toward successful mating. Therefore the changes in amygdala
activity that are observed here may have important ramifications for performance of social behaviors appropriate
to different phases of the estrous cycle, specifically mating. The measured endocrine parameters, estradiol levels,
uterine ballooning, and vaginal cell cytology, were not affected by stress; however, repeated stress did alter the
pattern of BLA activity that normally occurs across the estrous cycle. Earlier studies72,73 demonstrated that dis-
ruption of BLA activity influences the expression of mating-related behaviors but has only modulatory effects on
the timing of the luteinizing hormone (LH) with no significant impact on ovulation. Our data are in alignment
with this in that stress-induced alterations in BLA activity were not associated with acyclicity as assessed by our
measured parameters. The outcome is that, relative to coordinated changes in BLA function associated with a par-
ticular estrous phase in controls, BLA activity and function now align to different estrous phase, and presumably
no longer acts to facilitate adaptive behaviors that would be congruent with the hormonal rhythm. It is not clear
what drives the normal pattern of BLA function across the estrous cycle, though it may involve alterations in the
sensitivity of the amygdala to sex hormones as has been previously demonstrated47. Furthermore, mechanisms
contributing to the development of the abnormal patterning of LAT and BA neuronal function across estrous
after stress are not known. These changes are not likely due to disruption of the estrous cycle per se, since estrous
cyclicity continued unabated. One interesting hormonal response to stress was the overall increased circulating
levels of estradiol on both days of the cycle without affecting the typical estrous pattern (higher on proestrus);
stress-induced increases in estradiol have been noted in other studies74 (but see75,76). Discussionh The goals of these experiments were to test the effects of repeated stress on the activity of the BLA in females, and
determine if stress disrupts the normal fluctuations of BLA activity and morphology that occur over the estrous
cycle. In females, repeated stress reduced BLA neuronal firing rate, produced dendritic hypotrophy and a small
shift in the distribution of spines on LAT neurons, while increasing conditioned freezing and slowing acquisition
of extinction (Fig. 9). But in males, repeated stress increased BLA neuronal firing rate, produced BA dendritic
hypertrophy and increased dendritic spines alongside increased conditioned freezing (Fig. 9). Despite different
neuronal changes, both males and females displayed increased conditioned fear. It is possible that the sex dif-
ferences in the neuronal changes observed might translate into subtle aspects of conditioned fear that were not
examined here, as supported by differences in the effects of stress on extinction. We examined darting, but the
occurrence of darts was rare, possibly due to differences in conditioning procedures or attributes of the condition-
ing chambers67. Alternatively, the neuronal changes might be more important for other BLA-related behaviors.fi g
y
g
g
p
In female rats, BLA neurons exhibited different patterns of activity that were associated with specific days of
the estrous cycle. Exposure to repeated stress interfered with these cycle stage-dependent patterns of BLA activ-
ity with the LAT and BA being differentially altered by stress (Fig. 9). Within the LAT, repeated stress disrupted
the fluctuations of LAT neuron firing that occur over the estrous cycle. But stress enhanced the fluctuations of
BA neuron firing over the estrous cycle and flipped the pattern of morphological changes in BA neurons that
occurs over the estrous cycle. This disrupted pattern of neuronal activity and morphology paralleled the effects
of repeated stress on BLA-dependent conditioned freezing and extinction. In control animals, there is greater
conditioned freezing during diestrus compared to proestrus, but slower extinction in proestrus. Repeated stress
dampened the changes in cued conditioned fear and flipped the changes of fear extinction that occur over estrous
(Fig. 9). Because cued fear relies heavily on LAT and fear extinction involves the BA, these changes in the pattern
of behaviors over estrous caused by repeated stress may be related to the parallel changes of LAT and BA neuronal
function. Most studies of amygdala activation in humans after stress or depression find increased activity in males
and females68,69. Results
Eff
t All data mean ± s.e.m. *p < 0.05 Holm-Sidak’s multiple comparisons test after 2-way ANOVA. extinction to the estrous cycle, such that there was slower extinction in diestrus in control rats, but slower extinc-
tion in proestrus after repeated stress (Fig. 8F, right; p = 0.0180, t = 2.519, df = 27, two-tailed unpaired t-test). Repeated stress decreased the acquisition of fear extinction in female rats such that freezing during extinction was
high in diestrus and proestrus relative to the control group. extinction to the estrous cycle, such that there was slower extinction in diestrus in control rats, but slower extinc-
tion in proestrus after repeated stress (Fig. 8F, right; p = 0.0180, t = 2.519, df = 27, two-tailed unpaired t-test). Repeated stress decreased the acquisition of fear extinction in female rats such that freezing during extinction was
high in diestrus and proestrus relative to the control group. Results
Eff
t Repeated stress disrupts the estrous cyclicity of conditione
there was no significant difference in freezing during fear conditionin
significant difference in conditioned freezing during testing was obse
control rats, with greater freezing during diestrus. (C) After repeated
in freezing during fear conditioning across the estrous cycle. (D) A si
freezing during testing was observed across the estrous cycle after rep
proestrus. (E) The initial conditioned freezing during testing (trials 1
freezing was sensitive to the estrous cycle in controls, with greater fre
stress, this sensitivity was absent and freezing was high during diestru
conditioned response (trials 1–3) was decreased after repeated stress
session extinction (Trials 5–15) was compared. Freezing during extin
cycle in control groups, with greater freezing during diestrus. Freezin Figure 8. Repeated stress disrupts the estrous cyclicity of conditioned fear and extinction. (A) In controls,
there was no significant difference in freezing during fear conditioning between diestrus and proestrus. (B). A
significant difference in conditioned freezing during testing was observed between diestrus and proestrus in
control rats, with greater freezing during diestrus. (C) After repeated stress, there was no significant difference
in freezing during fear conditioning across the estrous cycle. (D) A significant difference in conditioned
freezing during testing was observed across the estrous cycle after repeated stress, with greater freezing during
proestrus. (E) The initial conditioned freezing during testing (trials 1–3) was compared. Initial conditioned
freezing was sensitive to the estrous cycle in controls, with greater freezing during diestrus. After repeated
stress, this sensitivity was absent and freezing was high during diestrus and proestrus. The cyclicity of the initial
conditioned response (trials 1–3) was decreased after repeated stress (right). (F) The acquisition of with-in
session extinction (Trials 5–15) was compared. Freezing during extinction trials was sensitive to the estrous
cycle in control groups, with greater freezing during diestrus. Freezing during extinction trials was also sensitive Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 www.nature.com/scientificreports/ to the estrous cycle in stress groups, but with higher freezing during proestrus, and overall higher levels of
freezing than controls. The cyclicity of extinction freezing was reversed between control and stress groups
(right). All data mean ± s.e.m. *p < 0.05 Holm-Sidak’s multiple comparisons test after 2-way ANOVA. to the estrous cycle in stress groups, but with higher freezing during proestrus, and overall higher levels of
freezing than controls. The cyclicity of extinction freezing was reversed between control and stress groups
(right). Discussionh Dendritic length of BA neurons shifts across estrous in a manner opposite to LAT, with
greater length during diestrus. This pattern is flipped after repeated stress. In males, a lower dendritic length
relative to females is increased after repeated stress. (C) Spine number of LAT neurons shifts across estrous,
with higher number of spines during proestrus. This pattern is maintained after repeated stress, but with greater
swings in spine number across estrous. In males, LAT neuron spine number is not greatly increased by repeated
stress. Spine number of BA neurons shifts across estrous, with higher number of spines during diestrus. This
pattern is flipped after repeated stress. In males, a lower number of BA neuron spines relative to females is
greatly increased after repeated stress. (D) Expression of condition fear shifts across estrous, with higher
freezing during diestrus. After repeated stress, conditioned freezing is uniformly high across estrous. In males,
conditioned freezing is higher after repeated stress. Acquisition of fear extinction shifts across the estrous cycle,
with better acquisition (lower freezing) in proestrus compared to diestrus. This pattern is flipped after repeated
stress, with overall decreased acquisition of extinction. In males, acquisition of fear extinction is not greatly
impacted after repeated stress. Figure 9. Summary of the effects of stress on BLA neurons and fear behaviors. The effects of stress on
neuronal properties and fear conditioning behaviors are represented across the estrous cycle. The pattern
in control animals (black line) often shows a shift between diestrus and proestrus (left to right). The pattern
after repeated stress (grey line) is represented similarly. For comparison, control and stress male groups are
summarized (right). These summaries are presented for LAT (left column) and BA (right column). (A) The
firing activity of LAT neurons is higher in diestrus than proestrus, and this pattern is flipped after repeated
stress (with an overall lower firing if collapsed across estrous). In males, a low LAT firing activity is greatly
increased after repeated stress. The firing activity of BA neurons is higher is proestrus than diestrus. While
this pattern is maintained after repeated stress, the firing of BA neurons is decreased across estrous. In males,
a low BA firing activity is greatly increased after repeated stress. (B) Dendritic length of LAT neurons shifts
across estrous, with greater length during proestrus. This pattern is maintained after repeated stress, but with
decreased dendritic length across estrous. Discussionh This pattern is maintained after repeated stress, but with
decreased dendritic length across estrous. In males, the dendritic length of LAT neurons is not greatly changed
after repeated stress. Dendritic length of BA neurons shifts across estrous in a manner opposite to LAT, with
greater length during diestrus. This pattern is flipped after repeated stress. In males, a lower dendritic length
relative to females is increased after repeated stress. (C) Spine number of LAT neurons shifts across estrous,
with higher number of spines during proestrus. This pattern is maintained after repeated stress, but with greater
swings in spine number across estrous. In males, LAT neuron spine number is not greatly increased by repeated
stress. Spine number of BA neurons shifts across estrous, with higher number of spines during diestrus. This
pattern is flipped after repeated stress. In males, a lower number of BA neuron spines relative to females is
greatly increased after repeated stress. (D) Expression of condition fear shifts across estrous, with higher
freezing during diestrus. After repeated stress, conditioned freezing is uniformly high across estrous. In males,
conditioned freezing is higher after repeated stress. Acquisition of fear extinction shifts across the estrous cycle,
with better acquisition (lower freezing) in proestrus compared to diestrus. This pattern is flipped after repeated
stress, with overall decreased acquisition of extinction. In males, acquisition of fear extinction is not greatly
impacted after repeated stress. Figure 9. Summary of the effects of stress on BLA neurons and fear behaviors. The effects of stress on
l
i
d f
di i
i
b h
i
d
h
l
Th Figure 9. Summary of the effects of stress on BLA neurons and fear behaviors. The effects of stress on
neuronal properties and fear conditioning behaviors are represented across the estrous cycle. The pattern
in control animals (black line) often shows a shift between diestrus and proestrus (left to right). The pattern
after repeated stress (grey line) is represented similarly. For comparison, control and stress male groups are
summarized (right). These summaries are presented for LAT (left column) and BA (right column). (A) The
firing activity of LAT neurons is higher in diestrus than proestrus, and this pattern is flipped after repeated
stress (with an overall lower firing if collapsed across estrous). In males, a low LAT firing activity is greatly
increased after repeated stress. The firing activity of BA neurons is higher is proestrus than diestrus. Discussionh These elevated levels of
estradiol could impact the BLA and therefore interfere with the normal cycle-related patterns of BLA activity and
BLA function. Additionally, other sex hormones (such as progesterone) could also contribute to the altered firing
patterns of LA and BA neurons at the different cycle times. f
y
A tight association of gonadal hormone levels and limbic function has been observed across the estrous cycle
and a mismatch between these rhythms is associated with the development of psychiatric disorders. Healthy
women in the luteal phase display higher left amygdala reactivity than in their follicular phase, but this pattern
of amygdala activity is reversed in women with premenstrual dysphoric disorder77, and the suppressive effects of
estradiol on amygdala activation across the menstrual cycle is impaired in women with major depression78. While
the functional importance of this mismatch in humans is not known, our results suggest that repeated stress can
cause a similar mismatch. Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 13 www.nature.com/scientificreports/ Figure 9. Summary of the effects of stress on BLA neurons and fear behaviors. The effects of stress on
neuronal properties and fear conditioning behaviors are represented across the estrous cycle. The pattern
in control animals (black line) often shows a shift between diestrus and proestrus (left to right). The patt
after repeated stress (grey line) is represented similarly. For comparison, control and stress male groups a
summarized (right). These summaries are presented for LAT (left column) and BA (right column). (A) T
firing activity of LAT neurons is higher in diestrus than proestrus, and this pattern is flipped after repeat
stress (with an overall lower firing if collapsed across estrous). In males, a low LAT firing activity is great
increased after repeated stress. The firing activity of BA neurons is higher is proestrus than diestrus. Wh
this pattern is maintained after repeated stress, the firing of BA neurons is decreased across estrous. In m
a low BA firing activity is greatly increased after repeated stress. (B) Dendritic length of LAT neurons sh
across estrous, with greater length during proestrus. This pattern is maintained after repeated stress, but
decreased dendritic length across estrous. In males, the dendritic length of LAT neurons is not greatly ch
after repeated stress. Dendritic length of BA neurons shifts across estrous in a manner opposite to LAT, w Figure 9. Discussionh Summary of the effects of stress on BLA neuro
neuronal properties and fear conditioning behaviors are
in control animals (black line) often shows a shift betwee
after repeated stress (grey line) is represented similarly. F
summarized (right). These summaries are presented for
firing activity of LAT neurons is higher in diestrus than p
stress (with an overall lower firing if collapsed across estr
increased after repeated stress. The firing activity of BA n
this pattern is maintained after repeated stress, the firing
a low BA firing activity is greatly increased after repeated
across estrous, with greater length during proestrus. This
decreased dendritic length across estrous. In males, the d
after repeated stress. Dendritic length of BA neurons shif
greater length during diestrus. This pattern is flipped afte
relative to females is increased after repeated stress. (C) S
with higher number of spines during proestrus. This patt
swings in spine number across estrous. In males, LAT ne
stress. Spine number of BA neurons shifts across estrous,
pattern is flipped after repeated stress. In males, a lower n
greatly increased after repeated stress (D) Expression of Figure 9. Summary of the effects of stress on BLA neurons and fear behaviors. The effects of stress on
neuronal properties and fear conditioning behaviors are represented across the estrous cycle. The pattern
in control animals (black line) often shows a shift between diestrus and proestrus (left to right). The pattern
after repeated stress (grey line) is represented similarly. For comparison, control and stress male groups are
summarized (right). These summaries are presented for LAT (left column) and BA (right column). (A) The
firing activity of LAT neurons is higher in diestrus than proestrus, and this pattern is flipped after repeated
stress (with an overall lower firing if collapsed across estrous). In males, a low LAT firing activity is greatly
increased after repeated stress. The firing activity of BA neurons is higher is proestrus than diestrus. While
this pattern is maintained after repeated stress, the firing of BA neurons is decreased across estrous. In males,
a low BA firing activity is greatly increased after repeated stress. (B) Dendritic length of LAT neurons shifts
across estrous, with greater length during proestrus. This pattern is maintained after repeated stress, but with
decreased dendritic length across estrous. In males, the dendritic length of LAT neurons is not greatly changed
after repeated stress. Discussionh In males, the dendritic length of LAT neurons is not greatly changed
after repeated stress. Dendritic length of BA neurons shifts across estrous in a manner opposite to LAT, with
greater length during diestrus. This pattern is flipped after repeated stress. In males, a lower dendritic length
relative to females is increased after repeated stress. (C) Spine number of LAT neurons shifts across estrous,
with higher number of spines during proestrus. This pattern is maintained after repeated stress, but with greater
swings in spine number across estrous. In males, LAT neuron spine number is not greatly increased by repeated
stress. Spine number of BA neurons shifts across estrous, with higher number of spines during diestrus. This
pattern is flipped after repeated stress. In males, a lower number of BA neuron spines relative to females is
greatly increased after repeated stress. (D) Expression of condition fear shifts across estrous, with higher
freezing during diestrus. After repeated stress, conditioned freezing is uniformly high across estrous. In males,
conditioned freezing is higher after repeated stress. Acquisition of fear extinction shifts across the estrous cycle,
with better acquisition (lower freezing) in proestrus compared to diestrus. This pattern is flipped after repeated
stress, with overall decreased acquisition of extinction. In males, acquisition of fear extinction is not greatly
impacted after repeated stress. Figure 9. Summary of the effects of stress on BLA neurons and fear behaviors. The effects of stress on
neuronal properties and fear conditioning behaviors are represented across the estrous cycle. The pattern
in control animals (black line) often shows a shift between diestrus and proestrus (left to right). The pattern
after repeated stress (grey line) is represented similarly. For comparison, control and stress male groups are
summarized (right). These summaries are presented for LAT (left column) and BA (right column). (A) The
firing activity of LAT neurons is higher in diestrus than proestrus, and this pattern is flipped after repeated
stress (with an overall lower firing if collapsed across estrous). In males, a low LAT firing activity is greatly
increased after repeated stress. The firing activity of BA neurons is higher is proestrus than diestrus. While
this pattern is maintained after repeated stress, the firing of BA neurons is decreased across estrous. In males,
a low BA firing activity is greatly increased after repeated stress. (B) Dendritic length of LAT neurons shifts
across estrous, with greater length during proestrus. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Among its initial effects, stress triggers the hypothalamic-pituitary-adrenal (HPA) axis. Numerous studies
demonstrate sex differences in the HPA axis and its response to stress79–83. Both human and rodent females have
higher levels of glucocorticoids compared to males under normal physiological conditions84–86. In response to
an acute stressor, females show a heightened glucocorticoid response, higher levels of ACTH, faster release of
glucocorticoids and a prolonged duration of elevated glucocorticoid levels compared to males86–89. This could be
the initial step in producing a sex difference in the effects of stress on BLA neurons. p
p
gff
Previous work has found that stress increases BLA neuron activity, excitability and spine number in
males38,39,42,61–63,90–93. Several mechanisms have been uncovered to explain the effects of stress on BLA activity in
males including increased excitatory input, decreased GABAergic regulation, and increased intrinsic membrane
excitability61,63,90–93. One consequence of many of the proposed mechanisms in males is a stress-induced imbal-
ance of neuronal inhibition and excitation. The balance between excitatory and inhibitory inputs determines
BLA neuron firing. While this balance was not directly measured in the current study, a change in spine number
implicates a change of excitatory synapses, while CV reflects the excitatory-inhibitory balance that promotes
firing of BLA neurons. These measures fluctuate across the estrous cycle and repeated stress altered the pattern of
fluctuation across the estrous cycle. This is preliminary support that a shift in the excitatory-inhibitory balance in
females may contribute to the effects of stress on the BLA neurons.fi yf
Changes in BLA neuron spine density were examined to reveal if stress had an effect on spines at specific loca-
tions from the cell soma. Differences in spine density from proximal to distal locations from the cell soma may
reflect impact on specific sources of afferents and will have different effects on the kinetic properties of excitatory
input and synaptic integration94–96. Spine density was increased on BA neurons at distances closer to the soma in
males38,62. In females, stress shifted spines toward more proximal locations and dampened the usual changes of
LAT neurons spine number that occur over the estrous cycle. In BA neurons however, stress led to a reversal in
the pattern of changes across estrous, such that spine number was greatest during proestrus (Fig. 9). www.nature.com/scientificreports/ Although
the specific topography of excitatory inputs to dendrites in BLA neurons is still not clear, the differential pattern
of the effects of stress on spine density in females and males may produce a sex difference in a specific source of,
or response to, afferent input to BLA.tf p
,f
p
Repeat exposure to stress is often a precipitating factor in the onset of affective disorders. Females are two
times more likely to suffer from affective disorders suggesting that there are sex-specific responses to stress in
brain regions implicated in these disorders, such as the amygdala. Although the impact of stress on the character-
istics and neuronal properties of amygdala neurons in males has been previously investigated (see above), this is
the first study to demonstrate that stress has opposing effects on BLA neuron activity between females and males. These results may help explain the disparity in cued and contextual fear conditioning and extinction observed
between sexes in response to stress and provide insight into a basis for sex differences in the effects of stress on
emotion, and perhaps in affective disorders.h p
pf
The datasets generated during and/or analyzed during the current study are available from the corresponding
author on reasonable request. References 1. Ferrari, A. J. et al. Global variation in the prevalence and incidence of major depressive disorder: a systematic review of the
epidemiological literature. Psychological Medicine 43, 471–481 (2012). p
g
y
g
2. Kessler, R. Sex and depression in the National Comorbidity Survey I: Lifetime prevalence, chronicity and recurrence. Journal o
Affective Disorders 29, 85–96 (1993). l
d
h
l
b d
h
f
l
h
(
) f
3. Kessler, R. C. Posttraumatic Stress Disorder in the National Comorbidity Survey. Archives of General Psychiatry 52, 1048 (1995). 4. Kornstein, S. G. et al. Gender differences in chronic major and double depression. Journal of Affective Disorders 60, 1–11 (2000). 4. Kornstein, S. G. et al. Gender differences in chronic major and double depression. Journal of Affective Disorders 60, 1 11 (2000
5. Scheibe, S., Preuschhof, C., Cristi, C. & Bagby, R. M. Are there gender differences in major depression and its respons
antidepressants? J Affect Disord 75, 223–235 (2003).ff p
ff
6. Weissman, M. M. et al. Sex differences in rates of depression: cross-national perspectives. J Affect Disord 29, 77–84 (1993).f ff
7. Felmingham, K. et al. Neural responses to masked fear faces: Sex differences and trauma exposure in posttraumatic stress
Journal of Abnormal Psychology 119, 241–247 (2010).f 8. Ruigrok, A. N. et al. A meta-analysis of sex differences in human brain structure. Neurosci Biobehav Rev 39, 34–50 (2014).i f
9. Schmaal, L. et al. Subcortical brain alterations in major depressive disorder: findings from the ENIGMA Major Depressive Disorder
working group. Mol Psychiatry 21, 806–812 (2016). g g
p
y
y
10. Tang, S. et al. Abnormal amygdala resting-state functional connectivity in adults and adolescents with major depressive disorder: A
comparative meta-analysis. EBioMedicine 36, 436–445 (2018).f p
y
1. Mothersill, O. & Donohoe, G. Neural effects of social environmental stress - an activation likelihood estimation meta-analysis
Psychol Med 46, 2015–2023 (2016). y
12. Breslau, N. et al. Trauma and Posttraumatic Stress Disorder in the Community. Archives of General Psychiatry 55, 626 (1998).hl 12. Breslau, N. et al. Trauma and Posttraumatic Stress Disorder i 13. Gerritsen, L. et al. The influence of negative life events on hippocampal and amygdala volumes in old age: a life-course perspe
Psychol Med 45, 1219–1228 (2015). 4. Gupta, A. et al. Early adverse life events are associated with altered brain network architecture in a sex- dependent manner. Neurobio
Stress 7, 16–26 (2017).ff 5. Merz, C. Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 Discussionh While
this pattern is maintained after repeated stress, the firing of BA neurons is decreased across estrous. In males,
a low BA firing activity is greatly increased after repeated stress. (B) Dendritic length of LAT neurons shifts
across estrous, with greater length during proestrus. This pattern is maintained after repeated stress, but with
decreased dendritic length across estrous. In males, the dendritic length of LAT neurons is not greatly changed
after repeated stress. Dendritic length of BA neurons shifts across estrous in a manner opposite to LAT, with
greater length during diestrus. This pattern is flipped after repeated stress. In males, a lower dendritic length
relative to females is increased after repeated stress. (C) Spine number of LAT neurons shifts across estrous,
with higher number of spines during proestrus. This pattern is maintained after repeated stress, but with greate
swings in spine number across estrous. In males, LAT neuron spine number is not greatly increased by repeate
stress. Spine number of BA neurons shifts across estrous, with higher number of spines during diestrus. This
pattern is flipped after repeated stress. In males, a lower number of BA neuron spines relative to females is
greatly increased after repeated stress. (D) Expression of condition fear shifts across estrous, with higher
freezing during diestrus. After repeated stress, conditioned freezing is uniformly high across estrous. In males,
conditioned freezing is higher after repeated stress. Acquisition of fear extinction shifts across the estrous cycle
with better acquisition (lower freezing) in proestrus compared to diestrus. This pattern is flipped after repeated
stress, with overall decreased acquisition of extinction. In males, acquisition of fear extinction is not greatly
impacted after repeated stress. Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 References Sex differences in the effects of acute and chronic stress and recovery after long-term stress on stress-related brain
regions of rats. Cereb Cortex 19, 1978–1989 (2009). g
6. Weathington, J. M., Puhy, C., Hamki, A., Strahan, J. A. & Cooke, B. M. Sexually dimorphic patterns of neural activity in response to
juvenile social subjugation. Behav Brain Res 256, 464–471 (2013).if 37. Westenbroek, C., Den Boer, J. A. & Ter Horst, G. J. Gender-specific effects of social housing on chronic stress-induced limbic Fos
expression. Neuroscience 121, 189–199 (2003).f 38. Padival, M. A., Blume, S. R. & Rosenkranz, J. A. Repeated restraint stress exerts different impact on structure of neurons in the lateral
and basal nuclei of the amygdala. Neuroscience 246, 230–242 (2013). and basal nuclei of the amygdala. Neuroscience 246, 230–242 (2013 yg
9. Zhang, W. & Rosenkranz, J. A. Repeated restraint stress increases basolateral amygdala neuronal activity in an age-dependen
manner. Neuroscience 226, 459–474 (2012).t manner. Neuroscience 226, 459 474 (2012). 0. Vyas, A., Pillai, A. G. & Chattarji, S. Recovery after chronic stress fails to reverse amygdaloid neuronal hypertrophy and enhanced
anxiety-like behavior Neuroscience 128 667–673 (2004) 40. Vyas, A., Pillai, A. G. & Chattarji, S. Recovery after chronic stress fails to reverse amygdaloid neuronal hypertrophy and enhanced
anxiety-like behavior. Neuroscience 128, 667–673 (2004). 41. Patel, D., Anilkumar, S., Chattarji, S. & Buwalda, B. Repeated social stress leads to contrasting patterns of structural plasticity in the
amygdala and hippocampus. Behav Brain Res 347, 314–324 (2018). yg
pp
p
42. Vyas, A., Mitra, R., Shankaranarayana Rao, B. S. & Chattarji, S. Chronic stress induces contrasting patterns of dendritic remo
in hippocampal and amygdaloid neurons. J Neurosci 22, 6810–6818 (2002).f pp
p
yg
J
(
)
43. Blume, S. R. et al. Sex- and Estrus-Dependent Differences in Rat Basolateral Amygdala. J Neurosci 37, 10567–10586 (2017).f f
44. Blaustein, J.D. In Handbook of Neurochemistry and Molecular Neurobiology, 2nd ed (ed Donald W. Pfaff, A.P.A., Anne M. E
Susan E. Fahrbach and Robert T. Rubin) 67–107 (Springer Science & Business Media, 2009). g
5. Hwang, M. J. et al. Contribution of estradiol levels and hormonal contraceptives to sex differences within the fear network during
fear conditioning and extinction. BMC Psychiatry 15, 295 (2015).hl ear conditioning and extinction. BMC Psychiatry 15, 295 (2015).hl 6. Milad, M. R. et al. The influence of gonadal hormones on conditioned fear extinction in healthy humans. References A. Contextual and Auditory Fear Conditioning are Mediated by the Lateral, Basal, and Central Amygdaloid Nuclei in
Rats. Learning &. Memory 8, 148–155 (2001). g
y
7. Nader, K. Damage to the Lateral and Central, but Not Other, Amygdaloid Nuclei Prevents the Acquisition of Auditory Fear
Conditioning. Learning &. Memory 8, 156–163 (2001). g
Conditioning. Learning &. Memory 8, 156–163 (2001). g
g
y
(
)
8. Onishi, B. K. A. & Xavier, G. F. Contextual, but not auditory, fear conditioning is disrupted by neurotoxic selective lesion of the basa
nucleus of amygdala in rats. Neurobiology of Learning and Memory 93, 165–174 (2010). g
g
y
28. Onishi, B. K. A. & Xavier, G. F. Contextual, but not auditory, fear conditioning is disrupted by ne
nucleus of amygdala in rats. Neurobiology of Learning and Memory 93, 165–174 (2010). 29. Bhatnagar, S. & Dallman, M. Neuroanatomical basis for facilitation of hypothalamic-pituitary-adrenal responses to a novel stressor
after chronic stress. Neuroscience 84, 1025–1039 (1998). t
(
)
30. Gerrits, M., Bakker, P. L., Koch, T. & Ter Horst, G. J. Stress-induced sensitization of the limbic system in ovariectomized rats is partly
restored by cyclic 17beta-estradiol administration. Eur J Neurosci 23, 1747–1756 (2006).t y y
1. Perrotti, L. I. et al. Induction of deltaFosB in reward-related brain structures after chronic stress. J Neurosci 24, 10594–10602 (2004) 31. Perrotti, L. I. et al. Induction of deltaFosB in reward-related brain structures after chronic stress. J Neurosci 24, 10594–10602 (2004). 32. Qi, C. et al. Anxiety-related behavioral inhibition in rats: a model to examine mechanisms underlying the risk to develop stress-
related psychopathology. Genes Brain Behav 9, 974–984 (2010). t
32. Qi, C. et al. Anxiety-related behavioral inhibition in rats: a model to examine mechanisms underlying the risk to develop st
related psychopathology. Genes Brain Behav 9, 974–984 (2010). p y
p
gy
(
)
33. Savonenko, A., Filipkowski, R. K., Werka, T., Zielinski, K. & Kaczmarek, L. Defensive conditioning-related functional heteroge
among nuclei of the rat amygdala revealed by c-Fos mapping. Neuroscience 94, 723–733 (1999). g
yg
y
pp g
34. Weinberg, M. S. et al. Inescapable but not escapable stress leads to increased struggling behavior and basolateral amygdala c-fos gene
expression in response to subsequent novel stress challenge. Neuroscience 170, 138–148 (2010).fft 35. Lin, Y. et al. References J. et al. Stress differentially affects fear conditioning in men and women. Psychoneuroendocrinology 38, 2529–2541 (2013) f
yf
g
y
gy
16. Bourke, C. H. & Neigh, G. N. Behavioral effects of chronic adolescent stress are sustained and sexually dimorphic. Horm Behav 60,
112–120 (2011). 17. Atchley, D., Hankosky, E. R., Gasparotto, K. & Rosenkranz, J. A. Pharmacological enhancement of calcium-activated potassium
channel function reduces the effects of repeated stress on fear memory. Behavioural Brain Research 232, 37–43 (2012). √ f
p
y
18. Conrad, C. D., Magariños, A. M., LeDoux, J. E. & McEwen, B. S. Repeated restraint stress facilitates fear conditi
independently of causing hippocampal CA3 dendritic atrophy. Behavioral Neuroscience 113, 902–913 (1999). p
y
g
pp
p
p y
19. Wood, G. E. & Shors, T. J. Stress facilitates classical conditioning in males, but impairs classical conditioning in females through
activational effects of ovarian hormones. Proc Natl Acad Sci USA 95, 4066–4071 (1998). 19. Wood, G. E. & Shors, T. J. Stress facilitates classical conditioning in males, but impairs classi
activational effects of ovarian hormones. Proc Natl Acad Sci USA 95, 4066–4071 (1998). f
20. Zhang, W. & Rosenkranz, J. A. Repeated restraint stress enhances cue-elicited conditioned freezing and impairs acquisition of
extinction in an age-dependent manner. Behav Brain Res 248, 12–24 (2013).f ction in an age-dependent manner. Behav Brain Res 248, 12–24 (20 g
p
1. Baran, S. E., Armstrong, C. E., Niren, D. C., Hanna, J. J. & Conrad, C. D. Chronic stress and sex differences on the recall of fea
conditioning and extinction. Neurobiol Learn Mem 91, 323–332 (2009). 15 Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 www.nature.com/scientificreports/ 2. Maeng, L. Y., Waddell, J. & Shors, T. J. The Prefrontal Cortex Communicates with the Amygdala to Impair Learning after Acute
Stress in Females but Not in Males. Journal of Neuroscience 30, 16188–16196 (2010).hf f
3. Waddell, J., Bangasser, D. A. & Shors, T. J. The basolateral nucleus of the amygdala is necessary to induce the opposing effects o
stressful experience on learning in males and females. J Neurosci 28, 5290–5294 (2008). p
g
24. Anglada-Figueroa, D. Lesions of the Basal Amygdala Block Expression of Conditioned Fear But Not Extinction. Journal of
Neuroscience 25, 9680–9685 (2005). 5. Bechara, A. et al. Double dissociation of conditioning and declarative knowledge relative to the amygdala and hippocampus in
humans. Science 269, 1115–1118 (1995). (
)
26. Goosens, K. References Neuroscience 168, 652–658
(2010). 7. Zeidan, M. A. et al. Estradiol modulates medial prefrontal cortex and amygdala activity during fear extinction in women and female
rats. Biol Psychiatry 70, 920–927 (2011). y
y
8. Graham, B. M. & Milad, M. R. Blockade of estrogen by hormonal contraceptives impairs fear extinction in female rats and women
Biol Psychiatry 73, 371–378 (2013).il y
y
49. Marcondes, F. K., Miguel, K. J., Melo, L. L. & Spadari-Bratfisch, R. C. Estrous cycle influences the respon
elevated plus-maze test. Physiol Behav 74, 435–440 (2001). Marcondes, F. K., Miguel, K. J., Melo, L. L. & Spadari-Bratfisch, R. C. Estrous cycle influences the response of female rats in the
evated plus-maze test. Physiol Behav 74, 435–440 (2001). p
y
50. Council, N. R. Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011).f 50. Council, N. R. Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011).f 1. Becker, J. B. et al. Strategies and methods for research on sex differences in brain and behavior. Endocrinology 146, 1650–1673
(2005). 52. Floresco, S. B. & Grace, A. A. Gating of hippocampal-evoked activity in prefrontal cortical neurons by inputs from the mediodorsal
thalamus and ventral tegmental area. J Neurosci 23, 3930–3943 (2003). Th
d
h d
(
d
) g
53. Paxinos, G. & Watson, C. The Rat Brain in Stereotaxic Coordinates, 6th edition (Academic Press, 2010). 54. Swanson, L. W. & Petrovich, G. D. What is the amygdala? Trends Neurosci 21, 323–331 (1998).h 5. McDonald, A. J. Neurons of the lateral and basolateral amygdaloid nuclei: A golgi study in the rat. The Journal of Comparative
Neurology 212, 293–312 (1982). 56. Barral, J. & Reyes, A. D. Optogenetic Stimulation and Recording of Primary Cultured Neurons with Spatiotemporal Control. Bio
Protoc 7 (2017) 57. Dorval, A. D. & White, J. A. Synaptic input statistics tune the variability and reproducibility of neuronal responses. Chaos 16, 026105
(2006).t (
)
8. Holt, G. R., Softky, W. R., Koch, C. & Douglas, R. J. Comparison of discharge variability in vitro and in vivo in cat visual cortex
neurons. J Neurophysiol 75, 1806–1814 (1996). p y
59. Nogueira, R., Lawrie, S. & Moreno-Bote, R. Neuronal Variability as a Proxy for Network State. Trends Neurosci 41, 170–173 (2018).li p y
59. Nogueira, R., Lawrie, S. & Moreno-Bote, R. Neuronal Variabilil 59. Nogueira, R., Lawrie, S. & Moreno-Bote, R. References Neuronal Variability as a Proxy for Network State. Trends Neurosci 41, 170–173 (2018). 60 Svirskis G & Rinzel J Influence of temporal correlation of synaptic input on the rate and variability of firing in neurons Biophys J 9. Nogueira, R., Lawrie, S. & Moreno Bote, R. Neuronal Variability as a Proxy for Network State. Trends Neurosci 41, 170 173 (2018)
0. Svirskis, G. & Rinzel, J. Influence of temporal correlation of synaptic input on the rate and variability of firing in neurons. Biophys
79, 629–637 (2000). 1. Suvrathan, A. et al. Stress enhances fear by forming new synapses with greater capacity for long-term potentiation in the amygdala
Philos Trans R Soc Lond B Biol Sci 369, 20130151 (2014).f 62. Padival, M., Quinette, D. & Rosenkranz, J. A. Effects of repeated stress on excitatory drive of basal amygdala neurons in vivo. Neuropsychopharmacology 38, 1748–1762 (2013). p y
p
gy
3. Rosenkranz, J. A., Venheim, E. R. & Padival, M. Chronic stress causes amygdala hyperexcitability in rodents. Biol Psychiatry 67
1128–1136 (2010). (
)
64. Brinley-Reed, M., Mascagni, F. & McDonald, A. J. Synaptology of prefrontal cortical projections to the basolateral amygdala: an
electron microscopic study in the rat. Neurosci Lett 202, 45–48 (1995). Scientific Reports | (2019) 9:12292 | https://doi.org/10.1038/s41598-019-48683-3 www.nature.com/scientificreports/ 5. Farb, C., Aoki, C., Milner, T., Kaneko, T. & LeDoux, J. Glutamate immunoreactive terminals in the lateral amygdaloid nucleus: a
possible substrate for emotional memory. Brain Res 593, 145–158 (1992). p
y
6. Rademacher, D. J., Rosenkranz, J. A., Morshedi, M. M., Sullivan, E. M. & Meredith, G. E. Amphetamine-associated contextua
learning is accompanied by structural and functional plasticity in the basolateral amygdala. J Neurosci 30, 4676–4686 (2010). 7. Gruene, T. M., Flick, K., Stefano, A., Shea, S. D. & Shansky, R. M. Sexually divergent expression of active and passive conditioned fea
responses in rats. Elife 4 (2015) 8. Sheline, Y. I. et al. Increased amygdala response to masked emotional faces in depressed subjects resolves with antidepressan
treatment: an fMRI study. Biol Psychiatry 50, 651–658 (2001). y
y
y
9. Silverman, M. E. et al. The neural processing of negative emotion postpartum: a preliminary study of amygdala function in
postpartum depression. Arch Womens Ment Health 14, 355–359 (2011). postpartum depression. Arch Womens Ment Health 14, 355–35 p
p
p
(
)
0. Holsen, L. M. et al. References Stress response circuitry hypoactivation related to hormonal dysfunction in women with major depression. Affect Disord 131, 379–387 (2011). ,
p
y yp
y
j
p
Affect Disord 131, 379–387 (2011). 1. Holsen, L. M. et al. Brain hypoactivation, autonomic nervous system dysregulation, and gonadal hormones in depression: a ff
1. Holsen, L. M. et al. Brain hypoactivation, autonomic nervous system dysregulation, and gonadal hormones in depression: a
preliminary study. Neurosci Lett 514, 57–61 (2012). p
y
y
72. Carrillo, A. J., Rabii, J., Carrer, H. F. & Sawyer, C. H. Modulation of the proestrous surge of luteinizing hormone by electrochemical
stimulation of the amygdala and hippocampus in the unanesthetized rat. Brain Res 128, 81–92 (1977).lf 3. Rodgers, C. H. & Schneider, V. M. Facilitatory influences on mating behavior in the female rat affected by lesions of the habenula or
the basolateral amygdaloid regions. Psychoneuroendocrinology 4, 237–244 (1979).f l
ral amygdaloid regions. Psychoneuroendocrinology 4, 237–244 (197 yg
g
y
gy
4. Goldman, P. R. & Vogel, W. H. Plasma estradiol and prolactin levels and their response to stress in two strains of rat with differen
sensitivities to 7,12-dimethylbenz[a]anthracene-induced tumors. Cancer Lett 25, 277–282 (1985).f y
75. Anderson, S. M. et al. Effects of chronic stress on food acquisition, plasma hormones, and the estrous cycle of female rats. Physiol
Behav 60, 325–329 (1996).f 76. Galea, L. A. et al. Sex differences in dendritic atrophy of CA3 pyramidal neurons in response to chronic restraint stress. Neuroscience
81, 689–697 (1997).f (
)
7. Gingnell, M., Morell, A., Bannbers, E., Wikström, J. & Sundström Poromaa, I. Menstrual cycle effects on amygdala reactivity to
emotional stimulation in premenstrual dysphoric disorder. Horm Behav 62, 400–406 (2012). 78. Jacobs, E. G. et al. 17β-estradiol differentially regulates stress circuitry activity in healthy and depressed women. Neuropsychopharmacology 40, 566–576 (2015). p y
p
gy
79. Goel, N., Workman, J. L., Lee, T. T., Innala, L. & Viau, V. 1121–1155 (John Wiley & Sons, Inc., 2014).f y
80. Kitay, J. I. Sex differences in adrenal cortical secretion in the rat. Endocrinology 68, 818–824 (1961).ff 80. Kitay, J. I. Sex differences in adrenal cortical secretion in the rat differences in adrenal cortical secretion in the rat. Endocrinology 6
k
ll
ll
h
l
d f
81. McCormick, C. M., Linkroum, W., Sallinen, B. J. & Miller, N. W. References Distinct effects of repeated restraint stress on basolateral amygdala neuronal membrane properties in
resilient adolescent and adult rats. Neuropsychopharmacology 39, 2114–2130 (2014). 91. Ehrlich, D. E. & Rainnie, D. G. Prenatal Stress Alters the Development of Socioemotional Behavior and Amygdala Ne
Excitability in Rats. Neuropsychopharmacology 40, 2135–2145 (2015).f y
p y
p
gy
2. Mozhui, K. et al. Strain differences in stress responsivity are associated with divergent amygdala gene expression and glutamate
mediated neuronal excitability. J Neurosci 30, 5357–5367 (2010). 93. Rau, A. R., Chappell, A. M., Butler, T. R., Ariwodola, O. J. & Weiner, J. L. Increased Basolateral Amygdala Pyramidal Cell Excitability
May Contribute to the Anxiogenic Phenotype Induced by Chronic Early-Life Stress. J Neurosci 35, 9730–9740 (2015). A d
b
d
h
ffi
f d
l
h
l CA
d l y
g
yp
y
y
94. Andreasen, M. & Lambert, J. D. Factors determining the efficacy of distal excitatory synapses in rat hippocampal CA1 pyramidal
neurones. J Physiol 507, 441–462 (1998). y
5. Larkum, M. E., Senn, W. & Lüscher, H. R. Top-down dendritic input increases the gain of layer 5 pyramidal neurons. Cereb Cortex
14, 1059–1070 (2004). 96. Magee, J. C. Dendritic integration of excitatory synaptic input. Nat Rev Neurosci 1, 181–190 (2000). 7. Swanson, L. W. Brain maps 4.0—Structure of the rat brain: An open access atlas with global nervous system nomenclature ontology
and flatmaps. J Comp Neurol 526, 935–943 (2018). Acknowledgementsh g
This work was supported by National Institutes of Health [grants R01MH084970 (JAR) and R01MH100536 (JAR,
JHU)]. The funding agency had no role in preparation or submission of this manuscript. SRB current address:
AbbVie 1 North Waukegan Road North Chicago IL 60064 g
This work was supported by National Institutes of Health [grants R01MH084970 (JAR) and R01MH100536 (JAR,
JHU)]. The funding agency had no role in preparation or submission of this manuscript. SRB current address:
AbbVie, 1 North Waukegan Road, North Chicago, IL 60064. References Peripheral and Central Sex Steroids Have Differential Effects o
HPA Axis of Male and Female Rats. Stress 5, 235–247 (2002).f 82. Mitsushima, D., Masuda, J. & Kimura, F. Sex Differences in the Stress-Induced Release of Acetylcholine in the Hippocampus and
Corticosterone from the Adrenal Cortex in Rats. Neuroendocrinology 78, 234–240 (2003). 82. Mitsushima, D., Masuda, J. & Kimura, F. Sex Differences in the Stress-Induced Release of Acetylch
Corticosterone from the Adrenal Cortex in Rats. Neuroendocrinology 78, 234–240 (2003). 83. Viau, V., Bingham, B., Davis, J., Lee, P. & Wong, M. Gender and puberty interact on the stress-induced activation of parvocellular
neurosecretory neurons and corticotropin-releasing hormone messenger ribonucleic acid expression in the rat. Endocrinology 146,
137–146 (2005).h (
)
84. Atkinson, H. C. & Waddell, B. J. The hypothalamic-pituitary-adrenal axis in rat pregnancy and lactation: circadian variation and
interrelationship of plasma adrenocorticotropin and corticosterone. Endocrinology 136, 512–520 (1995).f 5. Critchlow, V., Liebelt, R. A., Bar-Sela, M., Mountcastle, W. & Lipscomb, H. S. Sex difference in resting pituitary-adrenal function in
the rat. American Journal of Physiology-Legacy Content 205, 807–815 (1963). f
y
gy
g
y
86. Seale, J. V. et al. Gonadectomy reverses the sexually diergic patterns of circadian and stress-induced hypothalamic-pituitary-adrenal
axis activity in male and female rats. J Neuroendocrinol 16, 516–524 (2004). f
y
gy
g
y
86. Seale, J. V. et al. Gonadectomy reverses the sexually diergic patterns of circadian a
axis activity in male and female rats. J Neuroendocrinol 16, 516–524 (2004). 87. Iwasaki-Sekino, A., Mano-Otagiri, A., Ohata, H., Yamauchi, N. & Shibasaki, T. Gender differences in corticotropin and
corticosterone secretion and corticotropin-releasing factor mRNA expression in the paraventricular nucleus of the hypothalamus
and the central nucleus of the amygdala in response to footshock stress or psychological stress in rats. Psychoneuroendocrinology 34,
226–237 (2009). 88. Kant, G. J. et al. Comparison of stress response in male and female rats: Pituitary cyclic AMP and plasma prolactin, growth hormone
and corticosterone. Psychoneuroendocrinology 8, 421–428 (1983).f y
gy
9. Weinstock, M., Razin, M., Schorer-Apelbaum, D., Men, D. & McCarty, R. Gender differences in sympathoadrenal activity in rats a
rest and in response to footshock stress. Int J Dev Neurosci 16, 289–295 (1998).f 89. Weinstock, M., Razin, M., Schorer-Apelbaum, D., Men, D. & McCarty, R. Gender differences i
rest and in response to footshock stress. Int J Dev Neurosci 16, 289–295 (1998).f 90. Hetzel, A. & Rosenkranz, J. A. Author Contributions Author Contributions S.R.B., J.H.U. and J.A.R. Designed research; S.R.B., M.P., J.A.R. Performed research; S.R.B. and J.A.R. Analyzed
data; S.R.B., J.H.U. and J.A.R. Wrote the paper. S.R.B., J.H.U. and J.A.R. Designed research; S.R.B., M.P., J.A.R. Performed research; S.R.B. and J.A.R. Analyzed
data; S.R.B., J.H.U. and J.A.R. Wrote the paper. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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Plant Peroxisomes: A Factory of Reactive Species
|
Frontiers in plant science
| 2,020
|
cc-by
| 10,767
|
Plant Peroxisomes: A Factory of
Reactive Species
Francisco J. Corpas*, Salvador González-Gordo and José M. Palma
Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture, Department of Biochemistry,
Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas
(CSIC), Granada, Spain Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture, Department of Biochemistry,
Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas
(CSIC), Granada, Spain Plant peroxisomes are organelles enclosed by a single membrane whose biochemical
composition has the capacity to adapt depending on the plant tissue, developmental
stage, as well as internal and external cellular stimuli. Apart from the peroxisomal
metabolism of reactive oxygen species (ROS), discovered several decades ago, new
molecules with signaling potential, including nitric oxide (NO) and hydrogen sulfide
(H2S), have been detected in these organelles in recent years. These molecules
generate a family of derived molecules, called reactive nitrogen species (RNS) and
reactive sulfur species (RSS), whose peroxisomal metabolism is autoregulated through
posttranslational modifications (PTMs) such as S-nitrosation, nitration and persulfidation. The peroxisomal metabolism of these reactive species, which can be weaponized
against pathogens, is susceptible to modification in response to external stimuli. This
review aims to provide up-to-date information on crosstalk between these reactive
species families and peroxisomes, as well as on their cellular environment in light of
the well-recognized signaling properties of H2O2, NO and H2S. Edited by: Edited by:
Vasileios Fotopoulos,
Cyprus University of Technology,
Cyprus Reviewed by:
Christine Helen Foyer,
University of Birmingham,
United Kingdom
Olga V. Voitsekhovskaja,
Komarov Botanical Institute (RAS),
Russia Keywords: catalase, reactive oxygen, nitrogen and sulfur species, superoxide dismutase, nitric oxide,
S-nitrosation, persulfidation REVIEW
published: 03 July 2020
doi: 10.3389/fpls.2020.00853 REVIEW
published: 03 July 2020
doi: 10.3389/fpls.2020.00853 *Correspondence: For many years, peroxisomes in higher plants have been given different names, such as glyoxysomes
during seed germination and leaf senescence, as well as leaf, root and fruit peroxisomes according
to their presence in different organs and at different physiological stages (Tolbert and Essner, 1981;
Palma et al., 2018). This is explained by the presence of metabolic pathways which appear to be
specific to each type of peroxisome. However, peroxisomes, which share a number of metabolites
and enzymes common to all types of peroxisome, is now the preferred term regardless of their
specific metabolic characteristics (Pracharoenwattana and Smith, 2008). The most noteworthy
metabolites and enzymes include H2O2 and catalase, which are directly involved in the metabolism
of reactive oxygen species (ROS) (Su et al., 2018; Sousa et al., 2019). Specialty section:
This article was submitted to
Plant Physiology,
a section of the journal
Frontiers in Plant Science Received: 14 April 2020
Accepted: 27 May 2020
Published: 03 July 2020 Peroxisomes have a simple morphological constitution composed of a single membrane
surrounding an amorphous matrix. Over the last 30 years, an increasing number of new and
often unexpected components and processes in these organelles have been identified (Bueno
and del Río, 1992; del Río et al., 1992; Corpas et al., 1994, 2001, 2017a, 2019a, Barroso et al.,
1999; Reumann et al., 2009; Clastre et al., 2011; Simkin et al., 2011; Chowdhary et al., 2012;
Guirimand et al., 2012; Oikawa et al., 2015; Reumann and Bartel, 2016; Kao et al., 2018; Pan
et al., 2018, 2020; Borek et al., 2019), indicating that the plant peroxisomal metabolism and INTRODUCTION *Correspondence:
Francisco J. Corpas
javier.corpas@eez.csic.es *Correspondence:
Francisco J. Corpas
javier.corpas@eez.csic.es Citation: Corpas FJ, González-Gordo S
and Palma JM (2020) Plant
Peroxisomes: A Factory of Reactive
Species. Front. Plant Sci. 11:853. doi: 10.3389/fpls.2020.00853 July 2020 | Volume 11 | Article 853 1 Frontiers in Plant Science | www.frontiersin.org Plant Peroxisomes and Reactive Species Corpas et al. consequently
peroxisomal
enzymatic
and
non-enzymatic
components are more diverse than previously predicted. The
diverse complementary range of experimental approaches used
to identify these new peroxisomal constituents includes: (i)
the biochemical, proteomic and molecular analysis of purified
peroxisomes
combined
with
bioinformatics
methodologies
and (ii) cell biology studies of features such as immune
localization with the aid of electron microscopy and specific
fluorescent probes with appropriated controls. Although the
model plant Arabidopsis thaliana has increased our knowledge
of plant peroxisomes, it should be pointed out that studies of
peroxisomes from other plant species have been essential, as
the peroxisomal metabolism can be modulated depending on
the plant organ, development time and plant species involved. Therefore, this review principally aims to provide an update
of research on the metabolism of reactive species associated
with oxygen, nitrogen and, more recently, sulfur, as well as to
outline new challenges and possible future research perspectives
regarding crosstalk between peroxisomes and other subcellular
compartments such as oil bodies, mitochondria and plastids
which are closely related both biochemically and structurally
(Palma et al., 2006; Oikawa et al., 2019). Information on plant
peroxisomes could also be useful in relation to peroxisome
research into other organisms and vice versa. FIGURE 1 | Reactive oxygen species (ROS) produced from a sequential
one-electron reduction from oxygen. FIGURE 1 | Reactive oxygen species (ROS) produced from a sequential
one-electron reduction from oxygen. activity, leading to an increase in cellular H2O2 which acts as
a signaling molecule (Zhang et al., 2016; Kohli et al., 2019). Another sophisticated mechanism, involving the interaction of
the γb protein from the barley stripe mosaic virus with GOX,
has been reported to inhibit GOX and to facilitate infection
with the virus (Yang et al., 2018). More recently, Yamauchi
et al. (2019) observed a connection between the H2O2-generating
GOX and catalase, which is required in the stomatal movement. Thus, when there is an increase of oxidized peroxisomes they
were removed by pexophagy allowing an increase in H2O2
in guard cells which mediated the stomatal closure. This
mechanism of ROS homeostasis in guard cells seems to be
relevant in response to environmental changes. Citation: On the other
hand, the new peroxisomal small heat shock protein Hsp17.6CII,
capable of increasing catalase activity especially under stress
conditions, has been reported to be present in Arabidopsis plants
(Li et al., 2017). Frontiers in Plant Science | www.frontiersin.org PEROXISOMAL ROS METABOLISM Thus, peroxisomes
of watermelon cotyledons have two SOD isoenzymes, a
CuZn-SOD located in the matrix and a Mn-SOD that
is bound to the membrane (Bueno and del Río, 1992;
Rodríguez-Serrano et al., 2007); pea leaf peroxisomes have
a
Mn-SOD
present
in
the
matrix;
sunflower
cotyledon
peroxisomes have only a CuZn-SOD which is also located
in the matrix (Corpas et al., 1998); carnation petal and
pepper
fruit
peroxisomes
have
a
Mn-
and
an
Fe-SOD
(Droillard and Paulin, 1990; Palma et al., 2018); and olive
fruits peroxisomes contain four SOD isozymes, an Fe-SOD,
two CuZn-SOD and a Mn-SOD (López-Huertas and del
Río, 2014). Therefore, it could be hypothesized that the
presence of two or more types of SOD in peroxisomes
must have some physiological advantages. Thus, one of the
SOD isozymes could be constitutive while the other one
could be inducible under environmental or physiological
stimuli
such
as
seedling
development,
leaf
senescence
or fruit ripening. Given the capacity of ROS to mediate several PTMs,
particularly carbonylation and S-sulfenylation, certain amino
acid residues, especially arginine, lysine, threonine and proline,
are carbonylated, which affects target protein function in
many cases (Debska et al., 2012; Lounifiet al., 2013). Several
studies have identified peroxisomal proteins, such as catalase,
malate synthase and the fatty acid β-oxidation multifunctional
protein AIM1, which undergo carbonylation (Nguyen and
Donaldson, 2005; Anand et al., 2009; Mano et al., 2014;
Rodríguez-Ruiz et al., 2019). On the other hand, H2O2
can oxidize specific protein cysteine thiols to sulfenic acid
(SOH), a process known as S-sulfenylation, which usually
results in enzymatic inactivation. Using proteomic techniques,
approximately 2% of peroxisomal proteins have been reported
to be susceptible to S-sulfenylation (Akter et al., 2017; Huang
et al., 2019). This PTM has been observed to occur with
respect to fatty acid β-oxidation acyl-coenzyme A oxidase
1, the multifunctional proteins MFP2, and AIM1, as well as
amine oxidase, phosphomevalonate kinase, MDAR and NADP-
ICDH. Table 1 shows a summary of peroxisomal enzymes
targeted by carbonylation and S-sulfenylation, as well as other
PTMs mediated by RNS and RSS, a subject which will be
discussed below. In addition, it is worth noting the role of ascorbate-glutathione
cycle components, including ascorbate peroxidase (APX),
monodehydroascorbate reductase (MDAR), dehydroasrcorbate
reductase (DAR) and glutathione reductase (GR) (Jiménez
et al., 1998; Romero-Puertas et al., 2006; López-Huertas
and del Río, 2014; Corpas et al., 2017a). PEROXISOMAL ROS METABOLISM Several enzymes involved in
the
catabolism
of
polyamine,
including
H2O2-producing July 2020 | Volume 11 | Article 853 2 Plant Peroxisomes and Reactive Species Corpas et al. polyamine
oxidase
(PAO)
and
copper
amino
oxidase
(CuAO),
have
been
reported
to
be
present
in
plant
peroxisomes (Moschou et al., 2008; Kusano et al., 2015). These enzymes are also involved in the γ-aminobutyric acid
(GABA) biosynthesis signaling pathway (Zarei et al., 2015;
Corpas et al., 2009b). and Trelease, 1998) and leaf senescence (Ribeiro et al., 2017). To maintain the ascorbate-glutathione cycle at the GR level,
NADPH needs to be supplied by NADP-dependent endogenous
dehydrogenases including glucose-6-phosphate dehydrogenase
(G6PDH),
6-phosphogluconate
dehydrogenase
(6PGDH)
and isocitrate dehydrogenase (NADP-ICDH) (Leterrier et al.,
2016; Corpas and Barroso, 2018b and references therein). In
addition, Corpas et al. (2017b) have reported the presence of
a protein immunologically related to plant peroxiredoxins,
whose expression is differentially modulated under oxidative
stresses such as those induced by CdCl2 and the herbicide
2,4-dichlorophenoxyacetic
acid
(2,4-D);
however,
further
research is necessary to clarify this phenomenon. Figure 2
shows a working model of the ROS metabolism and its
interaction with other reactive species, including NO and H2S,
which modulate the activity of peroxisomal enzymes through
posttranslational modifications (PTMs), events which will be
further discussed below. In addition, peroxisomal xanthine oxidoreductase (XOR)
and superoxide dismutase (SOD), key enzymes in O2•−and
H2O2 metabolism, can be regulated by stress conditions such as
salinity, heavy metal and ozone stress (Corpas et al., 1993, 2008;
Ueda et al., 2013). Although catalase is the principal antioxidant enzyme
in the matrix of all types of peroxisome (Mhamdi et al.,
2010, 2012; Palma et al., 2020 and references therein),
other
enzymatic
antioxidants
are
present
in
both
the
matrix and the membrane. It is also important to highlight
the
role
of
SOD
isozymes,
which
differ
according
to
peroxisomal origin (del Río et al., 2018). PEROXISOMAL ROS METABOLISM Reactive oxygen species (ROS) are produced by a series of single-
electron reductions in molecular oxygen which sequentially form
superoxide (O2•−), hydrogen peroxide (H2O2) and hydroxyl
(HO•) radicals and ultimately ending in water (Figure 1). It
is worth noting that the term peroxisomes, formerly known as
microbodies, originates from their high H2O2 content (De Duve
and Baudhuin, 1966; Corpas, 2015). Plant peroxisomes contain a
significant number of enzymatic systems capable of generating
H2O2 such as glycolate oxidase (GOX), acyl-CoA oxidase
(AOX), urate oxidase (UO), polyamine oxidase, copper amine
oxidase (CuAO), sulfite oxidase (SO), sarcosine oxidase (SOX),
or superoxide dismutase (SOD) (Hauck et al., 2014; Corpas
et al., 2017a and references therein). These H2O2-generating
enzymes are involved in multiple biochemical pathways which
are essential not only for the endogenous metabolism of plant
peroxisomes but also for their interactions with other subcellular
compartments such as plastids, mitochondria, cytosols, oil bodies
and nuclei. In these subcellular interconnections, H2O2 itself
plays a highly important role as a signal molecule in crosstalk
between organelles in order to coordinate cell function. Acyl-CoA
oxidase
is
another
key
peroxisomal
H2O2-
generating enzyme involved in fatty acid β-oxidation which,
in collaboration with lipid bodies, enables triacylglyceride
mobilization especially during seed germination and is also
involved in the synthesis of signal molecules such as jasmonic
acid (Baker et al., 2006; Chen et al., 2019b; Wang X. et al.,
2019; Xin et al., 2019). However, under stress conditions such
as salinity, ROS generated by peroxisomal fatty acid β-oxidation
have a negative impact and contribute to oxidative damage
(Yu et al., 2019). Photorespiration has been estimated to be responsible for
70% of total H2O2 generated mainly from peroxisomal GOX
in photosynthetic tissues (Noctor et al., 2002). Zhang et al. (2016) have described an elegant dynamic physical GOX-catalase
association-dissociation mechanism that fine-tunes peroxisomal
H2O2 in rice plants. Although peroxisomal H2O2 is kept under
control when GOX and catalase are associated, under stress
conditions and when mediated by salicylic acid (SA), this
complex GOX-catalase dissociation mechanism inhibits catalase Polyamines such as putrescine, spermidine and spermine
are well known to be involved in multiple physiological
processes, as well as mechanisms of response to various
stress
conditions
(Wuddineh
et
al.,
2018;
Chen
et
al.,
2019a; Wang W. et al., 2019). PEROXISOMAL ROS METABOLISM The H2O2-generating sulfite oxidase (SO) converts sulfite (SO32-) to sulfate
(SO42-), which is a mechanism of protection because sulfite inhibits catalase activity. Red line denotes inhibition effect. the mechanism of response to plant stress in which several
subcellular compartments including peroxisomes are involved. The generation of singlet oxygen (1O2) has always been
associated with chloroplasts, particularly in photosystem II,
responsible for various types of photo-damage which triggers
distinct cellular responses (Wagner et al., 2004; Rosenwasser
et al., 2011; Chen and Fluhr, 2018; Dogra et al., 2018). Using the green fluorescence probe to detect 1O2, peroxisomes,
mitochondria and nuclei have been shown to be either the origin
or target of 1O2, suggesting that this ROS is generated in a
light-independent manner (Mor et al., 2014). These findings
open up new questions about the importance of
1O2 in PEROXISOMAL ROS METABOLISM An L-arginine (L-Arg) and Ca2+ dependent NOS-like activity generates NO which can react
chemically with O2- to produce peroxynitrite (ONOO-), a nitrating molecule that facilitates PTMs such as tyrosine nitration. NO can also interact with reduced
glutathione (GSH) to form S-nitrosoglutathione (GSNO), a NO donor which mediates S-nitrosation. GSH is regenerated by glutathione reductase (GR) which requires
NADPH supplied by several NADPH-generating enzymes (NADPH-ICDH, G6PDH, and 6PGDH). Uric acid is a ONOO- scavenger, this being a mechanism of
peroxisomal auto-regulation. With all these components, and according to reported data, the peroxisomal targets of NO-derived PTMs identified so far are CAT,
CuZn-SOD, and monodehydroascorbate reductase (MDAR) which can undergo an inhibitory effect either by nitration or S-nitrosation. Additionally, CAT and GOX can
be inhibited by hydrogen sulfide (H2S), and CAT is also inhibited by carbonylation. The H2O2-generating sulfite oxidase (SO) converts sulfite (SO32-) to sulfate
(SO42-), which is a mechanism of protection because sulfite inhibits catalase activity. Red line denotes inhibition effect. FIGURE 2 | Simple model of the global metabolism of reactive oxygen/nitrogen/sulfur species in plant peroxisomes. Peroxisomes have an important battery of
H2O2-generating enzymes, being the photorespiratory glycolate oxidase (GOX) one of the most relevant. Peroxisomal xanthine oxidoreductase (XOR) activity
generates uric acid which the concomitant generation of superoxide radical (O2•−) which is dismutated to H2O2 by superoxide dismutase (SOD). All three SOD
types have been described in plant peroxisomes from different origin, CuZ-SOD, Mn-SOD, and Fe-SOD. The H2O2 pool is mainly decomposed by catalase (CAT)
but also by the membrane-bound ascorbate peroxidase (APX). An L-arginine (L-Arg) and Ca2+ dependent NOS-like activity generates NO which can react
chemically with O2- to produce peroxynitrite (ONOO-), a nitrating molecule that facilitates PTMs such as tyrosine nitration. NO can also interact with reduced
glutathione (GSH) to form S-nitrosoglutathione (GSNO), a NO donor which mediates S-nitrosation. GSH is regenerated by glutathione reductase (GR) which requires
NADPH supplied by several NADPH-generating enzymes (NADPH-ICDH, G6PDH, and 6PGDH). Uric acid is a ONOO- scavenger, this being a mechanism of
peroxisomal auto-regulation. With all these components, and according to reported data, the peroxisomal targets of NO-derived PTMs identified so far are CAT,
CuZn-SOD, and monodehydroascorbate reductase (MDAR) which can undergo an inhibitory effect either by nitration or S-nitrosation. Additionally, CAT and GOX can
be inhibited by hydrogen sulfide (H2S), and CAT is also inhibited by carbonylation. PEROXISOMAL ROS METABOLISM While MDAR is
present in both matrix and membrane (Leterrier et al., 2005;
Lisenbee et al., 2005; Eastmond, 2007), APX is exclusively
located in the membrane (Corpas et al., 1994; Yamaguchi
et al., 1995; Bunkelmann and Trelease, 1996). With its
high affinity for H2O2 (low Km value around 74 µM),
membrane-bound APX appears to have fine-tuned control
of H2O2 (Ishikawa et al., 1998) as compared to catalase,
which, with a Km value in the mM range, is less efficient at
low concentrations of H2O2 (Huang et al., 1983; Mhamdi
et al., 2010). The Km values for plant catalase are reported to
vary quite considerably, with, for example, a Km of 50 mM
in Beta vulgaris (Dinçer and Aydemir, 2001), 100 mM in
rice (Ray et al., 2012) and 190 mM in pea (del Río et al.,
1977). Peroxisomal APX appears to be critical in a diverse
range of processes such as seedling development (Corpas Given growing awareness of the important role of ROS
peroxisomal
metabolism
in
combating
biotic
stress,
the
expression
of
genes
encoding
for
peroxisomal
proteins
involved
in
their
biogenesis,
fatty
acid
catabolism
and
the H2O2-generating glyoxylate cycle have been reported
to
increase
during
interactions
between
the
pathogen
Sclerotinia sclerotiorum and rapeseed (Brassica napus), thus
facilitating pathogen cell wall degradation and metabolism
detoxification (Chittem et al., 2020). On the other hand,
using the Arabidopsis nca1 mutant with no catalase activity
1, containing residual activity of the three catalase isozymes,
Hackenberg et al. (2013) identified a link between catalase
and
ROS
production
as
autophagy-dependent
cell
death
progresses. Table 2 shows some functional implications of
peroxisomal H2O2
and other signal molecules generated
in this organelle. July 2020 | Volume 11 | Article 853 Frontiers in Plant Science | www.frontiersin.org 3 Plant Peroxisomes and Reactive Species Corpas et al. p
p
FIGURE 2 | Simple model of the global metabolism of reactive oxygen/nitrogen/sulfur species in plant peroxisomes. Peroxisomes have an important battery of
H2O2-generating enzymes, being the photorespiratory glycolate oxidase (GOX) one of the most relevant. Peroxisomal xanthine oxidoreductase (XOR) activity
generates uric acid which the concomitant generation of superoxide radical (O2•−) which is dismutated to H2O2 by superoxide dismutase (SOD). All three SOD
types have been described in plant peroxisomes from different origin, CuZ-SOD, Mn-SOD, and Fe-SOD. The H2O2 pool is mainly decomposed by catalase (CAT)
but also by the membrane-bound ascorbate peroxidase (APX). Frontiers in Plant Science | www.frontiersin.org PEROXISOMAL REACTIVE NITROGEN
SPECIES (RNS) development, stomatal closure, senescence and fruit ripening, as
well as in the mechanism of response to many environmental
stresses including salinity, drought, heavy metals and extreme
temperature (Neill et al., 2008; León et al., 2014; Begara-Morales
et al., 2018; Kolbert et al., 2019; Wei et al., 2020). NO belongs
to a family of related molecules called reactive nitrogen species
(RNS), with peroxynitrite (ONOO−) and S-nitrosogluthione
(GSNO) being the most studied. Using various experimental
approaches including electron paramagnetic resonance (EPR)
spectroscopy, as well as biochemical and cellular biology, some
RNS including NO, ONOO−and GSNO have been detected in
plant peroxisomes (Barroso et al., 2013; Corpas and Barroso,
2014b; Corpas et al., 2019). Identification of peroxisomal proteins
undergoing PTMs mediated by these NO-derived species is
strong evidence of an active RNS metabolism in peroxisomes. Figures 3A–H shows in vivo images of NO and ONOO−in
Arabidopsis guard cell peroxisomes detected by confocal laser
scanning microscopy (CLSM) and specific fluorescent probes. oxidative addition of a nitro group (-NO2) to tyrosine residues,
a highly selective process which depends on factors such
as the protein environment of the Tyr and the nitration
mechanism (Bartesaghi and Radi, 2018). Table 1 shows some
nitrated proteins identified in plant peroxisomes and how
their function is affected. Interestingly, some of the proteins
affected are directly involved in the ROS metabolism, indicating
a close metabolic interconnection between both families of
reactive species. The antioxidant glutathione (GSH), a tripeptide (γ-Glu-Cys-
Gly), undergoes S-nitrosation in order to generate GSNO, a
low-molecular-weight NO reservoir, through a covalent addition
of NO to the thiol group of Cys residues in order to form
S-nitrosothiol (SNO) (Airaki et al., 2011). GSNO is a key
molecule given its dynamic interaction with free cysteines,
GSH and proteins through processes such as S-nitrosation,
S-transnitrosation and S-glutathionylation (Broniowska et al.,
2013; Corpas et al., 2013a,b). GSNO is enzymatically decomposed
by GSNO reductase (GSNOR; Leterrier et al., 2011), an enzyme
susceptible to S-nitrosation and consequently inhibition (Guerra
et al., 2016). An increase in Tyr nitration, an irreversible
process, is usually associated with nitro-oxidative stress; however,
protein S-nitrosation, a reversible process, is a regulatory
protein mechanism that occurs under physiological and stress
conditions. Table 1 shows some peroxisomal proteins targeted ONOO−results from a reaction between NO with O2•−,
considered one of the fastest chemical reactions with a rate
constant (k) of 1.9 × 1010 M−1 s−1 (Kissner et al., 1997). PEROXISOMAL REACTIVE NITROGEN
SPECIES (RNS) Nitric oxide (NO) metabolism has a significant impact on cellular
metabolisms due to its involvement in the important plant
physiological processes of seed and pollen germination, root July 2020 | Volume 11 | Article 853 Frontiers in Plant Science | www.frontiersin.org 4 Plant Peroxisomes and Reactive Species Corpas et al. TABLE 1 | Peroxisomal enzymes target of diverse posttranslational modifications (PTMs) whose activities are affected by either ROS, RNS, or RSS. Peroxisomal enzyme
Pathway/Reaction
PTM
Effect on activity
Catalase (CAT)
H2O2 decomposition
Carbonylation
Inhibition
Tyr-nitration
Inhibition
S-nitrosation
Inhibition
Persufidation
Inhibition
Monodehydroascorbate reductase (MDAR)
Ascorbate-glutathione cycle
Tyr-nitration
Inhibition
S-nitrosation
Inhibition
S-sulfenylationa
Not reported
Hydroxypyruvate reductase (HPR)
Photorespiration
Tyr-nitration
Inhibition
S-nitrosation
Inhibition
Glycolate oxidase (GOX)
Photorespiration
S-nitrosation
Inhibition
Persufidation
Inhibition
CuZn-superoxide dismutase (CSD3)
O2•−dismutation
Tyr-nitration
Inhibition
Malate dehydrogenase (MDH)
Fatty acid β-oxidation
Tyr-nitration
Inhibition
S-nitrosation
Inhibition
Malate synthase (MS)
Glyoxylate cycle
Carbonylation
Inhibition
Isocitrate lyase (ICL)
Glyoxylate cycle
S-nitrosationa
Not reported
Acyl-coenzyme A oxidase 1
Fatty acid β-oxidation
Persulfidationa
Not reported
S-sulfenylationa
Not reported
Multifunctional protein AIM1 isoform
Fatty acid β-oxidation
S-nitrosationa
Not reported
S-sulfenylationa
Not reported
Lon protease homolog 2
Peroxisomal protein import
S-nitrosationa
Not reported
Phosphomevalonate kinase
Isoprenoid biosynthesis
S-sulfenylationa
Not reported
NADP-isocitrate dehydrogenase
NADPH supply
Tyr-nitration
Inhibition
S-nitrosation
Inhibition
Persufidation
Inhibition
S-sulfenylationa
Not reported
aProteomic identification. sttranslational modifications (PTMs) whose activities are affected by either ROS, RNS, or RSS. aProteomic identification. development, stomatal closure, senescence and fruit ripening, as
well as in the mechanism of response to many environmental
stresses including salinity, drought, heavy metals and extreme
temperature (Neill et al., 2008; León et al., 2014; Begara-Morales
et al., 2018; Kolbert et al., 2019; Wei et al., 2020). NO belongs
to a family of related molecules called reactive nitrogen species
(RNS), with peroxynitrite (ONOO−) and S-nitrosogluthione
(GSNO) being the most studied. Using various experimental
approaches including electron paramagnetic resonance (EPR)
spectroscopy, as well as biochemical and cellular biology, some
RNS including NO, ONOO−and GSNO have been detected in
plant peroxisomes (Barroso et al., 2013; Corpas and Barroso,
2014b; Corpas et al., 2019). Identification of peroxisomal proteins
undergoing PTMs mediated by these NO-derived species is
strong evidence of an active RNS metabolism in peroxisomes. Figures 3A–H shows in vivo images of NO and ONOO−in
Arabidopsis guard cell peroxisomes detected by confocal laser
scanning microscopy (CLSM) and specific fluorescent probes. PEROXISOMAL REACTIVE NITROGEN
SPECIES (RNS) ONOO−, a strong oxidant and nitrating molecule involved in
protein tyrosine nitration (NO2-Tyr), modifies protein function,
mostly through inhibition (Corpas et al., 2009a; Mata-Pérez
et al., 2016). This NO-derived PTM involves the covalent July 2020 | Volume 11 | Article 853 Frontiers in Plant Science | www.frontiersin.org 5 Plant Peroxisomes and Reactive Species Corpas et al. TABLE 2 | Signal molecules generated in plant peroxisomes during different
processes and their functional implications. Peroxisomal signal
Functional implication
References
Hydrogen peroxide
(H2O2)
Plant development and
stress response
Zhang et al., 2016; Su
et al., 2019
Involved in peroxisome
abundance under drought
and heat stress
Hinojosa et al., 2019
Pexophagy
Yamauchi et al., 2019
Pathogen defense
Chittem et al., 2020
Nitric oxide (NO)
Pollen tube development
Prado et al., 2004
Leaf senescence
Corpas et al., 2019
Lateral root formation
Schlicht et al., 2013
Heavy metal and root
architecture
Piacentini et al., 2020
Hydrogen sulfide
Regulation of catalase
Corpas et al., 2019a
(H2S)
Herbicide glyphosate
response
Jasmonic acid (JA)
Plant growth
Wang X. et al., 2019
Environmental stimuli
Xin et al., 2019
Insect defense
γ-aminobutyric acid
(GABA)
Fruit flavor and flower
fragrance
Zarei et al., 2015
Abiotic stress tolerance
Shelp and Zarei, 2017 TABLE 2 | Signal molecules generated in plant peroxisomes during different
processes and their functional implications. The number of peroxisomal proteins targeted by NO-
mediated PTMs is growing continuously. Using the biotin-switch
technique and liquid chromatography/mass spectrometry/mass
spectrometry
(LC-MS/MS),
several
more
S-nitrosated
peroxisomal proteins have been identified during adventitious
root growth induced by treatment with NO (Niu et al., 2019). These proteins include the peroxisomal LON2 protease, which is
necessary for matrix protein import into peroxisomes (Lingard
and Bartel, 2009); isocitrate lyase (ICL), involved in the glyoxylate
cycle; and the multifunctional AIM1-like isoform, involved in
fatty acid β-oxidation. However, the source of enzymatic NO, as yet unelucidated,
is currently the most controversial aspect of NO metabolism
in higher plants (Kolbert et al., 2019). Two main candidates
have been proposed: nitrate reductase (NR) (Mohn et al., 2019)
and L-arginine-dependent NO synthase-like activity (Corpas
et al., 2017a). Although no evidence of NR has been found in
plant peroxisomes, NO synthase-like activity has been found
and characterized in peroxisomes purified from pea leaves
(Barroso et al., 1999). PEROXISOMAL REACTIVE NITROGEN
SPECIES (RNS) Though as yet unidentified, this protein is
called NOS-like activity, as peroxisomal NO generation requires
NOS proteins similar to those found in animals, including
L-arginine, NADPH, FMN, FAD, tetrahydrobiopterin, calcium,
and calmodulin (Corpas and Barroso, 2017b; Corpas et al., 2019). The protein responsible for NO generation is imported by a type
2 peroxisomal targeting signal involving a process dependent on
calmodulin and calcium (Corpas and Barroso, 2014a, 2018a). by S-nitrosation, as well as proteins involved in ROS metabolism
which are targeted by these NO-mediated PTMs. by S-nitrosation, as well as proteins involved in ROS metabolism
which are targeted by these NO-mediated PTMs. Peroxisomal NO metabolism is involved in processes such
as pollen tube germination (Prado et al., 2004), lateral FIGURE 3 | Representative images illustrating the CLSM in vivo detection of nitric oxide (NO) and peroxynitrite (ONOO-), peroxisomes (red) and chloroplasts (blue) in
guard cells of transgenic Arabidopsis seedlings expressing CFP-PTS1. (A,E) Fluorescence punctate (red) attributable to CFP-PTS1, indicating the localization of
peroxisomes in guard cells. (B,F) Fluorescence punctate (green) attributable to the detection in the same guard cells of NO and ONOO-, respectively. (C,G)
Chlorophyll autofluorescence (blue) attributable to the detection of chloroplasts. (D,H) Merged images for corresponding panels. Reproduced with permission from
Corpas et al. (2017a) provided by Elsevier. FIGURE 3 | Representative images illustrating the CLSM in vivo detection of nitric oxide (NO) and peroxynitrite (ONOO-), peroxisomes (red) and chloroplasts (blue) in
guard cells of transgenic Arabidopsis seedlings expressing CFP-PTS1. (A,E) Fluorescence punctate (red) attributable to CFP-PTS1, indicating the localization of
peroxisomes in guard cells. (B,F) Fluorescence punctate (green) attributable to the detection in the same guard cells of NO and ONOO-, respectively. (C,G)
Chlorophyll autofluorescence (blue) attributable to the detection of chloroplasts. (D,H) Merged images for corresponding panels. Reproduced with permission from
Corpas et al. (2017a) provided by Elsevier. July 2020 | Volume 11 | Article 853 Frontiers in Plant Science | www.frontiersin.org 6 Corpas et al. Plant Peroxisomes and Reactive Species FIGURE 4 | Representative images illustrating the CLSM in vivo detection of H2S (red color) and peroxisomes (green color) in root tips (A–C) and guard cells (D–G)
of 10 days old Arabidopsis seedlings expressing CFP-PTS1. (A,D) Fluorescence puncta (green) attributable to CFP-PTS1, indicating the localization of peroxisomes. (B,E) Fluorescence punctate (red) attributable to H2S detection in the same area. (C) Merged image of (A,B) showing colocalized fluorescence punctate (yellow). PEROXISOMAL REACTIVE NITROGEN
SPECIES (RNS) (F) Chlorophyll autofluorescence (blue) demonstrating location of chloroplasts. (G) Merged images of (D–F). H2S (red color) was detected by using 5 mM WSP-5, a
fluorescence probe for H2S. Arrows indicate representative punctate spots corresponding to the colocalization of H2S with peroxisomes. Asterisks indicate
localization of H2S in the cytosol. Reproduced with permission from Corpas et al. (2019a) provided by John Wiley and Sons. FIGURE 4 | Representative images illustrating the CLSM in vivo detection of H2S (red color) and peroxisomes (green color) in root tips (A–C) and guard cells (D–G)
of 10 days old Arabidopsis seedlings expressing CFP-PTS1. (A,D) Fluorescence puncta (green) attributable to CFP-PTS1, indicating the localization of peroxisomes. (B,E) Fluorescence punctate (red) attributable to H2S detection in the same area. (C) Merged image of (A,B) showing colocalized fluorescence punctate (yellow). (F) Chlorophyll autofluorescence (blue) demonstrating location of chloroplasts. (G) Merged images of (D–F). H2S (red color) was detected by using 5 mM WSP-5, a
fluorescence probe for H2S. Arrows indicate representative punctate spots corresponding to the colocalization of H2S with peroxisomes. Asterisks indicate
localization of H2S in the cytosol. Reproduced with permission from Corpas et al. (2019a) provided by John Wiley and Sons. root formation (Schlicht et al., 2013), and leaf senescence
(Corpas et al., 2019), as well as in responses to environmental and
heavy metal stresses such as salinity (Corpas et al., 2009b), lead
(Corpas and Barroso, 2017a), and cadmium (Corpas and Barroso,
2014b; Piacentini et al., 2020). sulfite to sulfate by producing H2O2. The functional relevance
of this enzyme is that it can protect catalase activity since sulfite,
at low concentration, has the capacity to inhibit catalase activity
(Veljovic-Jovanovic et al., 1998). Nevertheless, despite the greater
importance attributed to peroxisomal SO in a recent study,
mitochondrial SO in animal cells has the capacity to generate NO
from nitrite (Bender et al., 2019), while NO enzymatic generation
from SO in plant peroxisomes remains to be proven. An earlier
study confirmed the important role played by the peroxisomal
RSS metabolism (Corpas and Barroso, 2015). Frontiers in Plant Science | www.frontiersin.org REACTIVE SULFUR SPECIES (RSS) IN
PLANT PEROXISOMES Reactive sulfur species (RSS) are chemically comparable to ROS
(Olson, 2019) and can be generated from hydrogen sulfide
(H2S), some of these species are thiyl radical (HS•), hydrogen
persulfide (H2S2), persulfide radical (HS2•−), sulfite (SO32−)
or sulfate (SO42−) among others (Gruhlke and Slusarenko,
2012; Ono et al., 2014; Mishanina et al., 2015; Park et al.,
2015; Schöneich, 2016). However, the biochemistry of H2S
in cells, given its multiple interactions with other reactive
species, is more complex than previously thought (see Filipovic
et al., 2018 for a more in-depth review); for example, protein
thiyl radicals are generated during the reaction of H2O2
with heme proteins, possibly inducing protein degradation
(Schöneich, 2016). H2S has recently been proven to be present in plant
peroxisomes (Corpas et al., 2019a). Figures 4A–G shows
representative images of H2S in peroxisomes from the root tips
and guard cells of Arabidopsis seedlings detected by in vivo CLSM
and a specific fluorescent probe. Using proteomic techniques,
some peroxisomal enzymes have been identified as targets of
persulfidation (Aroca et al., 2015, 2017). On the other hand,
in vitro analysis shows that catalase activity from Arabidopsis and
sweet pepper fruits is inhibited in the presence of H2S (Corpas
et al., 2019a). Although, to our knowledge, the enzymatic source
of peroxisomal H2S remains unknown, previous studies have
proposed some potential candidates. For example, catalase, which
functions as a sulfide oxidase or sulfur reductase, is capable
of oxidizing or generating H2S (Olson et al., 2017). SOD has
also been reported to have the capacity to catalyze the reaction
between O2 and H2S to generate persulfide (Olson et al., 2018). In a previous study by Corpas and Barroso (2015), the presence
of these enzymatic and non-enzymatic components in plant Different molecules and enzymes, such as GSH (Müller
et al., 2004), glutathione reductase (Romero-Puertas et al., 2006),
and sulfite oxidase (Nowak et al., 2004; Hänsch and Mendel,
2005), involved in sulfur metabolism, are present in plant
peroxisomes. Sulfite oxidase (SO) catalyzes the conversion of July 2020 | Volume 11 | Article 853 Frontiers in Plant Science | www.frontiersin.org 7 Plant Peroxisomes and Reactive Species Corpas et al. targets. We also need to determine the effect of the different
PTMs, carbonylation, S-sulfenylation, S-nitrosation, tyrosine
nitration, and persulfidation, on target protein function and
peroxisomal metabolism. REACTIVE SULFUR SPECIES (RSS) IN
PLANT PEROXISOMES In addition, interactions with other
subcellular compartments which share biochemical pathways
such as photorespiration, fatty acid β-oxidation, isoprenoid
biosynthesis and purine and polyamine metabolism (Clastre
et al., 2011; Guirimand et al., 2012; Corpas et al., 2019b)
should be investigated. Similarly, the relationship between
reactive species and complex peroxisomal biogenesis, division
and matrix/membrane protein import mechanisms (Reumann
and Bartel, 2016; Kao et al., 2018) has been underexplored
(López-Huertas et al., 2000). Further research should also be
carried out to identify the proteins responsible for endogenous
peroxisomal generation of NO and H2S. This would increase our
knowledge of how organelle biochemistry is modulated within
the framework of the whole cell metabolism. This research could
lead to biotechnological applications given the important role of
peroxisomes in many physiological processes and in responses
to biotic and abiotic stresses. Furthermore, in addition to
harboring reactive species, with their known signaling properties,
peroxisomes are a source of other signaling molecules such as
jasmonic acid and γ-aminibutic acid (GABA), which extends the
functional role of plant peroxisomes. Table 2 shows signaling
molecules generated in the plant peroxisomal metabolism and
some examples of their role in various plant processes. peroxisomes indicates that, in addition to ROS and RNS, these
organelles also have an active RSS metabolism. peroxisomes indicates that, in addition to ROS and RNS, these
organelles also have an active RSS metabolism. CONCLUSION Much of our knowledge of reactive species metabolism in plant
peroxisomes is now well established. The three molecular families
ROS, RNS, and RSS are present in plant peroxisomes, which
are considered to be potential producers of reactive species
and to play an important role in the cell signaling network. However, our limited knowledge of reactive species families
needs to be expanded by identifying new peroxisomal protein ACKNOWLEDGMENTS SG-G acknowledges a “Formación de Personal Investigador”
contract (BES-2016-078368) from the Ministry of Economy and
Competitiveness, Spain. FUNDING This work was supported by the European Regional Development
Fund co-financed grant from the Ministry of Economy
and
Competitiveness
(AGL2015-65104-P
and
PID2019-
103924GB-I00), the Plan Andaluz de Investigación, Desarrollo
e Innovación (P18-FR-1359), and the Junta de Andalucía
(group BIO192), Spain. CROSSTALK BETWEEN PEROXISOMAL
REACTIVE SPECIES Functional interactions and inter-regulation through PTMs in
these families of reactive species are shown in Figure 2. In
this working model, under physiological conditions, catalase, the
main antioxidant enzyme, regulates levels of H2O2 generated by
different pathways, principally photorespiratory glycolate oxidase
(GOX) (Noctor et al., 2002). On the other hand, peroxisomal
xanthine oxidoreductase (XOR) activity involved in purine
catabolism generates uric acid, with the concomitant formation
of the O2•−(Corpas et al., 1997, 2008; Zarepour et al., 2010),
which, in turn, is dismutated to H2O2 by SOD. The pool of H2O2
is mainly decomposed by catalase (CAT) and also by membrane-
bound ascorbate peroxidase (APX). L-Arg-dependent NOS-like
activity generates NO (Corpas et al., 2019) which chemically
reacts with O2•−to produce peroxynitrite (ONOO−), a nitrating
molecule that facilitates PTMs such as tyrosine nitration. NO also interacts with reduced glutathione (GSH) to form
S-nitrosoglutathione (GSNO), a NO donor that mediates
S-nitrosation. Uric acid is a physiological ONOO−scavenger
(Alamillo and García-Olmedo, 2001) involved in endogenous
peroxisomal auto-regulation. Thus, the peroxisomal enzymes
targeted by NO-derived PTMs, catalase (CAT), CuZn-SOD,
and monodehydroascorbate reductase (MDAR), are inhibited by
nitration and S-nitrosation. Both CAT, and GOX are inhibited
by H2S; the former is also inhibited by carbonylation when
H2O2 is overproduced. In addition, H2O2-generating sulfite
oxidase (SO) is involved in the conversion of sulfite to sulfate
which, given sulfite’s ability to inhibit SO, has been reported
to be a catalase protection mechanism. These interconnections
highlight the biochemical complexity of this self-regulated plant
peroxisome network, in which the antioxidant catalase is one of
the most regulated peroxisomal enzymes (Palma et al., 2020). AUTHOR CONTRIBUTIONS Authors have made a collaborative, direct and intellectual
contribution to the work, and have approved it for publication. REFERENCES Peroxisomal plant metabolism - an
update on nitric oxide, Ca2+ and the NADPH recycling network. J. Cell Sci. 131:jcs202978. doi: 10.1242/jcs.202978 Broniowska, K. A., Diers, A. R., and Hogg, N. (2013). S-nitrosoglutathione. Biochim. Biophys. Acta 1830, 3173–3181. Corpas, F. J., Barroso, J. B., González-Gordo, S., Muñoz-Vargas, M. A., and Palma,
J. M. (2019a). Hydrogen sulfide: a novel component in Arabidopsis peroxisomes
which triggers catalase inhibition. J. Integr. Plant Biol. 61, 871–883. Bueno, P., and del Río, L. A. (1992). Purification and properties of glyoxysomal
cuprozinc superoxide dismutase from watermelon cotyledons (Citrullus
vulgaris Schrad). Plant Physiol. 98, 331–336. doi: 10.1104/pp.98.1.331 Corpas, F. J., del Río, L. A., and Palma, J. M. (2019b). Plant peroxisomes at the
crossroad of NO and H2O2 metabolism. J. Integr. Plant Biol. 61, 803–816. Bunkelmann, J. R., and Trelease, R. N. (1996). Ascorbate peroxidase. A prominent
membrane protein in oilseed glyoxysomes. Plant Physiol. 110, 589–598. doi:
10.1104/pp.110.2.589 Corpas, F. J., Barroso, J. B., Palma, J. M., and Rodríguez-Ruiz, M. (2017a). Plant
peroxisomes: a nitro-oxidative cocktail. Redox Biol. 11, 535–542. doi: 10.1016/
j.redox.2016.12.033 Chen, D., Shao, Q., Yin, L., Younis, A., and Zheng, B. (2019a). Polyamine function
in plants: metabolism, regulation on development, and roles in abiotic stress
responses. Front. Plant Sci. 9:1945. j
Corpas, F. J., Pedrajas, J. R., Palma, J. M., Valderrama, R., Rodríguez-Ruiz,
M., Chaki, M., et al. (2017b). Immunological evidence for the presence
of peroxiredoxin in pea leaf peroxisomes and response to oxidative stress
conditions. Acta Physiol. Plant 39:57. Chen, S., Lu, X., Ge, L., Sun, X., and Xin, Z. (2019b). Wound- and pathogen-
activated de novo JA synthesis using different ACX isozymes in tea plant
(Camellia sinensis). J. Plant Physiol. 243:153047. doi: 10.1016/j.jplph.2019. 153047 Corpas, F. J., Bunkelmann, J., and Trelease, R. N. (1994). Identification and
immunochemical characterization of a family of peroxisome membrane
proteins (PMPs) in oilseed glyoxysomes. Eur. J. Cell Biol. 65, 280–290. Chen, T., and Fluhr, R. (2018). Singlet oxygen plays an essential role in the root’s
response to osmotic stress. Plant Physiol. 177, 1717–1727. doi: 10.1104/pp.18. 00634 Corpas, F. J., Chaki, M., Leterrier, M., and Barroso, J. B. (2009a). Protein tyrosine
nitration: a new challenge in plants. Plant Signal. Behav. 4, 920–923. doi:
10.4161/psb.4.10.9466 Chittem, K., Yajima, W. R., Goswami, R. S., and Del Río Mendoza, L. E. (2020). Transcriptome analysis of the plant pathogen Sclerotinia sclerotiorum
interaction with resistant and susceptible canola (Brassica napus) lines. PLoS
One 15:e0229844. REFERENCES Akter,
S.,
Carpentier,
S.,
Van
Breusegem,
F.,
and
Messens,
J. (2017). Identification of dimedone-trapped sulfenylated proteins in plants under
stress. Biochem. Biophys. Rep. 9, 106–113. doi: 10.1016/j.bbrep.2016. 11.014 Airaki, M., Sánchez-Moreno, L., Leterrier, M., Barroso, J. B., Palma, J. M., and
Corpas, F. J. (2011). Detection and quantification of S-nitrosoglutathione
(GSNO) in pepper (Capsicum annuum L.) plant organs by LC-ES/MS. Plant
Cell Physiol. 52, 2006–2015. doi: 10.1093/pcp/pcr133 Airaki, M., Sánchez-Moreno, L., Leterrier, M., Barroso, J. B., Palma, J. M., and
Corpas, F. J. (2011). Detection and quantification of S-nitrosoglutathione
(GSNO) in pepper (Capsicum annuum L.) plant organs by LC-ES/MS. Plant
Cell Physiol. 52, 2006–2015. doi: 10.1093/pcp/pcr133 Alamillo, J. M., and García-Olmedo, F. (2001). Effects of urate, a natural inhibitor
of peroxynitrite-mediated toxicity, in the response of Arabidopsis thaliana to the July 2020 | Volume 11 | Article 853 Frontiers in Plant Science | www.frontiersin.org 8 Plant Peroxisomes and Reactive Species Corpas et al. bacterial pathogen Pseudomonas syringae. Plant J. 25, 529–540. doi: 10.1046/j. 1365-313x.2001.00984.x Corpas, F. J., Sandalio, L. M., del Río, L. A., Trelease, R. N. (1998). Copper–zinc
superoxide dismutase is a constituent enzyme of the matrix of peroxisomes in
the cotyledons of oilseed plants. New Phytol. 138, 307–314. doi: 10.1046/j.1469-
8137.1998.00899.x bacterial pathogen Pseudomonas syringae. Plant J. 25, 529–540. doi: 10.1046/j. 1365-313x.2001.00984.x Anand, P., Kwak, Y., Simha, R., and Donaldson, R. P. (2009). Hydrogen peroxide
induced oxidation of peroxisomal malate synthase and catalase. Arch. Biochem. Biophys. 491, 25–31. doi: 10.1016/j.abb.2009.09.019 Corpas, F J., Barroso, J B., del Río, L A. (2001). Peroxisomes as a source of reactive
oxygen species and nitric oxide signal molecules in plant cells. Trends Plant. Sci. 6, 145–50. doi: 10.1016/s1360-1385(01)01898-2 Aroca, A., Benito, J. M., Gotor, C., and Romero, L. C. (2017). Persulfidation
proteome reveals the regulation of protein function by hydrogen sulfide in
diverse biological processes in Arabidopsis. J. Exp. Bot. 68, 4915–4927. doi:
10.1093/jxb/erx294 Corpas, F. J., Alché, J. D., and Barroso, J. B. (2013a). Current overview
of
S-nitrosoglutathione
(GSNO)
in
higher
plants. Front. Plant
Sci. 4:126. Aroca, Á, Serna, A., Gotor, C., and Romero, L. C. (2015). S-sulfhydration: a cysteine
posttranslational modification in plant systems. Plant Physiol. 168, 334–342. doi: 10.1104/pp.15.00009 Corpas, F. J., Leterrier, M., Begara-Morales, J. C., Valderrama, R., Chaki, M.,
López-Jaramillo, J., et al. (2013b). Inhibition of peroxisomal hydroxypyruvate
reductase (HPR1) by tyrosine nitration. Biochim. Biophys. Acta 1830, 4981–
4989. doi: 10.1016/j.bbagen.2013.07.002 Baker, A., Graham, I. REFERENCES A., Holdsworth, M., Smith, S. M., and Theodoulou, F. L. (2006). Chewing the fat: beta-oxidation in signalling and development. Trends
Plant Sci. 11, 124–132. doi: 10.1016/j.tplants.2006.01.005 Corpas, F. J., and Barroso, J. B. (2014a). Peroxisomal plant nitric oxide
synthase (NOS) protein is imported by peroxisomal targeting signal type
2 (PTS2) in a process that depends on the cytosolic receptor PEX7
and calmodulin. FEBS Lett. 588, 2049–2054. doi: 10.1016/j.febslet.2014. 04.034 Barroso, J. B., Corpas, F. J., Carreras, A., Sandalio, L. M., Valderrama, R., Palma,
J. M., et al. (1999). Localization of nitric-oxide synthase in plant peroxisomes. J. Biol. Chem. 274, 36729–36733. Barroso, J. B., Valderrama, R., and Corpas, F. J. (2013). Immunolocalization of
S-nitrosoglutathione, S-nitrosoglutathione reductase and tyrosine nitration in
pea leaf organelles. Acta Physiol. Plant 35, 2635–2640. doi: 10.1007/s11738-
013-1291-0 Corpas, F. J., and Barroso, J. B. (2014b). Peroxynitrite (ONOO-) is endogenously
produced in Arabidopsis peroxisomes and is overproduced under cadmium
stress. Ann. Bot. 113, 87–96. doi: 10.1093/aob/mct260 Bartesaghi, S., and Radi, R. (2018). Fundamentals on the biochemistry of
peroxynitrite and protein tyrosine nitration. Redox Biol. 14, 618–625. doi:
10.1016/j.redox.2017.09.009 Corpas, F. J., and Barroso, J. B. (2015). Reactive sulfur species (RSS): possible new
players in the oxidative metabolism of plant peroxisomes. Front Plant Sci. 6:116. Corpas, F. J., and Barroso, J. B. (2017a). Lead-induced stress, which triggers the
production of nitric oxide (NO) and superoxide anion (O2·-) in Arabidopsis
peroxisomes, affects catalase activity. Nitric Oxide 68, 103–110. doi: 10.1016/j. niox.2016.12.010 Begara-Morales, J. C., Chaki, M., Valderrama, R., Sánchez-Calvo, B., Mata-Pérez,
C., Padilla, M. N., et al. (2018). Nitric oxide buffering and conditional nitric
oxide release in stress response. J. Exp. Bot. 69, 3425–3438. doi: 10.1093/jxb/
ery072 Corpas, F. J., and Barroso, J. B. (2017b). Nitric oxide synthase-like activity in higher
plants. Nitric Oxide 68, 5–6. doi: 10.1016/j.niox.2016.10.009 y
Bender, D., Tobias Kaczmarek, A., Niks, D., Hille, R., and Schwarz, G. (2019). Mechanism of nitrite-dependent NO synthesis by human sulfite oxidase. Biochem. J. 476, 1805–1815. doi: 10.1042/bcj20190143 Corpas, F. J., and Barroso, J. B. (2018a). Calmodulin antagonist affects peroxisomal
functionality by disrupting both peroxisomal Ca2+ and protein import. J. Cell
Sci. 131:jcs201467. doi: 10.1242/jcs.201467 Borek, S., Stefaniak, S., ´Sliwiñski, J., Garnczarska, M., and Pietrowska-Borek, M. (2019). Autophagic machinery of plant peroxisomes. Int. J. Mol. Sci. 20:4754. doi: 10.3390/ijms20194754 Corpas, F. J., and Barroso, J. B. (2018b). REFERENCES 41, 1727–1738. doi: 10.1111/pce.13332 Leterrier, M., Barroso, J. B., Valderrama, R., Begara-Morales, J. C., Sánchez-Calvo,
B., Chaki, M., et al. (2016). Peroxisomal NADP-isocitrate dehydrogenase is
required for Arabidopsis stomatal movement. Protoplasma 253, 403–415. doi:
10.1007/s00709-015-0819-0 Droillard, M. J., and Paulin, A. (1990). Isozymes of superoxide dismutase in
mitochondria and peroxisomes isolated from petals of carnation (Dianthus
caryophyllus) during senescence. Plant Physiol. 94, 1187–1192. doi: 10.1104/
pp.94.3.1187 Leterrier, M., Chaki, M., Airaki, M., Valderrama, R., Palma, J. M., Barroso, J. B.,
et al. (2011). Function of S-nitrosoglutathione reductase (GSNOR) in plant
development and under biotic/abiotic stress. Plant Signal. Behav. 6, 789–793. doi: 10.4161/psb.6.6.15161 Eastmond, P. J. (2007). MONODEHYROASCORBATE REDUCTASE4 is required
for seed storage oil hydrolysis and postgerminative growth in Arabidopsis. Plant
Cell 19, 1376–1387. doi: 10.1105/tpc.106.043992 Leterrier, M., Corpas, F. J., Barroso, J. B., Sandalio, L. M., and del Río,
L. A. (2005). Peroxisomal monodehydroascorbate reductase. Genomic clone
characterization and functional analysis under environmental stress conditions. Plant Physiol. 138, 2111–2123. doi: 10.1104/pp.105.066225 Filipovic, M. R., Zivanovic, J., Alvarez, B., and Banerjee, R. (2018). Chemical
biology of H2S signaling through persulfidation. Chem Rev. 118, 1253–1337. doi: 10.1021/acs.chemrev.7b00205 Gruhlke, M. C., and Slusarenko, A. J. (2012). The biology of reactive sulfur
species (RSS). Plant Physiol. Biochem. 59, 98–107. doi: 10.1016/j.plaphy.2012. 03.016 Li, G., Li, J., Hao, R., and Guo, Y. (2017). Activation of catalase activity by a
peroxisome-localized small heat shock protein Hsp17.6CII. J. Genet. Genomics
44, 395–404. doi: 10.1016/j.jgg.2017.03.009 Guerra, D., Ballard, K., Truebridge, I., and Vierling, E. (2016). S-Nitrosation of
conserved cysteines modulates activity and stability of S-nitrosoglutathione
reductase (GSNOR). Biochemistry 55, 2452–2464. doi: 10.1021/acs.biochem. 5b01373 Lingard, M. J., and Bartel, B. (2009). Arabidopsis LON2 is necessary for peroxisomal
function and sustained matrix protein import. Plant Physiol. 151, 1354–1365. doi: 10.1104/pp.109.142505 Lisenbee, C. S., Lingard, M. J., and Trelease, R. N. (2005). Arabidopsis
peroxisomes possess functionally redundant membrane and matrix isoforms
of monodehydroascorbate reductase. Plant J. 43, 900–914. doi: 10.1111/j.1365-
313x.2005.02503.x Guirimand, G., Simkin, A. J., Papon, N., Besseau, S., Burlat, V., St-Pierre, B., et al. (2012). Cycloheximide as a tool to investigate protein import in peroxisomes:
a case study of the subcellular localization of isoprenoid biosynthetic enzymes. J. Plant Physiol. 169, 825–829. doi: 10.1016/j.jplph.2012.01.020 Plant Physiol. 169, 825–829. doi: 10.1016/j.jplph.2012.01.020 López-Huertas, E., Charlton, W. L., Johnson, B., Graham, I. A., and Baker, A. (2000). Stress induces peroxisome biogenesis genes. EMBO J. 19, 6770–6777. REFERENCES doi: 10.1371/journal.pone.0229844 Corpas, F. J., Hayashi, M., Mano, S., Nishimura, M., and Barroso, J. B. (2009b). Peroxisomes are required for in vivo nitric oxide accumulation in the cytosol
following salinity stress of Arabidopsis plants. Plant Physiol. 151, 2083–2094. doi: 10.1104/pp.109.146100 One 15:e0229844. doi: 10.1371/journal.pone.0229844 Chowdhary, G., Kataya, A. R., Lingner, T., and Reumann, S. (2012). Non-canonical
peroxisome targeting signals: identification of novel PTS1 tripeptides and
characterization of enhancer elements by computational permutation analysis. BMC Plant Biol. 12:142. doi: 10.1186/1471-2229-12-142 Corpas, F. J., de la Colina, C., Sánchez-Rasero, F., and del Río, L. A. (1997). A role
for leaf peroxisomes in the catabolism of purines. J. Plant Physiol. 151, 246–250. doi: 10.1016/s0176-1617(97)80161-7 BMC Plant Biol. 12:142. doi: 10.1186/1471-2229-12-142 Corpas, F. J., del Río, L. A., and Palma, J. M. (2019). Impact of nitric oxide (NO) on
the ROS metabolism of peroxisomes. Plants 8:E37. Clastre, M., Papon, N., Courdavault, V., Giglioli-Guivarc’h, N., St-Pierre, B., and
Simkin, A. J. (2011). Subcellular evidence for the involvement of peroxisomes in
plant isoprenoid biosynthesis. Plant Signal. Behav. 6, 2044–2046. doi: 10.4161/
psb.6.12.18173 Corpas, F. J., Gómez, M., Hernández, J. A., and del Rio, L. A. (1993). Metabolism
of activated oxygen in peroxisomes from two Pisum salivum L. cultivars with
different sensitivity to sodium chloride. J. Plant Physiol. 141, 160–165. doi:
10.1016/s0176-1617(11)80753-4 Corpas, F. J. (2015). What is the role of hydrogen peroxide in plant peroxisomes? Plant Biol. 17, 1099–1103. doi: 10.1111/plb.12376 July 2020 | Volume 11 | Article 853 Frontiers in Plant Science | www.frontiersin.org 9 Plant Peroxisomes and Reactive Species Corpas et al. Corpas, F. J., Palma, J. M., Sandalio, L. M., Valderrama, R., Barroso, J. B., and del
Río, L. A. (2008). Peroxisomal xanthine oxidoreductase: characterization of the
enzyme from pea (Pisum sativum L.) leaves. J. Plant Physiol. 165, 1319–1330. doi: 10.1016/j.jplph.2008.04.004 Ishikawa, T., Yoshimura, K., Sakai, K., Tamoi, M., Takeda, T., and Shigeoka, S. (1998). Molecular characterization and physiological role of a glyoxysome-
bound ascorbate peroxidase from spinach. Plant Cell Physiol. 39, 23–34. doi:
10.1093/oxfordjournals.pcp.a029285 Corpas, F. J., and Trelease, R. N. (1998). Differential expression of ascorbate
peroxidase and a putative molecular chaperone in the boundary membrane of
differentiating cucumber seedling peroxisomes. J. Plant Physiol. 153, 332–338. doi: 10.1016/s0176-1617(98)80159-4 Jiménez, A., Hernández, J. A., Pastori, G., del Río, L. A., Sevilla, F. (1998). Role
of the ascorbate-glutathione cycle of mitochondria and peroxisomes in the
senescence of pea leaves. Plant Physiol. 118, 1327–35. REFERENCES doi: 10.1104/pp.118.4. 1327 De Duve, C., and Baudhuin, P. (1966). Peroxisomes (microbodies and related
particles). Physiol. Rev. 46, 323–357. doi: 10.1152/physrev.1966.46.2.323 Kao, Y. T., Gonzalez, K. L., and Bartel, B. (2018). Peroxisome function, biogenesis,
and dynamics in plants. Plant Physiol. 176, 162–177. doi: 10.1104/pp.17.01050 Debska, K., Bogatek, R., and Gniazdowska, A. (2012). Protein carbonylation and
its role in physiological processes in plants. Postepy Biochem. 58, 34–43. Kissner, R., Nauser, T., Bugnon, P., Lye, P. G., and Koppenol, W. H. (1997). Formation and properties of peroxynitrite as studied by laser flash photolysis,
high-pressure stopped-flow technique, and pulse radiolysis. Chem. Res. Toxicol. 10, 1285–1292. doi: 10.1021/tx970160x del Río, L. A., Ortega, M. G., López, A. L., and Gorgé, J. L. (1977). A more sensitive
modification of the catalase assay with the Clark oxygen electrode. Application
to the kinetic study of the pea leaf enzyme. Anal Biochem. 80, 409–415. doi:
10.1016/0003-2697(77)90662-5 Kohli, S. K., Khanna, K., Bhardwaj, R., Abd Allah, E. F., Ahmad, P., and Corpas,
F. J. (2019). Assessment of subcellular ROS and NO metabolism in higher
plants: multifunctional signaling molecules. Antioxidants 8:641. doi: 10.3390/
antiox8120641 del Río, L. A., Sandalio, L. M., Palma, J. M., Bueno, P., and Corpas, F. J. (1992). Metabolism of oxygen radicals in peroxisomes and cellular implications. Free
Radic. Biol. Med. 13, 557–580. doi: 10.1016/0891-5849(92)90150-f Kolbert, Z., Barroso, J. B., Brouquisse, R., Corpas, F. J., Gupta, K. J., Lindermayr,
C., et al. (2019). A forty year journey: the generation and roles of NO in plants. Nitric. Oxide 93, 53–70. del Río, L. A., Corpas, F. J., López-Huertas, E., Palma, J. M. (2018). “Plant superoxide dismutases: function under abiotic stress conditions,” in
Antioxidants and Antioxidant Enzymes in Higher Plants, (Cham: Springer
International Publishing), 1–26. doi: 10.1007/978?3?319?75088-0_1 Kusano, T., Kim, D. W., Liu, T., and Berberich, T. (2015). “Polyamine catabolism in
plants,” in Polyamines, eds T. Kusano, and H. Suzuki, (Tokyo: Springer), 77–88. doi: 10.1007/978-4-431-55212-3_6 Dinçer, A., and Aydemir, T. (2001). Purification and characterization of catalase
from chard (Beta vulgaris var. cicla). J. Enzyme Inhib. 16, 165–175. doi: 10. 1080/14756360109162366 León, J., Castillo, M. C., Coego, A., Lozano-Juste, J., and Mir, R. (2014). Diverse functional interactions between nitric oxide and abscisic acid in plant
development and responses to stress. J. Exp. Bot. 65, 907–921. doi: 10.1093/jxb/
ert454 Dogra, V., Rochaix, J. D., and Kim, C. (2018). Singlet oxygen-triggered chloroplast-
to-nucleus retrograde signalling pathways: an emerging perspective. Plant Cell
Environ. REFERENCES doi: 10.1016/j.abb.2005.04.018 Reumann, S., Quan, S., Aung, K., Yang, P., Manandhar-Shrestha, K., Holbrook,
D., et al. (2009). In-depth proteome analysis of Arabidopsis leaf peroxisomes
combined with in vivo subcellular targeting verification indicates novel
metabolic and regulatory functions of peroxisomes. Plant Physiol. 150, 125–143. doi: 10.1104/pp.109.137703 Niu, L., Yu, J., Liao, W., Xie, J., Yu, J., Lv, J., et al. (2019). Proteomic investigation of
S-nitrosylated proteins during NO-induced adventitious rooting of cucumber. Int. J. Mol. Sci. 20:5363. doi: 10.3390/ijms20215363 Noctor, G., Veljovic-Jovanovic, S., Driscoll, S., Novitskaya, L., Foyer, C H. (2002). Drought and oxidative load in the leaves of C3 plants: a predominant role for
photorespiration? Ann. Bot. 89, 841–50. doi: 10.1093/aob/mcf096 Ribeiro, C. W., Korbes, A. P., Garighan, J. A., Jardim-Messeder, D., Carvalho,
F. E. L., Sousa, R. H. V., et al. (2017). Rice peroxisomal ascorbate peroxidase
knockdown affects ROS signaling and triggers early leaf senescence. Plant Sci. 263, 55–65. doi: 10.1016/j.plantsci.2017.07.009 Nowak, K., Luniak, N., Witt, C., Wüstefeld, Y., Wachter, A., Mendel, R. R., et al. (2004). Peroxisomal localization of sulfite oxidase separates it from chloroplast-
based sulfur assimilation. Plant Cell Physiol. 45, 1889–1894. doi: 10.1093/pcp/
pch212 Rodríguez-Ruiz, M., González-Gordo, S., Cañas, A., Campos, M. J., Paradela, A.,
Corpas, F. J., et al. (2019). Sweet pepper (Capsicum annuum L.) fruits contain an
atypical peroxisomal catalase that is modulated by reactive oxygen and nitrogen
species. Antioxidants 8:374. doi: 10.3390/antiox8090374 Oikawa, K., Hayashi, M., Hayashi, Y., and Nishimura, M. (2019). Re-evaluation
of physical interaction between plant peroxisomes and other organelles using
live-cell imaging techniques. J. Integr. Plant Biol. 61, 836–852. Rodríguez-Serrano, M., Romero-Puertas, M. C., Pastori, G. M., Corpas, F. J.,
Sandalio, L. M., del Río, L. A., et al. (2007). Peroxisomal membrane manganese
superoxide dismutase: characterization of the isozyme from watermelon
(Citrullus lanatus Schrad.) cotyledons. J. Exp. Bot. 58, 2417–2427. doi: 10.1093/
jxb/erm095 Oikawa, K., Matsunaga, S., Mano, S., Kondo, M., Yamada, K., Hayashi, M., et al. (2015). Physical interaction between peroxisomes and chloroplasts elucidated
by in situ laser analysis. Nat. Plants. 1:15035. Olson, K. R. (2019). Hydrogen sulfide, reactive sulfur species and coping with
reactive oxygen species. Free Radic. Biol. Med. 140, 74–83. doi: 10.1016/j. freeradbiomed.2019.01.020 Romero-Puertas, M. C., Corpas, F. J., Sandalio, L. M., Leterrier, M., Rodríguez-
Serrano, M., del Río, L. A., et al. (2006). Glutathione reductase from pea leaves:
response to abiotic stress and characterization of the peroxisomal isozyme. New
Phytol. 170, 43–52. REFERENCES doi: 10.1111/j.1469-8137.2006.01643.x Olson, K. R., Gao, Y., Arif, F., Arora, K., Patel, S., DeLeon, E. R. et al. (2018). Metabolism of hydrogen sulfide (H2S) and production of reactive sulfur species
(RSS) by superoxide dismutase. Redox Biol. 15, 74–85. doi: 10.1016/j.redox. 2017.11.009 Rosenwasser, S., Rot, I., Sollner, E., Meyer, A. J., Smith, Y., Leviatan, N., et al. (2011). Organelles contribute differentially to reactive oxygen species-related events
during extended darkness. Plant Physiol. 156, 185–201. doi: 10.1104/pp.110. 169797 Olson, K. R., Gao, Y., DeLeon, E. R., Arif, M., Arif, F., Arora, N., et al. (2017). Catalase as a sulfide-sulfur oxido-reductase: an ancient (and modern?) regulator
of reactive sulfur species (RSS). Redox Biol. 12, 325–339. doi: 10.1016/j.redox. 2017.02.021 Schlicht, M., Ludwig-Müller, J., Burbach, C., Volkmann, D., and Baluska, F. (2013). Indole-3-butyric acid induces lateral root formation via peroxisome-derived
indole-3-acetic acid and nitric oxide. New Phytol. 200, 473–482. doi: 10.1111/
nph.12377 Ono, K., Akaike, T., Sawa, T., Kumagai, Y., Wink, D. A., Tantillo, D. J., et al. (2014). Redox chemistry and chemical biology of H2S, hydropersulfides, and derived
species: implications of their possible biological activity and utility. Free Radic. Biol. Med. 77, 82–94. doi: 10.1016/j.freeradbiomed.2014.09.007 Schöneich, C. (2016). Thiyl radicals and induction of protein degradation. Free
Radic. Res. 50, 143–149. doi: 10.3109/10715762.2015.1077385 Shelp, B. J., and Zarei, A. (2017). Subcellular compartmentation of 4-
aminobutyrate (GABA) metabolism in Arabidopsis: an update. Plant Signal. Behav. 12:e1322244. doi: 10.1080/15592324.2017.1322244 Biol. Med. 77, 82–94. doi: 10.1016/j.freeradbiomed.2014.09.007 Palma, J. M., de Morales, P. Á, del Río, L. A., and Corpas, F. J. (2018). The proteome
of fruit peroxisomes: sweet pepper (Capsicum annuum L.) as a model. Subcell
Biochem. 89, 323–341. doi: 10.1007/978-981-13-2233-4_14 Simkin, A. J., Guirimand, G., Papon, N., Courdavault, V., Thabet, I., Ginis, O., et al. (2011). Peroxisomal localisation of the final steps of the mevalonic acid pathway
in planta. Planta 234, 903–914. doi: 10.1007/s00425-011-1444-6 Palma, J. M., Jiménez, A., Sandalio, L. M., Corpas, F. J., Lundqvist, M., Gómez,
M., et al. (2006). Antioxidative enzymes from chloroplasts, mitochondria, and
peroxisomes during leaf senescence of nodulated pea plants. J. Exp. Bot. 57,
1747–1758. doi: 10.1093/jxb/erj191 Sousa, R. H. V., Carvalho, F. E. L., Lima-Melo, Y., Alencar, V. T. C. B., Daloso,
D. M., Margis-Pinheiro, M., et al. (2019). Impairment of peroxisomal APX and
CAT activities increases protection of photosynthesis under oxidative stress. J. Exp. Bot. 70, 627–639. doi: 10.1093/jxb/ery354 Palma, J. M., Mateos, R. REFERENCES doi: 10.1093/emboj/19.24.6770 Hackenberg, T., Juul, T., Auzina, A., Gwizdz, S., Malolepszy, A., Van Der Kelen, K.,
et al. (2013). Catalase and NO CATALASE ACTIVITY1 promote autophagy-
dependent cell death in Arabidopsis. Plant Cell 25, 4616–4626. doi: 10.1105/
tpc.113.117192 López-Huertas, E., and del Río, L. A. (2014). Characterization of antioxidant
enzymes and peroxisomes of olive (Olea europaea L.) fruits. J. Plant Physiol. 171, 1463–1471. doi: 10.1016/j.jplph.2014.06.014 Hänsch, R., and Mendel, R. R. (2005). Sulfite oxidation in plant peroxisomes. Photosynth Res. 86, 337–343. doi: 10.1007/s11120-005-5221-x Hauck, O. K., Scharnberg, J., Escobar, N. M., Wanner, G., Giavalisco, P., and Witte,
C. P. (2014). Uric acid accumulation in an Arabidopsis urate oxidase mutant
impairs seedling establishment by blocking peroxisome maintenance. Plant Cell
26, 3090–3100. doi: 10.1105/tpc.114.124008 Lounifi, I., Arc, E., Molassiotis, A., Job, D., Rajjou, L., and Tanou, G. (2013). Interplay between protein carbonylation and nitrosylation in plants. Proteomics
13, 568–578. doi: 10.1002/pmic.201200304 Mano, J., Nagata, M., Okamura, S., Shiraya, T., and Mitsui, T. (2014). Identification
of oxidatively modified proteins in salt-stressed Arabidopsis: a carbonyl-
targeted proteomics approach. Plant Cell Physiol. 55, 1233–1244. doi: 10.1093/
pcp/pcu072 Hinojosa, L., Sanad, M. N. M. E., Jarvis, D. E., Steel, P., Murphy, K., and Smertenko,
A. (2019). Impact of heat and drought stress on peroxisome proliferation in
quinoa. Plant J. 99, 1144–1158. doi: 10.1111/tpj.14411 Huang, A. H. C., Moore, T. S., and Trelease, R. N. (1983). Plant Peroxisomes. New York, NY: Academic Press. Mata-Pérez, C., Begara-Morales, J. C., Chaki, M., Sánchez-Calvo, B., Valderrama,
R., Padilla, M. N., et al. (2016). Protein tyrosine nitration during development
and abiotic stress response in plants. Front. Plant Sci. 7:1699. Huang, J., Willems, P., Wei, B., Tian, C., Ferreira, R. B., Bodra, N., et al. (2019). Mining for protein S-sulfenylation in Arabidopsis uncovers redox-sensitive
sites. Proc. Natl. Acad. Sci. U.S.A. 116, 21256–21261. doi: 10.1073/pnas. 1906768116 Mhamdi, A., Noctor, G., and Baker, A. (2012). Plant catalases: peroxisomal redox
guardians. Arch. Biochem. Biophys. 525, 181–194. doi: 10.1016/j.abb.2012. 04.015 July 2020 | Volume 11 | Article 853 Frontiers in Plant Science | www.frontiersin.org 10 Plant Peroxisomes and Reactive Species Corpas et al. Pan, R., Reumann, S., Lisik, P., Tietz, S., Olsen, L. J., and Hu, J. (2018). Proteome analysis of peroxisomes from dark-treated senescent Arabidopsis
leaves. J. Integr. Plant Biol. 60, 1028–1050. doi: 10.1111/jipb.12670 Mhamdi, A., Queval, G., Chaouch, S., Vanderauwera, S., and Van Breusegem,
F. (2010). REFERENCES Catalase function in plants: a focus on Arabidopsis mutants as
stress-mimic models. J. Exp. Bot. 61, 4198–4220. p
Mishanina, T. V., Libiad, M., and Banerjee, R. (2015). Biogenesis of reactive sulfur
species for signaling by hydrogen sulfide oxidation pathways. Nat. Chem. Biol. 11, 457–464. doi: 10.1038/nchembio.1834 Park, C. M., Weerasinghe, L., Day, J. J., Fukuto, J. M., and Xian, M. (2015). Persulfides: current knowledge and challenges in chemistry and chemical
biology. Mol. Biosyst. 11, 1775–1785. doi: 10.1039/c5mb00216h Mohn, M. A., Thaqi, B., and Fischer-Schrader, K. (2019). Isoform-specific NO
synthesis by Arabidopsis thaliana nitrate reductase. Plants 8:67. doi: 10.3390/
plants8030067 Piacentini, D., Corpas, F. J., D’Angeli, S., Altamura, M. M., and Falasca, G. (2020). Cadmium and arsenic-induced-stress differentially modulates Arabidopsis root
architecture, peroxisome distribution, enzymatic activities and their nitric oxide
content. Plant Physiol. Biochem. 148, 312–323. doi: 10.1016/j.plaphy.2020.01. 026 Mor, A., Koh, E., Weiner, L., Rosenwasser, S., Sibony-Benyamini, H., and Fluhr,
R. (2014). Singlet oxygen signatures are detected independent of light or
chloroplasts in response to multiple stresses. Plant Physiol. 165, 249–261. doi:
10.1104/pp.114.236380 Pracharoenwattana, I., and Smith, S. M. (2008). When is a peroxisome not
a peroxisome? Trends Plant Sci. 13, 522–525. doi: 10.1016/j.tplants.2008. 07.003 Moschou, P. N., Sanmartin, M., Andriopoulou, A. H., Rojo, E., Sanchez-Serrano,
J. J., and Roubelakis-Angelakis, K. A. (2008). Bridging the gap between plant
and mammalian polyamine catabolism: a novel peroxisomal polyamine oxidase
responsible for a full back-conversion pathway in Arabidopsis. Plant Physiol. 147, 1845–1857. doi: 10.1104/pp.108.123802 Prado, A. M., Porterfield, D. M., and Feijó, J. A. (2004). Nitric oxide is involved
in growth regulation and re-orientation of pollen tubes. Development 131,
2707–2714. doi: 10.1242/dev.01153 Ray, M., Mishra, P., Das, P., and Sabat, S. C. (2012). Expression and purification
of soluble bio-active rice plant catalase-A from recombinant Escherichia coli. J. Biotechnol. 157, 12–19. doi: 10.1016/j.jbiotec.2011.09.022 pp
Müller, M., Zechmann, B., and Zellnig, G. (2004). Ultrastructural localization of
glutathione in cucurbita pepo plants. Protoplasma 223, 213–219. g
p
Neill, S., Barros, R., Bright, J., Desikan, R., Hancock, J., Harrison, J., et al. (2008). Nitric oxide, stomatal closure, and abiotic stress. J. Exp. Bot. 59, 165–176. Reumann, S., and Bartel, B. (2016). Plant peroxisomes: recent discoveries in
functional complexity, organelle homeostasis, and morphological dynamics. Curr. Opt. Plant Biol. 34, 17–26. doi: 10.1016/j.pbi.2016.07.008 Nguyen, A. T., and Donaldson, R. P. (2005). Metal-catalyzed oxidation induces
carbonylation of peroxisomal proteins and loss of enzymatic activities. Arch. Biochem. Biophys. 2005, 25–31. REFERENCES M., López-Jaramillo, J., Rodríguez-Ruiz, M., González-
Gordo, S., Lechuga-Sancho, A. M., et al. (2020). Plant catalases as NO and H2S
targets. Redox Biol. 24, 131–137. doi: 10.1016/j.redox.2020.101525 Su, T., Li, W., Wang, P., and Ma, C. (2019). Dynamics of peroxisome homeostasis
and its role in stress response and signaling in Plants. Front. Plant Sci. 10:705. Pan, R., Liu, J., Wang, S., and Hu, J. (2020). Peroxisomes: versatile organelles with
diverse roles in plants. New Phytol. 225, 1410–1427. doi: 10.1111/nph.16134 July 2020 | Volume 11 | Article 853 Frontiers in Plant Science | www.frontiersin.org 11 Plant Peroxisomes and Reactive Species Corpas et al. membranes in pumpkin. Plant Cell Physiol. 36, 1157–1162. doi: 10.1093/
oxfordjournals.pcp.a078862 membranes in pumpkin. Plant Cell Physiol. 36, 1157–1162. doi: 10.1093/
oxfordjournals.pcp.a078862 Su, T., Wang, P., Li, H., Zhao, Y., Lu, Y., Dai, P., et al. (2018). The Arabidopsis
catalase triple mutant reveals important roles of catalases and peroxisome-
derived signaling in plant development. J. Integr. Plant Biol. 60, 591–607. doi:
10.1111/jipb.12649 Yamauchi, S., Mano, S., Oikawa, K., Hikino, K., Teshima, K. M., Kimori, Y.,
et al. (2019). Autophagy controls reactive oxygen species homeostasis in guard
cells that is essential for stomatal opening. Proc. Natl. Acad. Sci. U.S.A. 116,
19187–19192. doi: 10.1073/pnas.1910886116 Tolbert, N. E., and Essner, E. (1981). Microbodies: peroxisomes and glyoxysomes. J. Cell Biol. 91, 271s–283s. doi: 10.1083/jcb.91.3.271s Yang, M., Li, Z., Zhang, K., Zhang, X., Zhang, Y., Wang, X., et al. (2018). Barley
stripe mosaic virus γb interacts with glycolate oxidase and inhibits peroxisomal
ROS production to facilitate virus infection. Mol. Plant 11, 338–341. doi:
10.1016/j.molp.2017.10.007 Ueda, Y., Uehara, N., Sasaki, H., Kobayashi, K., and Yamakawa, T. (2013). Impacts
of acute ozone stress on superoxide dismutase (SOD) expression and reactive
oxygen species (ROS) formation in rice leaves. Plant Physiol. Biochem. 70,
396–402. doi: 10.1016/j.plaphy.2013.06.009 Veljovic-Jovanovic, S., Oniki, T., and Takahama, U. (1998). Detection of
monodehydroascorbic acid radical in sulfite-treated leaves and mechanism of
its formation. Plant Cell Physiol. 39, 1203–1208. doi: 10.1093/oxfordjournals. pcp.a029321 Yu, L., Fan, J., and Xu, C. (2019). Peroxisomal fatty acid β-oxidation negatively
impacts plant survival under salt stress. Plant Signal. Behav. 14:1561121. doi:
10.1080/15592324.2018.1561121 Zhang, Z., Xu, Y., Xie, Z., Li, X., He, Z H., Peng, X X. (2016). Association-
dissociation of glycolate oxidase with catalase in rice: a potential switch to
modulate intracellular H2O2 levels. Mol. Plant. 9, 737–748. doi: 10.1016/j.molp. REFERENCES 2016.02.002 Wagner, D., Przybyla, D., Op den Camp, R., Kim, C., Landgraf, F., Lee, K. P.,
et al. (2004). The genetic basis of singlet oxygen-induced stress responses of
Arabidopsis thaliana. Science 306, 1183–1185. doi: 10.1126/science.1103178 Wang, X., Zhu, B., Jiang, Z., and Wang, S. (2019). Calcium-mediation of jasmonate
biosynthesis and signaling in plants. Plant Sci. 287:110192. doi: 10.1016/j. plantsci.2019.110192 Zarei, A., Trobacher, C. P., Cooke, A. R., Meyers, A. J., Hall, J. C., and Shelp,
B. J. (2015). Apple fruit copper amine oxidase isoforms: peroxisomal MdAO1
prefers diamines as substrates, whereas extracellular MdAO2 exclusively utilizes
monoamines. Plant Cell Physiol. 56, 137–147. doi: 10.1093/pcp/pcu155 Wang, W., Paschalidis, K., Feng, J. C., Song, J., and Liu, J. H. (2019). Polyamine
catabolism in plants: a universal process with diverse functions. Front. Plant
Sci. 10:561. Zarepour, M., Kaspari, K., Stagge, S., Rethmeier, R., Mendel, R. R., and Bittner, F. (2010). Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a potent
producer of superoxide anions via its NADH oxidase activity. Plant Mol. Biol. 72, 301–310. doi: 10.1007/s11103-009-9570-2 Wei, L., Zhang, M., Wei, S., Zhang, J., Wang, C., and Liao, W. (2020). Roles of
nitric oxide in heavy metal stress in plants: cross-talk with phytohormones
and protein S-nitrosylation. Environ. Pollut. 259:113943. doi: 10.1016/j.envpol. 2020.113943 Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. Wuddineh, W., Minocha, R., and Minocha, S. C. (2018). Polyamines in the context
of metabolic networks. Methods Mol. Biol. 1694, 1–23. doi: 10.1007/978-1-
4939-7398-9_1 Copyright © 2020 Corpas, González-Gordo and Palma. 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. Xin, Z., Chen, S., Ge, L., Li, X., and Sun, X. (2019). The involvement of a herbivore-
induced acyl-CoA oxidase gene, CsACX1, in the synthesis of jasmonic acid and
its expression in flower opening in tea plant (Camellia sinensis). Plant Physiol. Biochem. 135, 132–140. doi: 10.1016/j.plaphy.2018.11.035 Biochem. 135, 132–140. doi: 10.1016/j.plaphy.2018.11.035 Yamaguchi, K., Mori, H., and Nishimura, M. (1995). Frontiers in Plant Science | www.frontiersin.org July 2020 | Volume 11 | Article 853 REFERENCES A novel isoenzyme
of ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal July 2020 | Volume 11 | Article 853 Frontiers in Plant Science | www.frontiersin.org 12
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An exploration of changes in plantar pressure distributions during walking with standalone and supported lateral wedge insole designs
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Journal of foot and ankle research
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RESEARCH Open Access Tse et al. Journal of Foot and Ankle Research (2021) 14:55
https://doi.org/10.1186/s13047-021-00493-5 Tse et al. Journal of Foot and Ankle Research (2021) 14:55
https://doi.org/10.1186/s13047-021-00493-5 (2021) 14:55 An exploration of changes in plantar
pressure distributions during walking with
standalone and supported lateral wedge
insole designs Calvin T. F. Tse1,2,3, Michael B. Ryan3,4, Jason Dien1, Alex Scott5 and Michael A. Hunt1,5* Abstract 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. * Correspondence: michael.hunt@ubc.ca
1Motion Analysis and Biofeedback Laboratory, University of British Columbia,
Vancouver, BC, Canada
5Department of Physical Therapy, University of British Columbia, Vancouver,
BC, Canada
Full list of author information is available at the end of the article * Correspondence: michael.hunt@ubc.ca
1Motion Analysis and Biofeedback Laboratory, University of British Columbia,
Vancouver, BC, Canada
5Department of Physical Therapy, University of British Columbia, Vancouver,
BC, Canada
Full list of author information is available at the end of the article BC, Canada
Full list of author information is available at the end of the article Abstract Background: Lateral wedge insoles (LWI), standalone or with medial arch support (supported-LWI), have been
thoroughly investigated for their effects on modifying gait biomechanics for people with knee osteoarthritis. However, plantar pressure distribution between these insole types has not been investigated and could provide
insight towards insole prescription with concomitant foot symptoms taken into consideration. Methods: In a sample of healthy individuals (n = 40), in-shoe plantar pressure was measured during walking with
LWI, with or without medial arch support (variable- and uniform-stiffness designs), and a flat control insole
condition. Pressure data from the plantar surface of the foot were divided into seven regions: medial/lateral
rearfoot, midfoot, medial/central/lateral forefoot, hallux. Plantar pressure outcomes assessed were the medial-lateral
pressure index (MLPI) for the whole foot, and the peak pressure, pressure-time integral (PTI), and contact area in
each plantar region. Comfort in each insole condition was rated as a change relative to the flat control insole
condition. Repeated-measures analyses of variance were calculated to compare the plantar pressure outcomes
between insole conditions. Results: Regionally, medial rearfoot and forefoot pressure were reduced by all wedged insoles, with the variable-
stiffness supported-wedge showing greater reductions than the standalone wedge. Lateral rearfoot and forefoot
pressure were reduced by both supported-LWI, but unchanged by the standalone wedge. In the midfoot, the
standalone wedge maintained pressure but reduced regional contact area, while both supported-LWI increased
midfoot pressure and contact area. All LWI increased the MLPI, indicating a lateral shift in plantar pressure
distribution throughout the weightbearing phase of gait. Comfort ratings were not significantly different between
insole conditions. © The Author(s). 2021 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/. Background
h
l demonstrated biomechanical efficacy, selecting an insole
for patients to avoid exacerbation of existing foot symp-
toms appears to be paramount. Shoe-worn lateral wedge insoles (LWI) are simple tools
used to modify gait biomechanics in people with medial
tibiofemoral osteoarthritis (knee OA). In this clinical
population,
conservative
biomechanical
interventions
typically target reductions in magnitudes of the knee ad-
duction moment (KAM), a surrogate of knee load distri-
bution linked to structural [1, 2] and clinical worsening
of knee OA [3]. LWI, as standalone insoles or combined
with medial arch supports (supported-LWI), have been
thoroughly investigated for their effect on the KAM,
with
a
recent
systematic
review
and
meta-analysis
reporting 5–10% reductions across various outcomes of
the KAM during walking with various LWI designs [4]. As a foot-based intervention, evaluating LWI for their
effects on modifying plantar pressure distribution would
help inform other clinically-relevant features, such as re-
gional loading and comfort. Evaluations of pressure distribution underfoot with
LWI have primarily reported lateral shifts in the centre
of pressure (CoP) [9, 14, 15]. Lateralization of CoP is hy-
pothesized to reduce the KAM by shifting the medially-
oriented ground reaction force to shorten the frontal
plane moment arm between the reaction force vector
and the knee joint. Shifts in CoP may not be a reliable
mechanism of KAM reduction, however, since medial
shifts in CoP have also been found to reduce the KAM,
as seen with medial thrust gait [16] and laterally-wedged
footwear (wedged externally on the outsole) [17]. Inves-
tigations of KAM reduction often report CoP measured
by floor-mounted force platforms, which provide high fi-
delity kinetic information about the interface between
footwear and the ground, necessary for calculating joint
moments. In comparison, flexible in-shoe pressure sen-
sors provide a less expensive and portable method of
measuring pressure between the plantar foot surface and
the insole, in particular those with non-planar designs. With a method for plantar pressure assessment that is
feasible for clinical settings, relevant information about
the foot-insole interface could inform insole prescription
when concomitant foot symptoms are present. There is growing interest in concomitant foot and
ankle symptoms and their link to clinical features of
knee OA. Recent analyses of the Osteoarthritis Initiative
database have highlighted relationships between symp-
tomatic knee OA and concomitant foot and ankle symp-
toms, defined as pain, aching, or stiffness for more than
half of the days in the past 30 days [5–7]. © The Author(s). 2021 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. Page 2 of 11 Tse et al. Journal of Foot and Ankle Research (2021) 14:55 Conclusions: Regional differences in plantar pressure may help determine an appropriate lateral wedge insole
variation to avoid exacerbation of concomitant foot symptoms by minimizing pressure in symptomatic regions. Lateral shifts in plantar pressure distribution were observed in all laterally wedged conditions, including one
supported-LWI that was previously shown to be biomechanically ineffective for modifying knee joint load
distribution. Thus, shifts in foot centre of pressure may not be a primary mechanism by which LWI can modify knee
joint load distribution for people with knee osteoarthritis. Keywords: Plantar pressure, Pressure distribution, Lateral wedge insole, Supported lateral wedge Background
h
l Patients with
knee OA and concomitant foot symptoms have exhib-
ited poorer outcomes of overall health and physical
function at baseline [5], and a higher risk of worsening
knee pain in the subsequent 4 years [7], compared to
knee OA patients without concomitant foot symptoms. Further, in individuals at risk of developing knee OA,
but asymptomatic and without radiographic signs at
baseline, the presence of foot and ankle symptoms in-
creased the likelihood of developing knee pain and
radiographic signs of OA over 4 subsequent years [6]. A
consistent effect of LWI is increased ankle eversion and
external eversion moment demands [4, 8, 9], with
greater angles of wedging also negatively affecting self-
reported comfort [10, 11]. Supported-LWI have been
shown to be effective at minimizing ankle eversion ef-
fects, while still reducing the KAM [8, 9, 12], and may
be preferred over a standalone LWI [13]. Considering
the clinical concerns of concomitant foot symptoms in
knee
OA
and
that
multiple
LWI
options
have Assessments of regional plantar pressure distribution
could provide insight into how redistribution of plantar
pressure with insoles could influence perceived symp-
toms. In people experiencing foot pain from work-
related prolonged standing, contoured insoles shifted re-
gional peak pressures from the rearfoot to the midfoot,
and reduced sensations of pain, discomfort, and fatigue
compared to not wearing the insoles [18]. A cross-
sectional exploration of patients with knee OA reported
that greater knee pain was associated with increased
medial plantar loading and midfoot contact area during
gait [19], which suggests plantar pressure distribution
could be linked to perceived symptoms in the lower limb
with knee OA. In-shoe measured plantar pressure effects
for varus and valgus wedged insoles [20] and externally-
wedged footwear [21] have reported lateral shifts in
pressure with lateral foot posting. However, the regional
plantar pressure effects have not been evaluated for Tse et al. Journal of Foot and Ankle Research (2021) 14:55 Page 3 of 11 Page 3 of 11 supported-LWI, nor interpreted with the intent of
informing insole treatment amongst concomitant foot
symptoms. Therefore, the aim of this study was to ex-
plore the effects of standalone LWI and supported-LWI
on plantar pressure distribution and perceived comfort
during gait in healthy adults. orthotic insole use in the 12 months prior to physical
screening, or any musculoskeletal or neurological condi-
tion that impaired gait at the time of testing. Orthotic insoles
F
i
f The current investigation is a complementary analysis of
plantar pressure and comfort data obtained during gait
analysis of various LWI designed for knee OA. A sum-
mary of the joint kinematic and kinetic outcomes for
these insoles were reported in Tse et al. [12], which
found standalone medial arch supports increased the
KAM. Similar findings of increased KAM with medial
arch supports have been reported [22, 23], despite Hin-
man et al. [23] reporting non-significant increases in a
small sample with large variability in KAM change. For
this reason, standalone medial arch supports were ex-
cluded from the current analysis of plantar pressure out-
comes. However, outcomes across all insole conditions
are reported in Appendix 1 of supplementary materials. Additionally, the current sample size was justified for de-
tecting a change in KAM with LWI from the affiliated
study [12]. Therefore, an additional sample size justifica-
tion was not conducted for the current exploratory as-
sessment of plantar pressure distribution and perceived
comfort with standalone LWI and supported-LWI. Four pairs of sulcus length orthotics were custom-
fabricated for each participant, using three-dimensional
laser volumetric casting by a Canadian Board-Certified
Pedorthist (Fig. 1). Non-contoured insoles fabricated
from ethyl-vinyl acetate foam (EVA) (Shore A stiffness
55) included a neutral 3 mm flat control (FLAT) and 5°
lateral wedge (WEDG). Two pairs of custom contoured
arch support insoles were fabricated from the volumetric
casts: (1) variable-stiffness (V-ARCH) was constructed
with plastazote foam laterally (Shore A stiffness 70) and
EVA medially (Shore A stiffness 20), (2) uniform-
stiffness (U-ARCH) was constructed with EVA (Shore A
stiffness 55). Two supported-LWI conditions were cre-
ated by affixing each custom arch support to the top of
the WEDG: WEDG+V-ARCH and WEDG+U-ARCH. All insoles were covered with a full-length piece of neo-
prene and secured into a standardized sandal during all
walking trials (Fig. 1). Background
h
l Ethics ap-
proval for this study was received from the institutional
Clinical Research Ethics Board. All participants received
written and verbal explanations of the details prior to
providing written consent for study enrolment. Participants All participants were randomly assigned a study limb of
interest and fitted with standardized sandals to match
their foot dimensions. The sandals had a neutral heel to
toe drop, Velcro straps to secure them to the feet, and
removeable footbeds, into which orthotic insoles could A convenience sample of healthy adults from the univer-
sity and surrounding community were recruited via elec-
tronic and print media and word of mouth. Exclusion
criteria for study participation included any history of Fig. 1 Insoles and standardized sandal set-up used for walking trials. A 3 mm flat control (FLAT). B 5° lateral wedge (WEDG). C Variable-stiffness
arch support (V-ARCH). D Uniform-stiffness arch support (U-ARCH). E Example of supported lateral wedge (WEDG+V-ARCH shown). F Sandal setup
with insole, neoprene topcover, and plantar pressure sensor Fig. 1 Insoles and standardized sandal set-up used for walking trials. A 3 mm flat control (FLAT). B 5° lateral wedge (WEDG). C Variable-stiffness
arch support (V-ARCH). D Uniform-stiffness arch support (U-ARCH). E Example of supported lateral wedge (WEDG+V-ARCH shown). F Sandal setup
with insole, neoprene topcover, and plantar pressure sensor Fig. 1 Insoles and standardized sandal set-up used for walking trials. A 3 mm flat control (FLAT). B 5° lateral wedge (WEDG). C Variable-stiffness
arch support (V-ARCH). D Uniform-stiffness arch support (U-ARCH). E Example of supported lateral wedge (WEDG+V-ARCH shown). F Sandal setup
with insole, neoprene topcover, and plantar pressure sensor Page 4 of 11 Tse et al. Journal of Foot and Ankle Research (2021) 14:55 Fig. 2 Representative plantar pressure map during a single stance phase. The coloured box overlays demarcate the complete pressure map into
multiple plantar regions used for calculating pressure outcomes. Red
dashed line between the heel centroid and second toe delineates the
midline of the foot. White crosses (+) denote the trajectory of the centre of
pressure (CoP) throughout stance phase. Green dashed lines demonstrate
how the time-series medial-lateral pressure index is calculated as the
distance between the CoP and the foot midline (normalized to foot width). Positive and negative MLPI indicate a CoP that is lateral and medial to the
foot midline, respectively be fitted. Prior to collecting data during walking in each
insole condition, participants were encouraged to accli-
mate to the insole and sandals by walking freely and re-
solving any fit abnormalities. Participants For all participants the
FLAT condition was tested first, and the five remaining
insole conditions were systematically randomized with a
Williams Design to account for possible order and
carry-over effects. Walking speed was measured using
two commercial photoelectric timing gates placed a
known distance apart, and self-selected walked speed
was established as the mean walking speed during walk-
ing trials with FLAT. Walking trials were deemed suc-
cessful if the speed was within 5% of the average walking
speed with FLAT for all other conditions, and if the
study limb foot struck the ground completely within the
boundary of a floor-embedded force platform. A mini-
mum of five successful walking trials along a 10 m walk-
way were recorded in each insole condition. Data collection and reduction Plantar pressure was measured bilaterally using flexible
shoe-embedded sensors (F-Scan, Tekscan, Boston, MA,
USA) at 100 Hz. All sensors were trimmed to match the
sandal size and secured to the neoprene topcover with
double-side tape to ensure consistent sensor placement
among insole conditions (Fig. 1). Each sensor detects
pressure as an array of sensing units, with a surface area
resolution of 0.258 cm2 per sensing unit. Only data col-
lected during the stance phase of gait were processed
and analyzed. For each calculated outcome, the average
of five successful walking trials was calculated to repre-
sent its value in each insole condition. Pressure data recorded by each sensing unit was fil-
tered using a zero-lag 4th order low-pass Butterworth
filter with a 25 Hz cut-off. For faulty sensing units that
detected non-physiological pressure, its data were re-
placed with the time-series average of its eight neigh-
bouring
sensing
units. A
semi-automated
masking
program was used to segment the foot into seven plantar
regions: hallux, forefoot (medial, central, lateral), mid-
foot, rearfoot (medial, lateral) (Fig. 2). All masks were
visually checked, and any erroneous regional masks were
manually corrected. For each plantar region, a represen-
tative time-series pressure signal was calculated as the Page 5 of 11 Tse et al. Journal of Foot and Ankle Research (2021) 14:55 Tse et al. Journal of Foot and Ankle Research (2021) 14:55 ARCH). An extended summary of discrete outcomes for
all insoles can be found in Appendix 1 of supplementary
materials. average pressure of all active sensing units within the re-
gion. From the regional time-series pressure data, the
discrete outcomes extracted for analysis included: peak
pressure (kPa), pressure-time integral (kPa*sec), and
time of peak pressure (% stance). The contact area in
each plantar region was calculated as the sum of active
sensing units in the region multiplied by the surface area
per unit. Results Forty healthy individuals participated in the study with
the following demographic information: 23 males, 17 fe-
males, mean (SD) age = 26.6 (2.9) years, height = 173.5
(8.6) cm, body mass = 71.2 (12.7) kg, BMI = 23.5 (2.8)
kg/m2. The median foot posture index was = 5 (25th per-
centile = 2; 75th percentile = 9). All evaluated outcomes
satisfied the assumptions for normality and homogeneity
of variance. The following summarizes the outcomes for
the four conditions that were statistically compared
(FLAT,
WEDG,
WEDG+V-ARCH,
and
WEDG+U- Statistical analysis Normality was evaluated via visual inspection of histo-
grams and supplemented with a Shapiro-Wilk test. Homogeneity of variance was evaluated via Mauchly’s
test of sphericity. For discrete outcomes of MLPI, re-
gional plantar pressure, and comfort change, repeated
measures analyses of variance were used to test for dif-
ferences between four insole conditions (FLAT, WEDG,
WEDG+V-ARCH, WEDG+U-ARCH). Significant main
effects of insole condition were followed-up with post
hoc Tukey’s HSD pairwise comparisons. Statistical cal-
culations were completed with jamovi version 1.6 [26],
at an alpha level of α = 0.05. Contact area in the medial rearfoot decreased with
WEDG, compared to FLAT (p < 0.05). The WEDG+V-
ARCH and WEDG+U-ARCH increased medial rearfoot
contact area when compared to WEDG (p < 0.05), but
were not different from FLAT. Medial forefoot contact
area was unchanged by any insole compared to FLAT
(p ≥0.32). Medial regional pressure Medial
rearfoot
and
forefoot
peak
pressure
and
pressure-time integral were reduced by WEDG, WEDG+
V-ARCH, and WEDG+U-ARCH (all p < 0.05) when
compared to FLAT. Both supported-LWI (WEDG+V-
ARCH and WEDG+U-ARCH) also reduced medial rear-
foot and forefoot peak pressure and pressure-time inte-
gral compared to WEDG (p < 0.05). Ensemble average
curves for the medial rear- and forefoot regions are
found in Fig. 3b. Regional plantar pressure
Lateral regional pressure g
p
Lateral
rearfoot
and
forefoot
peak
pressure
and
pressure-time integral were decreased with WEDG+V-
ARCH (p < 0.05), when compared to FLAT. WEDG+V-
ARCH also had lower peak pressure and pressure-time
integral compared to WEDG (p < 0.05). WEDG+U-
ARCH reduced the peak pressure in the lateral rearfoot
and the pressure-time integral in lateral forefoot, com-
pared to FLAT (both p < 0.05). Discrete plantar pressure
outcomes for all regions are reported in Table 1, and en-
semble average curves for the lateral rearfoot and fore-
foot regions are found in Fig. 3a. The medial-lateral pressure index (MLPI) represents
the medial-lateral plantar pressure distribution with re-
spect to the midline of the foot (heel centroid to 2nd
toe) throughout stance phase (Fig. 2). For each frame of
data, MLPI was calculated as the perpendicular distance
between the centre of pressure (CoP) and the midline of
the foot, normalized to the foot width [24]. Positive and
negative values indicate a CoP that is lateral or medial to
the midline of the foot, respectively. For each walking
trial, the mean MLPI and area under the MLPI curve
(AUC) during the first and second halves of stance phase
were calculated separately. Contact area in the lateral rearfoot was increased with
WEDG+V-ARCH and WEDG+U-ARCH compared to
FLAT (p < 0.05) and WEDG (p < 0.05). Lateral forefoot
contact area decreased with supported-LWI WEDG+V-
ARCH and WEDG+U-ARCH compared to FLAT (p <
0.05). WEDG did not significantly change contact area
in the lateral rear- or forefoot compared to FLAT (p ≥
0.06). Discrete contact area values for all regions are re-
ported in Table 1. Participants rated insole comfort on a 15-point global
rating of change scale [25]. Using the FLAT insole con-
dition as the reference comparator, + 7 and −7 repre-
sented
a
change
in
comfort
that
was
maximally
“improved” or “reduced”, respectively. Zero represented
no change in comfort (equivalent to FLAT). Following
completion of walking trials in all insole conditions, par-
ticipants selected one insole as their most preferred. Midfoot pressure Midfoot peak pressure and pressure-time integral were
unchanged with WEDG (p ≥0.13), and increased with
WEDG+V-ARCH (p < 0.05) and WEDG+U-ARCH (p <
0.05), compared to FLAT. Ensemble average curves for
the region are found in Fig. 4. Compared to FLAT, midfoot contact area was in-
creased with WEDG+V-ARCH and WEDG+U-ARCH
(p < 0.05), while decreased with WEDG (p < 0.05). Tse et al. Journal of Foot and Ankle Research (2021) 14:55 Page 6 of 11 Table 1 Regional plantar pressure outcomes by insole condition, reported as mean (standard deviation)
Plantar Region
Pressure Outcome
FLAT
WEDG
WEDG + V-ARCH
WEDG + U-ARCH
Lateral Rearfoot
Peak Pressure (kPa)
133.0 (24.1)
132.6 (26.0)
122.0 (22.6) a,b
126.1 (23.4) a
Pressure-Time Integral (kPa*sec)
30.0 (6.4)
29.0 (5.3)
26.7 (5.5) a,b
28.3 (6.9)
Contact Area (cm2)
18.7 (2.3)
18.1 (2.1)
19.5 (2.2) a,b
19.9 (2.6) a,b
Lateral Forefoot
Peak Pressure (kPa)
106.1 (31.8)
108.6 (33.3)
85.6 (26.7) a,b
100.7 (32.2)
Pressure-Time Integral (kPa*sec)
30.6 (10.4)
32.4 (10.7)
22.0 (7.3) a,b
26.8 (10.1) a,b
Contact Area (cm2)
12.9 (2.6)
12.6 (2.3)
12.2 (2.2) a
12.3 (2.2) a
Medial Rearfoot
Peak Pressure (kPa)
150.3 (23.2)
131.9 (26.1) a
105.3 (27.6) a,b
99.3 (21.5) a,b
Pressure-Time Integral (kPa*sec)
32.9 (6.7)
26.6 (5.8) a
21.4 (6.1) a,b
20.4 (5.2) a,b
Contact Area (cm2)
17.5 (2.1)
16.5 (2.1) a
17.9 (2.5) b
17.9 (2.8) b
Medial Forefoot
Peak Pressure (kPa)
125.3 (43.0)
105.4 (39.6) a
96.9 (34.2) a,b
93.4 (37.0) a,b
Pressure-Time Integral (kPa*sec)
30.1 (10.3)
23.4 (8.6) a
20.2 (7.3) a,b
18.3 (7.6) a,b
Contact Area (cm2)
14.4 (1.8)
13.9 (2.1)
14.5 (2.6)
14.7 (2.6)
Midfoot
Peak Pressure (kPa)
42.8 (11.8)
45.6 (10.6)
48.1 (12.9) a
48.0 (12.9) a
Pressure-Time Integral (kPa*sec)
14.1 (4.6)
15.5 (4.7)
16.2 (4.8) a
16.2 (5.2) a
Contact Area (cm2)
34.7 (8.7)
31.8 (7.7) a
50.9 (9.3) a,b
52.8 (9.1) a,b
All values reported as mean (standard deviation)
a denotes a significant difference from FLAT (p < 0.05)
b denotes a significant difference from WEDG (p < 0.05) g
(p
)
b denotes a significant difference from WEDG (p < 0.05) No differences in comfort were observed between insole
conditions. In light of the redistribution of plantar pres-
sure between LWI conditions, insole prescription may
be best informed by its appropriateness for pre-existing
foot symptoms. Medial-lateral pressure distribution Through early and late stance phase, all laterally-wedged
insole conditions (WEDG, WEDG+V-ARCH, WEDG+
U-ARCH) shifted the medial-lateral plantar distribution
laterally, exhibiting increased mean and AUC MLPI
compared to FLAT (p < 0.05). Discrete values of MLPI
are summarized in Table 2 and their corresponding en-
semble average curves for all insole conditions are found
in Fig. 5. g
A speculated mechanism by which LWI reduce the
KAM is through lateralization of the foot CoP, which re-
duces the frontal plane moment arm between the knee
joint and the medially-oriented ground reaction force
vector. We found the LWI and supported-LWI increased
the mean and AUC of MLPI by 1–4% of foot width (~
1–4 mm) throughout stance phase, which is in agree-
ment with the lateral shifts in CoP reported by previous
works that also evaluated gait with and without LWI,
but that used force platform technology [9, 14, 15]. While CoP lateralization appears to be involved in KAM
reduction with LWI, it is unlikely to be the primary
mechanism of effect. Hinman et al. reported only a weak
correlation (r = 0.25) between the lateral shift in CoP
with LWI and change in KAM peak [15]. In the current
study we also found WEDG+U-ARCH had the largest
lateral shifts in CoP, but was previously reported to be
ineffective at reducing the KAM [12]. In a different bio-
mechanical assessment of medial thrust gait, a medial
shift in CoP was found to be linearly related to KAM re-
duction
(r = 0.40–0.70)
[16]. Considering
these Comfort change Rated as a change in comfort compared to FLAT, the
comfort ratings for WEDG (mean [SD] = −1.2 [2.3]),
WEDG+V-ARCH
(mean
[SD] = −0.8
[2.3]),
and
WEDG+U-ARCH (mean [SD] = −0.7 [2.5]) were not sig-
nificantly different from each other (p ≥0.58). Midfoot pressure Especially in patients with concomitant
knee OA and foot symptoms, clinicians incorporating
insoles for biomechanical intervention of medial tibiofe-
moral OA should consider both kinetic effects at the
knee and effects on pressures in plantar regions in their
clinical decision making. Discussion In the current study we assessed plantar pressure distri-
butions of healthy individuals walking with LWI and
supported-LWI compared to a flat control insole. The
regional distribution of plantar pressure differed between
insole conditions. WEDG reduced medial rearfoot and
forefoot pressure, while maintaining lateral rearfoot and
forefoot
pressures
compared
to
FLAT. Meanwhile,
WEDG+V-ARCH exhibited greater reductions in medial
and lateral rearfoot and forefoot pressure than WEDG,
and increased midfoot pressure and contact area com-
pared to FLAT. Early and late stance phase MLPI were
increased by all wedged insole conditions, indicative of a
lateral shift in CoP throughout the stance phase of gait. Tse et al. Journal of Foot and Ankle Research (2021) 14:55 Page 7 of 11 Fig. 3 Ensemble average of the regional plantar pressure for the (a) lateral and (b) medial rearfoot (solid lines) and forefoot (dashed lines). The
colours representing each insole condition are as follows: FLAT (black), WEDG (green), WEDG+V-ARCH (red), WEDG+U-ARCH (yellow) ble average of the regional plantar pressure for the (a) lateral and (b) medial rearfoot (solid lines) and forefoot (dashed lines). The Fig. 3 Ensemble average of the regional plantar pressure for the (a) lateral and (b) medial rearfoot (solid lines) and forefoot (dashed lines). The
colours representing each insole condition are as follows: FLAT (black), WEDG (green), WEDG+V-ARCH (red), WEDG+U-ARCH (yellow) Fig. 3 Ensemble average of the regional plantar pressure for the (a) lateral and (b) medial rearfoot (solid lines) and forefoot (dashed lines). Th
colours representing each insole condition are as follows: FLAT (black), WEDG (green), WEDG+V-ARCH (red), WEDG+U-ARCH (yellow) Fig. 3 Ensemble average of the regional plantar pressure for the (a) lateral and (b) medial rearfoot (solid lines) and forefoot (dashed lines). Th
colours representing each insole condition are as follows: FLAT (black), WEDG (green), WEDG+V-ARCH (red), WEDG+U-ARCH (yellow) Tse et al. Journal of Foot and Ankle Research (2021) 14:55 Page 8 of 11 Fig. 4 Ensemble average of the regional plantar pressure for the midfoot. The colours representing each insole condition are as follows: FLAT
(black), WEDG (green), WEDG+V-ARCH (red), WEDG+U-ARCH (yellow) ig. 4 Ensemble average of the regional plantar pressure for the midfoot. Discussion The arch contouring and variable-stiffness design of
WEDG+V-ARCH appeared to be redistributing pressure
from the rearfoot and forefoot into the midfoot, which
was seen as greater midfoot peak pressure, PTI, and con-
tact area compared to FLAT (Fig. 4 & Table 1). Previ-
ously, increased medial and decreased lateral peak foot
forces during walking have been associated with greater
self-reported knee pain in symptomatic patients with
knee OA [19]. As such, the observed pattern for WEDG
and WEDG+V-ARCH to redistribute plantar pressure
laterally by reducing medial pressures may be relevant to
the treatment of medial knee OA. Selecting a LWI to
minimize regional pressures based on patients’ existing
foot symptoms may also be less likely to elicit adverse
reactions to insole treatment. With the pressure distri-
butions reported, patients experiencing symptoms in the
rearfoot or forefoot may be better suited for WEDG+V-
ARCH, since pressure in these affected plantar regions
are considerably lower, and less likely to exacerbate
symptoms. In contrast, patients experiencing symptoms
in the midfoot region may be better suited for WEDG
alone, since this insole does not significantly increase
midfoot pressure compared to FLAT. Future clinical trial work examining this potential impact on clinical care is
needed to confirm these preliminary findings from
young, healthy individuals. Ratings of comfort change relative to FLAT were not
significantly different between any of the LWI condi-
tions. These insoles were tested over a period of 10–15
min per insole condition, using a small sample (n = 40)
of healthy individuals that did not use orthotic insoles
habitually. As such, it is unsurprising that average com-
fort ratings for all LWI ranged between 0 (no change)
and −1 on a −7 to + 7 scale. Evaluating insole comfort
in healthy individuals provides foundational information
prior to use in clinical populations that could have con-
comitant foot pathologies. Future research in the com-
fort effects of these LWI would benefit from longer
study durations and using cohorts of patients with knee
OA. As previously mentioned, only WEDG and WEDG+
V-ARCH reduced the KAM [12]. Until we know other-
wise with data from patients with knee OA, these insoles
can perhaps be treated equivalently for their biomechan-
ical efficacy, and the prescription of either LWI should
prioritize minimizing regional pressures and improving
subjective comfort. Findings from this study should be interpreted within
the context of several limitations. Discussion Journal of Foot and Ankle Research (2021) 14:55 Page 9 of 11 Fig. 5 Ensemble average of the medial-lateral pressure index throughout stance phase for all insole conditions. Positive and negative MLPI indicate a
CoP that is lateral or medial to the foot midline, respectively. The colours representing each insole condition are as follows: FLAT (black), WEDG (green),
WEDG+V-ARCH (red), WEDG+U-ARCH (yellow) Fig. 5 Ensemble average of the medial-lateral pressure index throughout stance phase for all insole conditions. Positive and negative MLPI indicate a
CoP that is lateral or medial to the foot midline, respectively. The colours representing each insole condition are as follows: FLAT (black), WEDG (green),
WEDG+V-ARCH (red), WEDG+U-ARCH (yellow) medial rearfoot and forefoot, compared to FLAT. Mean-
while, WEDG+V-ARCH exhibited greater reductions in
the medial and lateral rearfoot and forefoot than WEDG,
with lower peak pressure and PTI (Fig. 3 & Table 1). The arch contouring and variable-stiffness design of
WEDG+V-ARCH appeared to be redistributing pressure
from the rearfoot and forefoot into the midfoot, which
was seen as greater midfoot peak pressure, PTI, and con-
tact area compared to FLAT (Fig. 4 & Table 1). Previ-
ously, increased medial and decreased lateral peak foot
forces during walking have been associated with greater
self-reported knee pain in symptomatic patients with
knee OA [19]. As such, the observed pattern for WEDG
and WEDG+V-ARCH to redistribute plantar pressure
laterally by reducing medial pressures may be relevant to
the treatment of medial knee OA. Selecting a LWI to
minimize regional pressures based on patients’ existing
foot symptoms may also be less likely to elicit adverse
reactions to insole treatment. With the pressure distri-
butions reported, patients experiencing symptoms in the
rearfoot or forefoot may be better suited for WEDG+V-
ARCH, since pressure in these affected plantar regions
are considerably lower, and less likely to exacerbate
symptoms. In contrast, patients experiencing symptoms
in the midfoot region may be better suited for WEDG
alone, since this insole does not significantly increase
midfoot pressure compared to FLAT. Future clinical trial medial rearfoot and forefoot, compared to FLAT. Mean-
while, WEDG+V-ARCH exhibited greater reductions in
the medial and lateral rearfoot and forefoot than WEDG,
with lower peak pressure and PTI (Fig. 3 & Table 1). Discussion The colours representing each insole condition ar
black), WEDG (green), WEDG+V-ARCH (red), WEDG+U-ARCH (yellow) discrepant shifts in foot CoP and KAM reduction, it is
possible lateralizing CoP with LWI is less important to
reducing the KAM than other alterations occurring be-
tween the foot and knee, such as frontal plane tibial in-
clination
or
knee
angle. The
frontal
plane
knee
alignment during gait has been shown to completely me-
diate the relationship between ankle eversion and magni-
tudes of the KAM [27], which is relevant to how LWI
can alter KAM as a foot-based biomechanical interven-
tion. It is important to acknowledge our investigation
measured CoP with flexible in-shoe sensors atop non-
planar insole surfaces, which may differ from CoP mea-
sured by floor-mounted force plates. Nonetheless, the
value of in-shoe plantar pressure assessment of LWI
may
be
more
relevant
to
considerations
regarding regional pressure changes that could impact comfort or
existing foot symptoms. The growing concern between concomitant foot and
ankle symptoms and poorer clinical features of knee OA
[5,
7]
highlights
a
need
for
LWI
intervention
to
minimize the likelihood of generating or exacerbating
foot symptoms. Our affiliated evaluation of the insoles
from this study found WEDG and WEDG+V-ARCH to
be effective at KAM reduction [12]. Differences in the
regional plantar pressure profiles may help inform which
of these two LWI could be more appropriate for bio-
mechanical intervention in patients experiencing con-
comitant foot symptoms. For example, in the WEDG
condition, peak pressure and PTI were maintained in
the lateral rearfoot and forefoot, but reduced in the Table 2 Medial-Lateral Pressure Index (MLPI) outcomes by insole condition
MLPI Outcome
FLAT
WEDG
WEDG + V-ARCH
WEDG + U-ARCH
Early Stance
Mean (% foot width)
3.2 (2.9)
5.5 (2.5) a
5.8 (3.0) a
6.9 (3.0) a,b
AUC (% foot width * sec)
1.0 (0.9)
1.8 (0.8) a
1.9 (1.0) a
2.3 (1.0) a,b
Late Stance
Mean (% foot width)
−2.5 (5.7)
0.5 (6.0) a
−0.4 (5.9) a
0.9 (6.2) a
AUC (% foot width * sec)
−0.8 (1.9)
0.2 (2.0) a
−0.1 (2.0) a
0.4 (2.1) a
All values reported as mean (standard deviation)
Positive (lateral) and negative (medial) values indicate the centre of pressure position relative to the midline of the foot
a denotes a significant difference from FLAT (p < 0.05)
b denotes a significant difference from WEDG (p < 0.05) Tse et al. Funding Funding
This study and contributing authors received funding from the Natural
Sciences and Engineering Research Council of Canada, Canadian Institutes of
Health Research, and the Michael Smith Foundation for Health Research. Pedorthic assessment services and orthotic insoles were provided by Kintec
Footlabs Inc. Ethics approval and consent to participate The study was approved by the University of British Columbia Clinical
Research Ethics Board (H16–01793). All participants provided written and
informed consent prior to study participation. Availability of data and materials
h d
d
d
l
d d The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request. Competing interests Authors CT and MR are employed by Kintec Footlabs Inc., a company
providing orthotic services. However, these authors do not receive any direct
benefit from this research that could potentially bias these study results. The
authors declare no other professional or financial affiliations that may be
potential conflicts of interest. Additional file 1. Additional file 1. Acknowledgments g
The authors would like to thank Natasha Krowchuk for assistance during the
data acquisition process. Discussion Firstly, healthy adults
without a history of orthotic use were recruited for this
exploratory study of plantar pressure changes with LWI Page 10 of 11 Page 10 of 11 Tse et al. Journal of Foot and Ankle Research (2021) 14:55 Tse et al. Journal of Foot and Ankle Research (2021) 14:55 and supported-LWI, intended for patients with knee
OA. While it is imperative future investigations be con-
ducted within the target clinical population to assess the
effects on pain and other clinically-relevant outcomes,
our findings did not demonstrate any immediate effects
of plantar pressure distribution or comfort that would
pose any major risk of adverse effects with insole use. In-
deed, current clinical guidelines [28] for management of
knee OA do not necessarily support the use of LWIs for
knee OA – based primarily on contradictory findings of
pain improvement [29]. While any biomechanical bene-
fits of these devices are generally not considered in these
guideline recommendations, our current findings do
provide new evidence that may be used in such deci-
sions. Next, we used flexible in-shoe pressure sensors to
measure plantar pressure distribution from a non-planar
surface at the foot-insole interface, which may not reflect
the CoP measured by floor-mounted force platforms or
pressure mats. Indeed, floor-measured data provides
CoP data necessary for evaluations of mechanisms of
KAM reduction. However, we demonstrated in-shoe
pressure sensors are a cost-effective method to obtain
regional pressure distribution information that could in-
form the insole prescription process amidst consider-
ations of existing foot symptoms. Finally, while longer
acclimatization periods with insoles are common for
clinical practice (~ 2–4 weeks), the number of insole
conditions insoles that we tested prevented us from
doing so within the current study design. However, par-
ticipants were encouraged to take as long as they felt
was necessary to adjust to the sensation of each new
orthotic, prior to collection of gait trials. Authors’ contributions CTFT, MBR, AS, MAH contributed to the study conceptualization,
methodology, and manuscript preparation (draft, review, edit). CTFT and JD
were responsible for the data acquisition, processing, and analysis. MBR and
MAH acquired study funding. The authors read and approved the final
manuscript. Abbreviations AUC: Area under curve; CoP: Centre of pressure; EVA: Ethyl vinyl acetate
foam; FLAT: 3 mm EVA flat control insole; KAM: Knee adduction moment;
Knee OA: Medial tibiofemoral osteoarthritis; kPa: kilopascal, unit of pressure;
kPa*sec: product of kilopascal and time, unit of cumulative pressure over
time; LWI: Lateral wedge insole; MLPI: Medial-lateral pressure index;
PTI: Pressure-time integral; Supported-LWI: Lateral wedge insole with medial
arch support; U-ARCH: Uniform-stiffness contoured arch support insole; V-
ARCH: Variable-stiffness contoured arch support insole; WEDG: 5° lateral
wedge insole; WEDG+U-ARCH: Lateral wedge with uniform-stiffness con-
toured arch support insole; WEDG+V-ARCH: Lateral wedge with variable-
stiffness contoured arch support insole Declarations In our exploration of plantar pressure in healthy adults
walking with LWI, with or without medial arch support,
the data demonstrated pressure distributions that may
streamline the selection process for which type of LWI
to implement for biomechanical intervention. All LWI
shifted the medial-lateral distribution laterally, including
the WEDG+U-ARCH which was previously shown to be
ineffective at reducing the KAM. Therefore, shifts in foot
CoP may not be the primary mechanism by which LWI
can reduce the KAM. Regional plantar pressure distribu-
tions point towards using the WEDG+V-ARCH for indi-
viduals experiencing foot symptoms in the rearfoot and
forefoot, and the WEDG for individuals experiencing
foot symptoms in the midfoot. Since comfort ratings did
not differ between insole conditions, the proposed ar-
rangement of LWI recommendations aims to reduce
pressures in the associated plantar region and minimize
the chance of exacerbating concomitant foot symptoms
in patients with knee OA. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s13047-021-00493-5. Additional file 1. Additional file 1. Author details 1Motion Analysis and Biofeedback Laboratory, University of British Columbia,
Vancouver, BC, Canada. 2Graduate Programs in Rehabilitation Sciences,
University of British Columbia, Vancouver, BC, Canada. 3Kintec Footlabs Inc,
Surrey, BC, Canada. 4Department of Biomedical Physiology and Kinesiology,
Simon Fraser University, Burnaby, BC, Canada. 5Department of Physical
Therapy, University of British Columbia, Vancouver, BC, Canada. Page 11 of 11 Page 11 of 11 Page 11 of 11 Tse et al. Journal of Foot and Ankle Research (2021) 14:55 Received: 9 April 2021 Accepted: 22 September 2021 References 18. Tarrade T, Doucet F, Saint-Lô N, Llari M, Behr M. Are custom-made foot
orthoses of any interest on the treatment of foot pain for prolonged
standing workers? Appl Ergon. 2019;80:130–5. https://doi.org/10.1016/j.a
pergo.2019.05.013. 1. Chang AH, Moisio KC, Chmiel JS, Eckstein F, Guermazi A, Prasad PV, et al. External knee adduction and flexion moments during gait and medial
tibiofemoral disease progression in knee osteoarthritis. Osteoarthr Cartil. 2015;23(7):1099–106. https://doi.org/10.1016/j.joca.2015.02.005. 1. Chang AH, Moisio KC, Chmiel JS, Eckstein F, Guermazi A, Prasad PV, et al. External knee adduction and flexion moments during gait and medial
tibiofemoral disease progression in knee osteoarthritis. Osteoarthr Cartil. 2015;23(7):1099–106. https://doi.org/10.1016/j.joca.2015.02.005. 19. Metcalf B, Paterson KL, Campbell PK, Wrigley TV, Kasza J, Bennell KL, et al. Relationship between static foot posture, in-shoe plantar forces and knee
pain in people with medial knee osteoarthritis. Osteoarthr Cartil. 2019;27:
S120–1. https://doi.org/10.1016/j.joca.2019.02.178. 2. Miyazaki T, Wada M, Kawahara H, Sato M, Baba H, Shimada S. Dynamic load
at baseline can predict radiographic disease progression in medial
compartment knee osteoarthritis. Ann Rheum Dis. 2002;61(7):617–23. https://doi.org/10.1136/ard.61.7.617. 20. Van Gheluwe B, Dananberg HJ. Changes in plantar foot pressure with in-
shoe varus or valgus wedging. J Am Podiatr Med Assoc. 2004;94(1):1–11. https://doi.org/10.7547/87507315-94-1-1. 3. Hatfield GL, Stanish WD, Hubley-Kozey CL. Three-dimensional biomechanical
gait characteristics at baseline are associated with progression to total knee
arthroplasty. Arthritis Care Res. 2015;67(7):1004–14. https://doi.org/10.1002/a
cr.22564. 3. Hatfield GL, Stanish WD, Hubley-Kozey CL. Three-dimensional biomechanical
gait characteristics at baseline are associated with progression to total knee
arthroplasty. Arthritis Care Res. 2015;67(7):1004–14. https://doi.org/10.1002/a
cr.22564. 21. van Tunen JAC, Paterson KL, Wrigley T V., Metcalf BR, Thorlund JB, Hinman
RS. Effect of knee unloading shoes on regional plantar forces in people with
symptomatic knee osteoarthritis - an exploratory study. J Foot Ankle Res
2018;11(1):1–8, doi: https://doi.org/10.1186/s13047-018-0278-x. 4. Shaw KE, Charlton JM, Perry CKL, De Vries CM, Redekopp MJ, White JA, et al. The effects of shoe-worn insoles on gait biomechanics in people with knee
osteoarthritis: a systematic review and meta-analysis. Br J Sports Med. 2018;
52(4):238–53. https://doi.org/10.1136/bjsports-2016-097108. 22. Franz JR, Dicharry J, Riley PO, Jackson K, Wilder RP, Kerrigan DC. The
influence of arch supports on knee torques relevant to knee osteoarthritis. Med Sci Sports Exerc. 2008;40(5):913–7. https://doi.org/10.1249/MSS.0b013
e3181659c81. 5. Paterson KL, Hinman RS, Hunter DJ, Wrigley TV, Bennell KL. Impact of
concurrent foot pain on health and functional status in people with knee
osteoarthritis: data from the osteoarthritis initiative. Arthritis Care Res. References 2019
American College of Rheumatology/Arthritis Foundation guideline for the
Management of Osteoarthritis of the hand, hip, and knee. Arthritis Care Res. 2020;72(2):149–62. https://doi.org/10.1002/acr.24131. 28. Kolasinski SL, Neogi T, Hochberg MC, Oatis C, Guyatt G, Block J, et al. 2019
American College of Rheumatology/Arthritis Foundation guideline for the
Management of Osteoarthritis of the hand, hip, and knee. Arthritis Care Res. 2020;72(2):149–62. https://doi.org/10.1002/acr.24131. 10. Tipnis RA, Anloague PA, Laubach LL, Barrios JA. The dose-response
relationship between lateral foot wedging and the reduction of knee
adduction moment. Clin Biomech. 2014;29(9):984–9. https://doi.org/10.1016/
j.clinbiomech.2014.08.016. 29. Parkes MJ, Maricar N, Lunt M, LaValley MP, Jones RK, Segal NA, et al. Lateral
wedge insoles as a conservative treatment for pain in patients with medial
knee osteoarthritis a meta-analysis. JAMA. 2013;310(7):722–30. https://doi. org/10.1001/jama.2013.243229. 11. Kerrigan DC, Lelas JL, Goggins J, Merriman GJ, Kaplan RJ, Felson DT. Effectiveness of a lateral-wedge insole on knee varus torque in patients
with knee osteoarthritis. Arch Phys Med Rehabil. 2002;83(7):889–93. https://
doi.org/10.1053/apmr.2002.33225. 11. Kerrigan DC, Lelas JL, Goggins J, Merriman GJ, Kaplan RJ, Felson DT. Effectiveness of a lateral-wedge insole on knee varus torque in patients
with knee osteoarthritis. Arch Phys Med Rehabil. 2002;83(7):889–93. https://
doi.org/10.1053/apmr.2002.33225. 12. Tse CTF, Ryan MB, Hunt MA. Influence of foot posture on immediate
biomechanical responses during walking to variable-stiffness supported
lateral wedge insole designs. Gait Posture. 2020;81:21–6. https://doi.org/10.1
016/j.gaitpost.2020.06.026. Received: 9 April 2021 Accepted: 22 September 2021 17. Erhart JC, Mündermann A, Mündermann L, Andriacchi TP. Predicting
changes in knee adduction moment due to load-altering interventions from
pressure distribution at the foot in healthy subjects. J Biomech. 2008;41(14):
2989–94. https://doi.org/10.1016/j.jbiomech.2008.07.021. 2989–94. https://doi.org/10.1016/j.jbiomech.2008.07.021. References 2015;
67(7):989–95. https://doi.org/10.1002/acr.22537. 23. Hinman RS, Bardin L, Simic M, Bennell KL. Medial arch supports do not
significantly alter the knee adduction moment in people with knee
osteoarthritis. Osteoarthr Cartil. 2013;21(1):28–34. https://doi.org/10.1016/j. joca.2012.10.009. 6. Paterson KL, Kasza J, Hunter DJ, Hinman RS, Menz HB, Peat G, et al. The
relationship between foot and ankle symptoms and risk of developing knee
osteoarthritis: data from the osteoarthritis initiative. Osteoarthr Cartil. 2017;
25(5):639–46. https://doi.org/10.1016/j.joca.2016.12.003. 24. Ferrigno C, Stoller IS, Shakoor N, Thorp LE, Wimmer MA. The feasibility of
using augmented auditory feedback from a pressure detecting insole to
reduce the knee adduction moment: a proof of concept study. J Biomech
Eng. 2016;138(2):1–7. https://doi.org/10.1115/1.4032123. 7. Paterson KL, Kasza J, Hunter DJ, Hinman RS, Menz HB, Peat G, et al. Longitudinal association between foot and ankle symptoms and worsening
of symptomatic radiographic knee osteoarthritis: data from the
osteoarthritis initiative. Osteoarthr Cartil. 2017;25(9):1407–13. https://doi. org/10.1016/j.joca.2017.05.002. 25. Kamper SJ, Maher CG, MacKay G. Global rating of change scales: a review of
strengths and weaknesses and considerations for design. J Man Manip Ther. 2012;17(3):163–70. https://doi.org/10.1179/jmt.2009.17.3.163. 8. Hatfield GL, Cochrane CK, Takacs J, Krowchuk NM, Chang R, Hinman RS,
et al. Knee and ankle biomechanics with lateral wedges with and without a
custom arch support in those with medial knee osteoarthritis and flat feet. J
Orthop Res. 2016;34(9):1597–605. https://doi.org/10.1002/jor.23174. 26. The jamovi project (2021). jamovi (Version 1.6) [Computer Software]. Retrieved from https://www.jamovi.org. 26. The jamovi project (2021). jamovi (Version 1.6) [Computer Software]. Retrieved from https://www.jamovi.org. 27. Hunt MA, Charlton JM, Felson DT, Liu A, Chapman GE, Graffos A, et al. Frontal plane knee alignment mediates the effect of frontal plane rearfoot
motion on knee joint load distribution during walking in people with
medial knee osteoarthritis. Osteoarthr Cartil. 2021;29(5):678–86. https://doi. org/10.1016/j.joca.2021.02.003. 27. Hunt MA, Charlton JM, Felson DT, Liu A, Chapman GE, Graffos A, et al. Frontal plane knee alignment mediates the effect of frontal plane rearfoot
motion on knee joint load distribution during walking in people with
medial knee osteoarthritis. Osteoarthr Cartil. 2021;29(5):678–86. https://doi. org/10.1016/j.joca.2021.02.003. 9. Chapman GJ, Parkes MJ, Forsythe L, Felson DT, Jones RK. Ankle motion
influences the external knee adduction moment and may predict who will
respond to lateral wedge insoles?: an ancillary analysis from the SILK trial. Osteoarthr Cartil. 2015;23(8):1316–22. https://doi.org/10.1016/j.joca.2015.02.164. 28. Kolasinski SL, Neogi T, Hochberg MC, Oatis C, Guyatt G, Block J, et al. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. 13. Hunt MA, Takacs J, Krowchuk NM, Hatfield GL, Hinman RS, Chang R. Lateral
wedges with and without custom arch support for people with medial
knee osteoarthritis and pronated feet: an exploratory randomized crossover
study. J Foot Ankle Res. 2017;10(1):1–10. https://doi.org/10.1186/s13047-017-
0201-x. 13. Hunt MA, Takacs J, Krowchuk NM, Hatfield GL, Hinman RS, Chang R. Lateral
wedges with and without custom arch support for people with medial
knee osteoarthritis and pronated feet: an exploratory randomized crossover
study. J Foot Ankle Res. 2017;10(1):1–10. https://doi.org/10.1186/s13047-017-
0201-x. 14. Sawada T, Tanimoto K, Tokuda K, Iwamoto Y, Ogata Y, Anan M, et al. Rear
foot kinematics when wearing lateral wedge insoles and foot alignment
influence the effect of knee adduction moment for medial knee
osteoarthritis. Gait Posture. 2017;57:177–81. https://doi.org/10.1016/j.ga
itpost.2017.06.009. 15. Hinman RS, Bowles KA, Metcalf BB, Wrigley TV, Bennell KL. Lateral wedge
insoles for medial knee osteoarthritis: effects on lower limb frontal plane
biomechanics. Clin Biomech. 2012;27(1):27–33. https://doi.org/10.1016/j. clinbiomech.2011.07.010. 16. Ferrigno C, Wimmer MA, Trombley RM, Lundberg HJ, Shakoor N, Thorp LE. A reduction in the knee adduction moment with medial thrust gait is
associated with a medial shift in center of plantar pressure. Med Eng Phys. 2016;38(7):615–21. https://doi.org/10.1016/j.medengphy.2016.03.008. 16. Ferrigno C, Wimmer MA, Trombley RM, Lundberg HJ, Shakoor N, Thorp LE. A reduction in the knee adduction moment with medial thrust gait is
associated with a medial shift in center of plantar pressure. Med Eng Phys. 2016;38(7):615–21. https://doi.org/10.1016/j.medengphy.2016.03.008.
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Correlates of weapon carrying among high school students in the United States
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BioMed Central BioMed Central Published: 7 July 2008 Received: 11 January 2008
Accepted: 7 July 2008 Annals of General Psychiatry 2008, 7:8
doi:10.1186/1744-859X-7-8
Ac ls of General Psychiatry 2008, 7:8
doi:10.1186/1744-859X-7-8 This article is available from: http://www.annals-general-psychiatry.com/content/7/1/8 This article is available from: http://www.annals-general-psychiatry.com/content/7/1/8
© 2008 M
l
l li
Bi M d C
l L d © 2008 Muula et al; licensee BioMed Central Ltd. © 2008 Muula et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creative
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Deaths and injuries arising from interpersonal violence among adolescents are
major public health concerns in the United States. The bearing of weapons among adolescents is a
critical factor in many of these deaths and injuries. Methods: A secondary analysis of the 2005 United States Youth Risk Behavior Surveillance System
Survey data was carried out to examine the variables associated with self-reported history of
weapon carrying on school property among high school students. We used logistic regression
analysis to assess the associations. Results: Of the 13,707 respondents who participated in the survey, 10.2% of males and 2.6% of
females reported carrying a weapon on school property. In multivariate logistic regression analysis,
males were more likely to report having carried a weapon than females (odds ratio (OR) = 5.58;
95% confidence interval (CI) [4.23, 7.62]). Self-reported race/ethnicity was also associated with
weapon carrying. Other variables positively associated with weapon carrying at school were
substance use (OR = 1.77; 95% CI [1.16, 2.68]), depression (OR = 1.44; 95% CI [1.10, 1.89]),
suicidal ideation (OR = 1.64; 95% CI [1.23, 2.19]), having had property stolen or deliberately
damaged at school (OR = 1.55; 95% CI [1.21, 1.98]), having been raped (OR = 1.70; 95% CI [1.22,
2.37]), having been threatened or injured with a weapon on school property (OR = 2.19; 95% CI
[1.63, 2.95]), and having engaged in physical fighting (OR = 2.02; 95% CI [1.56, 2.63]). Conclusion: This research identifies factors that are associated with weapon bearing among
adolescents in the United States. These factors may be important in the design of interventions
aimed at improving school safety and adolescent health. p
Primary research
Correlates of weapon carrying among high school students in the
United States
Adamson S Muula*1, Emmanuel Rudatsikira*2,3 and Seter Siziya4 Address: 1Department of Community Medicine and Public Health, University of Malawi, College of Medicine, Blantyre, Malawi, 2Department of
Global Health, School of Public Health, Loma Linda University, California, USA, 3Department of Epidemiology and Biostatistics, School of Public
Health, Loma Linda University, Loma Linda, California, USA and 4Department of Community Medicine, University of Zambia, School of
Medicine, Lusaka, Zambia Email: Adamson S Muula* - muula@email.unc.edu; Emmanuel Rudatsikira* - erudatsikira@llu.edu; Seter Siziya - ssiziya@yahoo.com
* C
di
h Email: Adamson S Muula* - muula@email.unc.edu; Emmanuel Rudatsikira* - erudatsikira@llu.edu; Seter Siziya - ssiziya@yahoo.com
* Corresponding authors * Corresponding authors Annals of General Psychiatry Open Access ronment within schools is disturbed if there is confirmed
or suspected carrying of weapons by students. through 12th grade students. In the 2005 survey, the tar-
get population consisted of all public, Catholic, and pri-
vate school students in grades 9–12. Two hundred and
three schools were sampled. In the first-stage, the sam-
pling frame consisted of 1,261 primary sampling units
(PSUs), consisting of counties, subareas of large counties,
or groups of smaller, adjacent counties. In the recent past, there have been several high profile
incidents in the United States that have highlighted the
public health concern of weapon carrying among stu-
dents. In 2004, the Centers for Disease Control and Pre-
vention (CDC) estimated that 6.1% of high school
students in the United States reported carrying weapons to
school [6]. The 1,261 PSUs were categorized into 16 strata according
to their metropolitan statistical area (MSA) status (that is,
urbanicity) and the percentages of minority race/ethnicity
groups (black and Hispanic students) in the PSUs. From
the 1,261 PSUs, 57 were selected with probability propor-
tional to overall school enrollment size for the PSU. In the
second stage of sampling, 203 schools with any of grades
9–12 were selected with probability proportional to
school enrollment size. The third stage of sampling con-
sisted of randomly selecting, in each chosen school and in
each of grades 9–12, one or two classrooms from either a
required subject (for example, English or social studies) or
a required period (for example, second period). Previous research has reported that several factors are
associated with carrying of weapons into schools. These
factors include neighborhood and community factors,
such as poverty and crime, family characteristics, school
organization and climate. Individual level attributes such
as gender, age and mental and psychological status of the
person involved have also been reported to be associated
with weapon bearing on school property [7-9]. In a study reported by Rudatsikira et al. [10] in which sev-
enth to eighth grade students in school districts of the
Southern California cities of Redlands and San Ber-
nardino in the United States participated, 13.8% of the
study participants reported having carried a weapon in the
past 30 days. These authors also reported that previous
victimization by the subject was associated with weapon
carrying. A CDC report based on the 2005 Youth Risk
Behavior Surveillance System (YRBSS) stated that "Over-
all, the prevalence of having carried a weapon was higher
among male (29.8%) than female (7.1%) students" [11]. ronment within schools is disturbed if there is confirmed
or suspected carrying of weapons by students. However, this CDC report did not assess whether being a
victim of rape, having suffered deliberate property dam-
age, having been threatened or injured and substance use
were associated with carrying a weapon by the adolescent. A weighting factor was applied to each student record to
adjust for non-response and the oversampling of black
and Hispanic students in the sample. The final, overall
weights were scaled so that the weighted count of students
was equal to the total sample size, and the weighted pro-
portions of students in each grade matched population
projections for each survey year. In the 2005 survey, all regular public, Catholic, and other
private school students in grades 9 through 12 in the 50
states and District of Columbia were included in the sam-
pling frame. Puerto Rico, the trust territories, and the Vir-
gin Islands were excluded from the sampling frame. Schools were selected systematically with probability pro-
portional to enrollment in grades 9 through 12 using a
random start. Based on the 2005 United States YRBSS, we carried out
this study to report the prevalence of self-reported weapon
carrying by in-school adolescents and to assess the associ-
ation between weapon carrying and a selected list of
explanatory variables such as race/ethnicity, sex, sub-
stance use, and previous victimization. The findings from
this study may assist in the informed design of interven-
tion programs aimed to reduce adolescent perpetrated
injury and death resulting from weapon use. All classes in a required subject or all classes meeting dur-
ing a particular period of the day, depending on the
school, were included in the sampling frame. Systematic
equal probability sampling with a random start was used
to select classes from each school that participated in the
survey. Schools' response rate was 78% while students'
response rate was 86%. The overall response rate was
67%. Background rying weapons on school property may cause physical
injury and deaths among students and school staff [4]. Victims and witnesses of weapon use may also suffer psy-
chological trauma from experiencing incidents where
such weapons are displayed or used [5]. The learning envi- g
There is growing interest in the study of interpersonal vio-
lence among adolescents [1-3]. The carrying of weapons
on school property by adolescents is an important aspect
of adolescent physical and mental health. Adolescents car- Page 1 of 8
(page number not for citation purposes) Page 1 of 8
(page number not for citation purposes) http://www.annals-general-psychiatry.com/content/7/1/8 http://www.annals-general-psychiatry.com/content/7/1/8 http://www.annals-general-psychiatry.com/content/7/1/8 Annals of General Psychiatry 2008, 7:8 ronment within schools is disturbed if there is confirmed
or suspected carrying of weapons by students. Questionnaire administration This study was based on secondary analysis of data from
the 2005 United States YRBSS. The YRBSS is an epidemio-
logical surveillance system established by the CDC to
monitor the prevalence of youth behaviors that most
influence health [12]. The survey uses a three-stage cluster
sample design to produce a representative sample of 9th Survey procedures for the national, state, and local surveys
were designed to protect students' privacy by allowing for
anonymous and voluntary participation. Before survey
administration, the CDC Institutional Review Board
approved the study protocol and parental permission was
obtained by participating schools. Students completed the Page 2 of 8
(page number not for citation purposes) http://www.annals-general-psychiatry.com/content/7/1/8 Annals of General Psychiatry 2008, 7:8 Statistical analysis self-administered questionnaire during one class period
and recorded their responses directly on a computer-scan-
nable booklet or answer sheet. The core questionnaire
contained 87 questions. States and cities could add or
delete questions from the core questionnaire. Skip pat-
terns were not included in any YRBSS questionnaire to
protect student privacy by ensuring all students took
about the same amount of time to complete the survey. self-administered questionnaire during one class period
and recorded their responses directly on a computer-scan-
nable booklet or answer sheet. The core questionnaire
contained 87 questions. States and cities could add or
delete questions from the core questionnaire. Skip pat-
terns were not included in any YRBSS questionnaire to
protect student privacy by ensuring all students took
about the same amount of time to complete the survey. y
We obtained frequencies of key variables in order to
describe the study sample. Logistic regression analysis was
conducted to determine associations between weapon
carrying on school property and a selected list of potential
explanatory variables. These variables, identified from
previous research on adolescent violence, included: sub-
stance use [13]; depression [14]; suicidal ideation [15];
physical fighting [16]; and prior victimization, such as
having had property stolen or deliberately damaged at
school, having been raped, and having been threatened or
injured with a weapon on school property [17-23]. Bivar-
iate logistic regression analyses were conducted between
each of the explanatory variables and the outcome
(weapon carrying). Further, multiple logistic regression
analysis was conducted with each of the explanatory vari-
ables as the main exposure while the other variables were
considered potential confounders. Data were analyzed
and weighted using the SUDAAN statistical software [24]. SUDAAN Programming statements for Taylor Lineariza-
tion (DESIGN = WR) allowed for accommodation of the
complex survey design [25] so that the results are repre-
sentative of students in grades 9–12 in public and private
schools in the US. Measures A full presentation of the questions asked in the 2005
YRBSS has been reported by the CDC [12]. For the pur-
pose of the current analysis, however, some of the ques-
tions of interest were as follow. With regards to weapon
carrying, "During the past 30 days, on how many days did
you carry a weapon such as a gun, knife, or club?" With
regards to weapon carrying at school, "During the past 30
days, on how many days did you carry a weapon such as
a gun, knife, or club on school property?" "During the
past 12 months, how many times has someone threat-
ened or injured you with a weapon such as a gun, knife,
or club on school property?" "During the past 12 months,
how many times has someone stolen or deliberately dam-
aged your property such as your car, clothing, or books on
school property?" "Have you ever been physically forced
to have sexual intercourse when you did not want to?" A
participant who reported "yes" to this last question was
deemed to have been raped within the reported time
period. The responses to these questions were dichot-
omized to yes or no; yes if the study participant reported
experiencing an incident on at least one day and no if oth-
erwise. Prevalence of weapon carrying on school property Islanders were more likely to carry weapons at school (OR
= 2.82; 95% CI [1.27, 6.27]). Other variables positively
associated with carrying weapons at school were sub-
stance use (OR = 2.35; 95% CI [1.65, 3.51]), depression
(OR = 2.43; 95% CI [1.94, 3.03]), suicidal ideation (OR =
2.75; 95% CI [1.86, 4.05]), having property stolen or
deliberately damaged by peers on school property (OR =
2.42; 95% CI [2.02, 2.90]), physical fighting (OR = 4.48;
95% CI [3.00, 6.70]), having been raped (OR = 3.51; 95%
CI [2.51, 4.90]), and having been threatened or injured
with a weapon on school property (OR = 5.43; 95% CI
[4.38, 6.74]). Islanders were more likely to carry weapons at school (OR
= 2.82; 95% CI [1.27, 6.27]). Other variables positively
associated with carrying weapons at school were sub-
stance use (OR = 2.35; 95% CI [1.65, 3.51]), depression
(OR = 2.43; 95% CI [1.94, 3.03]), suicidal ideation (OR =
2.75; 95% CI [1.86, 4.05]), having property stolen or
deliberately damaged by peers on school property (OR =
2.42; 95% CI [2.02, 2.90]), physical fighting (OR = 4.48;
95% CI [3.00, 6.70]), having been raped (OR = 3.51; 95%
CI [2.51, 4.90]), and having been threatened or injured
with a weapon on school property (OR = 5.43; 95% CI
[4.38, 6.74]). Of the 13,707 participants, 10.2% of males and 2.6% of
females reported carrying a weapon on school property. An estimated 29.8% (95% confidence interval (CI) [28.4,
31.2]) of males and 19.3% (95% CI [17.4, 21.3]) of
females had carried weapons anywhere. Characteristics of the study participants Characteristics of the study participants Data for 13,707 students who participated in the study
were analyzed; of these participants, 6,664 (50.5%) were
males and 7,193 (49.5%) were females (Table 1). The
median age was 16 years (Q1 = 15 years, Q3 = 17 years). Table 1: Characteristics of adolescents in the 2005 Youth Risk Behavior Surveillance System (United States)
Characteristic
Total
% (n)
Males
% (n)
Females
% (n)
Age (years)
<15
10.7 (1,221)
9.6 (520)
11.9 (701)
15
26.4 (3,170)
25.6 (1,450)
27.6 (1,720)
16
25.9 (3,535)
26.9 (1,718)
24.9 (1,817)
17
23.3 (3,661)
23.0 (1,780)
23.7 (1,881)
18+
13.6 (2,270)
14.9 (1,196)
12.3 (1,074)
Ethnicity
White
67.8 (6,117)
68.3 (3,015)
67.4 (3,102)
American Indian or Alaska native
1.1 (144)
1.1 (86)
1.2 (58)
Asian
3.7 (366)
4.0 (187)
3.4 (179)
Black
16.0 (3,343)
15.6 (1,575)
16.5 (1,768)
Native Hawaiian or other Pacific Islander
0.9 (90)
0.8 (44)
1.1 (46)
Hispanic
10.5 (2,063)
10.3 (920)
10.6 (1,143)
Substance use (tobacco, alcohol, and illegal drugs)
85.9 (6,969)
86.7 (3,541)
85.1 (3,428)
Depression
28.5 (4,136)
20.4 (1,477)
36.7 (2,719)
Suicidal ideation
16.9 (2,330)
12.0 (772)
21.8 (1,558)
Property stolen or deliberately damaged on school property
29.7 (4,132)
31.4 (2,105)
28.0 (2,027)
Rape
7.5 (1,046)
4.2 (308)
10.8 (738)
Threatened or injured with a weapon on school property
7.9 (1,083)
9.7 (649)
6.1 (434)
Physical fighting
35.8 (4,862)
43.4 (2,877)
28.2 (1,985)
Weapon carrying on school property
6.5 (815)
10.2 (625)
2.6 (190) Table 1: Characteristics of adolescents in the 2005 Youth Risk Behavior Surveillance System (United States) cents in the 2005 Youth Risk Behavior Surveillance System (United States) Annals of General Psychiatry 2008, 7:8 http://www.annals-general-psychiatry.com/content/7/1/8 Factors associated with carrying a weapon on school
property: bivariate analysis Table 2 indicates that, according to bivariate analysis,
there was no significant age difference in weapon carrying
at school. Compared to female respondents, males were
more than four times more likely to carry weapons to
school (odds ratio (OR) = 4.22; 95% CI [3.40, 5.23]). Compared to whites, native Hawaiians or other Pacific Table 2: Unadjusted associations between weapon carrying on school property and selected characteristics among US high school
students, 2005
OR [95% CI]
Characteristic
Total
Males
Females
Age (years)
<15
1.00
1.00
1.00
15
1.19 [0.83, 1.89]
1.08 [0.69, 1.68]
1.14 [0.54, 2.37]
16
1.31 [0.92, 1.87]
1.28 [0.84, 1.95]
1.18 [0.58, 2.40]
17
1.19 [0.83, 1.71]
1.20 [0.78, 1.85]
1.07 [0.52, 2.20]
18+
1.29 [0.88, 1.89]
1.30 [0.83, 2.04]
0.75 [0.32, 1.79]
Gender
Female
1.00
Male
4.22 [3.40, 5.23]
Ethnicity
White
1.00
1.00
1.00
American Indian or Alaska native
1.19 [0.66, 2.16]
1.47 [0.78, 2.78]
0.06 [0.01, 0.45]
Asian
0.44 [0.18, 1.07]
0.32 [0.10, 1.07]
0.98 [0.32, 2.98]
Black
0.83 [0.64, 1.06]
0.65 [0.48, 0.90]
1.64 [1.06, 2.55]
Native Hawaiian or other Pacific Islander
2.82 [1.27, 6.27]
4.43 [1.77, 11.07]
1.65 [0.22, 12.26]
Hispanic
1.08 [0.82, 1.42]
1.15 [0.84, 1.58]
0.85 [0.46, 1.56]
Substance use (tobacco, alcohol, and illegal drugs)
No
1.00
1.00
Yes
2.35 [1.65, 3.51]
2.40 [1.52, 3.78]
2.20 [1.17, 4.15]
Depression
No
1.00
1.00
1.00
Yes
2.43 [1.94, 3.03]
1.90 [1.58, 2.28]
3.67 [2.43, 5.55]
Suicidal ideation
No
1.00
1.00
1.00
Yes
2.75 [1.86, 4.05]
3.33 [2.60, 4.25]
2.29 [1.88, 2.79]
Property stolen or deliberately damaged on school property
No
1.00
1.00
Yes
2.42 [2.02, 2.90]
2.25 [1.82, 2.77]
2.78 [1.90, 4.06]
Physical fighting
No
1.00
1.00
1.00
Yes
4.48 [3.00, 6.70]
4.03 [3.20, 5.06]
4.84 [3.97, 5.89]
Rape
No
1.00
1.00
1.00
Yes
3.51 [2.51, 4.90]
4.05 [3.09, 5.32]
2.60 [2.13, 3.17]
Threatened or injured with a weapon on school property
No
1.00
1.00
1.00
Yes
5.43 [4.38, 6.74]
4.83 [3.75, 6.22]
5.79 [3.68, 9.12] http://www.annals-general-psychiatry.com/content/7/1/8 http://www.annals-general-psychiatry.com/content/7/1/8 Annals of General Psychiatry 2008, 7:8 Multivariate analysis of factors associated with carrying a
weapon on school property There were sex differences with regard to the prevalence
and effect of depression, suicidal ideation, physical fight-
ing, victimization rape and having been threatened or
injured with a weapon on school property. Females were
more than twice as likely (10.8%) to report having been
forced into sexual intercourse than males (4.2%). More
males, however, were more likely to have reported being
threatened or injured with a weapon compared to females
(9.7% versus 6.1%). Slightly more males than females
(86.7% versus 85.1%) were likely to have used substances
(alcohol, tobacco or illicit drugs). Overall, the main findings from the multivariate analysis
were unchanged for age, ethnicity, gender, substance use,
depression, suicidal ideation, property stolen or deliber-
ately damaged on school property, physical fighting, hav-
ing been raped, and having been threatened or injured
with a weapon on school property (Table 3). Discussion In a survey of US youth in 2005, 6.5% reported carrying a
weapon to school. More males (10.2%) compared to
females (2.6%) carried a weapon in the 30 days preceding
the survey. Another 7.9% reported having been threat-
ened or injured with a weapon, 7.5% having been raped
and 29.7% having ever had property deliberately dam-
aged or stolen at school. Discussion We take a
similar view in interpreting the results of the current
study. The present study confirms previous reports on the predic-
tors of weapon carrying among adolescents [10,13-23]. In
a study sample of 3,054 high school students in Massa-
chusetts, DuRant et al. [17] found that physical fighting
was positively associated with weapon carrying on school
property. Simon et al. [14] conducted a study of 2,200 par-
ticipants and reported that depression and drug use in the
9th grade were predictors of handgun carrying in 12th
grade. Using data from more limited sample sizes, Rudat-
sikira et al. [10] and Sullivan et al. [20] reported a positive
association between victimization and weapon carrying
among adolescents. While some of the previous studies
included much smaller sample sizes derived from specific
local settings, our study benefited from a much larger
national sample that could be perceived as representative
of the US school-going adolescent population. The sex differences where males were more likely to report
carrying weapons to school than females possibly has
multi-factorial influences. Bailey et al. [26] reported that
the perception that other students are carrying guns may
be a major factor in adolescent weapon bearing. It fol-
lows, therefore, that male students who bear weapons
may only be doing so out of belief that their peers are also
carrying weapons. Furthermore, numerous studies have
reported that males may be more likely to be violent and
engage in other unhealthy and antisocial behaviors, such
as cigarette smoking, alcohol and other substance use, and
physical fighting, than females [27-29]. This may reflect
the acceptance and/or expectation of the 'macho' status
that males are renowned for. We would have expected that minorities such as blacks
(African Americans), being largely disadvantaged in the
United States, would be more likely to bear weapons. This
reasoning comes from the literature, where the poor are
likely to live in violent neighborhoods and may be more
likely to feel unsafe and therefore carry weapons to
school. Our findings are therefore unexpected in as far as
we had hypothesized that black adolescents will be at
more risk of carrying weapons. There are possible explana-
tions for these findings. Firstly, data were based on self-
report. It is possible that black adolescents would under-
report their weapon carrying experiences. However, we do
not have evidence to suggest that this is the case. Discussion In bivariate logistic regression analysis, being male, native
Hawaiian or Pacific Islander (whites as referent), sub-
stance use, depression, suicidal ideation, having been vic-
timized (property stolen or deliberately damaged, raped
or threatened or injured with a weapon), and physical Table 3: Multivariate analysis of the associations between weapon carrying on school property and selected characteristics among US
high school students, 2005
Characteristic
OR [95% CI]
Age (years)
<15
1.00
15
0.91 [0.56, 1.47]
16
1.21 [0.76, 1.94]
17
1.18 [0.73, 1.94]
18+
1.21 [0.73, 2.03]
Gender
Female
1.00
Male
5.68 [4.23, 7.62]
Ethnicity
White
1.00
American Indian or Alaska native
0.82 [0.42, 1.60]
Asian
0.99 [0.35, 2.84]
Black
0.81 [0.58, 1.13]
Native Hawaiian or other Pacific Islander
4.67 [1.66, 13.14]
Hispanic
1.13 [0.82, 1.57]
Substance use (tobacco, alcohol, and illegal drugs)
No
1.00
Yes
1.77 [1.16, 2.68]
Depression
No
1.00
Yes
1.44 [1.10, 1.89]
Suicidal ideation
No
1.00
Yes
1.64 [1.23, 2.19]
Property stolen or deliberately damaged on school property
No
1.00
Yes
1.55 [1.21, 1.98]
Rape
No
1.00
Yes
1.70 [1.22, 2.37]
Threatened or injured with a weapon on school property
No
1.00
Yes
2.19 [1.63, 2.95]
Fighting
No
1.00
Yes
2.02 [1.56, 2.63] Table 3: Multivariate analysis of the associations between weapon carrying on school property and selected characteristics among US
high school students, 2005
Characteristic
OR [95% CI]
Age (years)
<15
1.00
15
0.91 [0.56, 1.47]
16
1.21 [0.76, 1.94]
17
1.18 [0.73, 1.94]
18+
1.21 [0.73, 2.03]
Gender
Female
1.00
Male
5.68 [4.23, 7.62]
Ethnicity
White
1.00
American Indian or Alaska native
0.82 [0.42, 1.60]
Asian
0.99 [0.35, 2.84]
Black
0.81 [0.58, 1.13]
Native Hawaiian or other Pacific Islander
4.67 [1.66, 13.14]
Hispanic
1.13 [0.82, 1.57]
Substance use (tobacco, alcohol, and illegal drugs)
No
1.00
Yes
1.77 [1.16, 2.68]
Depression
No
1.00
Yes
1.44 [1.10, 1.89]
Suicidal ideation
No
1.00
Yes
1.64 [1.23, 2.19]
Property stolen or deliberately damaged on school property
No
1.00
Yes
1.55 [1.21, 1.98]
Rape
No
1.00
Yes
1.70 [1.22, 2.37]
Threatened or injured with a weapon on school property
No
1.00
Yes
2.19 [1.63, 2.95]
Fighting
No
1.00
Yes
2.02 [1.56, 2.63] he associations between weapon carrying on school property and selected characteristics among US Page 5 of 8
(page number not for citation purposes) http://www.annals-general-psychiatry.com/content/7/1/8 http://www.annals-general-psychiatry.com/content/7/1/8 Annals of General Psychiatry 2008, 7:8 http://www.annals-general-psychiatry.com/content/7/1/8 Firstly, we need to state that we do not believe that there
are any inherent genetic differences that determine race
and that affect the way that adolescents behave. Discussion There has
been what we consider a healthy debate as to whether
racial/ethnic disparities in health are due to either innate
genetic differences or the biological impact of present and
past histories of racial discrimination and economic dep-
rivation [34-39]. While this debate has sometimes been
polarizing and emotional, there has not been enough evi-
dence to substantiate the claim that race has genetic roots
rather than being a social construct; however, even as a
social construct, it has enormous potential to influence
individual and societal behaviors and perceptions. We
take the view, therefore, that racial categorization has
facilitated the distribution of social and economic
resources (housing, school districts, wealth, social net-
works) that may consequently influence adolescent
behaviors and perceptions toward violent behavior [40-
43]. The perceptions that 'racial health disparities' are all
genetic in origin and that race exists because of genetic dif-
ferences have been discussed and possibly discarded by
Krieger [44] and Kaufman and Cooper [45,46]. We take a
similar view in interpreting the results of the current
study. fighting were all associated with weapon carrying on
school property. In multivariable analysis, the results
regarding the association between any of the explanatory
variables and weapon carrying at school remained virtu-
ally unchanged. Firstly, we need to state that we do not believe that there
are any inherent genetic differences that determine race
and that affect the way that adolescents behave. There has
been what we consider a healthy debate as to whether
racial/ethnic disparities in health are due to either innate
genetic differences or the biological impact of present and
past histories of racial discrimination and economic dep-
rivation [34-39]. While this debate has sometimes been
polarizing and emotional, there has not been enough evi-
dence to substantiate the claim that race has genetic roots
rather than being a social construct; however, even as a
social construct, it has enormous potential to influence
individual and societal behaviors and perceptions. We
take the view, therefore, that racial categorization has
facilitated the distribution of social and economic
resources (housing, school districts, wealth, social net-
works) that may consequently influence adolescent
behaviors and perceptions toward violent behavior [40-
43]. The perceptions that 'racial health disparities' are all
genetic in origin and that race exists because of genetic dif-
ferences have been discussed and possibly discarded by
Krieger [44] and Kaufman and Cooper [45,46]. Page 6 of 8
(page number not for citation purposes) Limitation of the study 3. Murakami S, Rappaport N, Penn JV: An overview of juveniles and
school violence. Psychiatr Clin North Am 2006, 29:725-741. Although the current analysis has strengths, there are also
some limitations that need consideration. Firstly, the data
used in the analysis were based on self-reports; therefore,
bias could result from possible mis-reporting by partici-
pants, either intentionally or inadvertently. Furthermore,
the response rate for this survey was 67% of all the eligible
students. The experiences of the students who did not
respond are not known. If, however, these non-respond-
ers had different experiences and exposures, our study
may not be representative of the situation among US ado-
lescents in schools. Finally, a small but not insignificant
percentage of adolescents in the US are either home
schooled, are truant or do not attend school altogether. Our findings may not be applicable to this group. It is also
important to highlight the fact that data for this study
were collected from a survey (cross-sectional methods)
and so it is not possible to attribute causation to any of the
factors so far identified as associated with weapon carry-
ing [57-60]. 4. Mercy JA, Rosenberg ML: Preventing firearm violence in and
around schools. In Violence in American Schools: A New Perspective
Edited by: Elliot DS, Hamburg BA, Williams KR. New York: Cam-
bridge University Press; 1998:159-187. g
y
5. Kingery PM, Pruitt BE, Heuberger G: A profile of rural Texas ado-
lescents who carry handguns to school. J Sch Health 1996,
66:18-24. g
y
5. Kingery PM, Pruitt BE, Heuberger G: A profile of rural Texas ado-
lescents who carry handguns to school. J Sch Health 1996,
66:18-24. 6. Centers for Disease Control and Prevention: Youth Risk Behavior Sur-
veillance – United States, 2003 Washington, DC: US Department of
Health and Human Services; 2004. 7. Afifi TO, Cox BJ, Katz LY: The associations between health risk
behaviours and suicidal ideation and attempts in a nationally
representative sample of young adolescents. Can J Psychiatry
2007, 52:666-674. 8. Khoury-Kassabri M, Benbenishty R, Astor RA: The effects of
school climate, socioeconomic, and cultural factors on
school victimization in Israel. Soc Work Res 2005, 29:165-180. 9. Khoury-Kassabri M, Benbenishty R, Zeira A, Astor RA: The contri-
butions of community, family, and school variables to stu-
dent victimization. Am J Community Psychol 2004, 34:187-204. J
y
y
10. Discussion Secondly,
if the black adolescents who are more likely to be carrying
weapons have dropped out of school, then it is possible
that the weapon-carrying adolescents would be under-
represented. Thirdly, if black students felt less threatened
at school, they may not thus carry weapons. Fourthly,
black students may not carry weapons to school if the
schools they attend are more vigilant in policing weapon
carrying as a possible result of high violence and weapon
bearing in black neighborhoods; if we knew this to be the
case, it would suggest that such an intervention was effec-
tive. Finally, we may have been biased into thinking that
blacks are more likely to carry weapons as a result of pre-
vailing prejudices. However, we also do not intend to
minimize the significantly higher risk of violent death by
firearms among blacks compared to whites in the United
States [47-50], although, with regard to our study, these
data are limited by being largely from situations outside We have also demonstrated that study participants who
had suffered forced sex or had been victimized in other
ways were more likely to carry weapons compared to
those who had not. Several studies have reported that per-
ceptions of fear and safety concerns, having being threat-
ened with a weapon and feelings of vulnerability are
associated with weapon bearing, possibly with the moti-
vation of self defense [30-33]. Native Hawaiian and other Pacific Islanders and Hispan-
ics were more likely to have reported having carried a
weapon on school grounds compared to whites. Blacks on
the other hand were less likely to carry weapons on school
grounds compared to whites. There was no difference
between whites and Asians. What do these results mean? Page 6 of 8
(page number not for citation purposes) Page 6 of 8
(page number not for citation purposes) http://www.annals-general-psychiatry.com/content/7/1/8 http://www.annals-general-psychiatry.com/content/7/1/8 http://www.annals-general-psychiatry.com/content/7/1/8 Annals of General Psychiatry 2008, 7:8 Annals of General Psychiatry 2008, 7:8 Annals of General Psychiatry 2008, 7:8 of school and among adults. These studies also report
solely firearm injuries and deaths while our study includes
all weapons, not just firearms. The higher likelihood of
carrying a weapon among Hispanics may have similar
associations with poverty or social factors. may enable the targeting of boys in prevention programs. Substance use prevention among adolescents may also be
associated with reduced weapon carrying. References References
1. Marsh L, McGee R, Nada-Raja S, Currey N: Adolescents' percep-
tions of violence and its prevention. Aust NZ J Public Health 2007,
31:224-229. 2. Cunningham R, Walton M, Trowbridge M, Weber J, Outman R, Ben-
way A, Maio R: Correlates of violent behavior among adoles-
cents presenting to an urban emergency department. J
Pediatr 2006, 149:770-776. 3. Murakami S, Rappaport N, Penn JV: An overview of juveniles and
school violence. Psychiatr Clin North Am 2006, 29:725-741. 4. Mercy JA, Rosenberg ML: Preventing firearm violence in and
around schools. In Violence in American Schools: A New Perspective
Edited by: Elliot DS, Hamburg BA, Williams KR. New York: Cam-
bridge University Press; 1998:159-187. 5. Kingery PM, Pruitt BE, Heuberger G: A profile of rural Texas ado-
lescents who carry handguns to school. J Sch Health 1996,
66:18-24. 6. Centers for Disease Control and Prevention: Youth Risk Behavior Sur-
veillance – United States, 2003 Washington, DC: US Department of
Health and Human Services; 2004. 7. Afifi TO, Cox BJ, Katz LY: The associations between health risk
behaviours and suicidal ideation and attempts in a nationally
representative sample of young adolescents. Can J Psychiatry
2007, 52:666-674. 8. Khoury-Kassabri M, Benbenishty R, Astor RA: The effects of
school climate, socioeconomic, and cultural factors on
school victimization in Israel. Soc Work Res 2005, 29:165-180. 9. Khoury-Kassabri M, Benbenishty R, Zeira A, Astor RA: The contri-
butions of community, family, and school variables to stu-
dent victimization. Am J Community Psychol 2004, 34:187-204. 10. Rudatsikira E, Singh P, Job J, Knutsen S: Variables associated with
weapon-carrying among young adolescents in Southern Cal-
ifornia. J Adolesc Health 2007, 40:470-473. 11. Eaton DK, Kann L, Kinchen S, Ross J, Hawkins J, Harris WA, Lowry
R, McManus T, Chyen D, Shanklin S, Lim C, Grunbaum JA, Wechsler
H: Youth Risk Behavior Surveillance – United States, 2005. MMWR 2006, 55:1-108. 12. Centers for Disease Control and Prevention: 2005 Youth Risk
Behavior Survey. [http://www.cdc.gov/yrbss]. Retrieved on
December 6, 2007 1. Marsh L, McGee R, Nada-Raja S, Currey N: Adolescents' percep-
tions of violence and its prevention. Aust NZ J Public Health 2007,
31:224-229. 1. Marsh L, McGee R, Nada-Raja S, Currey N: Adolescents' percep-
tions of violence and its prevention. Aust NZ J Public Health 2007,
31:224-229. 2. Authors' contributions ASM participated in the interpretation of the results and
drafting of the manuscript, ER conceived the analysis
plan, conducted the statistical analyses and participated in
the drafting of the manuscript, SS participated in the inter-
pretation of the results and drafting of the manuscript. All
authors approved the manuscript. The fact that adolescents who reported having suicidal
ideation and depressive symptoms and substance use
were also likely to bear weapons may be an example of
'clustering of harmful health behaviors' [27,52-56]; that
is, an adolescent who uses substances is also likely to bear
arms. As a policy direction, this calls for adolescent health
intervention program policy makers and implementers to
seriously consider that an adolescent who has one partic-
ular unhealthy behavior may also have other behaviors
that need attention. A multi-problem and multifaceted
approach has a better chance of promoting health than
single vertical interventions. References Cunningham R, Walton M, Trowbridge M, Weber J, Outman R, Ben-
way A, Maio R: Correlates of violent behavior among adoles-
cents presenting to an urban emergency department. J
Pediatr 2006, 149:770-776. Limitation of the study Rudatsikira E, Singh P, Job J, Knutsen S: Variables associated with
weapon-carrying among young adolescents in Southern Cal-
ifornia. J Adolesc Health 2007, 40:470-473. 11. Eaton DK, Kann L, Kinchen S, Ross J, Hawkins J, Harris WA, Lowry
R, McManus T, Chyen D, Shanklin S, Lim C, Grunbaum JA, Wechsler
H: Youth Risk Behavior Surveillance – United States, 2005. MMWR 2006, 55:1-108. Discussion We believe the
racial/ethnic differences may be indicative of general
socio-economic disparities existing across neighborhoods
in the US. The mechanisms behind the disparities in
weapon carrying with regard to socio-demographic char-
acteristics also need to be explored. Using students less than 15 years old as referents, we
found that older students were more likely to report hav-
ing carried weapons on school grounds. This could mean
several things: that older adolescents may be at higher risk
of perpetrating violence or being victimized; that older
adolescents may be more likely to resort to violence to
resolve conflicts [51]; or that peer influence or acceptance
of weapon bearing is higher in this age group. Further-
more, older adolescents may be more likely to be success-
ful in concealing weapons than much younger students. Acknowledgements We are grateful to the Centers for Disease Control and Prevention
(Atlanta, GA, USA) for providing us with the data from the Youth Risk
Behavior Surveillance System. p
g
The authors declare that they have no competing interests. p
g
The authors declare that they have no competing interests. Annals of General Psychiatry 2008, 7:8 Ann Epidemiol 2008, 18:119-123. 20. Sullivan TN, Farrell AD, Kliewer W: Peer victimization in early
adolescence: association between physical and relational vic-
timization and drug use, aggression, and delinquent behav-
iors among urban middle school students. Dev Psycholpathol
2006, 18(1):119-137. p
p
46. Kaufman JS, Cooper RS: Commentary: considerations for use of
racial/ethnic classification in etiologic research. Am J Epidemiol
2001, 154:291-298. ( )
21. Crick NR, Bigbee MA, Howes C: Gender differences in children's
normative beliefs about aggression: how do I hurt thee? Let
me count the ways. Child Dev 1996, 67:1003-1014. 47. Lemaire J: The effect of firearm deaths on life expectancy and
insurance premiums in the United States. LDI Issue Brief 2005,
11:1-4. 48. Guileyardo JM, Carmody TJ, Lene WJ, Stone IC: Racial and ethnic
patterns in firearms deaths. Am J Forensic Med Pathol 1994,
15:328-330. 22. Prinstein MJ, Boergers J, Vernberg EM: Overt and relational
aggression in adolescence: social-psychological adjustment
in aggressors and victims. J Clin Child Psychol 2001, 30:479-491. gg
J
y
23. Dowdell EB: The relationship between health risk behaviors
and fear in one urban seventh grade class. J Pediatr Nurs 2003,
18:187-194. 49. Centers for Disease Control and Prevention (CDC): Firearm-
related years of potential life lost before age 65 years –
United States, 1980–1991. MMWR Morb Mortal Wkly Rep 1994,
43:609-611. 24. Shah BV, Barnwell BG, Bieler GS: SUDAAN User's Manual, Release 7.5
Research Triangle Park, NC: Research Triangle Institute; 1997. 50. Centers for Disease Control and Prevention (CDC): Influence of
homicide on racial disparity in life expectancy – United
States, 1998. MMWR Morb Mortal Wkly Rep 2001, 0(36):780-783. 25. Demnati A, Rao JNK: Linearization variance estimators for sur-
vey data. Statistics Canada 2004, 3:17-26 [http://www.statcan.ca/
english/ads/12-001-XIE/12-001-XIE20040016991.pdf]. Retrieved 3
June, 2008 y
p
(
)
51. Blake CS, Hamrin V: Current approaches to the assessment
and management of anger and aggression in youth: a review. J Child Adolesc Psychiatr Nurs 2007, 20:209-221. J
26. Bailey SL, Flewelling RL, Rosenbaum DP: Characteristics of stu-
dents who bring weapons to school. J Adolesc Health 1997,
97(4):1-270. J
y
52. Holmberg LI, Hellberg D: Health, health-compromising behav-
ior, risk-taking behavior and sexuality in female and male
high school students in vocational compared with theoreti-
cal programs in Sweden. Int J Adolesc Med Health 2007,
19:459-472. 27. Siziya S, Muula AS, Kazembe LN, Rudatsikira E: Harmful lifestyles'
clustering among sexually active in-school adolescents in
Zambia. Annals of General Psychiatry 2008, 7:8 37. 37. Bloche MG: Race-based therapeutics. N Engl J Med 2004,
351:2035-2037. 37. Bloche MG: Race-based therapeutics. N Engl J Med 2004,
351:2035-2037. 13. DuRant RH, Krowchuk DP, Kreiter S, Sinal SH, Woods CR:
Weapon carrying on school property among middle school
students. Arch Pediatr Adolesc Med 1999, 153(1):21-26. 38. Cooper RS, Kaufman JS, Ward R: Race and genomics. N Engl J Med
2003, 248:1166-1170. ( )
14. Simon TR, Richardson JL, Dent CW, Chou CP, Flay BR: Prospective
psychosocial, interpersonal, and behavioral predictors of
handgun carrying among adolescents. Am J Public Health 1998,
88:960-963. 39. Kaplan JB, Bennett T: Use of race and ethnicity in biomedical
publication. JAMA 2003, 289:2709-2716. 40. Taylor TJ, Esbensen FA, Peterson D, Freng A: Putting youth vio-
lent victimization into context: sex, race/ethnicity, and com-
munity differences among a multisite sample of youths. Violence Vict 2007, 22:702-720. 15. Swahn MH, Bossarte RM: Gender, early alcohol use, and suicide
ideation and attempts: findings from the 2005 youth risk
behavior survey. J Adolesc Health 2007, 41:175-181. 41. Melzer-Lange MD, Van Thatcher CD, Liu J, Zhu S: Urban commu-
nity characteristics and adolescent assault victims. WMJ
2007, 106:394-396. y
J
16. Lowry R, Powell KE, Kann L, Collins JL, Kolbe LJ: Weapon-carry-
ing, physical fighting and fight-related injury among U.S. ado-
lescents. Am J Prev Med 1998, 1:122-129. 42. Molnar BE, Cerda M, Roberts AL, Buka SL: Effects of neighbor-
hood resources on aggressive and delinquent behaviors
among urban youths. Am J Public Health 2008, 98:1086-1093. J
17. DuRant RH, Kahn J, Beckford PH, Woods ER: The association of
weapon-carrying and fighting on school property and other
health risk and problem behaviors among high school stu-
dents. Arch Pediatr Adolesc Med 1997, 151:360-366. g
y
J
43. Masi CM, Hawkley LC, Piotrowski ZH, Pickett KE: Neighborhood
economic disadvantage, violent crime, group density, and
pregnancy outcomes in a diverse, urban population. Soc Sci
Med 2007, 65:2440-2457. 18. Leeb RT, Barker LE, Strine TW: The effect of childhood physical
and sexual abuse on adolescent weapon carrying. J Adolesc
Health 2007, 40:551-558. 44. Krieger N: Stormy weather: race, gene expression, and the
science of health disparities. Am J Public Health 2005,
95:2155-2160. 19. Malecki CK, Demaray MK: Carrying a weapon to school and
perceptions of social support in an urban middle school. J
Emot Behav Disord 2003, 11:169-178. 45. Kaufman JS, Cooper RS: Race in epidemiology: new tools, old
problems. Annals of General Psychiatry 2008, 7:8 BMC Pediatr 2008, 8:6. 28. Rudatsikira E, Dondog J, Siziya S, Muula AS: Prevalence and deter-
minants of adolescent cigarette smoking in Mongolia. Singa-
pore Med J 2008, 49:57-62. 53. Kim YS, Koh YJ, Leventhal B: School bullying and suicidal risk in
Korean middle school students. Pediatrics 2005, 115:357-363. ,
54. Saner H, Ellickson P: Concurrent risk factors for adolescent vio-
lence. J Adolesc Health 1996, 19:94-103. p
J
29. Kuzman M, Simetin IP, Franeliæ IP: Early sexual intercourse and
risk factors in Croatian adolescents. Coll Antropol 2007,
31(Suppl 2):121-130. J
55. Kshirsagar VY, Agarwal R, Bavdekar SB: Bullying in schools: prev-
alence and short-term impact. Indian Pediatr 2007, 44:25-28. (
pp
)
30. Simon TR, Dent CW, Sussman S: Vulnerability to victimization,
concurrent problem behaviors, and peer influence as predic-
tors of in-school weapon carrying among high school stu-
dents. Violence Vict 1997, 12:277-289. p
56. Park HS, Schepp KG, Jang EH, Koo HY: Predictors of suicidal ide-
ation among high school students by gender in South Korea. J Sch Health 2006, 76:181-188. J
57. Greenland S, Pearl J, Robins JM: Causal diagrams for epidemio-
logic research. Epidemiology 1999, 10:38-48. 31. Arria A, Borges G, Anthony JC: Fears and other suspected risk
factors for carrying lethal weapons among urban youths of
middle-school age. Arch Pediatr Adolesc Med 1997, 151:555-560. 58. Greenland S, Rothman KJ: Measures of effect and measures of
association. In Modern Epidemiology 2nd edition. Edited by: Rothman
KJ, Greenland S. Philadelphia, PA: Lippincott-Raven; 1998:48-66. 32. Arria A, Borges G, Anthony JC: Fears and other suspected risk
factors for carrying lethal weapons among urban youths of
middle-school age. Arch Pediatr Adolesc Med 1997, 151:555-560. 59. Kaufman JS, Cooper RS: Seeking causal explanations in social
epidemiology. Am J Epidemiol 1999, 150:113-120. g
33. McNabb SJ, Farley TA, Powell KE, Rolka HR, Horan JM: Correlates
of gun-carrying among adolescents in south Louisiana. Am J
Prev Med 1996, 12:96-102. p
g
J p
60. Rothman KJ: Causes. Am J Epidemiol 1976, 104:587-592. 34. Duster T: Race and reification in science. Science 2005,
307:1050-1051. 35. Hinds DA, Stuve LL, Nilsen GB, Halperin E, Eskin E, Ballinger DG,
Frazer KA, Cox DR: Whole-genome patterns of common DNA
variation in three human populations. Science 2005,
307:1072-1079. 36. Holden C: Race and medicine. Science 2003, 302:594-596. Conclusion 12. Centers for Disease Control and Prevention: 2005 Youth Risk
Behavior Survey. [http://www.cdc.gov/yrbss]. Retrieved on
December 6, 2007 This research identifies factors that may be used in the
design of interventions aimed at improving school safety. Knowledge that males are more likely to carry weapons Page 7 of 8
(page number not for citation purposes) http://www.annals-general-psychiatry.com/content/7/1/8 Annals of General Psychiatry 2008, 7:8 Page 8 of 8
(page number not for citation purposes) 36.
Holden C: Race and medicine. Science 2003, 302:594-596. Annals of General Psychiatry 2008, 7:8 Page 8 of 8
(page number not for citation purposes) Page 8 of 8
(page number not for citation purposes) Page 8 of 8
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https://dspace.lib.cranfield.ac.uk/bitstream/1826/12537/1/The_use_of_social_media_to_combat_research-isolation-2017.pdf
|
English
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The Use of Social Media to Combat Research-Isolation
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Annals of the Entomological Society of America
| 2,017
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cc-by
| 8,429
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Abstract Research-isolation is a common problem affecting many researchers who are disconnected from their research
communities. It can be caused by a number of factors, including physical isolation, unfamiliar research topics,
diversity, and the nature of the supervisory relationship. All of these aspects can have an impact on both work
and the mental health of researchers. Increasingly, researchers are turning to social media for support, by both
looking for communities and for increasing the impact of their work. In this paper, we set out a brief introduction
to a range of social media platforms used by researchers and present a discussion of the networks within those
platforms aimed at reducing research-isolation. These examples highlight just a few of the number of small
communities that have grown online to meet the needs of those seeking support through social media. We con-
clude with some recommendations for those affected by research-isolation and highlight the need for more re-
search into the role of social media on mental health in academics. Key words: social media, isolation, mental health, network, research course be fulfilled by these laboratory colleagues and supervisors, or
management structure. At an institutional level, support is often fo-
cused on early career researchers, as this period can be a particularly
stressful for the individual. Support in this sense can comprise of
counseling sessions or assigning mentors. Peers and supervisors can
introduce researchers to collaborators to expand their network, ei-
ther at conferences or during laboratory visits, and also help early
career researchers find postdoctoral and postgraduate positions. Isolation is commonly typified by either the inability of or unwill-
ingness to take part within the community around one’s work, in-
cluding both the local social community but also the wider
research community which a researcher may feel unable to access. Research-isolation is a very real problem in research institutes. Research-isolation is here used as a term that includes both social
disconnect and perceived isolation (Cornwell and Waite 2009) of
researchers in both academia and industrial settings. This impacts
not only the research itself, but many other aspects of a re-
searcher’s life, and can also negatively affect mental health, espe-
cially if prolonged (Shaw and Ward 2011, Shaw 2014). Finding
new methods of communication to break down isolation can pro-
vide a solution to this issue. Not every researcher has this experience as part of a research
group. V
C The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.
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 unre-
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449
m https://academic.oup.com/aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research
niversity user The Use of Social Media to Combat Research-Isolation
M. A. Reeve1,2 and M. Partridge3 1Institution of Environmental Sciences, Floor 3, 140 London Wall, London, EC2Y 5DN, United Kingdom (michelle.a.reeve@gmail.com),
2Corresponding author, e-mail: michelle.a.reeve@gmail.com, and 3Centre of Engineering Photonics, Cranfield University, Cranfield,
Bedfordshire MK42 0AL, United Kingdom (m.c.partridge@cranfield.ac.uk) j
g
y
Received 15 February 2017; Editorial decision 15 May 2017 munication
Annals of the Entomological Society of America, 110(5), 2017, 449–456
doi: 10.1093/aesa/sax051
Forum munication
Annals of the Entomological Society of America, 110(5), 2017, 449–456
doi: 10.1093/aesa/sax051
Forum munication
Annals of the Entomological Society of America, 110(5), 2017, 449–456
doi: 10.1093/aesa/sax051
Forum Forum Special Collection: Science Communication Abstract Many simply do not have, or are not comfortable accessing,
those kinds of internal support networks, for a variety of reasons
(Marshall et al. 2010). Some institutions may be lacking in support
facilities, or researchers may be working in a very small laboratory
group where networking and opportunities for advice are more lim-
ited. Researchers working remotely owing to necessity, such as com-
pleting fieldwork or through disability or illness, may miss
opportunities for engagement with their laboratory group, and upon
their return, this may cause them to may feel separated from their
peers. Individuals who may feel isolated owing to being a different
age, race, gender, or academic level to those around them might find
it harder to reach out for support, even if it is available. All of these
examples can lead to cases of research-isolation. Social media is one such communication tool that has impacted
a wide range of areas from politics (Loader and Mercea 2011) to
teaching (Tess 2013). Over the past 10 yr, social media use has
grown from 7% to 65% of adults worldwide (Perrin 2015). The
way people communicate and their networks are changing rapidly,
and academic research is just one of the areas affected by these
trends (Bik and Goldstein 2013). A wide array of social media platforms are available, all with dif-
ferent functions and aims. These cover a number of useful roles for
researchers: communicating their work, searching for jobs, network-
ing for potential collaborators, or asking for advice. Researchers of-
ten work in groups and are therefore surrounded by peers, relying
on them and their institution for advice, networking, and general
support. Many of the functions provided by social media can of Here, we discuss four key causes of research-isolation: physical
isolation; research topic, diversity, and the supervisory relationship. This list is by no means exhaustive, and each cause can contribute
independently or collectively to these feelings of isolation (Shaw
2014, The Thesis Whisperer 2015). Annals of the Entomological Society of America, 2017, Vol. 110, No. 5 450 limited, then the work itself can suffer. More importantly, pro-
longed research-isolation can have real implications on mental
health (Gewin 2012, Shaw 2014, Breines 2015). Abstract Academia is typi-
cally an industry where people work long, intense hours (Jacobs and
Winslow 2004); though the myths of needing to work 80-h weeks
are now being challenged (Duffy 2015), it is still prevalent, and
when coupled with isolation, can cause a lot of undue pressure. Perhaps the clearest of causes is that of physical isolation, where
the laboratory, university campus, or research institution is located
far away from other researchers. Physical isolation often directly af-
fects the more social side of a researcher’s life, and, especially if they
live in or close to the isolated area (Golden et al. 2008). Physical isola-
tion can also occur through extended home-working, for example
during thesis writing, through illness, or new parenthood (Moss
2016). Physical isolation can leave researchers feeling out-of-the-loop
or sidelined compared with their peers within the same research area. Researchers are increasingly using social media (Collins and
Hide 2010) as a way of combating isolation. Social media provides
an easily accessible means of communicating their work to a wider
and interested audience (Bukvova et al. 2010, Brossard and
Scheufele 2013), discussing experimental problems and asking for
advice, and even for recruiting subjects for experiments (O’Connor
et al. 2014, Shere et al. 2014, Yuan et al. 2014). However, even when physically located within a group, working
on a different topic to those around them can in itself cause isola-
tion. In this instance, the individual may be limited in who they can
ask for subject-specific advice, and may not have the “sounding
board” that lots of researchers have in their colleagues. Less experi-
enced early career researchers may be particularly affected in this sit-
uation, as it is reasonable to suggest that they would require more
time to compile literature reviews and plan experiments than would
be needed for others who are able to draw on knowledge of their
peers working in a similar area. This can introduce the feeling of be-
ing behind in their work, leaving the researcher feeling particularly
detached from those around them. In this paper, we set out a brief introduction to a range of social
media platforms used by researchers and present a discussion of the
networks within those platforms aimed at reducing research-
isolation. Downloaded from https://academic.oup.com/aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research
by Cranfield University user
on 08 September 2017 Social Media Platforms In addition to physical or topic isolation, diversity can be an iso-
lating issue in research (Smith and Calasanti 2005), as it is in many
industries. Those individuals who are in the minority within a labo-
ratory group, be that because of gender, race, sexuality, age or dis-
ability, or even political preference, can feel isolated and alone
(Powell 2007, Nivet 2010). Zimmerman et al. (2016) found that
women in academia report being ostracized in the workplace more
frequently than men and that faculty members of color report more
frequent information exclusion, defined as “situations where people
perceive being uninformed of information known mutually by oth-
ers” (Jones et al. 2009), than white faculty members. Some diversity
groups can be affected at different stages of academic careers; for ex-
ample, it is well-reported that women hold fewer senior researcher
or professor roles than men (EU 2009; van den Brink and Benschop
2012; Institute of Medicine, National Academy of Sciences and
National Academy of Engineering 2007), which could contribute to
isolation. New or single parents can also be isolated owing to diffi-
culties of being able to attend professional networking events, such
as conferences (Anonymous 2017). The growth in social media in the past decades has generated numer-
ous services, all of which have varying methods for social interac-
tion. They are constantly changing and evolving; services that were
built with a narrow focus and structure have often changed beyond
all recognition. Here, we present a number of the currently available
platforms as a summary of the state-of-the-art in social media. A vi-
sual summary can be seen in Fig. 1. We also examine the use of these
services in a research context, and how they might be used to pre-
vent or confront research-isolation. Profile Curation
h
f In the context of social media, we use profile curation as a term to
define platforms, which are primarily a way of hosting a public pro-
file of the user. They differ to discussion (section “Discussion
Platforms”) or content-based (section “Content Creation”) services,
which are more reliant on a steady stream of updates provided by
the user. Typically, the most basic information hosted on these ser-
vices consists of name, employment, and publications. These act as
virtual CVs, which are often linked to by other services. Finally, a key cause of isolation can be the quality of the stu-
dent–supervisor relationship during early academic career stages. There is anecdotal evidence (often anonymous) which suggests that
a suboptimal supervisory relationship can play a large role in exacer-
bating feelings of inadequacy and isolation. A supervisor’s leader-
ship style has even been cited as one factor which can negatively
impact PhD students’ mental health (Levecque et al. 2017). For ex-
ample, supervisors who fail to recognize stress (Anonymous 2014),
have different working styles to the student (Anonymous 2013), or
who give inappropriate, or minimal feedback on work (Anonymous
2015) can make an early career researcher unsure of themselves, and
reluctant to reach out for support, as doing so can often be perceived
as weakness (Anonymous 2015). Profile curation social media requires the lowest frequency of up-
dates, yet still acts as a valuable tool to provide a central place where
others can find individuals and their professional details. As a result,
use of curation kind of social media can lead to external network
building and collaboration. Some universities have begun requiring
researchers to sign up to one or more of these services, and some
have used these to replace their own profile database entirely
(Anonymous 2016c). ORCID
O
R Open Research ID, or ORCID, is a profile curation social media ser-
vice specifically aimed at academics. It allows users to host informa-
tion on employment history alongside various types of publications
and funding. There is no networking, and only a single, optional bi-
ography box to discuss a user’s work in more context. The ethos of
ORCID is to just provide the important information in a simple to
read easily accessible format. These are just a handful of examples of potential causes of
research-isolation. Many personal situations can also contribute to
feeling isolated, such as the previously discussed new parenthood,
illness, or disability, and sudden traumatic life events, for example
serious illness or death of family members. Events such as this can
affect academic performance and productivity (Gertler et al. 2004,
Servaty-Seib et al. 2006, Beach 2014), as well as life outside work. One of the great strengths of ORCID has been the growing inte-
gration with other services so that many of the fields can be popu-
lated automatically. In particular, integrating with larger paper
cataloging services, such as the SCOPUS publication library, means Research-isolation can affect many aspects of an academic’s life. No matter why the isolation is occurring, if support networks are m https://academic.oup.com/aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research
niversity user
r 2017 Annals of the Entomological Society of America, 2017, Vol. 110, No. 5 451 Fig. 1. Venn diagram showing the overlap between the various research-focused social media platforms. Fig. 1. Venn diagram showing the overlap between the various research-focused social media platforms. Content Creation There are a variety of social media platforms which can be used for
creating content about the researcher and their research, to engage
with others or simply communicate complex work. The type of con-
tent produced depends on the user’s preference, and also to some ex-
tent, the nature of the research itself—is it visually interesting, or
perhaps, it requires in-depth explanations? Different services cater
to these categories more readily, with options depending on what
media format users prefer. Some networks have more a supportive
nature, supporting each other as academics, often by using certain
hashtags or by interacting with groups which have been created and
are run specifically for the purpose of sharing content. ResearchGate
h Here, we describe a number of social media services which many
researchers use for communicating their work, engaging with others,
and seeking support, all of which are useful in combating research-
isolation. ResearchGate is similar to Academia.edu. Again, it allows users to
have a static academic profile showing off their employment history
and publications, and like Academia.edu, it asks for copies of re-
search papers which it can then make available to anyone looking at
the author’s profile. However, the networking opportunities on
ResearchGate are much wider, and there is more emphasis on en-
gagement with other people. In particular, there is the ability to post
public questions either asking for help within a topic or even just to
a particular researcher. Questions allow people to connect more eas-
ily both with colleagues but also other academics. The focus on con-
necting to people, as well as the suggestions to contact other
academics to ask questions or for copies of papers, does contribute
to users actively engaging with each other. LinkedIn that the curation aspect of ORCID is to some extent automatic. ORCID has also integrated with many journal services and some
even require an ORCID ID. Recently, ORCID has also started inte-
grating with other services commonly used in universities, such as
data access systems (e.g., figshare), and with university profile sys-
tems
(e.g.,
Current
Research
Information
System
or
CRIS). ORCID’s ability to automate data sharing between all these services
makes it a very popular system, with currently over 3 million users
(ORCID 2017). that the curation aspect of ORCID is to some extent automatic. ORCID has also integrated with many journal services and some
even require an ORCID ID. Recently, ORCID has also started inte-
grating with other services commonly used in universities, such as
data access systems (e.g., figshare), and with university profile sys-
tems
(e.g.,
Current
Research
Information
System
or
CRIS). ORCID’s ability to automate data sharing between all these services
makes it a very popular system, with currently over 3 million users
(ORCID 2017). LinkedIn
With 100 million users (Anonymous 2016a), LinkedIn is one of the
largest profile curation platforms, and is aimed at professionals rather
than specifically at researchers. Like ORCID, it provides a public page
showing some basic information on a user as well as their job history
and publications. LinkedIn, however, goes further and provides more
rich text areas for discussion of a user’s work and publications so that
users can see them in context. The resulting page is a profile which
reads as a fuller CV. In addition, LinkedIn also acts as a networking
tool and has several features specifically aimed at connecting its users. A user can add people to a network and categorize these people as col-
leagues, friends, etc. From these connections, LinkedIn will then pro-
duce updates and suggestions of new connections users might wish to
make from the existing people in their network. LinkedIn also provides
news feeds akin to Facebook for users to publish updates and comment
on other users’ pages. These news feeds offer an opportunity to engage
with others through their work and subsequently encourage interaction
with the user. Downloaded from https://academic.oup.com/aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research
by Cranfield University user
on 08 September 2017 Academia.edu On the surface, Academia.edu looks like an expanded equivalent of
ORCID. It has many of the same features and allows users to list
their job, publication list, and employment history. Academia.edu
allows for more network-style features, and it will link the details
that users add with other researchers so that users can, for example,
see all other people listed as employees of a particular employer. The other key difference from ORCID is in the way it handles publi-
cations. Academia.edu wants to host publications directly within its
servers and will prompt users to upload these. These are only avail-
able to other users of Academia.edu. The networking updates are
also driven by new uploads, so any paper that users repost on
Academia.edu will be shown to anyone in their network. It should
also be noted that Academia.edu is run with a different model to
ORCID and has come under significant criticism because of the way
it allows researchers to promote themselves within its search system
(Bond 2017). Discussion Platforms Discussion social media platforms are, unlike profile curation and
content creation platforms, more aimed at being places for dialog
and comment. These typically consist of regular short-form informal
updates and resharing of interesting content for discussion. Being
shorter and less formal means that replies and feedback on questions
and comments is often quicker than other network and from a wider
range of sources. There are also a number of research communities
that make active use of these platforms. All of these subreddits are community-managed and range from
“worldnews” to “catstandingup.” Some communities can be, by de-
sign, hostile places to contribute. However, Reddit is as diverse as
the rest of the Internet, and there are communities that are less toler-
ant to any abuse and very open to new members. There are some
key research communities discussed later in section “Advice,” but Instagram Facebook also has an increasing emphasis on providing a social
space with customizable privacy. Users can create a range of groups
(e.g., family, coworkers), then customize who can see which of their
updates. Customization can be a good way to ensure the right peo-
ple see a user’s updates and can help maintain a professional profile
separate to a personal profile on the same network. Instagram is primarily a photo-sharing site which allows users to take
and edit photos using filters and other image settings. Photo-sharing is
a rapid way of sharing something visual about a user’s research. It also
provides a visual insight into other research environments, which is an
advantage over text-based platforms (Pittman and Reich 2016). Popular and common things to share include behind-the-scenes photos
of methods or technology used, such as a 3D printer mid-print or
unidentifiable experimental equipment. Instagram also allows users to
share short videos (up to 60s; Lee 2016), and have recently introduced
a new “Stories” feature, where users can share a slideshow of short vid-
eos or images that disappears from their feed after 24h. Richer image
and video footage can be a good way to share a research method or
concept which ideally requires more than one image to explain, allow-
ing followers to view the images or clips in the intended order, without
then clogging up users feed. Hashtags can be used to signpost a particu-
lar field or area of science, as well as tapping in to the academic net-
work on Instagram itself. As it is more broadly focused, Facebook is not often considered a
“professional”
platform
but
rather
a
personal
platform. Nonetheless, it is increasingly being used for professional network-
ing and discussion of research, as discussed in section “Networking
through social media”. It is also a key part of disseminating any con-
tent such as images and videos owing to its broad reach. Reddit Reddit is different to other social media networks in a number of
ways. Firstly, it is rare to find anyone publicly identified, as most
users will be anonymous. Secondly, the network is highly variable in
terms of user experience. Reddit is a collection of subreddits, each
focusing on a narrow range of topics. In each subreddit, people can
post links, images, or videos which are then voted up and down and
commented on. Comments can also be voted up and down. The vot-
ing system has the effect of pushing certain comments or posts to the
top of the feed, making them more visible. Each subreddit has its
own rules about what can be posted and the kind of comments that
are allowed. Twitter Twitter is a social platform that is focused on short (140 characters)
status updates called Tweets. Users may upload short biographies
on their profile, but the biographies are also limited to just 160 char-
acters, and focuses on simply identifying the users. Many users stay
anonymous on Twitter, and it is not always expected that users iden-
tify themselves. YouTube YouTube, a platform for videos, is one such site which is suitable
for more visually engaging work. Video format is ideal for translat-
ing more complex scientific concepts relating to a user’s research,
but requires often expensive equipment and software to film and
edit. Perhaps for this reason, it tends to be institutions which pro-
duce video content rather than the individual researcher, often as
part of promotion for particular events, such as science festivals or m https://academic.oup.com/aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research
niversity user
r 2017 Annals of the Entomological Society of America, 2017, Vol. 110, No. 5 452 open days. It can also take a lot of time to plan, film, and edit video
content, which is another reason why a typically busy researcher
may not wish to pursue video-based communication. Time spent on
video creation can be a good investment; however, over 1 billion
people use YouTube, watching over 3 billion h of video every month
(Anonymous 2016d). personal information as well as professional information about em-
ployment and education. It is designed as a social media network for
everyone and so lacks some of the research-specific features of
ResearchGate, ORCID, or Academia.edu, such as adding publica-
tions and funding. There is also more emphasis on status updates
and discussion of current stories on a user’s news feed. Users typi-
cally update their feeds in the range of anything from once a month
to multiple times daily (Duggan et al. 2014). Blogs Blogs are typically presented in written form, and are a way of en-
gaging and communicating research with other scientists. There are
numerous blogging sites available, such as WordPress and Blogger,
which in a research context are primarily used for longer-form writ-
ten pieces interspersed with a few images, either about the research
itself, or about more personal experiences of academia. There are
also other blogging sites which are predominantly suited to sharing
media content, such as videos, GIFs, and even musical pieces. A
good example of this kind of mixed media site is Tumblr. The updates can be anything that fits the 140 character format and
can include photos, videos, and links. Perhaps owing to their short
length, the frequency of updates on Twitter is typically higher than
other platforms, with many people tweeting 10–20 times per day (Java
et al. 2007). Some people instead choose to use Twitter purely as a pas-
sive news feed to keep updated on people working in their field. The fo-
cus of updates varies from people sharing stories of their research,
commenting on news articles, to discussions about recipes. Twitter also relies on the resharing of other people’s status up-
dates via Retweeting. Resharing can mean that even a user with a
limited number of followers can quickly reach thousands of people. As discussed in section “Advice,” there are a number of support
groups
and
specifically
research-focused hashtags
on
Twitter
(Scobble 2016). Many researchers choose to start a blog as a way of practicing
and developing their writing skills (Kjellberg 2010), especially early
career researchers such as PhD students who may not have as much
writing experience. If writing about personal experiences of acade-
mia, it can also be an effective way of engaging with others in simi-
lar positions, creating support networks, as well as often being
cathartic for the writer. Depending on a researcher’s writing skills,
blogging can be a quick way of generating content for others to en-
gage with, though perhaps it is eclipsed by the large number of sci-
ence bloggers producing similar content, making it more difficult for
other users to find a user’s content just by chance. Downloaded from https://academic.oup.com/aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research
by Cranfield University user
on 08 September 2017 Online Journal Clubs
J
l l b Journal clubs are a common local support system, which are often
forums where students can discuss relevant papers and keep up-to-
date with the literature. The provision of journal clubs is sporadic,
however, depending on the institute and size of the research group. Social media has allowed journal clubs to expand from being a local
support network to being a wider online community support net-
work. Researchers joining a journal club can sign up to read or pro-
pose a paper that all those in the group then discuss via Twitter,
Facebook, or Skype. The online aspect allows researchers to tap into
a large club where they can hear from and discuss with a wider peer
group than may be possible locally. Previous work has indicated
that journal clubs not only help researchers connect but also foster
good research practices (Lizarondo et al. 2010). These communities tend to be based on a conserved membership
of people contributing and will have very loyal users. They are also
usually moderated by a select number of users to make sure com-
ments are on topic and not abusive. Research Support Research support groups and networks are places that actively work to
provide either collective services or research-targeted support. In many
cases, these are run or curated by other researchers, hoping to promote
good research habits and networks. There are a wide number of net-
works, ranging from assisting with mental health to even helping to
find the best paper-authoring software. Most communities are open
and do not require any membership to either participate or read the
available help. In almost all cases, actively participating can be more re-
warding, as the user can ask specific questions or for specific help based
on their own experiences and situation. Support groups like the ones described below provide two ser-
vices which are important for mental health. Firstly, they can give
isolated researchers support, which they may not be able to get more
locally. As discussed, student supervision and teaching can be vari-
able, and these groups provide critical help that students can search
for themselves. Secondly, the groups are often places that encourage
discussion and sharing of problems, which can provide researchers
with insight into the wider community and a sense of inclusivity
with others facing similar problems. Academic Blogs
l
f lf l Blogs fulfil several roles owing to the sheer diversity of blogs pro-
duced (Bukvova et al. 2010). As a free resource available to re-
searchers, they can be a vital source of information and tips about
research methods or even provide insight into research life (Reeve
2014). Although not as integrated as Facebook or Twitter, the com-
ment sections on blog posts can also be a focus of interesting discus-
sion and a place researchers can go to seek further advice and
support. Blogs are also easy to set up through some of the platforms
mentioned earlier, and many researchers may find it helpful to start
their own as a way of reaching out for support. Examples of best
practice in research blogs include Bug Squad (run by Garvey 2017,
an entomologist from UC Davis) and Don’t Forget About The
Roundabouts (run by Professor Leather 2017, an applied entomolo-
gist from Harper Adams University). There are a large number of
blogs online, some more active than others, and finding a blog that
gives a reader the information they are looking for can be difficult. There are some blog communities which bring together a number of
blogs into one place such as Occam’s Typewriter and Scientific
American Blogs. Facebook Facebook is the world’s largest social media network, with almost 2
billion users (Anonymous 2016b). It allows users to upload some tps://academic.oup.com/aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research
rsity user
17 Annals of the Entomological Society of America, 2017, Vol. 110, No. 5 453 there are a great many subreddit communities on Reddit, and mov-
ing beyond the default ones and searching for subreddits that match
the sort for information or communities a user wants to be a part of
is very important. Shut up and Write was founded to encourage researchers to over-
come issues with writing, which many find difficult. Started in 2013
by the Thesis Whisperer (The Thesis Whisperer 2013), it spans
across many social media platforms, and is an event where aca-
demics either come together physically or remotely at an agreed
time and write as much as possible in a given time span. Since its in-
ception, it has been expanded and copied by many organizations,
and several universities now have Shut Up and Write nights in the li-
braries. Through Twitter, there is also an account which prompts re-
searchers to do a fortnightly hour-long Shut Up and Write session,
and facilitates discussions before and after the event to talk about
writing tips and advice. Networking Through Social Media The previous section dealt primarily with the provision of social me-
dia services by companies and organizations. These are all services
which provide the platform on which to build a social network. However, one of the great strengths of social media for researchers
is not just in the platforms and their various pros and cons, but in
the networks and support available through them. These networks
can simply be a support network that grows slowly through contacts
and friends, but within all of these services, there are already proj-
ects specifically aimed at being a support network for researchers. One such club, Diversity Journal Club, is a multifaceted group,
existing on a WordPress blog, and on Twitter, with their own hash-
tag #DiversityJC. The purpose of the group is to discuss diversity in
STEM and academia, taking on a traditional “journal club” format,
where they invite participants to discuss a particular article or blog
post relating to diversity every month. Online forums such as jour-
nal clubs can be easy to join for short periods and get insight into lit-
erature that may be relevant to a researcher’s field. Here, we describe a selection of these networks chosen to illus-
trate the availability of support. It is not a comprehensive list of
these groups available. In this section, we have categorized these
into loose areas of focus; yet, much like the platforms they use, there
are networks that span multiple categories. Downloaded from https://academic.oup.com/aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research
by Cranfield University user
on 08 September 2017 Online Forums Forums are one of the longest standing forms of social media and
predate almost all of the other platforms. There is some debate as to
whether or not they can be defined as social media (Weinberg
2008), but within the context of researcher-isolation, they are cer-
tainly a social platform that can have many of the same features and
benefits of other social media. Forums are usually hosted websites where users login to post on
to a message board, typically organized by topic. Many are open to
read, but posting on them requires an account. Forums are simple to
set up and most are comparatively small communities with a partic-
ular focus. Different forums have different rules about the type of
content that can be posted, but typically they are text-based. Subreddits As previously mentioned, there are subreddits within Reddit, which
are communities aimed at providing a research or discussion area to
post questions, queries, or rants on any given subject. One example
is /r/AskAcademics, which is specifically geared toward academia. Another, /r/LabRats, has a broader remit for anyone working in re-
search. Both have a strong community of active users from a wide
range of backgrounds, who are always willing to share stories or ad-
vice. Researchers use subreddits to ask questions on topics spanning
simple experiment advice to more serious allegations of misconduct. The anonymous nature of Reddit encourages open discussion both
from the researchers asking questions and those replying. Users are
not required to interact, so some may only add their voice to an ex-
isting issue, often providing broad discussion on a given topic. There
are also some subject-specific subreddits such as/r/Entomology
which, though typically consist of small communities, cater to ques-
tions and support within a specific field. As previously mentioned, there are subreddits within Reddit, which
are communities aimed at providing a research or discussion area to
post questions, queries, or rants on any given subject. One example
is /r/AskAcademics, which is specifically geared toward academia. Another, /r/LabRats, has a broader remit for anyone working in re-
search. Both have a strong community of active users from a wide
range of backgrounds, who are always willing to share stories or ad-
vice. Researchers use subreddits to ask questions on topics spanning
simple experiment advice to more serious allegations of misconduct. Rotational curation (RoCur) social media accounts are accounts that
have a nominal subject which they discuss (such as biology or astro-
physics), but are run by a rotating host. One of the largest examples is
@RealScientists on Twitter, which has a different scientist running the
account every week. Originally, RoCur was unique to Twitter but
other platforms, such as Instagram, now also have rotational curation
accounts. From a reader perspective, these offer insight into a wide
range of other research activities and allow readers the opportunity to
ask curators about their research. As a networking tool, though,
RoCur is more effective when taking part as a curator. Being a curator
allows researchers to show off their work to a wide audience, gaining
immediate feedback and support. Groups As mentioned in section “Discussion Platforms,” many platforms of-
fer the ability to form or join groups. These groups range from
community-driven to professionally run and have a wide range of
focuses. LinkedIn typically has groups aimed, and expanding users
professionally network with researchers who can be of assistance. There are also networks aimed at connecting researchers with jobs. Facebook has a wider range of groups from local meetups (discussed
in more detail below) to equipment sharing. Finding groups can be
difficult because as they are easy to create, there are many to search
through. Good groups to look for are ones specifically linked to a
field and look for groups with the highest memberships, such as
TheEntomologyGroup on Facebook or Insects and Entomology on
LinkedIn. Some groups are also designed as “news feeds” of interest-
ing research information and others encourage active engagement. #PhDChat and #ECRChat These are hashtags that are used on Facebook, Tumblr, and Twitter
to
connect
early
career
researchers
discussing
research
life. #PhDChat is much more commonly used and almost entirely com-
prised of other PhD students talking about their life and research
during their PhD. #ECRChat has a similar aim, but is targeted at
early career researchers (ECR), which typically consists of people in
their first full time research position either as a postgraduate or post-
doc. As communities based on hashtags, there is no “membership”
as such, as people use it on an ad hoc basis. This results in a wide
range of topics being discussed, and replies to specific questions can
vary in quality and quantity depending on which researchers are on-
line at the time. Despite this, the size of the community means that
there is always ongoing discussion and frequently secondary hash-
tags for specific topics. There are also many subject-specific hashtags
such as #entomology or #arachnology, which are often good places
to look for advice on a niche topic. Advice There are places on social media to specifically ask for advice from
the community targeted in a similar way to research groups above. m https://academic.oup.com/aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research
niversity user
r 2017 Annals of the Entomological Society of America, 2017, Vol. 110, No. 5 454 Typically, these are often areas within the more discussion-style so-
cial media and are places where research can post questions or share
issues in their laboratory with other researchers. As these communi-
ties are based on the researchers within them, they can change fre-
quently. The communities and tags listed below are a selection of
the most consistently active groups. promoting professional networking and engagement between re-
searchers. Increased networking and engagement is particularly
valuable to researchers looking to expand their contacts beyond
those available through immediate peers. Social media also often
provides a route to networking with researchers in other fields
which users may not otherwise connect with. #ICanHazPDF
C Physical face-to-face meetups are the most active of all of the meth-
ods of improving researcher-isolation. Being face-to-face rather than
online, they are not obviously linked to social media; however, we
felt they should be included, as many researcher meetups are borne
out of social media campaigns and discussions. With respect to re-
search-isolation, meetups are particularly important because many
of these social media-prompted meetups were created in a direct re-
sponse to researcher isolation. On Twitter, these events are called
“Tweetups” and can range in size from countrywide events to small
local meetups of a couple of researchers. Many geographical regions
have hashtags helping to connect people to the event (e.g.,
#CamSciTweepup) or specific groups researchers can join to get no-
tified when there is an event in the local area (e.g., #UKSciMeetups). Typically these are community-driven and hosted on an ad hoc ba-
sis. There are groups dedicated to running regular meetups for scien-
tists and actively encouraging them to come meet other researchers,
as well as others working in science-related fields (e.g., London
SciComm Socials). It is also becoming more common to run social #ICanHazPDF is a good example of a network specifically targeting
a certain problem. In this case, the problem is the availability and ac-
cess to research papers and literature. Many researchers, particularly
those in industry, have limited access to research materials, and
#ICanHazPDF is a community of researchers which actively seek
out and share PDFs of these materials. On both Twitter and
Facebook, posting a paper title or link followed by #ICanHazPDF
will illicit replies from other academics with access to that paper, in-
cluding either a unpaywalled link or a request for an e-mail address
to send it to. Although this obviously has implications for copyright
with journals, this is still an example of a thriving community of re-
searchers helping each other out. In 2013, there were 10,000 tweets
tagged with #ICanHazPDF (Liu 2013). Subreddits It also helps highlight them as a re-
searcher and encourages people that may not have found them to fol-
low and engage after their week on the account has finished. Some of
the
largest
active
research-orientated
RoCur
accounts
are
@RealScientists,
@BioTweeps,
@WeHumanities,
@AstroTweeps,
@IamSciComm, and @IAmSciArt. Downloaded from https://academic.oup.com/aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research
by Cranfield University user
on 08 September 2017 Recommendations Anonymous. 2016c. Requiring ORCID in publication workflows: Open letter. (https://orcid.org/content/requiring-orcid-publication-workflows-open-letter) As discussed in the introduction, this summary of social media net-
works is designed to give readers an insight into the plethora of net-
works and platforms available. Based on the discussion set out in
this paper, we have two recommendations. Anonymous. 2016d. YouTube company statistics. (http://www.statisticbrain. com/youtube-statistics/) Anonymous 2017. Conferences are intellectual lifelines but as a single parent I
often miss out. (https://www.theguardian.com/higher-education-network/
2017/apr/14/anonymous-academic-single-parent-conferences-career) First, for any reader looking for social networking opportunities
to combat isolation, our strongest recommendation is to try any of
the mentioned networks. From our experience, every researcher has
different needs and expectation of the support available, and none
of the networks and communities discussed is a one-stop solution
for everyone. We hope that the discussion and summary has pro-
vided enough information to choose a network that best suits the
reader. For researchers who do not feel that the above choice of net-
works fulfils their needs, many of the projects and groups discussed
were founded by researchers who also felt that they wanted a differ-
ent kind of support, and so reached out to other researchers and
formed the network they needed. Social media makes starting a net-
work as easy as coining a new hashtag on one of the existing plat-
forms. Guides such as Ten Steps for Setting Up an Online Journal
Club (Chan et al. 2015) can provide a starting point for any re-
searchers interested in starting their own network. Beach, B. 2014. Dealing with grief while at university. (https://www.theguar
dian.com/education/mortarboard/2014/may/30/deal-with-grief-university) Bik, H. M., and M. C. Goldstein. 2013. An introduction to social media for
scientists. PLoS Biol. 11: Bond, S. 2017. Dear scholars, delete your account at academia.edu. (http://
www.forbes.com/forbes/welcome/?toURL¼http://www.forbes.com/sites/drsarah
bond/2017/01/23/dear-scholars-delete-your-account-at-academia-edu/
{&}refURL¼https://www.google.co.uk/{&}referrer¼https://www.google.co.uk/) {&}refURL¼https://www.google.co.uk/{&}referrer¼https://www.google.co.uk/) Breines, J. 2015. Graduate school and mental illness: Is there a link? (https://
www.psychologytoday.com/blog/in-love-and-war/201511/graduate-
school-and-mental-illness-is-there-link) Brossard, D., and D. A. Scheufele. 2013. Science, new media, and the public. Science 339: 40–41. Bukvova, H., H. Kalb, and E. Schoop. 2010. What we blog? A quantitative
analysis of researchers’ weblogs. In Publishing in the networked world:
transforming the nature of communication. 14th international conference
on electronic publishing, Edita Prima Ltd, Helsinki, Finland. Second is a recommendation for academics who specialize in
studying communities and mental health. Recommendations 2012. Mental health: Under a cloud. (https://www.nature.com/
naturejobs/science/articles/10.1038/nj7419-299a) Recommendations The summary provided in
this paper is based on all the available information that the authors
could find through asking academics, social media experts, and jour-
nal searches. In the opinion of the authors, there is currently a wor-
ryingly small number of high quality peer-reviewed studies in the
area of research isolation. It would be of great value to the research
community to better understand the effect of these social media net-
works and their role in mental health and work satisfaction. There
are signs that this need greater research is already being recognized
with the launch of journals, such as Research for All, which focuses
on the importance of public engagement in research, and we hope
that in future more literature will be available to researchers wishing
to make the best decisions about combating research-isolation. Chan, T. M., B. Thoma, R. Radecki, J. Topf, H. H. Woo, L. S. Kao, A. Cochran, S. Hiremath, and M. Lin. 2015. Ten steps for setting up an online
journal club. J. Cont. Educ. Health Prof. 35: 148–154. Collins, E., and B. Hide. 2010. Use and relevance of Web 2.0 resources for re-
searchers. In Publishing in the networked world: transforming the nature of
communication. 14th international conference on electronic publishing,
Edita Prima Ltd, Helsinki, Finland. Cornwell, E. Y., and L. J. Waite. 2009. Social disconnectedness, perceived iso-
lation, and health among older adults. J. Health Social Behav. 50: 31–48. Duffy, M. 2015. You do not need to work 80 hours a week to succeed in aca-
demia — Times Higher Education (THE). (https://www.timeshighereduca
tion.com/blog/you-do-not-need-work-80-hours-week-succeed-academia) Duggan, M., N. Ellison, A. Lenhart, and M. Madden. 2014. Frequency of so-
cial media use. (http://www.pewinternet.org/2015/01/09/frequency-of-so
cial-media-use-2/) Isolation is a difficult issue for many researchers around the world. Here, we have set out the roles that social media can play in providing
support networks to those affected. There are a wide range of plat-
forms and networks available for researchers, and we hope that by
highlighting them in this paper, we have demonstrated the potential of
social media in preventing and tackling isolation in academia. Entomology Today. 2016. Entomologists talk twitter at ICE 2016. (https://
entomologytoday.org/2016/09/29/entomologists-talk-twitter-at-ice-2016/) EU
2009. She
figures. Women
and
science,
statistics
and
indicators.[WorldCat]
(https://ec.europa.eu/research/swafs/pdf/pub_gen
der_equality/she_figures_2015-leaflet-web.pdf) der_equality/she_figures_2015-leaflet-web.pdf) Garvey, K. K. 2017. Bug squad. (http://ucanr.edu/blogs/bugsquad/) Gertler, P., D. I. Levine, and M. Ames. 2004. Schooling and parental death. Rev. Econ. Stat. 86: 211–225. Gewin, V. Acknowledgements The authors acknowledge funding from the Engineering and Physical Sciences
Research Council (EPSRC) UK, via grant EP/L010437. Golden, T. D., J. F. Veiga, and R. N. Dino. 2008. The impact of professional
isolation on teleworker job performance and turnover intentions: does time
spent
teleworking,
interacting
face-to-face,
or
having
access
to
communication-enhancing
technology
matter? J. Appl. Psychol. 93:
1412–1421. Building Networks In addition to the support and general advice groups, there are ser-
vices specifically aimed not at resolving specific issues but instead /aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research Annals of the Entomological Society of America, 2017, Vol. 110, No. 5 455 Anonymous. 2016a. Leading social networks worldwide as of September
2016, ranked by number of active users (in millions). (https://www.statista. com/statistics/272014/global-social-networks-ranked-by-number-of-users/) media meetups as part of conferences, for example, an entomology
“tweetup” at the International Congress of Entomology 2016
(Entomology Today 2016). Anonymous. 2016b. Number of monthly active Facebook users worldwide as
of 3rd quarter 2016 (in millions). (https://www.statista.com/statistics/
264810/number-of-monthly-active-facebook-users-worldwide/) Downloaded from https://academic.oup.com/aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research
by Cranfield University user
on 08 September 2017 References Cited Institute of Medicine, National Academy of Sciences and National Academy
of Engineering. 2007. Beyond bias and barriers: fulfilling the potential of
women in academic science and engineering. Washington, District of
Columbia: The National Academies Press. Anonymous. 2013. How I broke up with my supervisor. (https://thesiswhis
perer.com/2013/10/02/how-i-broke-up-with-my-supervisor/) Anonymous. 2013. How I broke up with my supervisor. (https://thesiswhis
perer.com/2013/10/02/how-i-broke-up-with-my-supervisor/) Anonymous. 2014. When good supervisors go bad. (https://thesiswhisperer. com/2014/08/27/when-good-supervisors-go-bad/) Anonymous. 2014. When good supervisors go bad. (https://thesiswhisperer. com/2014/08/27/when-good-supervisors-go-bad/) Jacobs, J. A., and S. E. Winslow. 2004. Overworked faculty: job stresses and
family demands. Ann. Am. Acad. Political Social Sci. 596: 104–129. Anonymous. 2015. Bad PhD supervisors can ruin research. So why aren’t they
accountable? (https://www.theguardian.com/higher-education-network/
2015/dec/11/bad-phd-supervisors-can-ruin-research-so-why-arent-they-
accountable) Anonymous. 2015. Bad PhD supervisors can ruin research. So why aren’t they
accountable? (https://www.theguardian.com/higher-education-network/
2015/dec/11/bad-phd-supervisors-can-ruin-research-so-why-arent-they-
accountable) Java, A., X. Song, T. Finin, and B. Tseng. 2007. Why we Twitter:
Understanding microblogging usage and communities. Proceedings of the Downloaded from https://academic.oup.com/aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research
by Cranfield University user
on 08 September 2017 456 Annals of the Entomological Society of America, 2017, Vol. 110, No. 5 Reeve, M. A. 2014. Taming monsters. (https://theresaspiderinthebath.word
press.com/2014/07/31/taming-monsters/) Reeve, M. A. 2014. Taming monsters. (https://theresaspiderinthebath.word
press.com/2014/07/31/taming-monsters/) 9th WebKDD and 1st SNA-KDD 2007 workshop on Web mining and social
network analysis - WebKDD/SNA-KDD ’07, 56–65. Scobble, J. 2016. Twitter for academics: a guide. 1st Edn. Kindle Jones, E. E., A. R. Carter-Sowell, J. R. Kelly, and K. D. Williams. 2009. ’I’m
out of the Loop’: Ostracism through information exclusion. Group Proc. Intergroup Relations 12: 157–174. Servaty-Seib, H. L., L. A. Hamilton, K. J. Doka, J. H. Harvey, E. D. Miller,
and K. Cook. 2006. Educational performance and persistence of bereaved
college students. J. College Student Dev. 47: 225–234. Kjellberg, S. 2010. I am a blogging researcher: Motivations for blogging in a
scholarly context. First Monday 15: Shaw, C. 2014. Overworked and isolated - work pressure fuels mental illness
in
academia. (https://www.theguardian.com/higher-education-network/
blog/2014/may/08/work-pressure-fuels-academic-mental-illness-guardian-
study-health) Leather, S. 2017. Don’t forget the roundabouts. (https://simonleather.word
press.com/) Lee, N. 2016. Instagram‘s big bet on video doesn‘t stop at 60 seconds). (https://www.engadget.com/2016/04/05/behind-instagram-video/) Shaw, C., and L. Ward. 2011. Dark thoughts: why mental illness is on the rise
in
academia. (https://www.theguardian.com/higher-education-network/
2014/mar/06/mental-health-academics-growing-problem-pressure-
university) Levecque, K., F. Anseel, A. De Beuckelaer, J. Van der Heyden, and L. Gisle. 2017. Work organization and mental health problems in PhD students. Res. Policy 46: 868–879. Liu, J. 2013. Interactions: The numbers behind #ICanHazPDF. (https://www. Downloaded from https://academic.oup.com/aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research
by Cranfield University user
on 08 September 2017 Downloaded from https://academic.oup.com/aesa/article-abstract/110/5/449/4103473/The-Use-of-Social-Media-to-Combat-Research
by Cranfield University user
on 08 September 2017 References Cited altmetric.com/blog/interactions-the-numbers-behind-icanhazpdf/) Shere, M., X. Y. Zhao, and G. Koren. 2014. The role of social media in recruit-
ing for clinical trials in pregnancy. PLoS ONE 9: Smith, J. W., and T. Calasanti. 2005. The influences of gender, race and eth-
nicity on workplace experiences of institutional and social isolation: an ex-
ploratory study of university faculty. Sociol. Spectrum 25: 307–334. Lizarondo, L., S. Kumar, and K. Grimmer-Somers. 2010. Online journal clubs:
an innovative approach to achieving. J. Allied Health 39: e17–e22. Loader, B. D., and D. Mercea. 2011. Networking democracy? Inf. Comm. Soc. 14: 757–769. Tess, P. A. 2013. The role of social media in higher education classes (real and
virtual)-A literature review. Comp. Human Behav. 29: A60–A68. Marshall, G. W., C. E. Michaels, and J. P. Mulki. 2010. Workplace isolation:
exploring the construct and its measurement. Psychol. Mkt. 24: 195–223. The Thesis Whisperer. 2013. Shut up and Write! (https://thesiswhisperer.com/
shut-up-and-write/) Moss, R. E. 2016. Only connect: Academia and isolation. (https://menysnowe
balles.wordpress.com/2016/10/07/only-connect-academia-and-isolation/) The Thesis Whisperer. 2015. Are most academics lonely at work? (https://the
siswhisperer.com/2015/08/05/are-most-academics-lonely-at-work/) Nivet, M. A. 2010. Minorities in academic medicine: Review of the literature. J. Vascular Surg. 51 (4 SUPPL): S53–S58. van den Brink, M., and Y. Benschop. 2012. Slaying the seven-headed dragon:
The quest for gender change in academia. Gender. Work Org. 19: 71–92. O’Connor, A., L. Jackson, L. Goldsmith, and H. Skirton. 2014. Can I get a
retweet please? Health research recruitment and the Twittersphere. J. Adv. Nursing 70: 599–609. Weinberg, T. 2008. Are forums” Social media” Sites? (https://www.seroundt
able.com/archives/017027.html) ORCID. 2017. ORCID Statistics. (https://orcid.org/statistics) Yuan, P., M. G. Bare, M. O. Johnson, and P. Saberi. 2014. Using online social
media for recruitment of human immunodeficiency virus-positive partici-
pants: A cross-sectional survey. J. Med. Internet Res. 16: 1–9. Perrin, A. 2015. Social media usage: 2005–2015. (http://www.pewinternet. org/2015/10/08/social-networking-usage-2005-2015/) Zimmerman, C. A., A. R. Carter-Sowell, and X. Xu. 2016. Examining
workplace ostracism experiences in academia: understanding how differences
in the faculty ranks influence inclusive climates on campus. Front. Psychol. 7:
1–9. Pittman, M., and B. Reich. 2016. Social media and loneliness: why an
Instagram picture may be worth more than a thousand Twitter words. Comp. Human Behav. 62: 155–167. Powell, K. 2007. Beyond the glass ceiling. Nature 448: 98–100.
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RETINAL FLECKS IN STARGARDT DISEASE REVEAL CHARACTERISTIC FLUORESCENCE LIFETIME TRANSITION OVER TIME
|
Retina
| 2,019
|
cc-by
| 7,206
|
RETINAL FLECKS IN STARGARDT
DISEASE REVEAL CHARACTERISTIC
FLUORESCENCE LIFETIME TRANSITION
OVER TIME YASMIN SOLBERG, MBBS, BSC (HONS),*† CHANTAL DYSLI, MD, PHD,*† PASCAL ESCHER, PHD,*†
LISA BERGER, MD,*† SEBASTIAN WOLF, MD, PHD,*† MARTIN S. ZINKERNAGEL, MD, PHD*† Purpose: Stargardt disease is the most common inherited juvenile macular dystrophy
and is characterized by yellowish flecks across the posterior pole. The purpose of this study
was to investigate fluorescence lifetime changes of retinal flecks over time using
fluorescence lifetime imaging ophthalmoscopy. g g
p
py
Methods: Longitudinal fluorescence lifetime data of 12 patients with Stargardt disease
(mean age ± SEM, 42.25 ± 2.1 years; range, 28–58 years) were acquired using a fluores-
cence lifetime imaging ophthalmoscope based on a Heidelberg Engineering Spectralis
system. Retinal autofluorescence was excited with a 470-nm laser. The emitted fluores-
cence was detected in two wavelength channels: a short spectral channel (498–560 nm)
and a long spectral channel (560–720 nm). The mean retinal autofluorescence lifetimes
were calculated and further analyzed with corresponding color fundus images, autofluor-
escence intensity images, and spectral domain optical coherence tomography. Patients
were classified into three subtypes. Results: All patients with Stargardt disease displayed characteristic autofluorescence
lifetime patterns. Mean fluorescence lifetime values within areas of yellow flecks were
significantly prolonged (long spectral channel 484 ps) compared with the surrounding
tissue (long spectral channel 297 ps). In 91.6% of the eyes, flecks with short fluorescence
lifetimes (long spectral channel 255 ps) were identified. Short lifetime flecks progressed to
flecks with characteristic long lifetimes in 75.1% of eyes within a mean interval of 29.2
months (range 3–45 months). Between baseline and follow-up, the rate of newly developed
short lifetime flecks (number/per year) based on subtypes was 2.62 in Group 1, 1.43 in
Group 2, and 0.81 in Group 3. Conclusion: Recent onset flecks in Stargardt disease display short fluorescence
lifetimes and convert into longer fluorescence lifetime flecks over time. This transition
may represent a change in the composition of retinal deposits with accumulation of
lipofuscin and retinoid by-products from the visual cycle. With emerging treatment options,
these findings may prove useful to monitor disease progression and therapeutic effects. RETINA 39 879 888 2019 RETINA 39:879–888, 2019 Over the course of the disease, initially well-defined
flecks progress outward from the central macula in
a centrifugal pattern. Y. Solberg: Heidelberg Engineering (nonfinancial support). C.
Dysli: Heidelberg Engineering (nonfinancial support). S. Wolf: Al-
lergan, Bayer, Novartis, Heidelberg Engineering, Hoya, Optos, and
Euretina. M. S. Zinkernagel: Allergan, Bayer, Novartis, and Hei-
delberg Engineering. The remaining authors have any financial/
conflicting interests to disclose. Supplemental digital content is available for this article. Direct
URL citations appear in the printed text and are provided in the
HTML and PDF versions of this article on the journal’s Web site
(www.retinajournal.com). RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2019 VOLUME 39 NUMBER 5 (A2PE-H2), or its oxidized form (A2PE).12,14 Char-
acteristic phenotypic features, such as pisciform
flecks and atrophy, are then believed to develop as
a result of the accumulation of these retinoid by-
products or lipofuscins in the RPE.10 In rare cases,
STGD is caused by dominantly inherited variants in
ELOVL4 encoding the elongation of very long chain
fatty acids protein 4 (STGD3; OMIM #600110) and
in PROM1 encoding the rim protein prominin-1
(STGD4; OMIM #603786). Fluorescence lifetime imaging ophthalmoscopy
(FLIO) measures lifetimes of endogenous retinal
fluorophores after excitation using a picosecond-
pulsed blue laser light. A previous report from our
group demonstrated characteristic fluorescence life-
time changes across the posterior pole in patients
with STGD. In this previous study, retinal flecks
with a broad range of autofluorescence lifetimes
were identified, and it was speculated that short
lifetime flecks represent recent lesions, whereas
flecks with longer lifetimes represent older lesions.20
The aim of this study was to investigate the dynam-
ics of fluorescence lifetimes within retinal flecks
over time. Several noninvasive imaging techniques have been
established in the diagnosis of STGD, and for
monitoring disease progression. Spectral domain opti-
cal coherence tomography (OCT) is a recognized
imaging tool that provides morphologic information
by visualizing the retinal architecture. Spectral domain g
p
p
y
Reprint requests: Martin S. Zinkernagel, MD, PhD, Department
of Ophthalmology, Freiburgstrasse 4, Inselspital, Bern University
Hospital, 3010 Bern, Switzerland; e-mail: martin.zinkernagel@
insel.ch This is an open access article distributed under the Creative
Commons Attribution License 4.0 (CCBY), which permits unre-
stricted use, distribution, and reproduction in any medium, pro-
vided the original work is properly cited. RETINAL FLECKS IN STARGARDT
DISEASE REVEAL CHARACTERISTIC
FLUORESCENCE LIFETIME TRANSITION
OVER TIME Flecks then fade, leaving poorly
demarcated yellow lesions and residual atrophy.2,5,6
Thus, at advanced stages of the disease, a progressive
bilateral atrophy of the RPE, photoreceptors, and cho-
roidal vasculature can be found.7 The severity of fun-
dus abnormalities seen on ophthalmoscopy is often not
directly associated with the visual acuity. However,
the overall prognosis of STGD is poor, and visual
acuity often deteriorates to 20/200 or less.8,9 S
t
h S
targardt disease (STGD) is the most common in-
herited macular dystrophy. It is a form of macular
degeneration initially described by Stargardt in 1909.1
Stargardt disease classically presents during the first
two decades of life and is characterized by a progres-
sive bilateral loss of central vision. Morphological fea-
tures include central macular atrophy and well-defined
yellowish deposits visible on the posterior pole around
the macular, known as “flecks.”1,2 Histologically,
flecks are located at the level of the retinal pigment
epithelium (RPE).3,4 879 From the *Department of Ophthalmology, Inselspital, Bern Uni-
versity Hospital, Bern, Switzerland; and †Department of BioMedical
Research, University of Bern, Bern, Switzerland.
Material presented on September 21, 2018 at Euretina 2018 in
Vienna, Austria.
Y. Solberg: Heidelberg Engineering (nonfinancial support). C.
Dysli: Heidelberg Engineering (nonfinancial support). S. Wolf: Al-
lergan, Bayer, Novartis, Heidelberg Engineering, Hoya, Optos, and
Euretina. M. S. Zinkernagel: Allergan, Bayer, Novartis, and Hei-
delberg Engineering. The remaining authors have any financial/
conflicting interests to disclose.
Supplemental digital content is available for this article. Direct
URL citations appear in the printed text and are provided in the
HTML and PDF versions of this article on the journal’s Web site
(www.retinajournal.com).
This is an open access article distributed under the Creative
Commons Attribution License 4.0 (CCBY), which permits unre-
stricted use, distribution, and reproduction in any medium, pro-
vided the original work is properly cited.
Reprint requests: Martin S. Zinkernagel, MD, PhD, Department
of Ophthalmology, Freiburgstrasse 4, Inselspital, Bern University
Hospital, 3010 Bern, Switzerland; e-mail: martin.zinkernagel@
insel.ch From the *Department of Ophthalmology, Inselspital, Bern Uni-
versity Hospital, Bern, Switzerland; and †Department of BioMedical
Research, University of Bern, Bern, Switzerland.
Material presented on September 21, 2018 at Euretina 2018 in
Vienna, Austria. From the *Department of Ophthalmology, Inselspital, Bern Uni-
versity Hospital, Bern, Switzerland; and †Department of BioMedical
Research, University of Bern, Bern, Switzerland. y
Material presented on September 21, 2018 at Euretina 2018 in
Vienna, Austria. RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2019 VOLUME 39 NUMBER 5 880 OCT may demonstrate thinning of retinal layers and
a loss of photoreceptor segment layers.15,16 Stargardt disease is predominately inherited in an
autosomal recessive trait, and most cases are caused
by pathogenic variants in the retinal-specific ATP-
binding cassette transporter gene ABCA4, located on
chromosome 1p13 (STGD1; OMIM #248200).10–12
ABCA4 expression is predominantly localized to the
rim of photoreceptor outer segments and at lower
levels in the retinal pigment epithelium.13 The AB-
CA4 transporter flips the N-retinylidene-phosphati-
dylethanolamine (N-ret-PE) that forms in the visual
cycle by reversible combination of toxic retinalde-
hyde with phosphatidylethanolamine (PE), from the
luminal to cytoplasmic surface of photoreceptor
outer disc membranes. In the absence of ABCA4
flippase activity, clearance of retinaldehyde is de-
layed, favoring a secondary condensation of N-ret-
PE with another retinaldehyde to form phospholipid-
conjugated
bisretinoids,
v.g. dihydro-N-
retinylidene-N-retinyl-phosphatidylethanolamine
(A2PE-H2), or its oxidized form (A2PE).12,14 Char-
acteristic phenotypic features, such as pisciform
flecks and atrophy, are then believed to develop as
a result of the accumulation of these retinoid by-
products or lipofuscins in the RPE.10 In rare cases,
STGD is caused by dominantly inherited variants in
ELOVL4 encoding the elongation of very long chain
fatty acids protein 4 (STGD3; OMIM #600110) and
in PROM1 encoding the rim protein prominin-1
(STGD4; OMIM #603786). Fundus autofluorescence (FAF) intensity imaging is
an established imaging technique providing informa-
tion on the retinal status by enabling the visualization
of hyperautofluorescent material such as bisretinoid
components, such as lipofuscin, originating from the
RPE.17 In early stage of STGD, FAF imaging reveals
hyperautofluorescent lesions consistent with visible
flecks on fundus examination. In later stages, hypoau-
tofluorescent areas appear, consistent with RPE atro-
phy and photoreceptor loss.6 Despite substantial research efforts in the past
decades, there are no approved therapeutic treat-
ments for STGD. The disease causes significant
morbidity with psychological and economic impli-
cations. However, promising new options, including
gene replacement therapy, stem cell transplantation,
and deuterated vitamin A, are emerging and are
currently being reviewed in clinical trials.18,19 A fur-
ther challenge researchers face in treating STGD is
the lack of a highly sensitive measurement tool to
monitor short-term changes in disease progression.19
This will be vital to assess effects of future therapies
accurately. Methods Twelve patients with a clinical diagnosis of STGD
were included in the study. The patients were consec-
utively recruited at the University Hospital in Bern,
Switzerland. The diagnosis of STGD was established
by findings on clinical examination and ancillary
studies including color fundus images, FAF intensity
images, and electroretinographic results.21 Moreover,
11 of 12 patients with STGD included had a molecular
diagnosis of STGD (see Table 1, Supplemental
Digital Content 1, http://links.lww.com/IAE/A990). FLUORESCENCE LIFETIME IMAGING IN STARGARDT DISEASE SOLBERG ET AL 881 All patients had a baseline and a follow-up exam-
ination. At each visit, best-corrected visual acuity
(BCVA; according to Early Treatment Diabetic Ret-
inopathy Study [ETDRS] letters22) was measured in
all patients, and a general dilated ophthalmologic
examination was performed. Maximal pupil dilation
was achieved using tropicamide 0.5% and phenyleph-
rine HCl 2.5%. Fundus color images (Zeiss FF
450plus; Zeiss, Oberkochen, Germany), OCT scans
of the macula (Heidelberg Spectralis HRA + OCT;
Heidelberg Engineering, Heidelberg, Germany), and
fluorescence lifetime images were obtained of both
eyes. requires scan duration of 90 seconds per eye approx-
imately in patients with STGD. During data accumu-
lation, an eye movement tracking system with a high-
contrast confocal infrared image ensures the correct
location of each detected photon within a field of
256 · 256 pixels. Statistical Data Analysis Fluorescence lifetime values for the SSC and the
LSC were analyzed separately. All data were presented
as mean ± SEM. Statistical analysis was performed
using GraphPad (Prism 6; GraphPad Software, Inc,
La Jolla, CA). To compare the results, the Mann–
Whitney test with a confidence interval of 95% was
used. P values ,0.05 were considered as statistically
significant. A stepwise forward regression analysis
was performed using SigmaPlot Version 12.3 (Systat
Software, Inc, San Jose, CA). Fluorescence Lifetime Imaging Ophthalmoscope Fluorescence
lifetime
imaging
ophthalmoscope,
based on an HRA Spectralis system (Heidelberg
Engineering), was used to obtain retinal fluorescence
lifetime data. The principles and details of FLIO have
been described elsewhere.22,24 In summary, the technique is based on the excitation
of retinal autofluorescence using a 470-nm pulsed laser
at 80-MHz repetition rate. Highly sensitive hybrid
photon-counting detectors (HPM-100–40; Becker &
Hickl, Berlin, Germany) were used for the registration
of emitted fluorescence light by time-correlated single-
photon counting modules (SPC-150; Becker & Hickl). Emitted fluorescence photons were measured in two
separate wavelength spectrums: a short spectral chan-
nel (SSC: 498–560 nm) and a long spectral channel
(LSC: 560–720 nm). In both wavelength channels, at
least 1,000 photons per pixel were obtained, which Fluorescence Lifetime Data Analysis Fluorescence lifetime data collection resulted in
a decay curve which was biexponentially approxi-
mated using SPCImage software version 4.6 (Becker
& Hickl). The chi-square value evaluated appropriate-
ness of the exponential fit. The resulting short and long
lifetime components (T1 and T2) along with their
respective relative amplitudes a1 and a2 were used
to calculate the mean fluorescence lifetime Tm, which
signifies the amplitude weighted mean fluorescence
lifetime. Patients with STGD were assigned to phenotypic
subtypes at baseline and follow-up. Based on the
clinical appearance of the color fundus and FAF
images, patients were assigned to one of three groups: 1. Group I: flecks and atrophy are confined to the
central macular area. The mean fluorescence lifetimes were analyzed
using “FLIO reader” software (ARTORG Center for
Biomedical Engineering Research, University of Bern,
Switzerland). To spatially quantify retinal autofluores-
cence lifetimes, a standard ETDRS grid was used with
following circle diameters: 1 mm for the central area,
3 mm for the inner ring, and 6 mm for the outer ring. Smaller region of interests with diameters of 0.16 mm
and 0.5 mm were used to analyze specific areas of
interest, for example, flecks. Fluorescence lifetimes
within flecks were analyzed by averaging three regions
of interest. 2. Group II: flecks are scattered, can spread nasally to
the optic disk, and/or exceed the vascular arcades. 3. Group III: flecks are ill-defined “resorbed” lesions,
and extensive RPE atrophy is visible.23 The study was conducted at the Department of
Ophthalmology at the University Hospital in Bern,
Switzerland, with the approval of the local ethics
committee and in accordance with the Declaration of
Helsinki. All participants provided written informed
consent before study entry. This study is registered at
ClinicalTrials.gov as “Measurement of Retinal Auto-
fluorescence with a Fluorescence Lifetime Imaging
Ophthalmoscope (FLIO Group),” with the identifier
number NCT01981148. RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2019 VOLUME 39 NUMBER 5 RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2019 VOLUME 39 NUMBER 5 882 Table 1. Baseline Patient Characteristics
Patient ID
Sex
Baseline Age, year
Age of Onset, y
Visual Acuity
Phenotype
Atrophy
Right
Left
Right
Left
Right
Left
STGD 1
M
34
28
80
81
2
2
—
—
STGD 2
M
33
30
79
84
2
2
—
—
STGD 3
M
53
40
35
34
2
2
Y
Y
STGD 4
F
43
12
32
34
3
3
GA
GA
STGD 5
M
41
34
38
40
2
2
Y
Y
STGD 6
M
45
18
19
17
3
3
GA
GA
STGD 7
F
45
40
82
83
2
3
—
—
STGD 8
F
56
33
34
36
2
2
GA
GA
STGD 9
M
47
42
70
84
1
2
—
—
STGD 10
M
41
18
44
47
2
2
GA
GA
STGD 11
F
26
18
34
34
2
2
GA
GA
STGD 12
F
43
43
83
83
2
2
—
—
Patient ID number, sex, age, age of onset, BCVA (ETDRS letters) at the last follow-up, fundus phenotype 1 to 3 according to Fishman
classification, presence of areas with geographic atrophy (GA), and presence of atrophy (Y). M, male; F, female. Table 1. Baseline Patient Characteristics Patient ID number, sex, age, age of onset, BCVA (ETDRS letters) at the last follow-up, fundus phenotype 1 to 3 according to Fishman
classification, presence of areas with geographic atrophy (GA), and presence of atrophy (Y). M, male; F, female. short and long fluorescence lifetimes, when compared
with the surrounding retina, and were represented as
red and blue lesions in color-coded images with
a range between 200 ps (red) and 600 ps (blue). various stages of hyperautofluorescent flecks and hy-
poautofluorescent
lesions,
consistent
with
RPE
changes and atrophy. All patients had a baseline and
at least one follow-up examination with a mean inter-
val of 29.2 months (range, 3–45 months). All patients
developed new flecks during the clinical follow-up
period. Most flecks displayed long (blue) fluorescence
lifetimes (SSC: 4,715 ± 17 ps; LSC: 484 ± 21 ps). However, in 91.7% (n = 22) of eyes, a smaller number
of flecks with shorter lifetimes, color-coded in red
(SSC: 265 ± 2 ps; LSC: 255 ± 4 ps), were identified
(Figure 2). RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2019 VOLUME 39 NUMBER 5 Mean age at the initial diagnosis of STGD was 29.7
± 3.1 years (range 12–43). The mean disease duration
at the time of the first and the last FLIO measurement
was 12.1 ± 3.1 years (range, 0.08–31), and 14.6 ± 3.24
years (range, 5–30), respectively. The mean BCVA at
baseline and at follow-up was 53.6 ± 4.9 (range
17–84) and 51.2 ± 4.8 (range 16–83) ETDRS letters,
respectively, with a mean EDTRS visual acuity reduc-
tion during the follow-up interval of 2.4 letters. There
was a significant correlation between the BCVA and
the mean fluorescence lifetime of the central ETDRS
subfield in both spectral channels when analyzing the
data of all study eyes at baseline (SSC: r2 = 0.35, P =
0.002; LSC: r2 = 0.32, P = 0.004) and follow-up (SSC:
r2 = 0.51, P , 0.0001; LSC: r2 = 0.45, P = 0.0001)
(Figure 1). A central atrophic lesion was found in
33.3% patients (10 eyes), with a mean size of
7.35 mm2 (range 0.48–20.3 mm2) at baseline, and
10.6 mm2 (range 1.69–28.7 mm2) at follow-up. The
mean progression rate of the atrophic area was
1.7 mm2 per year. Compared with the surrounding retina (SSC: 270 ±
6 ps; LSC: 297 ± 7 ps), Ƭm was significantly pro-
longed in long fluorescence lifetime flecks by +74%
in the SSC and +63% in the LCS (both P , 0.0001). While in short fluorescence lifetime flecks, Ƭm was
shortened by 22% in the SSC and 216% in the
LSC (SSC: P = 0.21, LSC: P , 0.0001) compared
with the surrounding retina. Fluorescence lifetime imaging ophthalmoscopy was
used to examine the progression of the disease in our
study eyes. Long fluorescence lifetime flecks pre-
sented as isolated deposits or as several flecks merging
with each other in a meshwork pattern. As expected,
fleck formation progressed centrifugal outward from
the central macular area as the disease advanced. On
follow-up, new flecks developed in all patients;
however, the rate and extent varied between patients. Short fluorescence lifetime flecks. A larger number
of short fluorescence lifetime flecks were identified in
patients at early stages of disease (Group 1 and Group
2), while at later stages, they were only found
occasionally. The ratio of short fluorescence lifetime
flecks identified at baseline between Group 1 versus
Group 3 was 9:5. Quantitative analysis of flecks with
short fluorescence lifetimes at two points in time Results Twenty-four eyes of 12 patients with a clinical
diagnosis of STGD were included in this study (53.8%
female). Patient characteristics are shown in Table 1. The mean age ± SEM at baseline was 42.25 ± 2.1
years (range, 26–56). All participants were white,
phakic with clear media, and had no concomitant oph-
thalmic diseases. The examined patients displayed FLUORESCENCE LIFETIME IMAGING IN STARGARDT DISEASE SOLBERG ET AL 883 Fig. 1. Correlation of BCVA (ETDRS letters) with mean fluorescence
lifetime (ps) within the central ETDRS subfield of the short (white
circle, SSC, 498–560 nm) and the long (black circle, LSC) spectral
channels at (A) baseline (SSC: r2 = 0.35, P = 0.002; LSC: r2 = 0.32, P =
0.004) and (B) follow-up (SSC: r2 = 0.51, P , 0.0001; LSC: r2 = 0.45,
P = 0.0001). Representative fluorescence lifetime (FLIO, LSC) images
of three identified groups and correlating OCT scan of the indicated
lines in the FLIO images are shown below. ps, vs. 522.6 ± 20, P , 0.0001) (LSC: 462.7 ± 14 ps,
vs. 548.3 ± 21 ps, P , 0.0001). Borders demon-
strated progression from sharp defined edges to
a more diffuse pattern (Figure 4). Correlation of Fluorescence Lifetime Data With
Optical Coherence Tomography Findings Colocalization of flecks in FLIO with OCT dis-
played hyperreflective material of variable size and
shape. At the level of the flecks, OCT bands displayed
convex, pyramidal-shaped, or ill-defined deposits,
interrupting the photoreceptor ellipsoid zone (EZ)
and/or external limiting membrane bands. Some
deposits displayed hyperreflective foci extending into
the outer nuclear layer, with a remaining connection to
the RPE. Depending on the location of hyperreflective
deposits in OCT, FLIO images displayed characteristic
lifetime patterns. Long fluorescence lifetime flecks
generally demonstrated intraretinal migration of de-
posits of varying extents (Figures 4 and 5), while short
fluorescence lifetime flecks usually presented as dome-
shaped deposits located within the outer retina at the
level of the RPE, with displacement or interruption of
the photoreceptor segments (Figure 3). Individual Fluorescence Lifetime Components As described in the method section, Tm is composed
of the separate lifetimes components T1 (short portion)
and T2 (long portion), along with their corresponding
amplitudes or intensities (a1 and a2). We analyzed
these individual fluorescence lifetime components
independently using 2D histograms to observe their
effect on Tm as described in previous publications.22,24 Fig. 1. Correlation of BCVA (ETDRS letters) with mean fluorescence
lifetime (ps) within the central ETDRS subfield of the short (white
circle, SSC, 498–560 nm) and the long (black circle, LSC) spectral
channels at (A) baseline (SSC: r2 = 0.35, P = 0.002; LSC: r2 = 0.32, P =
0.004) and (B) follow-up (SSC: r2 = 0.51, P , 0.0001; LSC: r2 = 0.45,
P = 0.0001). Representative fluorescence lifetime (FLIO, LSC) images
of three identified groups and correlating OCT scan of the indicated
lines in the FLIO images are shown below. Our data revealed that both short and long fluores-
cence lifetime flecks can be isolated from the surround-
ing
retina. Short
lifetime
flecks
characteristically
showed a shorter T1 and T2 component in comparison
to the surrounding retina and the long lifetime flecks
(Figure 5). (baseline and last follow-up) revealed that new flecks
visible in FLIO developed at a rate of 2.62/year in
Group 1, 1.43/year in Group 2, and 0.81/year in Group
3. Short lifetime lesion progressed to flecks with
characteristic long lifetimes in 75.1% (n = 22) within
29.2 months. In the transition phase, longer lifetimes
initiate in the center of the flecks, and radiate outward
with time. Borders of short lifetime flecks shifted to
long lifetimes last (Figure 3). (baseline and last follow-up) revealed that new flecks
visible in FLIO developed at a rate of 2.62/year in
Group 1, 1.43/year in Group 2, and 0.81/year in Group
3. Short lifetime lesion progressed to flecks with
characteristic long lifetimes in 75.1% (n = 22) within
29.2 months. In the transition phase, longer lifetimes
initiate in the center of the flecks, and radiate outward
with time. Borders of short lifetime flecks shifted to
long lifetimes last (Figure 3). Autofluorescence Lifetimes in Patients With
Stargardt Disease The white-yellow STGD flecks visible on fundus
examination correlated with hyperautofluorescent le-
sions in FAF intensity images and hyperreflective
changes seen in OCT. In FLIO, flecks exhibited both FLUORESCENCE LIFETIME IMAGING IN STARGARDT DISEASE SOLBERG ET AL RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2019 VOLUME 39 NUMBER 5 These results are in keeping with
our previous study of FLIO in STGD.20 In this
study, we provide evidence that flecks with short
lifetimes occur more frequently at earlier and more
active stages of the disease, and may be the result
from the buildup of intermediate components of the
retinoid cycle.11 On the other hand, blue flecks rep-
resent a more advanced stage of the lesion, possibly
indicating a more severe dysfunction of the under-
lying retina as by-products of the visual cycle accu-
mulate. Sparrow et al25 proposed that in STGD by-
products of the visual cycle can buildup in the outer
segment of photoreceptor cells. significantly correlated with longer mean fluorescence
lifetimes within the SSC (P = 0.006). Regression Analysis The influence of the independent variables patient’s
age, disease duration, and Fishman score was analyzed
against the dependent variables mean fluorescence
lifetime (Tm) in the SSC and the LSC. When analyz-
ing mean fluorescence lifetime values within the cen-
tral
ETDRS
grid
area,
the
disease
duration
significantly contributed to the ability of the equation
to predict lifetimes in the short and long wavelength
spectrum (SSC: P = 0.02 and LSC: P = 0.04). When
analyzing unaffected surrounding retina, only age Long fluorescence lifetime flecks. In general, there
was a predominance of long fluorescence lifetime
flecks in all stages of STGD in our cohort. On
analysis of long lifetime flecks, borders were identi-
fied to feature shorter mean fluorescence lifetime
values than the center of the fleck (SSC: 446.5 ± 17 RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2019 VOLUME 39 NUMBER 5 RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2019 VOLUME 39 NUMBER 5 884 significantly correlated with longer mean fluorescence
lifetimes within the SSC (P = 0.006). ill-defined on FAF intensity images, while flecks
with prolonged lifetimes were clearly identifiable. Interestingly, our follow-up measurements showed
Fig. 2. A. Quantitative analysis
of mean retinal autofluorescence
lifetime values in the SSC and
LSC in patients with STGD. Following areas were analyzed:
surrounding retinal tissue ( ),
flecks with long ( ) and short
( ) fluorescence lifetimes. B. A
small region of interest (circle
diameter: 0.16 mm) was used to
investigate mean retinal auto-
fluorescence
lifetimes
within
short lifetime flecks, long life-
time flecks, and the surrounding
retina. Areas of interest repre-
sent a mean value of three
individual
measurement
loca-
tions. Fluorescence lifetime data
of both spectral channels were
analyzed separately. ec s w t
o g ( ) a d s o t
( ) fluorescence lifetimes. B. A
small region of interest (circle
diameter: 0.16 mm) was used to
investigate mean retinal auto-
fluorescence
lifetimes
within
short lifetime flecks, long life-
time flecks, and the surrounding
retina. Areas of interest repre-
sent a mean value of three
individual
measurement
loca-
tions. Fluorescence lifetime data
of both spectral channels were
analyzed separately. g ( )
( ) fluorescence lifetimes. B. A
small region of interest (circle
diameter: 0.16 mm) was used to
investigate mean retinal auto-
fluorescence
lifetimes
within
short lifetime flecks, long life-
time flecks, and the surrounding
retina. Areas of interest repre-
sent a mean value of three
individual
measurement
loca-
tions. Fluorescence lifetime data
of both spectral channels were
analyzed separately. ill-defined on FAF intensity images, while flecks
with prolonged lifetimes were clearly identifiable. Interestingly, our follow-up measurements showed
that lesions with initially short lifetime values which
were faintly visible in FAF intensity measurements
progressed to flecks which were clearly visible as
hyperautofluorescent flecks and displayed long fluo-
rescence lifetimes. Discussion In this prospective study, we investigated retinal
fluorescence lifetimes in 24 eyes of patients with
STGD
using
fundus
autofluorescence
lifetime
(FLIO) imaging. Previously described characteristic
lifetime patterns20 were confirmed. Our data showed
that most retinal hyperautofluorescent flecks and de-
posits displayed longer lifetimes compared with the
surrounding retina. However, particularly at early
stages of disease (Groups 1 and 2), lesions with very
short fluorescence lifetimes were identified. These
lesions were more apparent in the LSC (560–720
nm). On correlation to other imaging modalities,
we observed that flecks with short fluorescence life-
times were either not yet visible, or faint and 885 FLUORESCENCE LIFETIME IMAGING IN STARGARDT DISEASE SOLBERG ET AL Fig. 3. Disease
progression
within 6- and 32-month follow-
up (Patient STGD 9). Color
fundus (CF), FAF intensity, and
fluorescence
lifetime
(FLIO,
LSC) images. Correlating OCT
scan of the indicated lines in the
FLIO images are shown below. From baseline (left) to 6-month
follow-up (middle), to 32-month
follow-up (right), clear disease
progression is visible with tran-
sition of flecks with short fluo-
rescence lifetimes (red arrows
and corresponding black arrows
in FLIO) to flecks with long
fluorescence lifetimes (blue ar-
rows and corresponding black
arrows in FLIO) and appearance
of new hyperfluorescent flecks. *Indicates a fleck with short
fluorescence
lifetime
initially
not visible on FAF intensity
image at baseline, but detectable
on follow-up. Hi
h l
i
l
di
h
h
h
fl
h
A2E (N
i
lid
N
i
l h
l
i
)
Fig. 3. Disease
progression
within 6- and 32-month follow-
up (Patient STGD 9). Color
fundus (CF), FAF intensity, and
fluorescence
lifetime
(FLIO,
LSC) images. Correlating OCT
scan of the indicated lines in the
FLIO images are shown below. From baseline (left) to 6-month
follow-up (middle), to 32-month
follow-up (right), clear disease
progression is visible with tran-
sition of flecks with short fluo-
rescence lifetimes (red arrows
and corresponding black arrows
in FLIO) to flecks with long
fluorescence lifetimes (blue ar-
rows and corresponding black
arrows in FLIO) and appearance
of new hyperfluorescent flecks. *Indicates a fleck with short
fluorescence
lifetime
initially
not visible on FAF intensity
image at baseline, but detectable
on follow-up. Fig. 3. Disease
progression
within 6- and 32-month follow-
up (Patient STGD 9). Color
fundus (CF), FAF intensity, and
fluorescence
lifetime
(FLIO,
LSC) images. Correlating OCT
scan of the indicated lines in the
FLIO images are shown below. Discussion Analysis
of
disease
progression of hyperfluorescent
flecks within 44-month follow-
up: autofluorescence intensity
image (FAF), and fluorescence
lifetime
(FLIO,
LSC)
with
selected hyperfluorescent flecks
(blue arrows) and corresponding
OCT scans. Follow-up exami-
nations show a clear progression
of flecks in different imaging
modalities,
with
changes
in
intensities and hyperreflective
material deposits on OCT. Fig. 4. Analysis
of
disease
progression of hyperfluorescent
flecks within 44-month follow-
up: autofluorescence intensity
image (FAF), and fluorescence
lifetime
(FLIO,
LSC)
with
selected hyperfluorescent flecks
(blue arrows) and corresponding
OCT scans. Follow-up exami-
nations show a clear progression
of flecks in different imaging
modalities,
with
changes
in
intensities and hyperreflective
material deposits on OCT. Information from endogenous fluorophores can be
obtained by analyzing the individual short (T1) and
long (T2) decay components, which contribute to the
mean fluorescence lifetime (Tm). As different fluoro-
phores have distinct lifetime patterns, specific retinal
structures can be demarcated. This technique can be
used to identify short and long decay times in separate
clusters (Figure 5). Furthermore, analysis of long life-
time flecks revealed that the borders display shorter
lifetimes than the center of a fleck. These findings
are in keeping with the observation that short lifetime
flecks progress to long lifetime flecks from the center
outward. This suggests that the short fluorescence life-
time flecks are composed of slightly different fluoro-
phore components than the long lifetime flecks. Sparrow et al33 identified an increased signal within
flecks using short-wavelength FAF. They purposed
that early on in STGD, the RPE cell layer is altered,
and that the enhanced short-wavelength FAF signal
could arise secondary to RPE atrophy and augmented
lipofuscin accumulation. These findings may provide
some insight into the possible sequence of retinal
changes in STGD. As described in previous clinical studies, our
longitudinal FLIO measurements revealed that fleck
formation
begins
centrally
in
the
foveal
area,
radiating outward as the disease progresses. In
addition, new lesions often develop in close prox-
imity to existing flecks, probably due to toxic effects
of visual by-products on neighboring RPE cells and
photoreceptors.5 Despite previous studies, the exact location of flecks
remains controversial. On colocalization of the flecks
to the OCT bands, we identified a variety of shapes
and sizes of hyperfluorescent accumulations at the EZ
and/or external limiting membrane bands. Discussion From baseline (left) to 6-month
follow-up (middle), to 32-month
follow-up (right), clear disease
progression is visible with tran-
sition of flecks with short fluo-
rescence lifetimes (red arrows
and corresponding black arrows
in FLIO) to flecks with long
fluorescence lifetimes (blue ar-
rows and corresponding black
arrows in FLIO) and appearance
of new hyperfluorescent flecks. *Indicates a fleck with short
fluorescence
lifetime
initially
not visible on FAF intensity
image at baseline, but detectable
on follow-up. Histopathological studies suggest that the hyper-
autofluorescent flecks originate from lipofuscin accu-
mulation in the RPE cells, which in STGD occurs at an
accelerated rate in comparison with the normal aging
process.26,27 Buildup of fluorescent lipofuscin in the
RPE is a hallmark of STGD, and the possible disease
mechanism due to the dysfunction of the ABCA4 flip-
pase could be the following.13 After photoactivation of
rhodopsin, all-trans retinal builds up in the photorecep-
tor outer segments and readily forms N-retinylidene-
phosphatidylethanolamine (N-ret-PE) by binding to
amine groups of phosphatidylethanolamine (PE). Loss
of ABCA4 flippase activity reduces clearance of all-
trans retinal and favors its condensation with a second
retinaldehyde to form the bisretinoids A2PE. On phago-
cytosis, A2PE is converted into the major lipofuscin fluorophore A2E (N-retinylidene-N-retinylethanolamine)
in the acidic environment of RPE phagosomes.26–28
The recent identification of ABCA4 in RPE internal
membranes is in further support that accumulation
of autofluorescent precursors may contribute to
both RPE- and outer photoreceptor segment dys-
function, leading to lipofuscin buildup.13,29 These
equilibriums possibly contribute to the shorter life-
times identified in the newly formed deposits in
STGD.20,22 In addition, other components such as melanin have
been suggested to be involved in the disease process. Melanin is concentrated at the fovea in the RPE cells
and has been shown to decrease in normal aging.30 It
has been speculated that lipofuscin buildup in the RPE
cells is partly influenced by melanin.31 RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2019 VOLUME 39 NUMBER 5 886 Fig. 4. Analysis
of
disease
progression of hyperfluorescent
flecks within 44-month follow-
up: autofluorescence intensity
image (FAF), and fluorescence
lifetime
(FLIO,
LSC)
with
selected hyperfluorescent flecks
(blue arrows) and corresponding
OCT scans. Follow-up exami-
nations show a clear progression
of flecks in different imaging
modalities,
with
changes
in
intensities and hyperreflective
material deposits on OCT. Fig. 4. Conclusions In STGD, retinal fluorescence lifetimes can be used
to analyze hyperfluorescent flecks. Fluorescence life-
time imaging ophthalmoscopy has the potential of
becoming a monitoring tool in retinal conditions by
illustrating metabolic malfunctions and providing
further information about the composition of endoge-
nous retinal fluorophores. This study establishes that
fundus
autofluorescence
lifetimes
demonstrate
explicit, reproducible patterns in STGD, and confirms
the diagnostic value of FLIO as a non-invasive
imaging modality. Using FLIO, we present a method of monitoring and
quantifying
disease
progression
in
patients
with
STGD. Patterns of disease progression, when quanti-
fied, could be useful in the development of new
outcome measurements for clinical trials testing novel
therapies for STGD. Since these changing autofluor-
escence lifetime patterns may reflect intracellular
events in RPE cells, they may be helpful in gauging
the biological effect of potential therapies and in
interpreting treatment effects. Key words: fluorescence lifetimes, fundus auto-
fluorescence, ophthalmic imaging, Stargardt disease,
macular dystrophy. Discussion A 2D analysis of short and long fluorescence lifetime compo
nents T1 and T2. Distribution histograms (bottom) are shown o
a patient with STGD with short and long fluorescence lifetime flecks
Specific areas are highlighted according to the lifetime distribution
clouds: surrounding retina, short fluorescence lifetime flecks, long
lifetime flecks, and optic nerve head. Corresponding fluorescence life
time image (FLIO, LSC), FAF intensity, and OCT scan. Discussion However,
an anatomical link to the RPE layer was present,
suggesting that the lipofuscin deposits begins in the
RPE and then migrates toward the inner retinal layers. As previously observed by Voigt et al,32 we hypothe-
size that the different forms of deposits observed in the
OCT bands represent different phases in the natural
history of fleck progression. Although flecks with var-
ied fluorescence intensities were identified, we could
not detect a correlation to the interruption of EZ, exter-
nal limiting membrane, or outer nuclear layer bands. On analysis of OCT sections, we observed that at
earlier stages of the disease, the hyperreflective mate-
rial was restricted to the area underneath the EZ,
whereas at later stages, lesions with longer lifetimes
tended to progress through the EZ into the outer retinal
layers. There are some limitations to this study. Primarily,
in one patient, the diagnosis of STGD was largely
based on clinical assessment, and in one patient, an
autosomal dominant inheritance trait was identified. Patients displayed a variable degree of disease severity
from early or childhood-onset to late-onset of STGD. We also acknowledge that the data provided in this
report showed a cohort of patients with an atypical A useful tool to investigate the characteristics and
progression of STGD is the 2D histogram analysis. FLUORESCENCE LIFETIME IMAGING IN STARGARDT DISEASE SOLBERG ET AL 887 g. 5. A 2D analysis of short and long fluorescence lifetime c
nts T1 and T2. Distribution histograms (bottom) are sho
patient with STGD with short and long fluorescence lifetime
ecific areas are highlighted according to the lifetime distr
ouds: surrounding retina, short fluorescence lifetime flecks
etime flecks, and optic nerve head. Corresponding fluorescen
me image (FLIO, LSC), FAF intensity, and OCT scan. presentation including an older age of onset of
symptoms and a higher progression rate of atrophic
lesions in comparison to the ProgStar cohort.34,35 Fur-
thermore, a larger sample size would have provided
more information. However, despite the small cohort,
the findings of conversion of retinal flecks were con-
sistent between patients. Concerns about the long-term
effects and the relationship between short-wavelength
light and the generation of toxic products have been
raised, especially patients with STGD. However, the
light exposure of the FLIO device is well below the
limits recommended by the ANSI standard for safe use
of lasers.36 Fig. 5. References 19. Campa C, Gallenga CE, Bolletta E, Perri P. The role of gene
therapy in the treatment of retinal diseases: a review. Curr
Gene Ther 2017;17:194–213. 1. Stargardt K. Ueber familiare progressive Degeneration in der
Makulagegend des Augues. Albrecht V Graefes Arch Optha-
mology 1909;71:534–550. 20. Dysli C, Wolf S, Hatz K, Zinkernagel MS. Fluorescence life-
time imaging in Stargardt disease: potential Marker for disease
progression. Invest Ophthalmol Vis Sci 2016;57:832–841. 2. Armstrong JD, Meyer D, Xu S, Elfervig JL. Long-term follow-
up of Stargardt’s disease and fundus flavimaculatus. Ophthal-
mology 1998;105:448–457. 21. Fujinami K, Zernant J, Chana RK, et al. Clinical and molecular
characteristics of childhood-onset Stargardt disease. Ophthal-
mology 2015;122:326–334. 3. Steinmetz RL, Garner A, Maguire JI, Bird AC. Histopathology
of incipient fundus flavimaculatus. Ophthalmology 1991;98:
953–956. 22. Dysli C, Wolf S, Berezin MY, et al. Fluorescence lifetime imag-
ing ophthalmoscopy. Prog Retin Eye Res 2017;60:120–143. 4. Sparrow JR, Boulton M. RPE lipofuscin and its role in retinal
pathobiology. Exp Eye Res 2005;80:595–606. 23. Lois N, Holder GE, Bunce C, et al. Phenotypic subtypes of
Stargardt macular dystrophy-fundus flavimaculatus. Arch Oph-
thalmol 2001;119:359–369. 5. Cukras CA, Wong WT, Caruso R, et al. Centrifugal expansion
of fundus autofluorescence patterns in Stargardt disease over
time. Arch Ophthalmol 2012;130:171–179. 24. Dysli C, Quellec G, Abegg M, et al. Quantitative analysis of
fluorescence lifetime measurements of the macula using the
fluorescence lifetime imaging ophthalmoscope in healthy sub-
jects. Invest Ophthalmol Vis Sci 2014;55:2106–2113. 6. Gomes NL, Greenstein VC, Carlson JN, et al. A comparison of
fundus autofluorescence and retinal structure in patients with Star-
gardt disease. Invest Ophthalmol Vis Sci 2009;50:3953–3959. 25. Sparrow JR, Fishkin N, Zhou J, et al. A2E, a byproduct of the
visual cycle. Vis Res 2003;43:2983–2990. 7. Rotenstreich Y, Fishman GA, Anderson RJ. Visual acuity loss
and clinical observations in a large series of patients with Star-
gardt disease. Ophthalmology 2003;110:1151–1158. 26. Maeda T, Golczak M, Maeda A. Retinal photodamage medi-
ated by all-trans-retinal. Photochem Photobiol 2012;88:1309–
1319. 8. Fishman GA, Stone EM, Grover S, et al. Variation of clinical
expression in patients with Stargardt dystrophy and sequence
variations in the ABCR gene. Arch Ophthalmol 1999;117:
504–510. 27. Ben-Shabat S, Parish CA, Vollmer HR, et al. Biosynthetic
studies of A2E, a major fluorophore of retinal pigment epithe-
lial lipofuscin. J Biol Chem 2002;277:7183–7190. 9. Fishman GA, Farber M, Patel BS, Derlacki DJ. References Visual acuity
loss in patients with Stargardt’s macular dystrophy. Ophthal-
mology 1987;94:809–814. 28. Mata NL, Weng J, Travis GH. Biosynthesis of a major lip-
ofuscin fluorophore in mice and humans with ABCR-mediated
retinal and macular degeneration. Proc Natl Acad Sci U S A
2000;97:7154–7159. 10. Smith RT, Gomes NL, Barile G, et al. Lipofuscin and auto-
fluorescence metrics in progressive STGD. Invest Ophthalmol
Vis Sci 2009;50:3907–3914. 29. Liu J, Itagaki Y, Ben-Shabat S, et al. The biosynthesis of A2E,
a fluorophore of aging retina, involves the formation of the
precursor, A2-PE, in the photoreceptor outer segment mem-
brane. J Biol Chem 2000;275:29354–29360. 11. Cideciyan AV, Aleman TS, Swider M, et al. Mutations in
ABCA4 result in accumulation of lipofuscin before slowing
of the retinoid cycle: a reappraisal of the human disease
sequence. Hum Mol Genet 2004;13:525–534. 30. Kennedy CJ, Rakoczy PE, Constable IJ, Lipofuscin of the
retinal pigment epithelium: a review. Eye (Lond) 1995;9:
763–771. 12. Allikmets R. A photoreceptor cell-specific ATP-binding trans-
porter gene (ABCR) is mutated in recessive Stargardt macular
dystrophy. Nat Genet 1997;17:122. 31. Sundelin SP, Nilsson SE, Brunk UT. Lipofuscin-formation in
cultured retinal pigment epithelial cells is related to their mel-
anin content. Free Radic Biol Med 2001;30:74–81. 13. Lenis TL, Hu J, Ng SY, et al. Expression of ABCA4 in the
retinal pigment epithelium and its implications for Stargardt
macular degeneration. Proc Natl Acad Sci USA 2018;115:
E11120–E11127. 32. Voigt M, Querques G, Atmani K, et al. Analysis of retinal
flecks in fundus flavimaculatus using high-definition spectral-
domain optical coherence tomography. Am J Ophthalmol
2010;150:330–337. 14. Allikmets R, Shroyer NF, Singh N, et al. Mutation of the
Stargardt disease gene (ABCR) in age-related macular degen-
eration. Science 1997;277:1805–1807. 33. Sparrow JR, Marsiglia M, Allikmets R, et al. Flecks in reces-
sive Stargardt disease: short-wavelength autofluorescence,
near-infrared autofluorescence, and optical coherence tomog-
raphy. Invest Ophthalmol Vis Sci 2015;56:5029–5039. 15. Chen Y, Roorda A, Duncan JL. Advances in imaging of Star-
gardt disease. Adv Exp Med Biol 2010;664:333–340. 16. Ergun E, Hermann B, Wirtitsch M, et al. Assessment of central
visual function in Stargardt’s disease/fundus flavimaculatus
with ultrahigh-resolution optical coherence tomography. Invest
Ophthalmol Vis Sci 2005;46:310–316. 34. Strauss RW, Muñoz B, Ho A, et al. Incidence of atrophic
lesions in Stargardt disease in the progression of atrophy sec-
ondary to Stargardt disease (ProgStar) study: report No. 5. JAMA Ophthalmol 2017;135:687–695. 17. RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2019 VOLUME 39 NUMBER 5 888 Acknowledgments The authors thank Jörg Fischer, PhD, Yoshihiko
Katayama, PhD, Kester Nahen, PhD,(all from Heidel-
berg Engineering GmBH, Heidelberg, Germany),
Bernhard Weber, PhD (Universität Würzburg), Sus-
anne Kohl, PhD (Universität Tübingen), Wolfgang
Berger, PhD (Universität Zürich), André Schaller,
PhD (Inselspital), and Salome Allemann (Inselspital). Fig. 5. A 2D analysis of short and long fluorescence lifetime compo-
nents T1 and T2. Distribution histograms (bottom) are shown of
a patient with STGD with short and long fluorescence lifetime flecks. Specific areas are highlighted according to the lifetime distribution
clouds: surrounding retina, short fluorescence lifetime flecks, long
lifetime flecks, and optic nerve head. Corresponding fluorescence life-
time image (FLIO, LSC), FAF intensity, and OCT scan. References Burke TR, Duncker T, Woods RL, et al. Quantitative fundus
autofluorescence in recessive Stargardt disease. Invest Oph-
thalmol Vis Sci 2014;55:2841–2852. 35. Strauss RW, Muñoz B, Ho A, et al. Progression of Stargardt
disease as determined by fundus autofluorescence in the retro-
spective progression of Stargardt disease study (ProgStar report
No. 9). JAMA Ophthalmol 2017;135:1232–1241. No. 9). JAMA Ophthalmol 2017;135:1232–1241. 18. Charbel Issa P, Barnard AR, Herrmann P, et al. Rescue of the
Stargardt phenotype in Abca4 knockout mice through inhibi-
tion of vitamin A dimerization. Proc Natl Acad Sci USA 2015;
112:8415–8420. p
36. Laser Institute of America. ANSI Z136.1—2007 American
National Standard for Safe Use of Lasers; 2007 http://www. z136.org.
|
https://openalex.org/W4244630309
|
https://zenodo.org/records/2230536/files/article.pdf
|
English
| null |
Southern Rhodesia
|
Geographical journal
| 1,910
|
public-domain
| 4,687
|
Review
Source: The Geographical Journal, Vol. 35, No. 2 (Feb., 1910), p. 188
Published by: geographicalj
Stable URL: http://www.jstor.org/stable/1776986
Accessed: 19-06-2016 13:54 UTC
Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at
http://about.jstor.org/terms Review
Source: The Geographical Journal, Vol. 35, No. 2 (Feb., 1910), p. 188
Published by: geographicalj
Stable URL: http://www.jstor.org/stable/1776986
Accessed: 19-06-2016 13:54 UTC
Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at
http://about.jstor.org/terms MEXICO.
MEXICO.
MEXICO.
MEXICO.
MEXICO.
MEXICO. ' Mexico.' By C. R. Enock. London: Fisher Unwin. 1909. Pp. xxxvi., 362. Maps and Illustrations. 10s. 6d. ' Mexico.' By C. R. Enock. London: Fisher Unwin. 1909. Pp. xxxvi., 362. Maps and Illustrations. 10s. 6d. ' Mexico.' By C. R. Enock. London: Fisher Unwin. 1909. Pp. xxxvi., 362. Maps and Illustrations. 10s. 6d. ' Mexico.' By C. R. Enock. London: Fisher Unwin. 1909. Pp. xxxvi., 362. Maps and Illustrations. 10s. 6d. ' Mexico.' By C. R. Enock. London: Fisher Unwin. 1909. Pp. xxxvi., 362. Maps and Illustrations. 10s. 6d. ' Mexico.' By C. R. Enock. London: Fisher Unwin. 1909. Pp. xxxvi., 362. Maps and Illustrations. 10s. 6d. Mr. Enock's work is already well known through his books on ' The Andes and
the Amazon' and ' Peru.' The present volume, like that on Peru, forms one of
the "South American Series," under the editorship of Mr. Martin Hume. Mr. E ume contributes an introduction, which consists of a brief survey of what is
much more fully elaborated in the text-the history of Mexico down to the
present time. To this subject Mr. Enock devotes six chapters, and, besides showing
himself to be amply grounded in its study,'has evidently a keen eye for its
romance. He has no less appreciation for the remarkable scenery and physical
peculiarities of the country, which are dealt with in one chapter. Further chapters
are devoted to the people, their cities and institutions, to life and travel, mineral
wealth, and other resources of the country, its finance, industries, and railways. When a number of good photographs and a large contoured map are taken into
consideration, it is seen that we have here an important general treatise on the
country, by a writer who has travelled widely in it, has given himself careful
training as an observer, and combines with this the interests of a historian and
of a business man, so that he is enabled properly to appraise the wealth and
Mr. Enock's work is already well known through his books on ' The Andes and
the Amazon' and ' Peru.' The present volume, like that on Peru, forms one of
the "South American Series," under the editorship of Mr. Martin Hume. Mr. E ume contributes an introduction, which consists of a brief survey of what is
much more fully elaborated in the text-the history of Mexico down to the
present time. MEXICO.
MEXICO.
MEXICO.
MEXICO.
MEXICO.
MEXICO. To this subject Mr. Enock devotes six chapters, and, besides showing
himself to be amply grounded in its study,'has evidently a keen eye for its
romance. He has no less appreciation for the remarkable scenery and physical
peculiarities of the country, which are dealt with in one chapter. Further chapters
are devoted to the people, their cities and institutions, to life and travel, mineral
wealth, and other resources of the country, its finance, industries, and railways. When a number of good photographs and a large contoured map are taken into
consideration, it is seen that we have here an important general treatise on the
country, by a writer who has travelled widely in it, has given himself careful
training as an observer, and combines with this the interests of a historian and
of a business man, so that he is enabled properly to appraise the wealth and
Mr. Enock's work is already well known through his books on ' The Andes and
the Amazon' and ' Peru.' The present volume, like that on Peru, forms one of
the "South American Series," under the editorship of Mr. Martin Hume. Mr. E ume contributes an introduction, which consists of a brief survey of what is
much more fully elaborated in the text-the history of Mexico down to the
present time. To this subject Mr. Enock devotes six chapters, and, besides showing
himself to be amply grounded in its study,'has evidently a keen eye for its
romance. He has no less appreciation for the remarkable scenery and physical
peculiarities of the country, which are dealt with in one chapter. Further chapters
are devoted to the people, their cities and institutions, to life and travel, mineral
wealth, and other resources of the country, its finance, industries, and railways. When a number of good photographs and a large contoured map are taken into
consideration, it is seen that we have here an important general treatise on the
country, by a writer who has travelled widely in it, has given himself careful
training as an observer, and combines with this the interests of a historian and
of a business man, so that he is enabled properly to appraise the wealth and
Mr. MEXICO.
MEXICO.
MEXICO.
MEXICO.
MEXICO.
MEXICO. Enock's work is already well known through his books on ' The Andes and
the Amazon' and ' Peru.' The present volume, like that on Peru, forms one of
the "South American Series," under the editorship of Mr. Martin Hume. Mr. E ume contributes an introduction, which consists of a brief survey of what is
much more fully elaborated in the text-the history of Mexico down to the
present time. To this subject Mr. Enock devotes six chapters, and, besides showing
himself to be amply grounded in its study,'has evidently a keen eye for its
romance. He has no less appreciation for the remarkable scenery and physical
peculiarities of the country, which are dealt with in one chapter. Further chapters
are devoted to the people, their cities and institutions, to life and travel, mineral
wealth, and other resources of the country, its finance, industries, and railways. When a number of good photographs and a large contoured map are taken into
consideration, it is seen that we have here an important general treatise on the
country, by a writer who has travelled widely in it, has given himself careful
training as an observer, and combines with this the interests of a historian and
of a business man, so that he is enabled properly to appraise the wealth and
Mr. Enock's work is already well known through his books on ' The Andes and
the Amazon' and ' Peru.' The present volume, like that on Peru, forms one of
the "South American Series," under the editorship of Mr. Martin Hume. Mr. E ume contributes an introduction, which consists of a brief survey of what is
much more fully elaborated in the text-the history of Mexico down to the
present time. To this subject Mr. Enock devotes six chapters, and, besides showing
himself to be amply grounded in its study,'has evidently a keen eye for its
romance. He has no less appreciation for the remarkable scenery and physical
peculiarities of the country, which are dealt with in one chapter. Further chapters
are devoted to the people, their cities and institutions, to life and travel, mineral
wealth, and other resources of the country, its finance, industries, and railways. Official considerations prevent him from dealing in detail
with the discussions of the convention, but he is able to write with peculiar autho-
rity about the whole circumstances of the union, surveys the historical circum-
stances which led up to it, deals in detail with the executive and legislative
arrangements, and indicates with first-hand knowledge the popular feeling on the
various leading questions of the union. dangers on his journeys. ' The Union of South Africa.' By the Hon. R. H. Brand. (Oxford: Clarendon
Press. 1909. Pp. 192. 6s.) This volume, appearing at an appropriate time, is
by a writer who served as secretary to the Transvaal delegates at the South African
National Convention. Official considerations prevent him from dealing in detail
with the discussions of the convention, but he is able to write with peculiar autho-
rity about the whole circumstances of the union, surveys the historical circum-
stances which led up to it, deals in detail with the executive and legislative
arrangements, and indicates with first-hand knowledge the popular feeling on the
various leading questions of the union. various leading questions of the union. 'Southern Rhodesia.' By Percy F. Hone. (London: Bell. 1909. Pp. xv.,
406. Maps and Illustrations. 10s. 6d.) We have here an ample volume on
"the administrative and industrial development of Southern Rhodesia since its
occupation by the British South Africa Company." The writer appears to have
had ample assistance from official sources, and quotes fully from original documents. In view of the possible subsequent relations of the territory with the newly formed
union, the book appears at an opportune time
various leading questions of the union. 'Southern Rhodesia.' By Percy F. Hone. (London: Bell. 1909. Pp. xv.,
406. Maps and Illustrations. 10s. 6d.) We have here an ample volume on
"the administrative and industrial development of Southern Rhodesia since its
occupation by the British South Africa Company." The writer appears to have
had ample assistance from official sources, and quotes fully from original documents. In view of the possible subsequent relations of the territory with the newly formed
union, the book appears at an opportune time
various leading questions of the union. 'Southern Rhodesia.' By Percy F. Hone. (London: Bell. 1909. Pp. xv.,
406. Maps and Illustrations. 10s. Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted
digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about
JSTOR, please contact support@jstor.org. Wiley, The Royal Geographical Society (with the Institute of British Geographers) are
collaborating with JSTOR to digitize, preserve and extend access to The Geographical Journal Wiley, The Royal Geographical Society (with the Institute of British Geographers) are
collaborating with JSTOR to digitize, preserve and extend access to The Geographical Journal This content downloaded from 146.189.156.77 on Sun, 19 Jun 2016 13:54:13 UTC
All use subject to http://about.jstor.org/terms 188
188
188
188
188
188 REVIEWS. REVIEWS. REVIEWS. REVIEWS. REVIEWS. REVIEWS. author's experiences and adventures are well told, and he met with not a few
dangers on his journeys
author's experiences and adventures are well told, and he met with not a few
dangers on his journeys
author's experiences and adventures are well told, and he met with not a few
dangers on his journeys
author's experiences and adventures are well told, and he met with not a few
dangers on his journeys
author's experiences and adventures are well told, and he met with not a few
dangers on his journeys
author's experiences and adventures are well told, and he met with not a few
dangers on his journeys dangers on his journeys. ' The Union of South Africa.' By the Hon. R. H. Brand. (Oxford: Clarendon
Press. 1909. Pp. 192. 6s.) This volume, appearing at an appropriate time, is
by a writer who served as secretary to the Transvaal delegates at the South African
National Convention. Official considerations prevent him from dealing in detail
with the discussions of the convention, but he is able to write with peculiar autho-
rity about the whole circumstances of the union, surveys the historical circum-
stances which led up to it, deals in detail with the executive and legislative
arrangements, and indicates with first-hand knowledge the popular feeling on the
various leading questions of the union. dangers on his journeys. ' The Union of South Africa.' By the Hon. R. H. Brand. (Oxford: Clarendon
Press. 1909. Pp. 192. 6s.) This volume, appearing at an appropriate time, is
by a writer who served as secretary to the Transvaal delegates at the South African
National Convention. Official considerations prevent him from dealing in detail
with the discussions of the convention, but he is able to write with peculiar autho-
rity about the whole circumstances of the union, surveys the historical circum-
stances which led up to it, deals in detail with the executive and legislative
arrangements, and indicates with first-hand knowledge the popular feeling on the
various leading questions of the union. dangers on his journeys. ' The Union of South Africa.' By the Hon. R. H. Brand. (Oxford: Clarendon
Press. 1909. Pp. 192. 6s.) This volume, appearing at an appropriate time, is
by a writer who served as secretary to the Transvaal delegates at the South African
National Convention. Official considerations prevent him from dealing in detail
with the discussions of the convention, but he is able to write with peculiar autho-
rity about the whole circumstances of the union, surveys the historical circum-
stances which led up to it, deals in detail with the executive and legislative
arrangements, and indicates with first-hand knowledge the popular feeling on the
various leading questions of the union. dangers on his journeys. ' The Union of South Africa.' By the Hon. R. H. Brand. (Oxford: Clarendon
Press. 1909. Pp. 192. 6s.) This volume, appearing at an appropriate time, is
by a writer who served as secretary to the Transvaal delegates at the South African
National Convention. Official considerations prevent him from dealing in detail
with the discussions of the convention, but he is able to write with peculiar autho-
rity about the whole circumstances of the union, surveys the historical circum-
stances which led up to it, deals in detail with the executive and legislative
arrangements, and indicates with first-hand knowledge the popular feeling on the
various leading questions of the union. dangers on his journeys. ' The Union of South Africa.' By the Hon. R. H. Brand. (Oxford: Clarendon
Press. 1909. Pp. 192. 6s.) This volume, appearing at an appropriate time, is
by a writer who served as secretary to the Transvaal delegates at the South African
National Convention. This content downloaded from 146.189.156.77 on Sun, 19 Jun 2016 13:54:13 UTC
All use subject to http://about.jstor.org/terms (London: Smith, Elder. 1909. Pp. xxvi., 291. Maps. 6s.) Mr. Morel's attacks on the Belgian "Congo
system " command wide attention, and in this strongly worded exposition he is
supported by Sir A. Conan Doyle in an introduction. 6d.) We have here an ample volume on
"the administrative and industrial development of Southern Rhodesia since its
occupation by the British South Africa Company." The writer appears to have
had ample assistance from official sources, and quotes fully from original documents. In view of the possible subsequent relations of the territory with the newly formed
union, the book appears at an opportune time
various leading questions of the union. 'Southern Rhodesia.' By Percy F. Hone. (London: Bell. 1909. Pp. xv.,
406. Maps and Illustrations. 10s. 6d.) We have here an ample volume on
"the administrative and industrial development of Southern Rhodesia since its
occupation by the British South Africa Company." The writer appears to have
had ample assistance from official sources, and quotes fully from original documents. In view of the possible subsequent relations of the territory with the newly formed
union, the book appears at an opportune time
various leading questions of the union. 'Southern Rhodesia.' By Percy F. Hone. (London: Bell. 1909. Pp. xv.,
406. Maps and Illustrations. 10s. 6d.) We have here an ample volume on
"the administrative and industrial development of Southern Rhodesia since its
occupation by the British South Africa Company." The writer appears to have
had ample assistance from official sources, and quotes fully from original documents. In view of the possible subsequent relations of the territory with the newly formed
union, the book appears at an opportune time
various leading questions of the union. 'Southern Rhodesia.' By Percy F. Hone. (London: Bell. 1909. Pp. xv.,
406. Maps and Illustrations. 10s. 6d.) We have here an ample volume on
"the administrative and industrial development of Southern Rhodesia since its
occupation by the British South Africa Company." The writer appears to have
had ample assistance from official sources, and quotes fully from original documents. In view of the possible subsequent relations of the territory with the newly formed
union, the book appears at an opportune time union, the book appears at an opportune time. ' The Ore Deposits of South Africa.' Part ii. By J. P. Johnson. (London:
Crosby Lockwood. 1909. Pp. 51. Diagrams. 5s.) This work, of which only
the second part is before us, deals in the first with the distribution and character
of " base " metals in South Africa. The second deals with the Witwatersrand and
Pilgrimsrest goldfields and others. It is a strictly technical work, copiously
illustrated with diagrams, including distribution maps, which, however, would
hardly be of service to any but metallurgists. union, the book appears at an opportune time. ' The Ore Deposits of South Africa.' Part ii. By J. P. Johnson. (London:
Crosby Lockwood. 1909. Pp. 51. Diagrams. 5s.) This work, of which only
the second part is before us, deals in the first with the distribution and character
of " base " metals in South Africa. The second deals with the Witwatersrand and
Pilgrimsrest goldfields and others. It is a strictly technical work, copiously
illustrated with diagrams, including distribution maps, which, however, would
hardly be of service to any but metallurgists. union, the book appears at an opportune time. ' The Ore Deposits of South Africa.' Part ii. By J. P. Johnson. (London:
Crosby Lockwood. 1909. Pp. 51. Diagrams. 5s.) This work, of which only
the second part is before us, deals in the first with the distribution and character
of " base " metals in South Africa. The second deals with the Witwatersrand and
Pilgrimsrest goldfields and others. It is a strictly technical work, copiously
illustrated with diagrams, including distribution maps, which, however, would
hardly be of service to any but metallurgists. union, the book appears at an opportune time. ' The Ore Deposits of South Africa.' Part ii. By J. P. Johnson. (London:
Crosby Lockwood. 1909. Pp. 51. Diagrams. 5s.) This work, of which only
the second part is before us, deals in the first with the distribution and character
of " base " metals in South Africa. The second deals with the Witwatersrand and
Pilgrimsrest goldfields and others. It is a strictly technical work, copiously
illustrated with diagrams, including distribution maps, which, however, would
hardly be of service to any but metallurgists. union, the book appears at an opportune time. ' The Ore Deposits of South Africa.' Part ii. By J. P. Johnson. (London:
Crosby Lockwood. 1909. Pp. 51. Diagrams. 5s.) This work, of which only
the second part is before us, deals in the first with the distribution and character
of " base " metals in South Africa. The second deals with the Witwatersrand and
Pilgrimsrest goldfields and others. It is a strictly technical work, copiously
illustrated with diagrams, including distribution maps, which, however, would
hardly be of service to any but metallurgists. union, the book appears at an opportune time. y
y
g
'Great Britain and the Congo.' By E. D. Morel. (London: Smith, Elder. 1909. Pp. xxvi., 291. Maps. 6s.) Mr. Morel's attacks on the Belgian "Congo
system " command wide attention, and in this strongly worded exposition he is
supported by Sir A. Conan Doyle in an introduction. y
y
g
'Great Britain and the Congo.' By E. D. Morel. (London: Smith, Elder. 1909. Pp. xxvi., 291. Maps. 6s.) Mr. Morel's attacks on the Belgian "Congo
system " command wide attention, and in this strongly worded exposition he is
supported by Sir A. Conan Doyle in an introduction. y
y
g
'Great Britain and the Congo.' By E. D. Morel. (London: Smith, Elder. 1909. Pp. xxvi., 291. Maps. 6s.) Mr. Morel's attacks on the Belgian "Congo
system " command wide attention, and in this strongly worded exposition he is
supported by Sir A. Conan Doyle in an introduction. g
'Great Britain and the Congo.' By E. D. Morel. (London: Smith, Elder. 1909. Pp. xxvi., 291. Maps. 6s.) Mr. Morel's attacks on the Belgian "Congo
system " command wide attention, and in this strongly worded exposition he is
supported by Sir A. Conan Doyle in an introduction. g
'Great Britain and the Congo.' By E. D. Morel. (London: Smith, Elder. 1909. Pp. xxvi., 291. Maps. 6s.) Mr. Morel's attacks on the Belgian "Congo
system " command wide attention, and in this strongly worded exposition he is
supported by Sir A. Conan Doyle in an introduction. g
'Great Britain and the Congo.' By E. D. Morel. (London: Smith, Elder. 1909. Pp. xxvi., 291. Maps. 6s.) Mr. Morel's attacks on the Belgian "Congo
system " command wide attention, and in this strongly worded exposition he is
supported by Sir A. Conan Doyle in an introduction. g
'Great Britain and the Congo.' By E. D. Morel. (London: Smith, Elder. 1909. Pp. xxvi., 291. Maps. 6s.) Mr. Morel's attacks on the Belgian "Congo
system " command wide attention, and in this strongly worded exposition he is
supported by Sir A. Conan Doyle in an introduction. g
'Great Britain and the Congo.' By E. D. Morel. (London: Smith, Elder. 1909. Pp. xxvi., 291. Maps. 6s.) Mr. Morel's attacks on the Belgian "Congo
system " command wide attention, and in this strongly worded exposition he is
supported by Sir A. Conan Doyle in an introduction. g
'Great Britain and the Congo.' By E. D. Morel. ' The Ore Deposits of South Africa.' Part ii. By J. P. Johnson. (London:
Crosby Lockwood. 1909. Pp. 51. Diagrams. 5s.) This work, of which only
the second part is before us, deals in the first with the distribution and character
of " base " metals in South Africa. The second deals with the Witwatersrand and
Pilgrimsrest goldfields and others. It is a strictly technical work, copiously
illustrated with diagrams, including distribution maps, which, however, would
hardly be of service to any but metallurgists. union, the book appears at an opportune time. ' The Ore Deposits of South Africa.' Part ii. By J. P. Johnson. (London:
Crosby Lockwood. 1909. Pp. 51. Diagrams. 5s.) This work, of which only
the second part is before us, deals in the first with the distribution and character
of " base " metals in South Africa. The second deals with the Witwatersrand and
Pilgrimsrest goldfields and others. It is a strictly technical work, copiously
illustrated with diagrams, including distribution maps, which, however, would
hardly be of service to any but metallurgists. union, the book appears at an opportune time. ' The Ore Deposits of South Africa.' Part ii. By J. P. Johnson. (London:
Crosby Lockwood. 1909. Pp. 51. Diagrams. 5s.) This work, of which only
the second part is before us, deals in the first with the distribution and character
of " base " metals in South Africa. The second deals with the Witwatersrand and
Pilgrimsrest goldfields and others. It is a strictly technical work, copiously
illustrated with diagrams, including distribution maps, which, however, would
hardly be of service to any but metallurgists. union, the book appears at an opportune time. ' The Ore Deposits of South Africa.' Part ii. By J. P. Johnson. (London:
Crosby Lockwood. 1909. Pp. 51. Diagrams. 5s.) This work, of which only
the second part is before us, deals in the first with the distribution and character
of " base " metals in South Africa. The second deals with the Witwatersrand and
Pilgrimsrest goldfields and others. It is a strictly technical work, copiously
illustrated with diagrams, including distribution maps, which, however, would
hardly be of service to any but metallurgists. union, the book appears at an opportune time. ' The Ore Deposits of South Africa.' Part ii. By J. P. Johnson. (London:
Crosby Lockwood. 1909. Pp. 51. Diagrams. 5s.) This work, of which only
the second part is before us, deals in the first with the distribution and character
of " base " metals in South Africa. The second deals with the Witwatersrand and
Pilgrimsrest goldfields and others. It is a strictly technical work, copiously
illustrated with diagrams, including distribution maps, which, however, would
hardly be of service to any but metallurgists. union, the book appears at an opportune time. ' The Ore Deposits of South Africa.' Part ii. By J. P. Johnson. (London:
Crosby Lockwood. 1909. Pp. 51. Diagrams. 5s.) This work, of which only
the second part is before us, deals in the first with the distribution and character
of " base " metals in South Africa. The second deals with the Witwatersrand and
Pilgrimsrest goldfields and others. It is a strictly technical work, copiously
illustrated with diagrams, including distribution maps, which, however, would
hardly be of service to any but metallurgists. union, the book appears at an opportune time. ' The Ore Deposits of South Africa.' Part ii. By J. P. Johnson. (London:
Crosby Lockwood. 1909. Pp. 51. Diagrams. 5s.) This work, of which only
the second part is before us, deals in the first with the distribution and character
of " base " metals in South Africa. The second deals with the Witwatersrand and
Pilgrimsrest goldfields and others. It is a strictly technical work, copiously
illustrated with diagrams, including distribution maps, which, however, would
hardly be of service to any but metallurgists. y
y
g
'Great Britain and the Congo.' By E. D. Morel. (London: Smith, Elder. 1909. Pp. xxvi., 291. Maps. 6s.) Mr. Morel's attacks on the Belgian "Congo
system " command wide attention, and in this strongly worded exposition he is
supported by Sir A. Conan Doyle in an introduction. y
y
g
'Great Britain and the Congo.' By E. D. Morel. (London: Smith, Elder. 1909. Pp. xxvi., 291. Maps. 6s.) Mr. Morel's attacks on the Belgian "Congo
system " command wide attention, and in this strongly worded exposition he is
supported by Sir A. Conan Doyle in an introduction. y
y
g
'Great Britain and the Congo.' By E. D. Morel. (London: Smith, Elder. 1909. Pp. xxvi., 291. Maps. 6s.) Mr. Morel's attacks on the Belgian "Congo
system " command wide attention, and in this strongly worded exposition he is
supported by Sir A. Conan Doyle in an introduction. MEXICO.
MEXICO.
MEXICO.
MEXICO.
MEXICO.
MEXICO. When a number of good photographs and a large contoured map are taken into
consideration, it is seen that we have here an important general treatise on the
country, by a writer who has travelled widely in it, has given himself careful
training as an observer, and combines with this the interests of a historian and
of a business man, so that he is enabled properly to appraise the wealth and
Mr. Enock's work is already well known through his books on ' The Andes and
the Amazon' and ' Peru.' The present volume, like that on Peru, forms one of
the "South American Series," under the editorship of Mr. Martin Hume. Mr. E ume contributes an introduction, which consists of a brief survey of what is
much more fully elaborated in the text-the history of Mexico down to the
present time. To this subject Mr. Enock devotes six chapters, and, besides showing
himself to be amply grounded in its study,'has evidently a keen eye for its
romance. He has no less appreciation for the remarkable scenery and physical
peculiarities of the country, which are dealt with in one chapter. Further chapters
are devoted to the people, their cities and institutions, to life and travel, mineral
wealth, and other resources of the country, its finance, industries, and railways. When a number of good photographs and a large contoured map are taken into
consideration, it is seen that we have here an important general treatise on the
country, by a writer who has travelled widely in it, has given himself careful
training as an observer, and combines with this the interests of a historian and
of a business man, so that he is enabled properly to appraise the wealth and
|
W4236787376.txt
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https://www.qeios.com/read/2800Z4/pdf
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de
|
RASSF1 wt Allele
|
Definitions
| 2,020
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cc-by
| 96
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Qeios · Definition, February 7, 2020
Ope n Pe e r Re v ie w on Qe ios
RASSF1 wt Allele
National Cancer Institute
Source
National Cancer Institute. RASSF1 wt Allele. NCI T hesaurus. Code C60666.
Human RASSF1 wild-type allele is located in the vicinity of 3p21.3 and is approximately 11
kb in length. T his allele, which encodes Ras association domain-containing protein 1, is
involved in death receptor-dependent apoptosis. Loss or altered expression of the gene
is associated with the pathogenesis of a variety of cancers, including lung cancer.
Qeios ID: 2800Z4 · https://doi.org/10.32388/2800Z4
1/1
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https://openalex.org/W2280323653
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https://europepmc.org/articles/pmc4766703?pdf=render
|
English
| null |
The anti-oxidative transcription factor Nuclear factor E2 related factor-2 (Nrf2) counteracts TGF-β1 mediated growth inhibition of pancreatic ductal epithelial cells -Nrf2 as determinant of pro-tumorigenic functions of TGF-β1
|
BMC cancer
| 2,016
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cc-by
| 10,425
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The anti-oxidative transcription factor
Nuclear factor E2 related factor-2 (Nrf2)
counteracts TGF-β1 mediated growth
inhibition of pancreatic ductal epithelial
cells -Nrf2 as determinant of
pro-tumorigenic functions of TGF-β1 Geeske Genrich1†, Marcus Kruppa1†, Lennart Lenk1, Ole Helm1, Anna Broich1, Sandra Freitag-Wolf2,
Christoph Röcken3, Bence Sipos4, Heiner Schäfer5 and Susanne Sebens1* * Correspondence: susanne.sebens@email.uni-kiel.de
†Equal contributors
1Group Inflammatory Carcinogenesis, Institute for Experimental Cancer
Research, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 17,
24105 Kiel, Germany
Full list of author information is available at the end of the article Genrich et al. BMC Cancer (2016) 16:155
DOI 10.1186/s12885-016-2191-7 Genrich et al. BMC Cancer (2016) 16:155
DOI 10.1186/s12885-016-2191-7 Open Access Background Smad4/DPC4 gene [12]. Thus, the function of TGF-β1
is a double-edged sword and the switch from tumor sup-
pressor to a tumor promoter seems to be context
dependent, albeit the exact underlying mechanisms are
still poorly understood [12, 13]. Pancreatic ductal adenocarcinoma (PDAC) is still a lead-
ing cause of cancer related deaths in western countries
with a poor 5-year survival rate of 6 % [1]. This can be
mainly explained by the late diagnosis when the disease
has been already progressed to an advanced stage and a
profound therapy resistance. Accordingly, much effort is
given to a better understanding of the early steps of
PDAC development in order to identify targets that
can be used for screening tests, early diagnosis and/or
chemoprevention. p
y
[
,
]
A similar dual role in tumorigenesis has been de-
scribed for the antioxidative transcription factor Nuclear
factor E2 related factor-2 (Nrf2) [14–16]. In response to
metabolic, xenobiotic or oxidative stress (e.g. in the
course of inflammation), Nrf2 becomes activated leading
to transcription of a variety of genes contributing to res-
toration of redox and cell homeostasis, e.g. antioxidant
enzymes NAD(P)H dehydrogenase [quinone] 1 (NQO1),
Hemoxygenase (HO)-1, anti-apoptotic proteins such as
Bcl-2 or metabolic enzymes [16]. However, constitutive
high expression and activity of Nrf2 have been described
for several tumors including PDAC [17] contributing to
chemo-/radioresistance [18–21], enhanced cell motility
[22], metabolic reprogramming [23], maintenance of
self-renewal of cancer stem cells [24] as well as en-
hanced proliferation [17, 25]. In an endogenous PDAC
mouse model it was shown that oncogenic kras signaling
leads to tumor cell proliferation and tumorigenesis via
elevation of Nrf2 activity [25]. The fact that TGF-β1 and
Nrf2 both become upregulated upon persistent inflam-
mation suggests that these two factors may virtually im-
pact on their signaling pathways paving the way for their
switch from tumor suppressor to tumor promoter. Ac-
cordingly, it has been shown that Nrf2 can inhibit the
profibrotic action of TGF-β1 by preventing Smad3 acti-
vation [26, 27]. Thus, the present study intends to inves-
tigate whether Nrf2 contributes to the pro-tumorigenic
switch of TGF-β1 in PDAC by antagonizing the TGF-β1
mediated growth inhibiting effect on pancreatic ductal
epithelial cells thereby undergoing a functional switch it-
self. (Continued from previous page) (Continued from previous page) Results: Significantly elevated expression of activated Nrf2 and Ki67 could be detected in PanINs but not in
normal pancreatic ductal epithelium. While the effect of Nrf2 on basal cell growth of H6c7-pBp, H6c7-kras and
Colo357 cells was minor, it clearly attenuated the growth inhibiting effects of TGF-β1 in all cell lines. This enhanced
Nrf2-mediated cell survival was predominantly based on an enhanced proliferative activity. Accordingly, expression
of p21 expression along with expression of phospho-p38 and phospho-Smad3 was diminished whereas
Erk-phosphorylation was enhanced under these conditions. Conclusions: Overall, our data demonstrate that Nrf2 being elevated in early precursor lesions counteracts the
growth inhibiting function of TGF-β1 already in benign and premalignant pancreatic ductal epithelial cells. This
could represent one fundamental mechanism underlying the functional switch of both- TGF-β1 and Nrf2 – which
may manifest already in early stages of PDAC development. Keywords: Chronic pancreatitis, Pancreatic cancer precursor lesion, Oxidative stress, TGF-β1, Proliferation,
Pancreatic cancer Abstract Background: Nuclear factor E2 related factor-2 (Nrf2) is an oxidative stress inducible transcription factor being
essential in regulating cell homeostasis. Thus, acute induction of Nrf2 in epithelial cells exposed to inflammation
confers protection from oxidative cell damage and mutagenesis supporting an anti-tumorigenic role for Nrf2. However, pancreatic ductal adenocarcinoma (PDAC) is characterized by persistent Nrf2 activity conferring therapy
resistance which points to a pro-tumorigenic role of Nrf2. A similar dichotomous role in tumorigenesis is described
for the Transforming Growth Factor-beta 1 (TGF-β1). The present study therefore aimed at elucidating whether the
switch of Nrf2 function towards a tumor promoting one relates to the modulation of TGF-β1 induced cell responses
and whether this might occur early in PDAC development. Methods: In situ analysis comprised immunohistochemical stainings of activated (phosphorylated) Nrf2 and Ki67 in
pancreatic tissues containing normal ducts and pancreatic intraepithelial neoplasia (PanINs). In vitro, Nrf2 levels in
benign (H6c7-pBp), premalignant (H6c7-kras) and malignant (Colo357) pancreatic ductal epithelial cells were
modulated by Nrf2 specific siRNA or Nrf2 overexpression. Then, the effect of Nrf2 alone and in combination with
TGF-β1 on cell growth and survival was investigated by cell counting, Ki67 staining and apoptosis assays. The
underlying cell signaling was investigated by western blotting. Statistical analysis was performed by Shapiro-Wilk
test for normal distribution. Parametric data were analyzed by one-way ANOVA, while non-parametric data were
analyzed by Kruskal-Wallis one-way ANOVA on ranks. (Continued on next page) * Correspondence: susanne.sebens@email.uni-kiel.de
†Equal contributors
1Group Inflammatory Carcinogenesis, Institute for Experimental Cancer
Research, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 17,
24105 Kiel, Germany
Full list of author information is available at the end of the article © 2016 Genrich 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. Page 2 of 14 Genrich et al. BMC Cancer (2016) 16:155 Methods Preparation of whole cell lysates and nuclear extracts as
well as electrophoresis and western blotting have been
described elsewhere [31, 32]. The following antibodies
were used according to the manufacturer’s instructions:
rabbit anti-HSP90α/β (clone H-114), mouse anti-lamin-
A/C (clone 346), goat anti-Smad2/3 (clone E-20) (all
from Santa Cruz, Heidelberg, Germany), rabbit anti-
Erk1/Erk2, rabbit anti-phospho Erk1/Erk2 (T204/Y209),
rabbit anti-p38, mouse anti-phospho p38 (T180/Y182,
clone D3F9), rabbit anti-phospho Smad3 (Ser423/425),
rabbit anti-PARP (all from Cell Signaling via New Eng-
land Biolabs, Frankfurt/a.M., Germany), mouse anti-p21
(clone 187) (BD Biosciences, Heidelberg, Germany),
monoclonal rabbit anti-Nrf2 (clone EP1808Y, Abcam,
Berlin, Germany), mouse anti-tubulin (clone B-5-1-2)
and rabbit anti-HA (both from Sigma-Aldrich, Tauf-
kirchen, Germany). Primary antibodies were incubated
overnight at 4 ° C and detected by anti-rabbit, anti-goat
or anti-mouse HRP-linked antibodies (Cell Signaling) at
room temperature for 1 h. After washing in TBST, blots
were developed with SuperSignal West Dura Extended
Duration Substrate (Perbio Sciences, Bonn, Germany). Average band intensities were determined by densitom-
etry using ImageI 1.47v software (National Institute of
Health). Values of the proteins of interest were divided
by the values of the corresponding loading control
(Hsp90). Additionally, values of phosphorylated proteins
were divided by the values of the corresponding total
protein (data are presented in Additional file 1: Figure
S2A + C). Determination of vital cell number After detachment with trypsin-EDTA (PAA, Pasching,
Austria), cells were stained with trypan blue (Sigma-Al-
drich, Munich, Germany) and counted using a Neubauer
counting chamber. For quantification of vital cells, blue
stained cells were excluded from counting. Cell lines and cell culture As model for benign pancreatic ductal epithelium, the
human pancreatic ductal epithelial cell line H6c7-pBp
and as model for premalignant pancreatic ductal epithe-
lium harboring a krasG12V mutation, the cell line H6c7-
kras [28] were used, both well-established cell models
and kindly provided by M.S. Tsao (Ontario Cancer Insti-
tute, Toronto, Canada). Both cell lines were cultured in
H6c7-medium (50 % RPMI 1640 medium (Biochrom,
Berlin, Germany) and 50 % KSF-medium (Gibco Life
Technologies, Darmstadt, Germany) supplemented with
5 % fetal calf serum, 0.5 % L-glutamine (both Biochrom),
50 μg/mL bovine pituitary extract, 5 ng/mL epidermal
growth factor (both Gibco Life Technologies) + 0.5 μg/
mL puromycin (Invitrogen, Darmstadt, Germany). The
human pancreatic ductal epithelial cell line Colo357 was
kindly provided by H. Kalthoff, Institute of Experimental
Cancer Research, Kiel, Germany) and kept in Colo357-
medium which is composed of RPMI-1640 medium
supplemented with 1 % L-glutamine, 10 % FCS and 1 %
sodium pyruvate. Colo357 cells used in this study harbor
a wild type Smad4/DPC4 genotype [29] and are genetic-
ally distinct from those Colo357 cells having a homozy-
gous deletion of the Smad4 gene [30]. Background In order to verify whether this switch might occur
at early stages of PDAC development, particular em-
phasis was given to the in situ analyses of activated Nrf2 Precursor lesions of PDAC predominantly originate
from ductal cells with pancreatic intraepithelial neoplasia
(PanIN) being the most frequent and best characterized
precursor lesions of PDAC [2]. One of the earliest gen-
etic alterations which is present in 99 % of even early
PanINs (PanIN1) is the mutation of the oncogene kras
[3] being an essential driver of PDAC development [4]. Besides the genetic alterations, one hallmark of PDAC is
its pronounced stromal microenvironment comprising
stellate cells, myofibroblasts and diverse immune cells
together with extracellular matrix [5–8] which start to
accumulate in earliest PanINs [9]. Besides cancer cells themselves, myofibroblasts and
immune cells such as macrophages are a main source of
Transforming Growth Factor-beta1 (TGF-β1) [5, 10]. Al-
though TGF-β1 is able to potently inhibit the growth of
cells including transformed cells and thereby acts as
tumor suppressor, it also represents an important key
driver in tumor development, e.g. of PDAC, by promot-
ing invasion, metastasis and chemoresistance of tumor
cells as well as immunosuppression and angiogenesis
[10, 11]. TGF-β1 can exert its pleiotropic functions via
the Smad-dependent (canonical) signaling pathway or
via signaling through various Smad-independent path-
ways e.g. the Mitogen-activated protein kinases (MAPK)
p38 and Erk1/2, the latter ones contributing to TGF-β1
responsiveness even in the presence of mutations in the Genrich et al. BMC Cancer (2016) 16:155 Page 3 of 14 replaced by 1,6 mL fresh medium. Then, 100 μL/well
EC-puffer, 8 μL/μg plasmid Enhancer and 20 μL/μg plas-
mid Effectene (all from Qiagen) together with 0,3 μg/
well of either pcDNA3.1 control vector (pcDNA3.1; Invi-
trogen) or pcDNA3.1 encoding Nrf2-HA were mixed
and added to the cells. After 16 h, medium was replaced
by 2 mL/well of fresh H6c7- or Colo357 medium and
cells were either left untreated or treated with 10 ng/mL
TGF-β1 for 48 h. and Ki67 in early PanINs. In vitro, the interplay of Nrf2
and TGF-β1 on cell growth was investigated on three
pancreatic ductal epithelial cell lines resembling different
stages of PDAC development, namely benign H6c7-pBp,
premalignant H6c7-kras and malignant Colo357 cells. Overall, our study provides ample evidence that the
functional switch of Nrf2 occurs early in PDAC develop-
ment based on its ability to counteract the growth inhi-
biting function of TGF-β1. Knock-down of Nrf2 To suppress endogenous Nrf2 expression, 1 × 105 cells/
well of H6c7-pBp, H6c7-kras and Colo357 cells were
seeded in 12-well plates containing 1 mL medium/well. For transfection, medium was removed and 1 mL fresh
medium was added. Then, 6 μL/well HiperFect reagent
(Qiagen, Hilden, Germany) and 75 ng/well of either
negative control siRNA or specific Nrf2 siRNA (no. SI03246614, both from Qiagen) were mixed with 100 μL
FCS-free medium and added to the cells. Specificity of
the siRNA was confirmed previously [18]. After 24 h,
cells were either left untreated or treated with 10 ng/mL
TGF-β1 (BioLegend, Fell, Germany) for 48 h. Evaluation Stained tissue sections were evaluated twice in a blinded
manner by scoring the extent of distribution (given as
%-positivity of the whole section). In case of two dis-
crepant results, sections were evaluated by a second in-
vestigator. If possible, 10 normal ducts and PanINs were
analysed in each pancreatic tissue. Each parameter was
evaluated using a 5-score system (1 = negative, 2 = <10 %
positive, 3 = 11–50 % positive, 4 = 51–90 % positive, 5 =
more than 90 % positive). To enable performance of
statistical analysis, the created groups of tissue samples
per score were then dichotomized by the median score
and refitted into two groups of < median (low expres-
sion) and ≥median (high expression). Evaluation of the
sections was carried out using an Axioplan 2.0 micro-
scope (Zeiss, Jena, Germany). Pictures were taken using
a Keyence BZ9000 microscope (Keyence, Neu-Isenburg,
Germany). Immunohistochemistry Paraffin-embedded and formalin-fixed postmortem pan-
creatic tissues of 22 individuals that had died of non-
pancreatic diseases were used for immunohistochemical
analysis. The research was approved by the ethics com-
mittee of the Semmelweis University, Budapest, Hungary
(140-1/1996). The need for an informed consent was
waived by the ethics committee of the Semmelweis
University, Budapest, Hungary according to national reg-
ulations. Only tissues that have been extensively charac-
terized were used [34, 35]. Consecutive 3 μm thick
tissue sections were deparaffinized and rehydrated as
previously described [8]. Staining for Ki67 (clone SP6,
Fisher/Thermo Scientific) was performed at the Institute
of Pathology using an automated routine procedure. For
staining of p-Nrf2, antigen retrieval was performed with
1:10 diluted citrate buffer at pH 6.0 for 20 min. After
washing, sections were incubated either with a monoclo-
nal rabbit anti-phospho(Ser40)-Nrf2 (1 μg/ml; clone
EP18094, Abcam) diluted in 1 % BSA and 0.3 % Triton- Statistical analysis Relationships between data from immunohistochemical
stainings of pancreatic tissues were categorized and
compared between groups by Fisher’s exact test using
SPSS 17.0 (IBM, Ehningen, Germany). Statistical analysis
of in vitro data was performed using SigmaPlot Software
12.5 (Systat Software GmbH, Erkrath, Germany). The
Shapiro-Wilk test was used to test for normal distribu-
tion. Parametric data were analyzed by one-way RM
ANOVA, while non-parametric data were analyzed by
Kruskal-Wallis one-way ANOVA on ranks test. P-values
< 0.05 were regarded as statistically significant and are
indicated with an asterisk (*). Overexpression of Nrf2 For Ki67 staining, cells were seeded on cover slips
(ThermoScientific, Schwerte, Germany) before transfec-
tion and stimulation procedures. Then, medium was
removed, cells were washed with PBS and fixed with ice- To overexpress Nrf2 in H6c7-pBp and H6c7-kras cells,
either cell line was seeded into 6 well plates (2 x 105
cells/well). After 24 h, medium was removed and Genrich et al. BMC Cancer (2016) 16:155 Page 4 of 14 Page 4 of 14 cold acetone + 0.3 % H2O2 for 10 min. After washing in
PBS, cells were blocked in 4 % BSA/PBS for 20 min. Cells were incubated with 2 μg/mL mouse IgG1 anti-
Ki67 antibody (BD Biosciences, Heidelberg, Germany)
diluted in 1 % BSA/PBS at RT for 45 min. Cells were
washed with PBS and incubated with EnVision-HRP
anti-mouse (Dako, Hamburg, Germany) for 30 min,
washed with PBS and incubated with AEC Substrate
(Dako) for 2-10 min. After final washing in PBS, cells
were stained in Mayer’s Haemalaun (AppliChem, Darm-
stadt, Germany) for 2 min. After washing in water for
10 min, cover slips were fixed with Kaiser’s glycerine gel-
atine
(Waldeck,
Münster,
Germany). Initial
isotype
control stainings were performed with a mouse IgG1
antibody (R&D Systems, Wiesbaden, Germany) under
identical conditions revealing no staining. Evaluation
was done using an EvosxL Core microscope (AMG,
Bothell, USA). Quantification of Ki67 positive cells was
performed at a 200-fold magnification by choosing 5
representative visual fields in each counting positively
stained as well as negative cells along a diagonal line
using Microsoft Powerpoint 2007 in order to calculate
the percentage of Ki67 positive cells. If less than ten cells
touched the line, all captured cells of the visual field
were counted. X-100/PBS at 4 °C overnight. After washing, sections
were incubated with EnVision-HRP anti-rabbit (Dako)
for 45 min at room temperature. Substrate reaction was
performed with AEC Substrate (Dako) for 10 min. After
washing, cells were stained in Mayer’s Haemalaun
(Merck, Darmstadt, Germany) for 2 min. After washing
in water for 10 min, sections were covered with Kaiser’s
glycerine gelatine (Roth, Karlsruhe, Germany). Usage of
control antibodies revealed no or only weak background
staining. Measurement of apoptosis Determination of caspase-3/7 activity was performed in
transfected and stimulated H6c7-kras and Colo357 cells
using a Caspase-Glo® 3/7 assay (Promega, Mannheim,
Germany), according to the manufacturer’s instructions
and as described [32, 33]. All samples were measured in
duplicates. Expression of activated Nrf2 is elevated in early PanINs
correlating with an increased proliferative activity To elucidate whether early PanIN lesions already exhibit
greater Nrf2 activity and whether this correlates with an
increased proliferative activity, phospho-Nrf2 (p-Nrf2),
representing activated Nrf2, as well as Ki67 were immu-
nohistochemically detected and scored in normal ducts
and PanINs of pancreatic tissues from 22 individuals
which had died from non-pancreatic diseases (Tables 1 Genrich et al. BMC Cancer (2016) 16:155 Page 5 of 14 creatic ducts and PanINs
anINs
core 1
Score 2
Score 3
Score 4
Score 5
Median score
13
4
0
0
2
15
4
0
0
2
Fig. 1 Expression of activated Nrf2 is elevated in PanINs correlating with
an increased proliferative activity. Representative immunhistochemical
stainings of activated (phosphorylated) Nrf2 (p-Nrf2) and Ki67 in (a)
normal pancreatic ducts and (b) PanINs of two individuals. Magnification
x400 and x800 (frames) Table 1 Initial scoring of p-Nrf2 and Ki67 expression in normal pancreatic ducts and PanINs Table 1 Initial scoring of p-Nrf2 and Ki67 expression in normal pancreatic ducts and PanINs
Normal ducts
PanINs
Marker
Score 1
Score 2
Score 3
Score 4
Score 5
Score 1
Score 2
Score 3
Score 4
p-Nrf2
16
5
0
0
0
3
13
4
0
Ki67
12
10
0
0
0
0
15
4
0 and 2). While the majority of normal ducts showed no
or only weak p-Nrf2 expression (16/21 with low expres-
sion, Fig. 1a), significantly elevated p-Nrf2 expression (=
high expression) was detected in PanINs of 17/20 tissues
(Fig. 1b). Ki67 expression was elevated in 45 % (10/22
with high expression) of normal ducts, but in all ana-
lyzed PanINs (Fig. 1, Tables 1 and 2). Moreover, this ele-
vated proliferative activity of PanINs was significantly
associated with increased p-Nrf2 expression (Table 3)
supporting the view that Nrf2 activity accounts for
higher proliferation of pancreatic ductal epithelial cells
already in early PanINs. Nrf2 antagonizes the growth inhibiting effect of TGF-β1
on benign, premalignant and malignant pancreatic ductal
epithelial cells Animal based studies revealed that oncogene activation
can lead to enhanced Nrf2 mediated gene transcription
accounting for increased proliferation and tumorigenesis
[25]. To investigate the impact of Nrf2 on cell growth of
human pancreatic ductal epithelial cells, pancreatic ductal
epithelial cell lines were used resembling different stages
of PDAC development: 1) benign H6c7-pBp cells lacking
any of the known mutations driving pancreatic tumori-
genesis [28], 2) premalignant H6c7-kras cells harboring a
krasG12V mutation [28] and 3) malignant Colo357 cells
[36]. Since TGF-β1 is elevated at early stages of PDAC de-
velopment, too, the investigations focused on the interplay
between Nrf2 and TGF-β1. As demonstrated by immun-
cytochemical staining (Fig. 2a,b) and western blotting
(Fig. 2c), basal nuclear Nrf2 expression could be detected
in all three pancreatic duct cell lines with the lowest ex-
pression in benign H6c7-pBp cells and the highest nuclear
Nrf2 expression in malignant Colo357 cells (Fig. 2a-c). In
line with our recent findings [37], treatment with 10 ng/ml Fig. 1 Expression of activated Nrf2 is elevated in PanINs correlating with
an increased proliferative activity. Representative immunhistochemical
stainings of activated (phosphorylated) Nrf2 (p-Nrf2) and Ki67 in (a)
normal pancreatic ducts and (b) PanINs of two individuals. Magnification
x400 and x800 (frames) Table 2 Interrelationship of p-Nrf2 and Ki67 expression in normal
pancreatic ducts and PanINs, initial scores were dichotomized by
the median score and refitted into 2 groups of low and high
expression pancreatic ducts and PanINs, initial scores were dichotomized by
the median score and refitted into 2 groups of low and high
expression
Normal ducts (n = 22)
PanINs (n = 20)
p-value
Marker
Low
High
Low
High
p-Nrf2
16/21
5/21
3/20
17/20
0.002
Ki67
12/22
10/22
0/19
19/19
0.001
In two cases, no PanINs were detectable Normal ducts (n = 22)
PanINs (n = 20)
p-value
Marker
Low
High
Low
High
p-Nrf2
16/21
5/21
3/20
17/20
0.002
Ki67
12/22
10/22
0/19
19/19
0.001
In two cases, no PanINs were detectable Fig. 1 Expression of activated Nrf2 is elevated in PanINs correlating with
an increased proliferative activity. Representative immunhistochemical
stainings of activated (phosphorylated) Nrf2 (p-Nrf2) and Ki67 in (a)
normal pancreatic ducts and (b) PanINs of two individuals. Magnification
x400 and x800 (frames) Fig. 1 Expression of activated Nrf2 is elevated in PanINs correlating with
an increased proliferative activity. Nrf2 antagonizes the growth inhibiting effect of TGF-β1
on benign, premalignant and malignant pancreatic ductal
epithelial cells In order to verify the interference of Nrf2 with the growth
inhibitory effect of TGF-β1 in all three pancreatic ductal
epithelial cell lines, Nrf2 siRNA knockdown experiments
were conducted. As demonstrated in Fig. 3b, knockdown of
Nrf2 clearly enhanced the growth inhibiting effect of TGF-
β1 in H6c7-pBp, H6c7-kras and Colo357 cells. Transfection
efficiency was confirmed by detecting endogenous total-
Nrf2 by western blotting (Fig. 3b, bottom panel) and real-
time PCR based determination of reduced expression of the
Nrf2 target gene NQO1 (data not shown). Overall, these
data indicate that Nrf2 potently antagonizes the growth
inhibiting function of TGF-β1 in human pancreatic ductal
epithelial cells. Next it was analyzed how Nrf2 impacts on cell growth of
pancreatic ductal epithelial cells. For this purpose, H6c7-
pBp and H6c7-kras cells exhibiting low and moderate basal
Nrf2 expression, respectively (see Fig. 2), were transfected
with Nrf2-HA and being either left untreated or treated
with 10 ng/ml TGF-β1. After 48 h, vital cell numbers were
determined. Compared to control transfected cells, in which
TGF-β1 treatment significantly reduced the number of vital
H6c7-pBp and H6c7-kras cells by 30 and 46.6 %, respect-
ively, overexpression of Nrf2 clearly diminished the growth
inhibiting effect of TGF-β1 treatment on both cell lines (10
and 31 %, respectively, compared to untreated cells) (Fig. 3a). The transfection efficiency was confirmed by detecting the
HA-tag by western blotting using an HA-specific antibody
for specific detection of recombinant Nrf2 (Fig. 3a bottom
panel) and realtime PCR based determination of elevated
expression of the Nrf2 target gene NQO1 (data not shown). Nrf2 only slightly impact on basal and TGF-β1 induced
apoptosis in pancreatic ductal epithelial cells Overall, these
data indicate that Nrf2 potently antagonizes the growth
inhibiting function of TGF-β1 in human pancreatic ductal
epithelial cells. Nrf2 increases proliferation of benign, premalignant and
malignant pancreatic ductal epithelial cells
To elucidate how Nrf2 antagonizes the growth inhibiting
effect of TGF-β1, proliferation of H6c7-pBp, H6c7-kras
and Colo357 cells was analyzed in dependence on Nrf2
and TGF-β1. As demonstrated in Fig. 4a-c, TGF-β1 Nrf2 antagonizes the growth inhibiting effect of TGF-β1
on benign, premalignant and malignant pancreatic ductal
epithelial cells Representative immunhistochemical
stainings of activated (phosphorylated) Nrf2 (p-Nrf2) and Ki67 in (a)
normal pancreatic ducts and (b) PanINs of two individuals. Magnification
x400 and x800 (frames) Page 6 of 14 Genrich et al. BMC Cancer (2016) 16:155 Table 3 Association between p-Nrf2 and Ki67 expression in
normal pancreatic ducts and PanINs, expressed as p-values from
chi-square test Table 3 Association between p-Nrf2 and Ki67 expression in
normal pancreatic ducts and PanINs, expressed as p-values from
chi-square test Table 3 Association between p-Nrf2 and Ki67 expression in
normal pancreatic ducts and PanINs, expressed as p-values from
chi-square test treatment drastically reduced the overall cell number
as well as the number of Ki67 positive H6c7-pBp,
H6c7-kras and Colo357 cells that had been trans-
fected with the empty vector. In contrast, overexpres-
sion of Nrf2 clearly attenuated this effect of TGF-β1
and increased the cell density along with the number
of
Ki67
positive
H6c7-pBp
and
H6c7-kras
cells
(Fig. 4a). In line with these findings, knockdown of
Nrf2 in H6c7-pBp, H6c7-kras and Colo357 cells in-
creased the antiproliferative effect of TGF-β1 as indi-
cated by a further reduced cell number and less Ki67
expressing cells (Fig. 4b, c). Similar results were ob-
tained by MTT assay (data not shown) supporting the
view that Nrf2 antagonizes the growth inhibiting ef-
fect of TGF-β1 by counteracting its antiproliferative
activity in pancreatic ductal epithelial cells. TGF-β1 for 48 h led to an induction of Nrf2 expression in
all three cell lines (Fig. 2a-c). Next it was analyzed how Nrf2 impacts on cell growth of
pancreatic ductal epithelial cells. For this purpose, H6c7-
pBp and H6c7-kras cells exhibiting low and moderate basal
Nrf2 expression, respectively (see Fig. 2), were transfected
with Nrf2-HA and being either left untreated or treated
with 10 ng/ml TGF-β1. After 48 h, vital cell numbers were
determined. Compared to control transfected cells, in which
TGF-β1 treatment significantly reduced the number of vital
H6c7-pBp and H6c7-kras cells by 30 and 46.6 %, respect-
ively, overexpression of Nrf2 clearly diminished the growth
inhibiting effect of TGF-β1 treatment on both cell lines (10
and 31 %, respectively, compared to untreated cells) (Fig. 3a). The transfection efficiency was confirmed by detecting the
HA-tag by western blotting using an HA-specific antibody
for specific detection of recombinant Nrf2 (Fig. 3a bottom
panel) and realtime PCR based determination of elevated
expression of the Nrf2 target gene NQO1 (data not shown). Nrf2 only slightly impact on basal and TGF-β1 induced
apoptosis in pancreatic ductal epithelial cells p p
p
p
Since the growth inhibiting effect of TGF-β1 may also
involve an increase of apoptosis, we next investigated
the impact of Nrf2 on basal and TGF-β1 mediated apop-
tosis in the three pancreatic ductal epithelial cell lines. As shown in Fig. 5a, the basal apoptotic rate as deter-
mined by caspase-3/7 activity correlated well with the
basal Nrf2 expression in the three pancreatic ductal epi-
thelial cell lines. Hence, H6c7-pBp cells with the lowest
basal Nrf2 activity (see also Fig. 2) exhibited the highest
basal caspase-3/7 activity while Colo357 cells - showing
the highest Nrf2 expression - were characterized by the
lowest caspase-3/7 activity. Accordingly, when overex-
pressing Nrf2 in H6c7-pBp cells the basal (Fig. 5b) as
well as the TGF-β1 mediated (Fig. 5c) apoptotic rate was
clearly reduced, whereas Nrf2 overexpression in H6c7-
kras cells only slightly affected basal and TGF-β1
dependent apoptosis. In line with the overexpression ex-
periments, suppression of Nrf2 by siRNA transfection
mainly increased the basal caspase-3/7 activity of H6c7-
pBp cells but only slightly in H6c7-kras and Colo357
cells (Fig. 5d). Moreover, the TGF-β1 mediated apoptosis
was marginally affected by Nrf2 suppression in all three
cell lines (Fig. 5e). In order to validate these moderate
Nrf2 mediated effects on apoptosis induction observed
in H6c7-pBp and H6c7-kras cells by caspase-3/7 activity
assay, PARP cleavage was analysed in the same cells by
western blotting. Even though slight differences in the
full length form of PARP were observed, cleaved PARP
could be detected in neither sample (Additional file 2:
Figure S1). These data indicate that the interference of
Nrf2 with the growth suppression by TGF-β1 mainly re-
lies on the reversal of its antiproliferative activity (see
above) and to lesser extent on the protection from TGF-
β1 dependent apoptosis p
g
g
Q
(
)
In order to verify the interference of Nrf2 with the growth
inhibitory effect of TGF-β1 in all three pancreatic ductal
epithelial cell lines, Nrf2 siRNA knockdown experiments
were conducted. As demonstrated in Fig. 3b, knockdown of
Nrf2 clearly enhanced the growth inhibiting effect of TGF-
β1 in H6c7-pBp, H6c7-kras and Colo357 cells. Transfection
efficiency was confirmed by detecting endogenous total-
Nrf2 by western blotting (Fig. 3b, bottom panel) and real-
time PCR based determination of reduced expression of the
Nrf2 target gene NQO1 (data not shown). Nrf2 increases proliferation of benign, premalignant and
malignant pancreatic ductal epithelial cells To elucidate how Nrf2 antagonizes the growth inhibiting
effect of TGF-β1, proliferation of H6c7-pBp, H6c7-kras
and Colo357 cells was analyzed in dependence on Nrf2
and TGF-β1. As demonstrated in Fig. 4a-c, TGF-β1 Page 7 of 14 Genrich et al. BMC Cancer (2016) 16:155 Fig. 2 Different Nrf2 expression in benign, premalignant and
malignant pancreatic ductal epithelial cells. H6c7-pBp, H6c7-kras and
Colo357 cells were either left untreated or were treated with 10 ng/
ml TGF-β1 for 48 h. Representative immunocytochemical stainings
of (a) total (t)-Nrf2 and (b) phospho (p)-Nrf2. Magnification x 280
(magnification of images in frames x 540). c Representative western
blots showing t-Nrf2 and p-Nrf2 expression in nuclear extracts of
H6c7-pBp, H6c7-kras and Colo357 cells either left untreated or
treated with 10 ng/ml TGF-β1 for 48 h. Lamin-A, tubulin and Hsp90
were detected as loading control for nuclear, cytosolic and total cell
extracts, respectively Nrf2 modulates MAPK and Smad signaling in benign,
premalignant and malignant pancreatic ductal epithelial
cells Next it was investigated how Nrf2 antagonizes TGF-β1
mediated growth inhibition. For this purpose the impact
of Nrf2 on relevant signaling pathways, such as MAPK
and Smad signaling as well as the expression of the
cyclin-dependent kinase inhibitor 1 (p21) was analysed. As shown in Fig. 6a, overexpression of Nrf2 which was
demonstrated by detecting the HA-tag of recombinant
Nrf2 (see Fig. 3a) reduced basal as well as TGF-β1 medi-
ated phosphorylation of p38. Likewise, Smad3 phosphor-
ylation was also reduced by Nrf2 overexpression, albeit
at lesser extent (Fig. 6a, Additional file 1: Figure S2A +
B). Conversely, Nrf2 overexpression increased phosphor-
ylation of the proliferation associated MAPK Erk in
H6c7-pBp and H6c7-kras cells (Fig. 6a, Additional file 1:
Figure S2A+B). Moreover, expression levels of p21 were
reduced by Nrf2 overexpression in H6c7-kras cells in the
absence and presence of TGF-β1 while in H6c7-pBp cells
a marked effect of Nrf2 overexpression was observed
solely in the presence of TGF-β1 (Fig. 6a, Additional file 1:
Figure S2A + B). To confirm the effects of Nrf2 on these growth control-
ling mediators, Nrf2 knock-down experiments were con-
ducted with all three pancreatic ductal epithelial cell lines. siRNA mediated suppression of Nrf2 in H6c7-pBp, H6c7-
kras and Colo357 cells, as demonstrated by detection of
reduced levels of endogenous total-Nrf2 (see Fig. 3b), en-
hanced basal as well as TGF-β1 induced phosphorylation
of p38 and Smad3 along with an elevated expression of
p21. Nrf2 increases proliferation of benign, premalignant and
malignant pancreatic ductal epithelial cells By contrast, TGF-β1 mediated phosphorylation of
Erk was clearly reduced in the absence of Nrf2 (Fig. 6b,
Additional file 1: Figure S2C + D). Overall, these data sup-
port the view that Nrf2 antagonizes TGF-β1 mediated
growth suppression by attenuating the growth inhibiting
(p38 and Smad3) signaling pathways and concomitantly
enhancing growth promoting Erk-signaling. Discussion The dual role of TGF-β1 in tumorigenesis of epithelial tu-
mors is well established. Under physiological conditions, Page 8 of 14 Genrich et al. BMC Cancer (2016) 16:155 TGF-β1 is an essential cell growth inhibitor and thereby
oncogene [37, 39]. This functional switch of TGF-β1 lead-
Fig. 3 Nrf2 antagonizes the growth inhibiting effect of TGF-β1 on pancreatic ductal epithelial cells. a H6c7-pBp and H6c7-kras cells that were
transfected with a control vector (pcDNA3.1) or Nrf2-HA and left untreated or were treated with 10 ng/ml TGF-β1 for 48 h. Determination of vital
cell numbers by trypan blue exclusion and cell counting. Verification of Nrf2 overexpression was performed by western blotting using an HA-specific
antibody and Hsp90 as loading control. b H6c7-pBp, H6c7-kras and Colo357 cells were transfected either with control siRNA or Nrf2 siRNA and left
untreated or were treated with 10 ng/ml TGF-β1 for 48 h. Determination of vital cell numbers by trypan blue exclusion and cell counting. Verification
of Nrf2 knockdown was performed by western blotting using a t-Nrf2 antibody and Hsp90 as loading control. Data of vital cell numbers are presented
as Box-Plots of 4-6 independent experiments. * = p < 0.05 Nrf2 antagonizes the growth inhibiting effect of TGF-β1 on pancreatic ductal epithelial cells. a H6c7-pBp and H6c7-kras cells that were
ted with a control vector (pcDNA3.1) or Nrf2-HA and left untreated or were treated with 10 ng/ml TGF-β1 for 48 h. Determination of vital
mbers by trypan blue exclusion and cell counting. Verification of Nrf2 overexpression was performed by western blotting using an HA-specific
y and Hsp90 as loading control. b H6c7-pBp, H6c7-kras and Colo357 cells were transfected either with control siRNA or Nrf2 siRNA and left Fig. 3 Nrf2 antagonizes the growth inhibiting effect of TGF-β1 on pancreatic ductal epithelial cells. a H6c7-pBp and H6c7-kras cells that were
transfected with a control vector (pcDNA3.1) or Nrf2-HA and left untreated or were treated with 10 ng/ml TGF-β1 for 48 h. Determination of vital
cell numbers by trypan blue exclusion and cell counting. Verification of Nrf2 overexpression was performed by western blotting using an HA-specific
antibody and Hsp90 as loading control. b H6c7-pBp, H6c7-kras and Colo357 cells were transfected either with control siRNA or Nrf2 siRNA and left
untreated or were treated with 10 ng/ml TGF-β1 for 48 h. Determination of vital cell numbers by trypan blue exclusion and cell counting. Discussion Verification
of Nrf2 knockdown was performed by western blotting using a t-Nrf2 antibody and Hsp90 as loading control. Data of vital cell numbers are presented
as Box-Plots of 4-6 independent experiments. * = p < 0.05 oncogene [37, 39]. This functional switch of TGF-β1 lead-
ing to reprogramming of its activated signaling pathways
seems to be context dependent, albeit the underlying
mechanisms are still not fully understood. One mechan-
ism explaining the TGF-β1 paradox might be the differen-
tial activation of MAPK such as Erk in benign and
malignant epithelial cells [12]. Many tumors exhibit con-
stitutive high Erk activation due to a mutation in the ras
oncogene, leading to sustained TGF-β1 synthesis and sig-
naling. In line with this, Gotzmann et al. showed that TGF-β1 is an essential cell growth inhibitor and thereby
prevents epithelial hyperproliferation and the onset of epi-
thelial tumors. However, TGF-β1 becomes a potent tumor
promoter through its capacity to initiate EMT, to enhance
cell migration and invasion and to favour chemoresistance
[12, 13], applying also to PDAC [38–40]. Moreover, ex-
perimental evidence indicate that this functional switch
might occur already at early stages in tumorigenesis when
epithelial cells still do not harbour any of the driver muta-
tions or at least only single mutations, such as in the k-ras Genrich et al. BMC Cancer (2016) 16:155 Page 9 of 14 Fig. 4 Nrf2 increases proliferation of benign, premalignant and
malignant pancreatic ductal epithelial cells. Representative Ki67
stainings of (a) H6c7-pBp and H6c7-kras cells that were transfected
with a control vector (pcDNA3.1) or Nrf2-HA and left untreated or
were treated with 10 ng/ml TGF-β1 for 48 h, and (b) H6c7-pBp,
H6c7-kras and (c) Colo357 cells that were transfected either with
control siRNA or Nrf2 siRNA and left untreated or were treated with
10 ng/ml TGF-β1 for 48 h. Magnification x280 (magnification of
images in frames x540). Quantification of Ki67 positive cells was
performed at a 200-fold magnification by choosing 5 representative
visual fields, in each counting positively stained as well as negative
cells along a diagonal line using Microsoft Powerpoint 2007 in order
to calculate the percentage of Ki67 positive cells. If less than ten cells
touched the line, all captured cells of the visual field were counted. Data are expressed as mean ± SD of counted cells from 5 visual
fields. Discussion * = p < 0.05 cooperation of TGF-β1 signaling and oncogenic expres-
sion of Ha-ras promotes a mesenchymal and invasive
phenotype in hepatocytes [41]. Furthermore, sustained
MAPK activation can also result from prolonged exposure
to a plethora of inflammatory mediators (e.g. IL-6, TNF-α,
TGF-β1) upon chronic inflammation [42, 43]. Thus, our
study extends this view by demonstrating that sustained
high expression and activity of the antioxidative transcrip-
tion factor Nrf2 essentially contributes to deregulation of
TGF-β1 signaling by reducing Smad3 and p38 activation
and augmenting Erk activation resulting in reduced p21
expression and enhanced proliferation of pancreatic ductal
epithelial cells. One can speculate that an elevated Nrf2
activity leads to changes in the assembly of TGF-β I (Alk1
and Alk5) and TGF-β II receptors, thereby favouring an
altered recruitment of downstream mediators that can
initiate Smad independent/non-canonical pathways like
Ras-ERKs. Thus, further studies have to unravel in
more detail how Nrf2 suppresses TGF-β1 mediated
Smad3 and p38 activation and concomitantly enhance
Erk signaling. Interestingly, we could observe these ef-
fects likewise in benign, premalignant and malignant
pancreatic ductal epithelial cells suggesting that high
Nrf2 activity can promote this functional switch of
TGF-β1 already early in tumorigenesis. Favoring this
hypothesis, we could detect enhanced expression of ac-
tivated (phosphorylated) Nrf2 in nuclei of early PanINs
being significantly associated with a higher proliferative
activity exemplified by elevated Ki67 expression. More-
over, these data are in line with findings from an en-
dogenous PDAC mouse model showing that oncogenic
kras activation is one key mechanism leading to sus-
tained Nrf2 expression and activity driving pancreatic
tumorigenesis [25]. Hence, pancreata of Nrf2 deficient
mice exhibited fewer PanINs with lower proliferative
activity than the wildtype mice whereas no differences
in the extent of apoptotic cells could be observed [25]. Thus, activation of the ras-raf-Erk pathway [25, 44] Genrich et al. BMC Cancer (2016) 16:155 Page 10 of 14 Fig. 5 (See legend on next page.) Fig. 5 (See legend on next page.) e.) Page 11 of 14 Genrich et al. BMC Cancer (2016) 16:155 (See figure on previous page.)
Fig. 5 Nrf2 hardly impact on basal and TGF-β1 induced apoptosis in pancreatic ductal epithelial cells. a, d, e H6c7-pBp, H6c7-kras and Colo357
cells were transfected either with control siRNA or Nrf2 siRNA or (b, c) H6c7-pBp and H6c7-kras cells were transfected with a control vector
(pcDNA3.1) or Nrf2-HA. Discussion Hence, the growing inflammatory micro-
environment together with oncogenic kras activation in
ductal epithelial cells, which is present in almost all early
PanINs [3], may promote upregulation of both Nrf2 and
TGF-β1 activity already at this early stage of PDAC devel-
opment. Supporting this view, we previously demon-
strated that predominantly proinflammatory macrophages
releasing ROS at high levels lead to nuclear accumulation
of Nrf2 in colonic epithelial cells conferring apoptosis
resistance towards TRAIL or the chemotherapeutic drug
irinotecan [47]. This Nrf2 mediated apoptosis resistance
relied on Nrf2 enhanced proteasomal gene expression
[47], a mechanism that apparently also operates in PDAC
cells [18]. of Nrf2 expression. Nrf2 essentially impacts on cell sur-
vival of pancreatic ductal epithelial cells by antagonizing
the growth inhibitory effect of TGF-β1 by diminishing
TGF-β1 induced p21 expression and accelerating Erk
signaling resulting in increased proliferation. Besides a
pro-proliferative function, Nrf2 has been described to
confer profound protection from apoptosis by e.g. up-
regulation of anti-apoptotic proteins such as bcl-2 or
bcl-xL [19, 20] or proteasomal genes such as s5a/psmd4
or α5/psma5 [18]. Thus, elevated Nrf2 expression is one
pivotal mechanism underlying chemoresistance of many
tumors, e.g. PDAC [18–21]. Accordingly, in this study
the highest Nrf2 expression/activity could be correlated
with the lowest apoptosis rate in Colo357 cells and vice
versa. However, overexpression or knock down of Nrf2 in
pancreatic ductal epithelial cells only slightly modulated
TGF-β1 mediated apoptosis induction. Thus, Nrf2 appar-
ently enhances the growth of pancreatic ductal epithelial
cells predominantly by antagonizing the proliferation inhi-
biting effect of TGF-β1. Since this phenomenon could
be observed not only in malignant but also in premalig-
nant and benign epithelial cells, one can speculate that
the switch of Nrf2 from tumor suppressor to a tumor
promoter might occur quite early in tumorigenesis and
in particular in the concomitant presence of TGF-β1. represents an important mechanism leading to en-
hanced Nrf2 expression and activity in tumor cells,
applying also to pancreatic ductal epithelial cells. Ac-
cordingly, we could demonstrate higher basal Nrf2 ex-
pression and activity in H6c7-kras and Colo357 cells
compared to benign H6c7-pBp cells. Discussion Important to note,
constitutive Nrf2 activation is caused to a lesser extent
by genetic alterations affecting either the Nrf2 inhibitor
Keap1 or Nrf2 itself (accounting for 10–15 % in gall-
bladder and ovarian cancer or in lung cancer up to
30 % of enhanced Nrf2 activation) as well as epigenetic
mechanisms accounting for enhanced Nrf2 activity in
20–30 % of tumors [45, 46]. Accordingly, we previously
showed that PDAC cells with different Nrf2 activity
exhibit similar Keap1 expression levels [18] supporting the
view that sustained Nrf2 activation in PDAC cells is not
caused by epigenetic or genetic alterations of Keap1 and
rather than other factors. Besides the above mentioned
oncogene induced Nrf2 activation, the exposure of epithe-
lial cells to persistent metabolic and/or oxidative stress,
e.g. caused by the presence of ROS releasing macrophages
and neutrophils during the course of a chronic inflamma-
tion [47] is a major cause for constitutive elevated Nrf2 ac-
tivity. Macrophages are not only abundant in chronic
pancreatitis and PDAC [8] but start to accumulate around
early PanINs. Hence, the growing inflammatory micro-
environment together with oncogenic kras activation in
ductal epithelial cells, which is present in almost all early
PanINs [3], may promote upregulation of both Nrf2 and
TGF-β1 activity already at this early stage of PDAC devel-
opment. Supporting this view, we previously demon-
strated that predominantly proinflammatory macrophages
releasing ROS at high levels lead to nuclear accumulation
of Nrf2 in colonic epithelial cells conferring apoptosis
resistance towards TRAIL or the chemotherapeutic drug
irinotecan [47]. This Nrf2 mediated apoptosis resistance
relied on Nrf2 enhanced proteasomal gene expression
[47], a mechanism that apparently also operates in PDAC
cells [18]. In contrast to the findings of Lister et al in our stud- Discussion Then, cells were either left untreated or were treated with 10 ng/ml TGF-β1 for 48 h. Caspase-3/7 activity was determined
and normalized to the cell number. a Basal caspase-3/7 activity of control siRNA, untreated H6c7-pBp, H6c7-kras and Colo357 cells, expressed as
n-fold caspase-3/7 activity of H6c7-pBp cells which were set as 1. b Caspase-3/7 activity in H6c7-pBp and H6c7-kras after overexpression of
Nrf2-HA or (d) siRNA mediated Nrf2 suppression, expressed as n-fold caspase-3/7 activity of the corresponding control transfected cell line which
was set as 1. c + e Caspase-3/7 activity in TGF-β1 treated H6c7-pBp and H6c7-kras after (c) overexpression of Nrf2-HA or (e) siRNA mediated Nrf2
suppression, expressed as n-fold caspase-3/7 activity of the corresponding control transfected, TGF-β1 treated cell line which was set as 1. Data
are presented as mean ± SD of 3-5 independent experiments. * = p < 0.05 represents an important mechanism leading to en-
hanced Nrf2 expression and activity in tumor cells,
applying also to pancreatic ductal epithelial cells. Ac-
cordingly, we could demonstrate higher basal Nrf2 ex-
pression and activity in H6c7-kras and Colo357 cells
compared to benign H6c7-pBp cells. Important to note,
constitutive Nrf2 activation is caused to a lesser extent
by genetic alterations affecting either the Nrf2 inhibitor
Keap1 or Nrf2 itself (accounting for 10–15 % in gall-
bladder and ovarian cancer or in lung cancer up to
30 % of enhanced Nrf2 activation) as well as epigenetic
mechanisms accounting for enhanced Nrf2 activity in
20–30 % of tumors [45, 46]. Accordingly, we previously
showed that PDAC cells with different Nrf2 activity
exhibit similar Keap1 expression levels [18] supporting the
view that sustained Nrf2 activation in PDAC cells is not
caused by epigenetic or genetic alterations of Keap1 and
rather than other factors. Besides the above mentioned
oncogene induced Nrf2 activation, the exposure of epithe-
lial cells to persistent metabolic and/or oxidative stress,
e.g. caused by the presence of ROS releasing macrophages
and neutrophils during the course of a chronic inflamma-
tion [47] is a major cause for constitutive elevated Nrf2 ac-
tivity. Macrophages are not only abundant in chronic
pancreatitis and PDAC [8] but start to accumulate around
early PanINs. Fig. 6 (See legend on next page.) References
R h b 1. Rahib L, Smith BD, Aizenberg R, Rosenzweig AB, Fleshman JM, Matrisian LM. Projecting cancer incidence and deaths to 2030: the unexpected burden of
thyroid, liver, and pancreas cancers in the United States. Cancer Res. 2014;
74:2913–21. 2. Esposito I, Konukiewitz B, Schlitter AM, Klöppel G. Neue Einblicke in die
Entstehung des Pankreaskarzinoms. Pathologe. 2012;33:189–93. 3. Kanda M, Matthaei H, Wu J, Hong SM, Yu J, Borges M, et al. Presence of
somatic mutations in most early-stage pancreatic intraepithelial neoplasia. Gastroenterology. 2012;142:730–3. 4. Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, Jacobetz MA, et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in
the mouse. Cancer Cell. 2003;4:437–50. 5. Kleeff J, Beckhove P, Esposito I, Herzig S, Huber PE, Löhr JM, et al. Pancreatic
cancer microenvironment. Int J Cancer. 2007;121:699–705. 6. Protti MP, De Monte L. Immune infiltrates as predictive markers of survival
in pancreatic cancer patients. Front Physiol. 2013;4:210. 7. Ino Y, Yamazaki-Itoh R, Shimada K, Iwasaki M, Kosuge T, Kanai Y, et al. Immune cell infiltration as an indicator of the immune microenvironment of
pancreatic cancer. Br J Cancer. 2013;108:914–23. 8. Helm O, Mennrich R, Petrick D, Goebel L, Freitag-Wolf S, Röder C, et al. Comparative characterization of stroma cells and ductal epithelium in
chronic pancreatitis and pancreatic ductal adenocarcinoma. PloS ONE. 2014;
9:e94357. 9. Shi C, Washington MK, Chaturvedi R, Drosos Y, Revetta FL, Weaver CJ, et al. Fibrogenesis in pancreatic cancer is a dynamic process regulated by
macrophage-stellate cell interaction. Lab Invest. 2014;94:409–21. 10. Hernández-Muñoz I, Skoudy A, Real FX, Navarro P. Pancreatic ductal
adenocarcinoma: cellular origin, signaling pathways and stroma
contribution. Pancreatology. 2008;8:462–9. 11. Pickup M, Novitskiy S, Moses HL. The roles of TGFβ in the tumour
microenvironment. Nat Rev Cancer. 2013;13:788–99. 12. Principe DR, Doll JA, Bauer J, Jung B, Munshi HG, Bartholin L, et al. TGF-β:
duality of function between tumor prevention and carcinogenesis. J Natl
Cancer Inst. 2014;106:djt369. 13. Inman GJ. Switching TGFβ from a tumor suppressor to a tumor promoter. Curr Opin Genet Dev. 2011;21:93–9. 1. Rahib L, Smith BD, Aizenberg R, Rosenzweig AB, Fleshman JM, Matrisian LM. Projecting cancer incidence and deaths to 2030: the unexpected burden of
thyroid, liver, and pancreas cancers in the United States. Cancer Res. 2014;
74:2913–21. 1. Rahib L, Smith BD, Aizenberg R, Rosenzweig AB, Fleshman JM, Matrisian LM. Conclusions Overall this study provides a novel mechanism explain-
ing the functional switch of Nrf2 and TGF-β1, both fac-
tors
that
exert
anti-tumorigenic
functions
and
contribute to cell/tissue homeostasis under physio-
logical and acute inflammatory conditions but which
likewise are able to promote tumor development. Hence, concomitant and persistent elevation of both
factors leads to deregulation of the activated signaling
pathways thereby fostering survival and growth of
transformed cells as well as cell invasion [37]. Thus,
this might also explain why therapeutic strategies tar-
geting only either factor have been proven less effective. Therefore, future studies have to investigate whether
concomitant targeting of Nrf2 and TGF-β1 will be
more efficient in the treatment of highly malignant tu-
mors such as PDAC. In contrast to the findings of Lister et al., in our stud-
ies the effect of Nrf2 per se on cell growth of pancreatic
ductal epithelial cells was minor [17] which might be ex-
plained by different cultivation periods after modulation Page 12 of 14 Genrich et al. BMC Cancer (2016) 16:155 Fig. 6 (See legend on next page.) ext page.) Fig. 6 (See legend on next page.) ge ) Fig. 6 (See legend on next page.) Page 13 of 14 Genrich et al. BMC Cancer (2016) 16:155 (See figure on previous page.)
Fig. 6 Nrf2 modulates MAPK and Smad signaling in benign, premalignant and malignant pancreatic ductal epithelial cells. a H6c7-pBp and H6c7-kras
cells were transfected with a control vector (pcDNA3.1) or Nrf2-HA or (b) H6c7-pBp, H6c7-kras and Colo357 cells were transfected either with control
siRNA or Nrf2 siRNA. Then, cells were either left untreated or were treated with 10 ng/ml TGF-β1 for 48 h. Representative western blots of 3-4
independent experiments showing expression of phosphorylated and total p38 (p-/t-p38), phosphorylated Smad3 (p-Smad3, marked by arrow) and
total-Smad2/3 (t-Smad3 marked by arrow), phosphorylated and total Erk (p-/t-Erk) and p21. Hsp90 was detected as loading control. Numbers above
each band indicate average band intensities determined by densitometry. Values of the proteins of interest were divided by the values of the
corresponding loading control (Hsp90) Acknowledgements The authors thank Dagmar Leisner for excellent technical assistance. Funding
is acknowledged by the Lieselotte Beutel Stiftung (SS), the Deutsche
Forschungsgemeinschaft (DFG)-funded Cluster of Excellence “Inflammation
at Interfaces” and the DFG-funded Pancreas Cancer Consortium-Kiel (HS, SS). Additional file 1: Figure S2. Nrf2 modulates MAPK and Smad
signaling in benign, premalignant and malignant pancreatic ductal
epithelial cells. A + B) H6c7-pBp and H6c7-kras cells were transfected
with a control vector (pcDNA3.1) or Nrf2-HA or (C + D) H6c7-pBp,
H6c7-kras and Colo357 cells were transfected either with control siRNA
or Nrf2 siRNA. Then, cells were either left untreated or were treated
with 10 ng/ml TGF-β1 for 48 h. A + C) Densitometric analysis of p-p38,
p-Smad3, p-Erk and p21 expression normalised to Hsp90 expression in
the indicated samples. Data are presented as mean ± SD of three
independent experiments. B + D) Representative western blots of 3-4
independent experiments showing expression of phosphorylated and
total p38 (p-/t-p38), phosphorylated Smad3 (p-Smad3 marked by arrow)
and total-Smad3 (t-Smad3 marked by arrow) and phosphorylated and
total Erk (p-/t-Erk). Hsp90 was detected as loading control. Numbers
above each band indicate average band intensities determined by
densitometry. Values of phosphorylated proteins were divided by the
values of the corresponding total protein. Nrf2 overexpression was
confirmed by detecting the HA-tag of recombinant Nrf2 (see Fig. 3a)
and siRNA mediated suppression of endogenous Nrf2 was confirmed
by detection of total-Nrf2 (t-Nrf2, see Fig. 3b). (PDF 273 kb) Received: 15 October 2015 Accepted: 17 February 2016 Received: 15 October 2015 Accepted: 17 February 2016 The authors disclose no conflict of interest. 9. Shi C, Washington MK, Chaturvedi R, Drosos Y, Revetta FL, Weaver CJ, et al. Fibrogenesis in pancreatic cancer is a dynamic process regulated by
macrophage-stellate cell interaction. Lab Invest. 2014;94:409–21. Author details 1Group Inflammatory Carcinogenesis, Institute for Experimental Cancer
Research, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 17,
24105 Kiel, Germany. 2Institute of Medical Informatics and Statistics, UKSH
Campus Kiel, Brunswiker Str. 10, 24105 Kiel, Germany. 3Department of
Pathology, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 14,
24105 Kiel, Germany. 4Department of Pathology and Neuropathology, 1Group Inflammatory Carcinogenesis, Institute for Experimental Cancer
Research, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 17,
24105 Kiel, Germany. 2Institute of Medical Informatics and Statistics, UKSH
Campus Kiel, Brunswiker Str. 10, 24105 Kiel, Germany. 3Department of
Pathology, Christian-Albrechts-University Kiel, Arnold-Heller-Str. 3, Building 14,
24105 Kiel, Germany. 4Department of Pathology and Neuropathology,
University Hospital Tübingen, Liebermeisterstr. 8, 72076 Tübingen, Germany. 5Laboratory of Molecular Gastroenterology & Hepatology, Institute for
Experimental Cancer Research, Christian-Albrechts-University Kiel,
Arnold-Heller-Str. 3, Building 6, 24105 Kiel, Germany. References
R h b 5. Kleeff J, Beckhove P, Esposito I, Herzig S, Huber PE, Löhr JM, et al. Pancreatic
cancer microenvironment. Int J Cancer. 2007;121:699–705. cancer microenvironment. Int J Cancer. 2007;121:699–705. 6. Protti MP, De Monte L. Immune infiltrates as predictive markers of survival
in pancreatic cancer patients. Front Physiol. 2013;4:210. Abbreviations
CP
h
i 6. Protti MP, De Monte L. Immune infiltrates as predictive markers of survival
in pancreatic cancer patients. Front Physiol. 2013;4:210. CP: chronic pancreatitis; HO-1: Hemoxygenase-1; MAPK: Mitogen-activated
protein kinase; NQO1: NAD(P)H dehydrogenase [quinone] 1; Nrf2: Nuclear
factor E2 related factor-2; PanIN: pancreatic intraepithelial neoplasia;
PDAC: pancreatic ductal adenocarcinoma; TGF-β1: Transforming Growth
Factor beta-1. CP: chronic pancreatitis; HO-1: Hemoxygenase-1; MAPK: Mitogen-activated
protein kinase; NQO1: NAD(P)H dehydrogenase [quinone] 1; Nrf2: Nuclear
factor E2 related factor-2; PanIN: pancreatic intraepithelial neoplasia;
PDAC pancreatic d ctal adenocarcinoma TGF β1 Transforming Gro th 7. Ino Y, Yamazaki-Itoh R, Shimada K, Iwasaki M, Kosuge T, Kanai Y, et al. Immune cell infiltration as an indicator of the immune microenvironment of
pancreatic cancer. Br J Cancer. 2013;108:914–23. PDAC: pancreatic ductal adenocarcinoma; TGF-β1: Transforming Growth
Factor beta-1. 8. Helm O, Mennrich R, Petrick D, Goebel L, Freitag-Wolf S, Röder C, et al. Comparative characterization of stroma cells and ductal epithelium in
chronic pancreatitis and pancreatic ductal adenocarcinoma. PloS ONE. 2014;
9:e94357. Competing interests The authors disclose no conflict of interest. References
R h b Projecting cancer incidence and deaths to 2030: the unexpected burden of
thyroid, liver, and pancreas cancers in the United States. Cancer Res. 2014;
74:2913–21. Additional file 2: Figure S1. Nrf2 hardly impact on basal and TGF-β1
induced apoptosis in pancreatic ductal epithelial cells. H6c7-pBp and
H6c7-kras cells were transfected either with (A) a control vector
(pcDNA3.1) or Nrf2-HA or (B) control siRNA or Nrf2 siRNA. Then, cells were
either left untreated or were treated with 10 ng/ml TGF-β1 for 48 h. Cells
were detached and one part was used for determining caspase-3/7 activity. The other part was used for cell counting and normalization of caspase-3/7
activity. Then, these cells were lysed in Laemmli buffer and analysed for
PARP (full length 116 kD form and cleaved 89 kD form) by western blotting. Hsp90 was detected as loading control. One representative result is shown. (PDF 159 kb) Additional file 2: Figure S1. Nrf2 hardly impact on basal and TGF-β1
induced apoptosis in pancreatic ductal epithelial cells. H6c7-pBp and
H6c7-kras cells were transfected either with (A) a control vector
(pcDNA3.1) or Nrf2-HA or (B) control siRNA or Nrf2 siRNA. Then, cells were
either left untreated or were treated with 10 ng/ml TGF-β1 for 48 h. Cells
were detached and one part was used for determining caspase-3/7 activity. The other part was used for cell counting and normalization of caspase-3/7
activity. Then, these cells were lysed in Laemmli buffer and analysed for 2. Esposito I, Konukiewitz B, Schlitter AM, Klöppel G. Neue Einblicke in die
Entstehung des Pankreaskarzinoms. Pathologe. 2012;33:189–93. 3. Kanda M, Matthaei H, Wu J, Hong SM, Yu J, Borges M, et al. Presence of
somatic mutations in most early-stage pancreatic intraepithelial neoplasia. Gastroenterology. 2012;142:730–3. 3. Kanda M, Matthaei H, Wu J, Hong SM, Yu J, Borges M, et al. Presence of
somatic mutations in most early-stage pancreatic intraepithelial neoplasia. Gastroenterology. 2012;142:730–3. PARP (full length 116 kD form and cleaved 89 kD form) by western blotting. Hsp90 was detected as loading control. One representative result is shown. (PDF 159 kb) 4. Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, Jacobetz MA, et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in
the mouse. Cancer Cell. 2003;4:437–50. 4. Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, Jacobetz MA, et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in
the mouse. Cancer Cell. 2003;4:437–50. Authors’ contributions 10. Hernández-Muñoz I, Skoudy A, Real FX, Navarro P. Pancreatic ductal
adenocarcinoma: cellular origin, signaling pathways and stroma
contribution. Pancreatology. 2008;8:462–9. GG, MK, LL, OH and AB have made substantial contribution to the
acquisition and interpretation of the data. SF-W, LL and OH have substantial
contribution in the statistical analysis of the data. CR and BS have provided
tissues and helped by immunohistochemical stainings and their evaluations. HS has substantial contribution in the conception of the study, interpretation
of the data as well as in critical revising of the manuscript. SS has substantial
contribution in the conception and design of the study, interpretation of the
data and has written the manuscript. All authors have given final approval of
the version to be published and agree to be accountable for all aspects of
the work. 11. Pickup M, Novitskiy S, Moses HL. The roles of TGFβ in the tumour
microenvironment. Nat Rev Cancer. 2013;13:788–99. 12. Principe DR, Doll JA, Bauer J, Jung B, Munshi HG, Bartholin L, et al. TGF-β:
duality of function between tumor prevention and carcinogenesis. J Natl
Cancer Inst. 2014;106:djt369. 13. Inman GJ. Switching TGFβ from a tumor suppressor to a tumor promoter. Curr Opin Genet Dev. 2011;21:93–9. 13. Inman GJ. Switching TGFβ from a tumor suppressor to a tumor promoter. Curr Opin Genet Dev. 2011;21:93–9. Page 14 of 14 Page 14 of 14 Genrich et al. BMC Cancer (2016) 16:155 Page 14 of 14 14. Geismann C, Arlt A, Sebens S, Schäfer H. Cytoprotection “gone astray”: Nrf2
and its role in Cancer. Onco Targets Ther. 2014;7:1497–518. 37. Arfmann-Knübel S, Struck B, Genrich G, Helm O, Sipos B, Sebens S, et al. The
Crosstalk between Nrf2 and TGF-β1 in the Epithelial-Mesenchymal
Transition of Pancreatic Duct Epithelial Cells. PLoS ONE. 2015;10:e0132978. 15. Sporn MB, Liby KT. NRF2 and cancer: the good, the bad and the importance
of context. Nat Rev Cancer. 2012;12:564–71. 38. Rane SG, Lee JH, Lin HM. Transforming growth factor-beta pathway: role in
pancreas development and pancreatic disease. Cytokine Growth Factor Rev. 2006;17:107–19. 16. Shelton P, Jaiswal AK. The transcription factor NF-E2-related factor 2 (Nrf2): a
protooncogene? FASEB J. 2013;27:414–23. 39. Geismann C, Morscheck M, Koch D, Bergmann F, Ungefroren H, Arlt A, et al. Up-regulation of L1CAM in pancreatic duct cells is transforming growth
factor beta1- and slug-dependent: role in malignant transformation of
pancreatic cancer. Cancer Res. 2009;69:4517–26. 17. Authors’ contributions Lister A, Nedjadi T, Kitteringham NR, Campbell F, Costello E, Lloyd B, et al. Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation
and therapy. Mol Cancer. 2011;10:37. 18. Arlt A, Sebens S, Krebs S, Geismann C, Grossmann M, Kruse M-L, et al. Inhibition of the Nrf2 transcription factor by the alkaloid trigonelline renders
pancreatic cancer cells more susceptible to apoptosis through decreased
proteasomal gene expression and proteasome activity. Oncogene. 2013;32:
4825–35. 40. Birnbaum DJ, Mamessier E, Birnbaum D. The emerging role of the TGFβ
tumor suppressor pathway in pancreatic cancer. Cell Cycle. 2012;11:683–6. 41. Gotzmann J, Huber H, Thallinger C, Wolschek M, Jansen B, Schulte-Hermann
R, et al. Hepatocytes convert to a fibroblastoid phenotype through the
cooperation of TGF-beta1 and Ha-Ras: steps towards invasiveness. J Cell Sci. 2002;115:1189–202. 19. Niture SK, Jaiswal AK. Nrf2 protein up-regulates antiapoptotic protein Bcl-2
and prevents cellular apoptosis. J Biol Chem. 2012;287(13):9873–86. 42. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell. 2010;140:883–99. 20. Niture SK, Jaiswal AK. Nrf2-induced antiapoptotic Bcl-xL protein enhances
cell survival and drug resistance. Free Radic Biol Med. 2013;57:119–31. 43. Kundu JK, Surh YJ. Emerging avenues linking inflammation and cancer. Free
Radic Biol Med. 2012;52:2013–37. 21. Kim WD, Kim YW, Cho IJ, Lee CH, Kim SG. E-cadherin inhibits nuclear
accumulation of Nrf2: implications for chemoresistance of cancer cells. J Cell
Sci. 2012;125:1284–95. 44. Banerjee P, Basu A, Datta D, Gasser M, Waaga-Gasser AM, Pal S. The heme
oxygenase-1 protein is overexpressed in human renal cancer cells following
activation of the Ras-Raf-ERK pathway and mediates anti-apoptotic signal. J Biol Chem. 2011;286:33580–90. 22. Shen H, Yang Y, Xia S, Rao B, Zhang J, Wang J. Blockage of Nrf2 suppresses
the migration and invasion of esophageal squamous cell carcinoma cells in
hypoxic microenvironment. Dis Esophagus. 2014;27:685–92. 45. Hayes JD, McMahon M. NRF2 and KEAP1 mutations: permanent activation
of an adaptive response in cancer. Trends Biochem Sci. 2009;34:176–88. 23. Mitsuishi Y, Taguchi K, Kawatani Y, Shibata T, Nukiwa T, Aburatani H, et al. Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic
reprogramming. 2407-13-380. Cancer Cell. 2012;22:66–79. 46. Ganan-Gomez I, Wei Y, Yang H, Boyano-Adanez MC, Garcia-Manero G. Oncogenic functions of the transcription factor Nrf2. Free Radic Biol Med. 2013;65:750–64. 24. Zhu J, Wang H, Sun Q, Ji X, Zhu L, Cong Z, et al. Nrf2 is required to
maintain the self-renewal of glioma stem cells. BMC Cancer. 2013;13:380. 47. Authors’ contributions Sebens S, Bauer I, Geismann C, Grage-Griebenow E, Ehlers S, Kruse M-L,
et al. Inflammatory macrophages induce Nrf2 dependent proteasome
activity in colonic NCM460 cells and thereby confer anti-apoptotic
protection. J Biol Chem. 2011;286:40911–21. 25. DeNicola GM, Karreth FA, Humpton TJ, Gopinathan A, Wei C, Frese K, et al. Oncogene-induced Nrf2 transcription promotes ROS detoxification and
tumorigenesis. Nature. 2011;475:106–9. 26. Choi HK, Pokharel YR, Lim SC, Han HK, Ryu CS, Kim SK, et al. Inhibition of
liver fibrosis by solubilized coenzyme Q10: Role of Nrf2 activation in
inhibiting transforming growth factor-beta1 expression. Toxicol Appl
Pharmacol. 2009;240:377–84. 27. Oh CJ, Kim JY, Min AK, Park KG, Harris RA, Kim HJ, et al. Sulforaphane attenuates
hepatic fibrosis via NF-E2-related factor 2-mediated inhibition of transforming
growth factor-β/Smad signaling. Free Radic Biol Med. 2012;52:671–82. 28. Qian J, Niu J, Li M, Chiao PJ, Tsao MS. In vitro modeling of human
pancreatic duct epithelial cell transformation defines gene expression
changes induced by Kras oncogenic activation in pancreatic carcinogenesis. Cancer Res. 2005;65:5045–53. 29. Moore PS, Sipos B, Orlandini S, Sorio C, Real FX, Lemoine NR, et al. Genetic
profile of 22 pancreatic carcinoma cell lines. Analysis of Kras, p53, p16 and
DPC4/Smad4. Virchows Arch. 2001;439:798–802. 30. Dai JL, Turnacioglu KK, Schutte M, Sugar AY, Kern SE. Dpc4 transcriptional
activation and dysfunction in cancer cells. Cancer Res. 1998;58:4592–7. 31. Sebens Müerköster S, Rausch AV, Isberner A, Minkenberg J, Blasczcuk E, Witt
M, et al. The apoptosis-inducing effect of gastrin on colorectal cancer cells
relates to an increased IEX-1 expression mediating NF-kB inhibition. Oncogene. 2008;27:1122–34. 32. Sebens Muerkoster S, Werbing V, Sipos B, Debus MA, Witt M, Grossmann M,
et al. Drug-induced expression of the cellular adhesion molecule L1CAM
confers anti-apoptotic protection and chemoresistance in pancreatic ductal
adenocarcinoma cells. Oncogene. 2007;26:2759–68. 33. Müerköster S, Wegehenkel K, Arlt A, Witt M, Sipos B, Kruse ML, et al. Tumor
stroma interactions induce chemoresistance in pancreatic ductal carcinoma
cells involving increased secretion and paracrine effects of nitric oxide and
interleukin-1beta. Cancer Res. 2004;64:1331–7. Submit your next manuscript to BioMed Central
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p y
y
p 34. Detlefsen S, Sipos B, Feyerabend B, Klöppel G. Pancreatic fibrosis associated
with age and ductal papillary hyperplasia. Virchows Arch. 2005;447:800–5. 35. Lüttges J, Reinecke-Lüthge A, Möllmann B, Menke MA, Clemens A,
Klimpfinger M, et al. Duct changes and Kras mutations in the disease-free
pancreas: analysis of type, age relation and spatial distribution. Virchows
Arch. 1999;435:461–8. 36. Sipos B, Möser S, Kalthoff H, Török V, Löhr M, Klöppel G. A comprehensive
characterization of pancreatic ductal carcinoma cell lines: towards the
establishment of an in vitro research platform. Virchows Arch. 2003;442:444–52. 36. Sipos B, Möser S, Kalthoff H, Török V, Löhr M, Klöppel G. A comprehensive
characterization of pancreatic ductal carcinoma cell lines: towards the
establishment of an in vitro research platform. Virchows Arch. 2003;442:444–52.
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Analysis of intestinal flora and cognitive function in maintenance hemodialysis patients using combined 16S ribosome DNA and shotgun metagenome sequencing
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Aging Clinical and Experimental Research
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cc-by
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Abstract Abstract
Background Cognitive impairment is widely prevalent in maintenance hemodialysis (MHD) patients, and seriously affects
their quality of life. The intestinal flora likely regulates cognitive function, but studies on cognitive impairment and intestinal
flora in MHD patients are lacking. l
Methods MHD patients (36) and healthy volunteers (18) were evaluated using the Montreal Cognitive Function Scale,
basic clinical data, and 16S ribosome DNA (rDNA) sequencing. Twenty MHD patients and ten healthy volunteers were
randomly selected for shotgun metagenomic analysis to explore potential metabolic pathways of intestinal flora. Both16S
rDNA sequencing and shotgun metagenomic sequencing were conducted on fecal samples.i Results Roseburia were significantly reduced in the MHD group based on both 16S rDNA and shotgun metagenomic
sequencing analyses. Faecalibacterium, Megamonas, Bifidobacterium, Parabacteroides, Collinsella, Tyzzerella, and Phas-
colarctobacterium were positively correlated with cognitive function or cognitive domains. Enriched Kyoto Encyclopedia
of Genes and Genomes (KEGG) pathways included oxidative phosphorylation, photosynthesis, retrograde endocannabinoid
signaling, flagellar assembly, and riboflavin metabolism. ll
Conclusion Among the microbiota, Roseburia may be important in MHD patients. We demonstrated a correlation between
bacterial genera and cognitive function, and propose possible mechanisms. Keywords Cognitive function · Intestinal flora · Microbiota · Maintenance dialysis · 16S rDNA · Shotgun metagenome
sequencing Keywords Cognitive function · Intestinal flora · Microbiota · Maintenance dialysis · 16S rDNA · Shotgun metagenome
sequencing Research Foundation of Educational department of Liaoning
Province, China. Qiuyi Gao and Dianshi Li have contributed equally to this work and
jointly hold the first-author position. * Hongli Lin
linhongli@vip.163.com
* Nan Wang
pyran78@163.com
1
Department of Nephrology, The First Affiliated Hospital
of Dalian Medical University, Dalian, China
2
Centre for Empirical Legal Studies, Faculty of Law,
University of Macau, Macau, China
3
Department of Nephrology, Binzhou Medical University
Affiliated Shengli Oilfield Central Hospital, Binzhou, China
4
School of Graduate, Dalian Medical University, Dalian,
China Research Foundation of Educational department of Liaoning
Province, China. Aging Clinical and Experimental Research (2024) 36:28
https://doi.org/10.1007/s40520-023-02645-y Aging Clinical and Experimental Research (2024) 36:28
https://doi.org/10.1007/s40520-023-02645-y (2024) 36:28 ORIGINAL ARTICLE ORIGINAL ARTICLE Qiuyi Gao1 · Dianshi Li2 · Yue Wang3 · Chunhui Zhao1 · Mingshuai Li4 · Jingwen Xiao4 · Yan Kang4 · Hongli Lin1 ·
Nan Wang1 Received: 16 August 2023 / Accepted: 8 December 2023
© The Author(s) 2024 Received: 16 August 2023 / Accepted: 8 December 2023
© The Author(s) 2024 Analysis of intestinal flora and cognitive function in maintenance
hemodialysis patients using combined 16S ribosome DNA
and shotgun metagenome sequencing nshi Li2 · Yue Wang3 · Chunhui Zhao1 · Mingshuai Li4 · Jingwen Xiao4 · Yan Kang4 · Hongli Lin1 · Research Foundation of Educational department of Liaoning
Province, China. Criteria for patient recruitment The inclusion criteria for the MHD group met the KDIGO
criteria of CKD stage 5; estimated glomerular filtration rate
(eGFR) < 15 ml/(min‧1.73m2), and regular hemodialysis
at least three times a week for more than 3 months. The
exclusion criteria were as follows: (1) patients with eating
or digestive system disorders; (2) patients with cerebrovas-
cular disease (serious cerebral infarction, cerebral hemor-
rhage), Parkinson’s disease (PD), depression, autism, or
a history of substance abuse; (3) patients who had taken
antibiotics, immunosuppressants, or glucocorticoids in the
past 3 months; (4) patients suffering from a life-threatening
disease. The gut microbiota is known to participate in substance
synthesis, nutrient absorption, regulation of immunity, and
preventing infection [10, 11]. In recent years, the microbi-
ota–gut–brain axis theory has gained wide acceptance. This
theory describes the bidirectional connection between the
microbiota and psychiatric disorders through nervous, endo-
crine, and immune mechanisms. Also, a change in the com-
position of the gut microbiota occurs in patients with CKD
or MHD, including an increase in potentially pathogenic
species and a decrease in beneficial species [12]. However,
to our knowledge, research on the relationship between the
microbiota and cognitive function in MHD patients has not
been reported. Eighteen sex-, age-, and education-matched healthy vol-
unteers who were not taking any medication and who had
not previously reported any other disease were recruited
(hemodialysis: healthy = 2:1). All participants gave written
informed consent. This research protocol passed the ethical
review of the Ethics Committee of Dalian Medical Univer-
sity (ethics number PJ-KS-KY-2018-74). Clinic data collection and evaluation of cognitive
function Both 16S rDNA and shotgun metagenome sequencing are
widely employed to investigate the microbiota. These meth-
ods are based on high-throughput sequencing technology,
but they are quite different in principle, purpose, sequencing
depth, and cost. Specifically, 16S rDNA sequencing involves
PCR amplification of specific hypervariable regions, which
is cheaper and widely used to analyze bacterial diversity
[13–15]. By contrast, shotgun metagenomic sequencing
amplifies all microbial genomic DNA, then assembles
genomic fragments for gene prediction [16]. Herein, we
employed a combined approach to evaluate the cognitive
functions of MHD patients in our clinic. First, 16S rDNA
sequencing technology was used to characterize the micro-
biota in MHD patients. Next, the microbiota was correlated
with cognitive function. Finally, shotgun metagenome
sequencing was used to identify the functional pathways
potentially involved. We collected basic data including height, weight, age, gen-
der, education level, systolic blood pressure, and diastolic
blood pressure for all enrolled patients. MHD patients also
included information on primary disease, duration of dialy-
sis, frequency of dialysis, and adequacy of dialysis. All par-
ticipants were required to maintain normal eating habits, and
blood samples were taken following fasting for more than
8 h. Blood samples were immediately stored at 0–4 degrees
and tested by the Department of Clinical Laboratory of the
First Affiliated Hospital of Dalian Medical University for
glucose, lipids, renal function, electrolyte, and high-sensi-
tivity C-reactive protein. On the same day, all participants
were analyzed using the Montreal Cognitive Assessment
(MoCA) scale by the same trained operator following the
operating instructions before receiving hemodialysis treat-
ment. The maximum score is 30, and 26 was considered the
cut-off value (i.e., a score < 26 was considered to indicate
cognitive dysfunction, while > 26 was considered to indi-
cate normal cognitive function). MHD patients underwent
cognitive function evaluation 2 h before hemodialysis. We
collected feces from participants 2 days before blood col-
lection to ensure the reliability of the results. Feces were Introduction As a result of improved dialysis techniques and reduced
mortality, the number and lifespan of patients undergoing
maintenance hemodialysis (MHD) is growing globally [1,
2]. However, the quality of life (Qol) for MHD patients is
less than satisfactory. Mild cognitive impairment has an
adverse effect on Qol, while severe cases can lead to dis-
ability and an increasingly heavy healthcare burden [3]. Cognitive impairment is diagnosed when cognitive func-
tions decline in at least one or more cognitive domains,
including attention, memory, language, comprehension and
judgment, executive function, and others. It can range from
mild forms of forgetfulness to severe dementia [4]. As the
incidence of chronic kidney disease increases, the degree
of cognitive impairment becomes more severe. Cognitive
impairment incidence for MHD is 30–60.9%, which is twice Qiuyi Gao and Dianshi Li have contributed equally to this work and
jointly hold the first-author position. * Hongli Lin
linhongli@vip.163.com * Nan Wang
pyran78@163.com * Nan Wang
pyran78@163.com 1
Department of Nephrology, The First Affiliated Hospital
of Dalian Medical University, Dalian, China 2
Centre for Empirical Legal Studies, Faculty of Law,
University of Macau, Macau, China 3
Department of Nephrology, Binzhou Medical University
Affiliated Shengli Oilfield Central Hospital, Binzhou, China 4
School of Graduate, Dalian Medical University, Dalian,
China (012 3456789) 3456789) Aging Clinical and Experimental Research (2024) 36:28 Page 2 of 13 28 the incidence in the age-matched general population [5–8]. Diagnosis of mild cognitive impairment (MCI, a transi-
tional state with minimal cognitive impairment and rela-
tively intact daily functioning) or Alzheimer’s disease (AD)
is mainly based on clinical manifestations [9]. However,
the mechanisms of cognitive impairment in MHD patients
remain unclear. Besides genetic factors, non-genetic related
factors play an important role in the development of cogni-
tive dysfunction. These include decreased cerebral blood
flow caused by chronic kidney disease (CKD)-related vascu-
lar factors (such as atherosclerosis), glymphatic fluid trans-
port issues caused by endothelial dysfunction, and uremic
toxin factors that cannot be excreted from the body because
of renal damage. These can result in neuronal damage and
lead to cognitive dysfunction. work took place from December 2018 to January 2019. To
eliminate the endogenous differences caused by age and sex
at maximum extent, we matched 18 normal persons to the
36 patients at a 1:2 ratio. The basic experimental scheme is
shown in Fig. 1. Materials and methods In our cross-sectional study, 36 dialysis patients from the
Blood Purification Center of the First Affiliated Hospital
of Dalian Medical University were included, all of whom
gave informed consent form and agreed to participate. This Page 3 of 13
28 Aging Clinical and Experimental Research (2024) 36:28 Aging Clinical and Experimental Research 3 of 13
28 Fig. 1 Basic experimental scheme Fig. 1 Basic experimental scheme Fig. 1 Basic experimental scheme Fig. 1 Basic experimental scheme study are publicly available. The 16r DNA sequencing data
can be found at [https://www.ncbi.nlm.nih.gov/sra?LinkN
ame=biosample_sra&from_uid=29625227 under accession
code BioProject PRJNA860849. The shotgun metagenomic
sequencing data can be found at [https://www.ncbi.nlm.nih.
gov/bioproject/PRJNA849628 under accession code BioPro-
ject PRJNA849628. collected by multi-point sampling, stored on ice throughout
transportation, and sent for 16S rDNA and metagenomic
sequence analysis. 16S rDNA amplifiers and shotgun metagenomic
sequencing We performed 16S rDNA amplification for all participants. We used microPITA (Microbiomes: Picking Interesting
Taxa for Analysis) [17] software in a representative way
to automatically and randomly generate the selected sam-
ple IDs for shotgun metagenomic sequencing (20 dialysis
patients and 10 healthy people). Beijing Novogene Technol-
ogy Co., Ltd (Beijing, China) performed sequencing and
analysis of 16S rDNA and shotgun metagenomic sequencing
data from fecal samples. Details of 16S rDNA and shotgun
metagenomic sequencing including DNA extraction, PCR
amplification, sequencing and bioinformatics analysis can
be found in the additional material. Row data used in the Statistical analyses We analyzed differences in general conditions, cognitive
function scale scores, and laboratory indices between dialy-
sis patient and healthy groups. The Kolmogorov–Smirnov
Z test was used to assess the normality of quantitative data. When the data obeyed a normal distribution and the vari-
ance was uniform, two independent sample T test was used,
otherwise Mann–Whitney non-parametric test was used. For
the microbiota, T test was used to analyze differences in
alpha-diversity between the two groups, while differences in Aging Clinical and Experimental Research (2024) 36:28 Page 4 of 13 28 weight, education level, and diastolic blood pressure. How-
ever, the systolic pressure (SP) was higher in MHD patients
(p < 0.05). MHD patients had a higher prevalence of cog-
nitive dysfunction (61% vs. 28% in healthy volunteers,
p < 0.05), and the difference was mainly manifested in the
total MoCA score, visual space/executive function, naming,
and language domains (all p < 0.05), but there were no dif-
ferences in attention, abstract ability, memory or orientation. Further details are shown in Table 1. beta-diversity were analyzed by unweighted Wilcoxon test. Linear discriminant analysis effect size (LEfSe) was used to
identify meaningful biomarkers with statistical differences
(LEfSe < 4) between groups. Spearman correlation analy-
sis was performed between the 35 most abundant intestinal
microflora components of MHD patients and the general
condition or Montreal Cognitive Assessment (MoCA) scale
(p < 0.05). Intestinal flora data were analyzed using R (Ver-
sion2.15.3, the R software can be found at https://www.r-
project.org/) and other data were analyzed by the software
of Statistical Package for Social Sciences 22.0. 16S rDNA sequencing A total of 2 kingdoms, 21 phyla, 34 classes, 70 orders, 130
families, 283 genera, and 266 species were identified by the
16S rDNA technique. f
cation level (1 = primary school, 2 = junior high school, 3 = senior high school, 4 = junior college, 5 = undergraduate) ** and * represent differences between MHD and healthy groups at p < 0.01 and p < 0.05 es between MHD and healthy groups at p < 0.01 and p < 0.05 Alpha–beta‑diversity analysis A total of 36 MHD patients and 18 healthy volunteers
were recruited, and there were no statistical differences
between the 2 groups in terms of gender, age, height, Chao1, Observed_species, and Shannon index were calcu-
lated based on different principles for α-diversity (within-
habitat diversity) evaluation of the microbiota [18]. The Table 1 Basic clinical characteristics and MoCA scores for cognitive function
** and * represent differences between MHD and healthy groups at p < 0.01 and p < 0.05
Parameters (units)
MHD group (n = 36)
Healthy group (n = 18)
Statistical value
(T or Z value)
Significance
(double tail)
Gender (male/female)
(18/18)
(9/9)
0.00
1
Age (years)
61.06 ± 11.42
60.28 ± 7.06
0.26
0.793
Height (cm)
168 ± 7.87
167.44 ± 5.55
0.27
0.79
Weight (kg)
67.74 ± 11.06
69.35 ± 9.16
0.53
0.596
Education level
3.5 (2, 5)
3.5 (2, 5)
1.23
0.218
Systolic blood pressure (mmHg)
147.5 (130,155)
120 (119.25, 130)
4.27
0.000**
Diastolic blood pressure (mmHg)
80 (80, 90)
80 (77.75, 81.25)
1.47
0.143
Kt/v
1.35 ± 0.79
–
–
–
Dialysis age (years)
5.15 ± 4.12
–
–
–
Primary disease
Primary glomerulonephritis
23 (63.9%)
–
–
–
Hypertensive renal damage
6 (16.67%)
–
–
–
Diabetic nephropathy
4 (11.1%)
–
–
–
Polycystic kidney
2 (5.5%)
–
–
–
Other
1 (2.8%)
–
–
–
Total Montreal Cognitive Scale (MoCA) score
25 (23, 26.75)
27.5 (24.75, 28)
2.03
0.042*
Percentage of cognitive impairment (normal/abnormal)
61% (14/22)
28% (13/5)
–
–
Visual space/executive function
3 (3, 4)
4.5 (3, 5)
2.23
0.026*
Naming
3 (2, 3)
3 (3, 3)
2.07
0.038*
Attention
6 (5, 6)
6 (5, 6)
0.03
0.974
Language
1.5 (1, 2)
2 (1, 3)
2.14
0.033*
Abstract ability
2 (1, 2)
2 (1, 2)
0.38
0.701
Memory
4 (3, 5)
4 (4, 5)
1.27
0.205
Orientation
6 (6, 6)
6 (6, 6)
0.28
0.777 Table 1 Basic clinical characteristics and MoCA scores for cognitive function Aging Clinical and Experimental Research (2024) 36:28 Page 5 of 13 Aging Clinical and Experimental Research 28 results showed that Chao1 and Observed_species indices
were significantly increased in MHD patients (Fig. 2a and b,
p = 0.000 and 0.001, respectively). Meanwhile, the Shannon
index, which reflects species richness and evenness, showed
no significant difference (p = 0.54) in community diversity
between MHD patient and healthy groups (Fig. 2c). Alpha–beta‑diversity analysis We
performed principal component analysis (PCA) based on
variance decomposition, and selected the largest contribu-
tion rate for graph plotting. The principal component con-
tribution values for horizontal and vertical axes were 6.57%
and 6.07%, respectively (Fig. 2d). The results revealed huge
inter-individual differences for the horizontal axes (com-
position of the intestinal flora) between MHD and healthy
groups. annotation and functional genes was performed between
MHD patients and healthy people. Relative abundance and diversity of the microbiota A total of one phylum, two classes, three orders, seven fami-
lies, seven genera, and eight species were identified from T
test statistical analysis of the relative abundances of micro-
biota components between the two groups (all p < 0.05). Fir-
micutes, Bacteroidetes, Proteobacteria, and Actinobacteria
were the main components, and their relative abundances
decreased successively (Fig. 2e and f). Compared with the
healthy group, the relative abundance of the Proteobacteria
phylum was highest in the MHD group (p < 0.01, Fig. 2g). At the genus level, Bacteroides was dominant in both
groups. Roseburia was significantly reduced in the MHD
group, while Blautia, Erysipelatoclostridium, Phascolarc-
tobacterium, Sellimonas, Hungatella, and Stenotrophomonas
were increased significantly (all p < 0.01; Fig. 2h). LEfSe is an efficient tool for finding biomarkers with
statistical differences between groups, and it works by
combining parametric and non-parametric testing. Our
results showed that the relative abundance of Roseburia_
inulinivorans, uncultured_Butyricicoccus_sp, and Dialis-
ter_sp_CAG_357 belonging to Firmicutes, and Haemophi-
lus_parainfluenzae belonging to Proteobacteria, was more
abundant in the healthy group (Fig. 4d). KEGG pathway function annotation of shotgun
metagenomic sequencing data Analysis of correlations between the microbiota and cogni‑
tive function A spearman correlation analysis between the
top 35 most abundant bacterial genera, general conditions,
and cognitive function was conducted on all MHD patients
(n = 36). A total of 12 bacterial genera showed a significant
correlation, among which Faecalibacterium, Megamonas,
Bifidobacterium, Parabacteroides, Collinsella, Tyzzerella,
and Phascolarctobacterium were positively correlated with
cognitive function or cognitive domains (all p < 0.05). Phas-
colarctobacterium showed a positive correlation with vis-
ual space/executive power (r = 0.37, p = 0.027; Fig. 3) and
language function (r = 0.34, p = 0.042; Fig. 3), but showed
a negative correlation with the duration of dialysis (r = −
0.40, p = 0.018; Fig. 3). Erysipelatoclostridium was posi-
tively correlated with age (r = 0.39, p = 0.018; Fig. 3). Figure 5a shows an overview of the results of functional
gene annotation. The box plot shows the differences in the
number of genes between MHD and healthy groups. The
MHD group had fewer functional genes (Fig. 5b). Figure 5c
shows an overview of the results of KEGG pathway function
annotation. The results reveal differences in functional clas-
sification, and the numbers on the bar graph represent the
number of unigenes for the annotation, while the other axis
lists the codes for each level 1 class in the KEGG pathway
database. PCA revealed differences in KEGG pathway func-
tion annotation between MHD and healthy groups (Fig. 5d). Functional genes related to lipid transport and metabolism,
nucleotide transport and metabolism, extracellular struc-
tures, posttranslational modification, protein turnover,
chaperones, and replication-recombination and repair were
enriched in the healthy group (Fig. 5e). Genes associated
with the ko00190 (oxidative phosphorylation), ko00195
(photosynthesis), ko04723 (retrograde endocannabinoid
signaling), ko02040 (flagellar assembly), and ko00740 (ribo-
flavin metabolism) were especially enriched (Fig. 5f). Taxonomic annotation of shotgun metagenomic
sequencing data Figure 4a and b shows the relative abundance of intestinal
flora using shotgun metagenomic sequencing technology at
phylum and genus levels, respectively. Firmicutes, Bacteroi-
detes, Proteobacteria, and Actinobacteria remain the main
bacterial flora components. At the genus level, Bacteroides,
Prevotella, Ruminococcus, Megamonas, and Faecalibacte-
rium constitute the main components of the intestinal flora. l
We performed PCA on the shotgun metagenomic
sequencing data annotation results and selected the main
contributors for graphical display. At the phylum level, there
was a difference in the composition of the intestinal flora
between MHD and healthy groups, and the principal compo-
nent contribution values for the horizontal and vertical axes
were 9.66% and 7.04%, respectively (Fig. 4c).fii Shotgun metagenomic sequencing e and f The main components of the
microbiota at phylum and genus levels, respectively. g and h Differ-
ences in the microbiota between the two groups at phylum and genus
level, respectively
◂ The Chao1 index was found to be decreased in intesti-
nal flora of end-stage of renal disease (ESRD) patients in
southern China [20]. While, our results revealed increased
biodiversity in MHD patients, as evidenced by an increase
in Chao1 index and Observed_species index. Analyzing the
reasons for this phenomenon in our study, we believe that
one reason is that the intestinal flora is affected by many
factors, such as diet, region, and some confounding factors. Compared with the southern study, the subjects participating
in our study are all from northern China and have started
hemodialysis treatment. We also have more participants in
our experiment. These different factors may explain the dif-
ferent results between our results and those of Jiang et al. (2017). Of the 20 MHD patients, 15 were cognitively abnor-
mal (HD.UN) and 5 were cognitively normal (HD.N). We
conducted differential metagenomic analysis between the
two groups, and Chao1, Observed_species, and Shannon
index showed a significant increase in MHD.N patients
(Online Resource 1 a, b, c). LEfSe analysis revealed that
the relative abundances of S_Megamonas_funiformis,
0_Verrucomicrobiales, and f_Akkermansiaceae were
higher in the HD.UN group. Meanwhile, S_ Sutterella_
wadsworthensis, S_Bacteroidales_bacterium_43_36,
g_Chryseobacterium, S_Azobacteroides_phage_PrpJPt_
Bp1, S__Porphyromonadaceae_bacterium_KH3CP3RA,
g_Sutterella, and S_Veillonella_sp_CAG_933 were elevated
in the HD. N group (Online Resource Fig. 1d). However, we
found no significant differences in KEGG pathway func-
tional genes between intestinal flora and cognitive function
in MHD patients. In addition, our Shannon index shows that there is no dif-
ference in microbial diversity between the MHD group and
the healthy group, which is consistent with those of Noriaki
et al. [23] and Wu et al. [24]. The difference between Shan-
non and Chao1 index as evaluating microbial diversity tools
in bacterial diversity is that, in addition to being consistent
with Chao1’s role in evaluating species richness, Shannon
also considers community evenness. Combining the high
species richness of the MHD patients in our study and the
no difference in Shannon index, we infer that the community
evenness of the MHD is poor, which is consistent with the
significant intra-group differences shown in our Fig. 2d. f Shotgun metagenomic sequencing In our study, 20 MHD patients and 10 healthy people were
randomly selected by microPITA [17] to explore functional
genes and metabolic pathways. A comparison of taxonomic Aging Clinical and Experimental Research (2024) 36:28 Page 6 of 13 Aging Clinical and Experimental Research 28 28 Aging Clinical and Experimental Research (2024) 36:28
Page 6 of 13 Page 7 of 13 Aging Clinical and Experimental Research (2024) 36:28 Aging Clinical and Experimental Research 28 was 25 points, and the prevalence of cognitive impairment
was 61%. The Montreal score of MHD patients was signifi-
cantly lower than that of the healthy group, mainly in terms
of visuospatial/executive function, naming, and language. The results of prevalence and the specific domains of cogni-
tive impairment among MHD patients were consistent with
previous studies [5–8]. However, the median scores for our
cohort were slightly higher than in previous studies [6]. Fig. 2 Analysis of differences in the microbiota based on 16S ribo-
some DNA (rDNA) sequencing between MHD patients and healthy
people. a–c Chao1, Observed_species. and Shannon indices, respec-
tively. Chao1 and Observed_species explain α-diversity (within-hab-
itat diversity), and both indices were significantly increased in MHD
patients (Figure a and b, p = 0.000 and 0.001, respectively). Mean-
while, the Shannon index c showed no significant difference between
the two groups (p = 0.541). d Principal component analysis (PCA)
of differences in the composition of the intestinal flora. The princi-
pal component contribution values of horizontal and vertical axes are
6.57% and 6.07%, respectively. e and f The main components of the
microbiota at phylum and genus levels, respectively. g and h Differ-
ences in the microbiota between the two groups at phylum and genus
level, respectively
◂ Fig. 2 Analysis of differences in the microbiota based on 16S ribo-
some DNA (rDNA) sequencing between MHD patients and healthy
people. a–c Chao1, Observed_species. and Shannon indices, respec-
tively. Chao1 and Observed_species explain α-diversity (within-hab-
itat diversity), and both indices were significantly increased in MHD
patients (Figure a and b, p = 0.000 and 0.001, respectively). Mean-
while, the Shannon index c showed no significant difference between
the two groups (p = 0.541). d Principal component analysis (PCA)
of differences in the composition of the intestinal flora. The princi-
pal component contribution values of horizontal and vertical axes are
6.57% and 6.07%, respectively. Discussion Due to the limitations of experimental funds and difficulties
in collecting clinical data, there is no uniform standard for
determining an appropriate sample size. Furthermore, the
traditional formula for sample size estimation is not very
applicable. Therefore, we referred to literature exploring the
role of gut microbiota in disease models, such as a study
of 41 pre-dialysis diabetic kidney disease patients [19] and
51 end-stage renal disease patients [20]. On this basis, we
selected an appropriate sample size for our studies. if
The relative abundance and statistical differences in the
relative abundance were analyzed separately in 16S rDNA
and shotgun metagenomic sequencing. Both sets of results
showed that Firmicutes, Bacteroidetes, Proteobacteria, and
Actinobacteria were the main components of the micro-
biota. These results are consistent with those of previous
research on Western and Asian countries [20, 26]. We were
also surprised to observe a significant reduction in Rose-
buria in MHD patients. A reduction of Roseburia has been
reported previously in studies on patients with early-stage
CKD [23] and end-stage renal disease [19]. Roseburia mem-
bers are likely to be beneficial bacteria due to their anti-
inflammatory effects and ability to modulate sugar and pro-
tein metabolism [27]. Roseburia bind to Toll-like receptor
5 on intestinal epithelium cells through the flagellum, stim-
ulating dendritic cells to secrete cytokines and promoting
differentiation of regulatory T cells, thereby exerting anti-
inflammatory effects. Also, butyrate produced by Roseburia
binds to G-protein-coupled receptors and participates in the Cerebrovascular disease, age, gender, and education
level are important factors affecting cognitive dysfunction
[21, 22]; hence, all people involved in our experiment were
free from cerebrovascular disease, and both groups were
recruited by matching age, sex, and education to avoid
potential confounding factors, and to discover other factors
affecting cognitive function. We applied the MoCA test to
assess cognitive function because it is rapid, comprehensive,
and widely applied. The mean age of maintenance hemodi-
alysis (MHD) patients was 61.06 years, the Montreal score Aging Clinical and Experimental Research (2024) 36:28 Aging Clinical and Experimental Research Page 8 of 13 28 Aging Clinical and Experimental Research (2024) 36:28
28
Page 8 of 13
regulation of glycoprotein metabolism pathways. Wang et al. [28] identified two major enterotypes (Faecalibacterium
prausnitzii and Prevotella) and Roseburia were associated
with reduced production of short-chain fatty acids. Thus,
this microbial imbalance further aggravates inflammatory
responses and promotes the progression of kidney disease. Fig. 3 Spearman correlation
analysis between the microbiota
and cognitive function. Twelve
of the top thirty-five bacterial
genera show a significant posi-
tive correlation with cognitive
function or cognitive domains
(all p < 0.05) Discussion In the present work, Blautia, Erysipelatoclostridium, Phas-
colarctobacterium, Sellimonas, and Butyricicoccus were
identified, all of which can produce short-chain fatty acids
(SCFAs) [29], which indicates that SCFAs are also involved
in the progression of advanced CKD. In addition, such dif-
ferential bacterial flora may provide a potential therapeutic
Fig. 3 Spearman correlation
analysis between the microbiota
and cognitive function. Twelve
of the top thirty-five bacterial
genera show a significant posi-
tive correlation with cognitive
function or cognitive domains
(all p < 0.05) Fig. 3 Spearman correlation
analysis between the microbiota
and cognitive function. Twelve
of the top thirty-five bacterial
genera show a significant posi-
tive correlation with cognitive
function or cognitive domains
(all p < 0.05) In the present work, Blautia, Erysipelatoclostridium, Phas-
colarctobacterium, Sellimonas, and Butyricicoccus were
identified, all of which can produce short-chain fatty acids
(SCFAs) [29], which indicates that SCFAs are also involved
in the progression of advanced CKD. In addition, such dif-
ferential bacterial flora may provide a potential therapeutic regulation of glycoprotein metabolism pathways. Wang et al. [28] identified two major enterotypes (Faecalibacterium
prausnitzii and Prevotella) and Roseburia were associated
with reduced production of short-chain fatty acids. Thus,
this microbial imbalance further aggravates inflammatory
responses and promotes the progression of kidney disease. Aging Clinical and Experimental Research (2024) 36:28
Page 9 of 13
28
strategy for CKD management through supplementation of
SCFAs combined with selected prebiotics or probiotics. Based on correlation analysis of cognitive function
and intestinal flora between dialysis patients and healthy
people, Faecalibacterium, Megamonas, Bifidobacterium,
Parabacteroides, Collinsella, Tyzzerella, and Phasco-
larctobacterium were positively correlated with cognitive
function or cognitive domains. This may provide evidence
for the gut–brain axis modulating cognitive function,
even in relatively young patients with stage 5 CKD. A
Fig. 4 Differences in α-diversity
and β-diversity between MHD
patients and healthy peo-
ple based on metagenomic
sequencing. a and b The main
components of the micro-
biota at phylum and genus
levels, respectively. Firmicutes,
Bacteroidetes, Proteobacteria,
Actinobacteria and Bacteroides
are the main components of the
microbiota. c PCA of differ-
ences in intestinal flora between
MHD and healthy control
groups. The principal com-
ponent contribution values of
horizontal and vertical axes are
9.66% and 7.04%, respectively. Discussion d Roseburia_inulinivorans,
uncultured_Butyricicoccus_sp,
Dialister_sp_CAG_357 and
Haemophilus_parainfluenzae
are the most abundant compo-
nent of the intestinal microbiota
in the healthy group based on
linear discriminant analysis
effect size (LEfSe) Page 9 of 13
28 Aging Clinical and Experimental Research (2024) 36:28 28 Fig. 4 Differences in α-diversity
and β-diversity between MHD
patients and healthy peo-
ple based on metagenomic
sequencing. a and b The main
components of the micro-
biota at phylum and genus
levels, respectively. Firmicutes,
Bacteroidetes, Proteobacteria,
Actinobacteria and Bacteroides
are the main components of the
microbiota. c PCA of differ-
ences in intestinal flora between
MHD and healthy control
groups. The principal com-
ponent contribution values of
horizontal and vertical axes are
9.66% and 7.04%, respectively. d Roseburia_inulinivorans,
uncultured_Butyricicoccus_sp,
Dialister_sp_CAG_357 and
Haemophilus_parainfluenzae
are the most abundant compo-
nent of the intestinal microbiota
in the healthy group based on
linear discriminant analysis
effect size (LEfSe) strategy for CKD management through supplementation of
SCFAs combined with selected prebiotics or probiotics. strategy for CKD management through supplementation of
SCFAs combined with selected prebiotics or probiotics. Based on correlation analysis of cognitive function
and intestinal flora between dialysis patients and healthy
people, Faecalibacterium, Megamonas, Bifidobacterium, strategy for CKD management through supplementation of
SCFAs combined with selected prebiotics or probiotics. Based on correlation analysis of cognitive function
and intestinal flora between dialysis patients and healthy
people, Faecalibacterium, Megamonas, Bifidobacterium,
Parabacteroides, Collinsella, Tyzzerella, and Phasco-
larctobacterium were positively correlated with cognitive
function or cognitive domains. This may provide evidence
for the gut–brain axis modulating cognitive function,
even in relatively young patients with stage 5 CKD. A strategy for CKD management through supplementation of
SCFAs combined with selected prebiotics or probiotics. Based on correlation analysis of cognitive function
and intestinal flora between dialysis patients and healthy
people, Faecalibacterium, Megamonas, Bifidobacterium, Parabacteroides, Collinsella, Tyzzerella, and Phasco-
larctobacterium were positively correlated with cognitive
function or cognitive domains. This may provide evidence
for the gut–brain axis modulating cognitive function,
even in relatively young patients with stage 5 CKD. A Based on correlation analysis of cognitive function
and intestinal flora between dialysis patients and healthy
people, Faecalibacterium, Megamonas, Bifidobacterium, Aging Clinical and Experimental Research (2024) 36:28 Page 10 of 13 28 Fig. 5 KEGG pathway function
annotation of metagenomic
sequencing data. a Basic
statistical information for
metagenomic sequencing analy-
sis. b Box plot showing fewer
functional genes in the MHD
group. Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s40520-023-02645-y. Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s40520-023-02645-y. Acknowledgements We thank colleagues from the Department of
Nephrology and the Department of Laboratory Medicine at the First
Affiliated Hospital of Dalian Medical University for their hardwork. We also thank Beijing Novogene Technology Co., Ltd. for carrying out
16S rDNA and shotgun metagenome sequencing.i Our functional gene annotation based on shotgun
metagenomic sequencing results identified metabolic path-
ways that maybe related to the occurrence of cognitive dis-
order. Lipopolysaccharide biosynthesis is enriched in the
microbiome at the advanced stage of CKD [25], and oxida-
tive phosphorylation affects cognitive function by causing
the accumulation of iron complexes [38]. The retrograde
endocannabinoid signaling pathway is essential for core cog-
nitive processes in schizophrenia, such as working memory
[39] and involved in the regulation of anxiety and depres-
sion-like behaviors [40]. Riboflavin metabolism is linked
to vitamin B, and vitamin B deficiency is associated with
neurocognitive disorders [41].f Contribution to the field statement: As a result of improved dialysis
techniques and reduced mortality, the number and lifespan of patients
undergoing maintenance hemodialysis is growing globally. Loss of
attention, memory, language and communication, comprehension and
judgment, executive function, and other cognitive domains loss are
considered as mild cognitive impairment. Mild cognitive impairment is
prevalent in maintenance hemodialysis patients and affects their quality
of life. However, the mechanisms of cognitive impairment in hemodi-
alysis patients remain unclear. Since the intestinal flora likely regulates
cognitive function, we combined the 16S ribosome DNA and shotgun
metagenome sequencing techniques to analyze the differences and
correlations between cognitive function and intestinal flora in mainte-
nance hemodialysis patients. Roseburia were significantly reduced in
the hemodialysis group based on both 16S rDNA and shotgun metagen-
omic sequencing analyses. Twelve bacterial genera showed a positive
correlation with cognitive function or cognitive domains. Oxidative
phosphorylation, photosynthesis, retrograde endocannabinoid signal-
ing, flagellar assembly, and riboflavin metabolism were linked to cogni-
tive dysfunction. In summary, we demonstrated a correlation between
bacterial genera and cognitive function in MHD patients and propose
a possible mechanism that may provide therapeutic targets for cogni-
tive dysfunction. Herein, we performed a simple metagenomic differ-
ence analysis between HD.UN and HD.N to explore links
between intestinal flora and cognitive function. We found
that Megamonas_funiformis and Akkermansiaceae of Ver-
rucomicrobiales were more abundant in the HD.UN group. Discussion In the
future, we will increase the sample size of HD-UN and
HD-N to explore the underlying mechanisms linking intes-
tinal flora and cognitive function. blood circulation. Eventually, excessive activation of inflam-
mation and accumulation of toxic metabolites promote the
progression of kidney disease [30]. F. prausnitzii, one of the most important bacteria in the
human intestinal flora, plays a beneficial probiotic role in
intestinal physiology and maintains host health by produc-
ing butyric acid via G-protein-coupled receptor 43 Axis
[31], and accounts for 5–15% of total bacteria detected
in healthy human stool samples [32]. A study on healthy
Dutch adults (65–79 years old) exploring the relationships
between diet, gastrointestinal microbiota composition, and
cognitive function found that F. prausnitzii and Roseburia
exhibit anti-inflammatory properties and are associated with
beneficial health effects [33]. Herein, we were not surprised
to observe a correlation between Bifidobacteria and cog-
nitive function since numerous intervention studies found
that Bifidobacteria supplementation can improve cogni-
tive function [34, 35]. We are very interested in Phasco-
larctobacterium because members were both differentially
expressed in MHD patients, and correlated with cognitive
function. Although the exact mechanisms through which
Phascolarctobacterium affects cognitive function are not
known, members play a key role in various disease models. In autism spectrum disorder, Phascolarctobacterium are
increased in abundance and their metabolites may contribute
to the occurrence and severity of the disorder [36]. Phas-
colarctobacterium is involved in intestinal flora processes,
neuroactive metabolites, and brain functional connectivity
networks in bipolar depression [37]. In any case, these intes-
tinal floras provide evidence for a functional gut–brain axis,
and a potential treatment strategy for alleviating cognitive
dysfunction in patients. l
There are some shortcomings with our study. First, the
sample size was relatively small. Although we accounted
for the effects of some known factors, including age and
education, we cannot avoid the interference of some uncer-
tain factors. We only explored the correlation between
intestinal flora and cognitive function in dialysis patients,
but not CKD patients at different stages. We predicted
functional genes of metabolic pathways via which the
intestinal flora may affect cognitive function, but the
results require validation in vivo. In summary, we carried out a detailed exploration of
the cognitive functions of MHD patients, and investigated
potential links between changes in the intestinal microflora
and cognitive function in MHD patients. Discussion c Statistical analysis of
the number of KEGG unigene
annotations. The numbers on
the bar chart represent the num-
ber of unigenes on the annota-
tions, and the other axis lists the
codes for each level 1 functional
class in each database. d PCA
showing differences in KEGG
pathway function annotation
between MHD and healthy
groups. e and f Differences in
KEGG pathways between the
two groups at KEGG pathway
levels 2 and 3, respectively brain–gut–kidney axis has been proposed involving neural,
immune, and metabolic interactions [30]. After the brain
is stimulated by factors such as the environment, diet, and
CKD-related stimulatory factors, it activates the sympathetic
nervous system throughout the body. On the one hand, the
sympathetic nervous system promotes the activation of
immune cells in bone marrow, leading to an increase in the
level of inflammation; on the other hand, it also alters the
permeability of the intestines, allowing intestinal bacteria
and increased intestinal flora metabolites to participate in
group. c Statistical analysis of
the number of KEGG unigene
annotations. The numbers on
the bar chart represent the num-
ber of unigenes on the annota-
tions, and the other axis lists the
codes for each level 1 functional
class in each database. d PCA
showing differences in KEGG
pathway function annotation
between MHD and healthy
groups. e and f Differences in
KEGG pathways between the
two groups at KEGG pathway
levels 2 and 3, respectively sympathetic nervous system promotes the activation of
immune cells in bone marrow, leading to an increase in the
level of inflammation; on the other hand, it also alters the
permeability of the intestines, allowing intestinal bacteria
and increased intestinal flora metabolites to participate in brain–gut–kidney axis has been proposed involving neural,
immune, and metabolic interactions [30]. After the brain
is stimulated by factors such as the environment, diet, and
CKD-related stimulatory factors, it activates the sympathetic
nervous system throughout the body. On the one hand, the Page 11 of 13 Aging Clinical and Experimental Research (2024) 36:28 Aging Clinical and Experimental Research 28 non-alcoholic steatohepatitis (NASH) without metabolic
syndrome (MS) [43]. However, we failed to find any mean-
ingful KEGG pathway functional genes linking intestinal
flora and cognitive function in MHD patients. This may
be due to the limited sample size, which may affect our
ability to identify metabolic pathways of intestinal flora
involved in the process of cognitive dysfunction. Discussion By combining
16SrDNA and shotgun metagenomic sequencing, we found a
correlation between changes in intestinal flora and cognitive
function in MHD patients, and propose a possible mecha-
nism for the first time. Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s40520-023-02645-y. Megamonas_funiformis plays a pivotal role in the progres-
sion of colorectal cancer (CRC) [42]. Verrucomicrobiales
is associated with inflammatory activity in patients with Aging Clinical and Experimental Research (2024) 36:28 Aging Clinical and Experimental Research Page 12 of 13 28 5. Davey A, Elias M, Robbins M et al (2012) Decline in renal func-
tioning is associated with longitudinal decline in global cognitive
functioning, abstract reasoning and verbal memory. Nephrol, Dial,
Transplant 28:1810–1819. https://doi.org/10.1093/ndt/gfs470 Author contributions QG was responsible for the experiments design
and writing this paper. DL was responsible for the analysis of the exper-
imental data and for revising the format of the paper. QG and DL have
contributed equally to this work and share first authorship. YW, CZ,
ML, JX, and YK assisted in specimen collection, recruited the patients,
and experimental organization. HL and WN was responsible for the
support of the experimental fund, design and revision of the manu-
script. HL and WN were co-corresponding authors. The manuscript
has been read and approved for submission by all authors. All persons
listed as authors have contributed to preparing the manuscript, and the
editorial criteria for authorship have been met. 6. Pei X, Lai S, He X et al (2018) Mild cognitive impairment in
maintenance hemodialysis patients: a cross-sectional survey and
cohort study. Clin Interv Aging 14:27–32. https://doi.org/10.2147/
cia.s178854f 7. Kurella-Tamura M, Vittinghoff E, Hsu C-Y et al (2017) Loss of
executive function after dialysis initiation in adults with chronic
kidney disease. Kidney Int 91:948–953. https://doi.org/10.1016/j.
kint.2016.11.015 Funding This work was supported by the Key Project of Basic Sci-
entific Research [grant number LJKZZ20220099] of the Educational
Department of Liaoning Province. 8. Bugnicourt JM, Godefroy O, Chillon JM et al (2013) Cognitive
disorders and dementia in CKD: the neglected kidney-brain axis. J
Am Soc Nephrol 24:353–363. https://doi.org/10.1681/ASN.20120
50536 Data availability The data used in this article come from the author's
own collection and analysis of data. The sequencing data used in this
study have been uploaded to the Internet and are publicly accessible. Specific URLs of the data are given in the Materials and Methods sec-
tion. The format and content of the data are consistent with the analyzes
and results presented in this article. We guarantee the integrity and
accuracy of the data and are responsible for the quality of the data. 9. Declarations 12. Stadlbauer V, Horvath A, Ribitsch W et al (2017) Structural and
functional differences in gut microbiome composition in patients
undergoing haemodialysis or peritoneal dialysis. Sci Rep 7:15601. https://doi.org/10.1038/s41598-017-15650-9 Conflict of interest The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that
could be construed as a potential conflict of interest. This article has
never been published in public. 13. Alkema W, Boekhorst J, Wels M et al (2016) Microbial bioinfor-
matics for food safety and production. Brief Bioinform 17:283–
292. https://doi.org/10.1093/bib/bbv034i 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/. 14. Chen Z, Cheng H, Cai Z et al (2021) Identification of Microbiome
Etiology Associated With Drug Resistance in Pleural Empyema. Front Cell Infect Microbiol 11:637018. https://doi.org/10.3389/
fcimb.2021.637018 15. Wang K, Huang Y, Zhang Z et al (2019) A Preliminary Study of
Microbiota Diversity in Saliva and Bronchoalveolar Lavage Fluid
from Patients with Primary Bronchogenic Carcinoma. Medical
science monitor : international medical journal of experimental
and clinical research 25:2819–2834. https://doi.org/10.12659/
MSM.915332 16. Quince C, Walker AW. (2017) Shotgun metagenomics, from sam-
pling to analysis 35(9):833–844. doi:https://doi.org/10.1038/nbt.
3935 17. Tickle TL, Segata N, Waldron L et al (2013) Two-stage microbial
community experimental design. ISME J 7:2330–2339. https://
doi.org/10.1038/ismej.2013.139 Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s40520-023-02645-y. Lin C-H, Chiu C-C, Huang C-H et al (2019) pLG72 levels increase
in early phase of Alzheimer’s disease but decrease in late phase. Sci Rep 9:13221. https://doi.org/10.1038/s41598-019-49522-1 10. Grantham JP, Staub K, Rühli FJ et al (2014) Modern diet and
metabolic variance–a recipe for disaster? Nutr J 3:15. https://doi.
org/10.1186/1475-2891-13-15 g
11. Volovat SR, Volovat C, Miron I et al (2020) Oncogenic mecha-
nisms in renal insufficiency 14:507–515. https://doi.org/10.1093/
ckj/sfaa122 References 18. Li B, Zhang X, Guo F et al (2013) Characterization of tetracycline
resistant bacterial community in saline activated sludge using
batch stress incubation with high-throughput sequencing analysis. Water Res 47:4207–4216. https://doi.org/10.1016/j.watres.2013.
04.021 1. Couser WG, Remuzzi G, Mendis S et al (2011) The contribution
of chronic kidney disease to the global burden of major noncom-
municable diseases. Kidney Int 80:1258–1270. https://doi.org/10.
1038/ki.2011.368 19. Zhang Q, Zhang Y, Zeng L et al (2021) The Role of Gut Micro-
biota and Microbiota-Related Serum Metabolites in the Progres-
sion of Diabetic Kidney Disease. Front Pharmacol 12:757508. https://doi.org/10.3389/fphar.2021.757508 2. Lysaght MJ (2002) Maintenance dialysis population dynamics:
current trends and long-term implications. J Am Soc Nephrol
13:S37-40 3. Iyasere O, Okai D, Brown E (2017) Cognitive function and
advanced kidney disease: longitudinal trends and impact on deci-
sion-making. Clin Kidney J 10:89–94. https://doi.org/10.1093/ckj/
sfw128 20. Jiang S, Xie S, Lv D et al (2017) Alteration of the gut micro-
biota in Chinese population with chronic kidney disease. Sci Rep
7:2870. https://doi.org/10.1038/s41598-017-02989-2 p
g
21. Kurella M, Mapes DL, Port FK et al (2006) Correlates and out-
comes of dementia among dialysis patients: the Dialysis Outcomes
and Practice Patterns Study. Nephrology, dialysis, transplantation :
official publication of the European Dialysis and Transplant Asso-
ciation - European Renal Association 21:2543–2548. https://doi.
org/10.1093/ndt/gfl275 4. Chua SIL, Tan NC, Wong WT et al (2019) Virtual reality for
screening of cognitive function in older persons: comparative
study. J Med Internet Res 21:e14821–e14821. https://doi.org/10.
2196/14821 4. Chua SIL, Tan NC, Wong WT et al (2019) Virtual reality for
screening of cognitive function in older persons: comparative
study. J Med Internet Res 21:e14821–e14821. https://doi.org/10.
2196/14821 Aging Clinical and Experimental Research Page 13 of 13 Aging Clinical and Experimental Research (2024) 36:28 28 (2024) 36:28 rch (2024) 36:28 adults: the NU-AGE study. Nutrients 12:3471. https://doi.org/10.
3390/nu12113471 22. Tollitt J, Odudu A, Montaldi D et al (2020) Cognitive impairment
in patients with moderate to severe chronic kidney disease: the
Salford kidney cohort study. Clin Kidney J 14:1639–1648. https://
doi.org/10.1093/ckj/sfaa178i 34. Kim CS, Cha L, Sim M et al (2021) Probiotic supplementation
improves cognitive function and mood with changes in gut micro-
biota in community-dwelling older adults: a randomized, double-
blind, placebo-controlled, multicenter trial. J Gerontol A Biol Sci
Med Sci 76:32–40. https://doi.org/10.1093/gerona/glaa090i g
j
23. References Sato N, Kakuta M, Hasegawa T et al (2020) Metagenomic profil-
ing of gut microbiome in early chronic kidney disease. Nephrol-
ogy, dialysis, transplantation : official publication of the European
Dialysis and Transplant Association - European Renal Association
36:1675–1684. https://doi.org/10.1093/ndt/gfaa122 35. Shi S, Zhang Q, Sang Y et al (2022) Probiotic Bifidobacterium
longum BB68S improves cognitive functions in healthy older
adults: a randomized, double-blind placebo-controlled trial. Nutri-
ents 15:51. https://doi.org/10.3390/nu15010051 p
g
g
24. Wu HY, Lin YT, Tsai WC, et al. (2023) Microbiota analysis in
the hemodialysis population - Focusing on Enterobacteriaceae. Journal of microbiology, immunology, and infection = Wei mian
yu gan ran za zhi 56(2):311–323. doi:https://doi.org/10.1016/j.
jmii.2022.12.001 36. Iglesias-Vázquez L, Van Ginkel RG, Arija V et al (2020) Composi-
tion of gut microbiota in children with autism spectrum disorder:
a systematic review and meta-analysis. Nutrients 12:792. https://
doi.org/10.3390/nu12030792 j
25. Wu IW, Gao SS, Chou HC et al (2020) Integrative metagenomic
and metabolomic analyses reveal severity-specific signatures of
gut microbiota in chronic kidney disease. Theranostics 10:5398–
5411. https://doi.org/10.7150/thno.41725 37. Li Z, Lai J, Zhang P et al (2022) Multi-omics analyses of serum
metabolome, gut microbiome and brain function reveal dysregu-
lated microbiota-gut-brain axis in bipolar depression. Mol Psychi-
atry 27:4123–4135. https://doi.org/10.1038/s41380-022-01569-9 p
g
26. (2012) Structure, function and diversity of the healthy human
microbiome. Nature 486(7402):207–214. doi:https://doi.org/10.
1038/nature11234 38. Ward RJ, Zucca FA, Duyn JH et al (2014) The role of iron in
brain ageing and neurodegenerative disorders. Lancet Neurol
13:1045–1060. https://doi.org/10.1016/s1474-4422(14)70117-6 27. Nie K, Ma K, Luo W et al (2021) Roseburia intestinalis: A Ben-
eficial Gut Organism From the Discoveries in Genus and Species. Front Cell Infect Microbiol 11:757718. https://doi.org/10.3389/
fcimb.2021.757718 39. Volk DW, Lewis DA (2015) The role of endocannabinoid signal-
ing in cortical inhibitory neuron dysfunction in schizophrenia. Biol Psychiat 79:595–603. https://doi.org/10.1016/j.biopsych.
2015.06.015 28. Wang X, Yang S, Li S et al (2020) Aberrant gut microbiota alters
host metabolome and impacts renal failure in humans and rodents. Gut 69:2131–2142. https://doi.org/10.1136/gutjnl-2019-319766 40. Ueda A, Shinkai S, Shiroma H et al (2021) Identification of Fae-
calibacterium prausnitzii strains for gut microbiome-based inter-
vention in Alzheimer’s-type dementia. Cell Rep Med 2:100398. https://doi.org/10.1016/j.xcrm.2021.100398 29. Devriese S, Eeckhaut V, Geirnaert A et al (2017) Reduced
Mucosa-associated Butyricicoccus Activity in Patients with
Ulcerative Colitis Correlates with Aberrant Claudin-1 Expres-
sion. J Crohns Colitis 11:229–236. https://doi.org/10.1093/ecco-
jcc/jjw142 41. Mikkelsen K, Apostolopoulos V (2018) B vitamins and ageing. Sub-Cell Biochem 90:451–470. References https://doi.org/10.1007/978-981-
13-2835-0_15 42. Ren X, Xu J, Zhang Y et al (2020) Bacterial alterations in post-
cholecystectomy patients are associated with colorectal cancer. Front Oncol 10:1418. https://doi.org/10.3389/fonc.2020.01418 30. Yang T, Richards EM, Pepine CJ et al (2018) The gut microbiota
and the brain-gut-kidney axis in hypertension and chronic kidney
disease. Nat Rev Nephrol 14:442–456. https://doi.org/10.1038/
s41581-018-0018-2 43. De Oliveira JM, Pace FL, Ghetti FF et al (2020) Non-alcoholic
steatohepatitis: comparison of intestinal microbiota between dif-
ferent metabolic profiles a pilot study. J Gastrointest Liver Dis
JGLD 29:369–376. https://doi.org/10.15403/jgld-497 31. Li HB, Xu ML, Xu XD et al (2022) Faecalibacterium praus-
nitzii Attenuates CKD via Butyrate-renal GPR43 axis. Circ Res
131:e120–e134. https://doi.org/10.1161/circresaha.122.320184 32. Miquel S, Martín R, Rossi O et al (2013) Faecalibacterium praus-
nitzii and human intestinal health. Curr Opin Microbiol 16:255–
261. https://doi.org/10.1016/j.mib.2013.06.003 Publisher's Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. 33. van Soest APM, Hermes GDA, Berendsen AAM et al (2020)
Associations between pro- and anti-inflammatory gastro-intestinal
microbiota, diet, and cognitive functioning in Dutch healthy older
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Factors affecting students’ entrepreneurial intentions: a systematic review (2005–2022) for future directions in theory and practice
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Management Review Quarterly (2023) 73:1903–1970
https://doi.org/10.1007/s11301-022-00289-2 Management Review Quarterly (2023) 73:1903–1970
https://doi.org/10.1007/s11301-022-00289-2 * Greeni Maheshwari
greeni.maheshwari@rmit.edu.vn 1
Economics and Finance Department, RMIT University, Ho Chi Minh City, Vietnam
2
School of Economics and Management, Xiamen University, Sepang, Selangor, Malaysia Factors affecting students’ entrepreneurial intentions:
a systematic review (2005–2022) for future directions
in theory and practice Greeni Maheshwari1 · Khanh Linh Kha1 · Anantha Raj A. Arokiasamy Received: 12 January 2022 / Accepted: 12 July 2022 / Published online: 15 August 2022
© The Author(s) 2022 Keywords Entrepreneurial intentions · Systematic literature review · Citation
analysis · Thematic analysis · Entrepreneurship 1
Economics and Finance Department, RMIT University, Ho Chi Minh City, Vietnam 1 Introduction Entrepreneurship has been viewed as a critical contributor and an economic engine
of every country as it helps in creating new jobs, and increases innovation and com-
petitiveness in the labor market (Barba-Sánchez et al. 2022). Entrepreneurship activ-
ities have been given importance in many Western countries and are also gaining
more attention in developing countries. Many studies have identified entrepreneurial
intention (EI) as one of the most significant predictors of entrepreneurial activities
and behaviors (Krueger et al. 2000; Autio et al. 2001; Arasti et al. 2012). Hence,
the focus of various contemporary research has shifted from entrepreneurship to EI
(Yu et al. 2021). Indeed, the number of studies using EI as a research framework has
increased since the early 90s, confirming the importance of EI aspect in several set-
tings (Liñán and Fayolle 2015). In addition, it is crucial for graduates to eventually
alter their mindset from searching for jobs to creating jobs as a country’s govern-
ment will not be able to ensure sufficient job provision for all tertiary-level graduates
in the future (Reuel Johnmark et al. 2016). University students should shift their
focus towards entrepreneurial revolution (Nuan and Xin 2012; Jiang and Sun 2015). Considering this, it is important to understand the factors that affect the EI of stu-
dents in order to nurture their future entrepreneurialism in their respective countries. There have been several studies conducted by scholars to examine the factors that
impact EI of higher education students. Cognitive and personality factors, such as
self-efficacy, individual attitudes, desire for achievement and behavioral control,
have significant influence on students’ intentions towards entrepreneurship (Nasip
et al. 2017; Shah and Soomro 2017; Biswas and Verma 2021). Social and environ-
mental researchers have identified elements such as prior experience, family back-
ground, regional culture and government support as critical factors that affect EI of
students (Ahamed and Rokhman 2015; Ali et al. 2019; Tiwari et al. 2020). Another
fundamental factor contributing to the formation of students’ EI is entrepreneurial
education. Entrepreneurial education in higher education plays an important role in
enhancing foundational entrepreneurial knowledge and various cognitive and non-
cognitive skills by stimulating students’ entrepreneurial activities (Walter and Block
2016; Brüne and Lutz 2020). This will further motivate students towards entrepre-
neurship, help improve entrepreneurship quality, and lead to entrepreneurial success
(Galloway and Brown 2002). Abstract Entrepreneurship has been viewed as a critical contributor and an economic engine
in a country for creating new jobs and it is crucial for graduates to alter their mind-
set to become self-employed. Thus, it is necessary to synthesize the factors that
impact the entrepreneurial intentions (EI) of students at tertiary level. The aim of
this research is twofold; first to identify the factors which have been most studied in
the literature and second, to determine which factors are less explored to measure
the EI of students. This research adopts the systematic review approach to identify
various studies conducted between 2005 to June 2022. The paper further adopted
citation analysis and identified the 36 most impactful studies in this area of research. Next, the thematic analysis was conducted and seven main themes (factors) (cogni-
tive, personality, environmental, social, educational, contextual and demographic) of
EI determinants were identified.The analysis of the papers clearly demonstrated that
the TPB model and cognitive factors dominate this area of research. Furthermore,
over half of the studies are conducted in Asia, hence it is important to explore other
regions such as Africa, America and Europe and other comparative studies between
various regions. The study offers avenues for future research and practical implica-
tions of the study for the practitioners. Keywords Entrepreneurial intentions · Systematic literature review · Citation
analysis · Thematic analysis · Entrepreneurship Keywords Entrepreneurial intentions · Systematic literature review · Citation
analysis · Thematic analysis · Entrepreneurship JEL Classification A22 · A23 · L26 · O50 456789)
3 1904 G. Maheshwari et al. 1 Introduction Many entrepreneurship models and theories have been
developed to investigate the impact of factors on EI of an individual. Among those
proposed models, most of the papers in this research area used theory of planned
behaviour (TPB) model (as highlighted by analysis from this review discussed next
in the paper) to study the EI of the students. The EI of students is not only affected
by factors from TPB model, but there are various other models affecting the EI of
students as discussed later in the paper. Based on the different models developed on entrepreneurship (as discussed fur-
ther in the paper), there are various factors that affect the entrepreneurial inten-
tions of university students, such as educational factors, contextual factors, envi-
ronmental factors, psychological factors, and personality factors but little work is
done to understand which factors scholars considered the most in measuring the 1 3 3 Factors affecting students’ entrepreneurial intentions:… 1905 entrepreneurial intentions of the university students. This review of literature is
based on the synthesis of papers and will provide an overview of (1) which fac-
tors the scholars have paid the most attention to measure EI of the students and (2)
what factors are understudied in the literature to determine the factors affecting EI of
students.f This study will contribute to the growing body of literature on the factors affect-
ing entrepreneurial intentions of university students by providing theoretical and
practical contributions. The study will be particularly beneficial to researchers work-
ing in this research area as this paper also provides gaps for future research. The
study results would also help educational institutions to support and encourage stu-
dents towards their entrepreneurial intentions and policy makers who will be able to
understand how they can support the development of entrepreneurship activities that
in turn, enhance the economic growth of the country. Despite entrepreneurship being considered a key contributor for sustained growth
and development of countries, and EI being regarded as the dominant influence of
an individual’s entrepreneurship, the non-quantitative studies pertaining to deter-
mine the factors affecting EI have not been paid much attention. There are some
scholars who have conducted systematic reviews of study in this area; for example,
Pittaway and Cope (2007) examined the interface between higher education institu-
tions and business sector. The study by Bae et al. 2.2 The expectancy theory Expectancy Theory (known as Theory of Motivation or the Rational Intention The-
ory) developed by (Vroom (1964) states that conscious choice of an individual to
maximize satisfaction and minimize adversity will lead to a person’s behavior. In the
theory, motivation is defined as a result of an expectancy that greater effort will fos-
ter greater performance, instrumentality refers to the expectation of an individual to
receive a certain outcome when they make the effort, and valence implies the degree
to which the person values the outcome. The Expectancy Theory has been used as
a framework in many studies to explore people’s motivation for becoming entrepre-
neurs (Locke and Baum 2007). The three variables: expectancy, instrumentality, and
valence, were confirmed to increase entrepreneurship motivation, further concluding
that apart from ability and aptitude, motivation could enhance the entrepreneurial
intentions of an individual (Barba-Sánchez and Atienza-Sahuquillo 2017, 2018). 1 Introduction (2014) was regarding a systematic
review of literature in order to find the correlation between entrepreneurial educa-
tion and EI of students. Nabi et al. (2017) provided a systematic review of literature
to study the impact on the EI of students considering entrepreneurial education. Wu
and Wu (2017) systematically reviewed the effect of entrepreneurial education on
the EI of students, particularly in the Asia–Pacific region. It is important to synthe-
size the literature to get the holistic picture and contribute towards this research field
(Kuckertz and Block 2021). Hence, in this paper, the synthesis of factors that impact
the EI of students is carried out and further analyze the extent they have been used
by various scholars in this field. i
This research adopts the systematic review approach, which is important for syn-
thesizing knowledge to identify numerous studies conducted between 2005 and June
2022. The studies on the EI of students have been receiving a lot of attention since
2016 (as determined by the analysis in this paper) and hence it is clearly visible that
this area triggered the researchers’ interest and therefore is important to understand
which factors are considered by different scholars to measure the EI of university
students in the studies conducted so far. The aim of this research is twofold; first to
identify the factors affecting the EI of students which have been studied the most
in literature in previous years (from 2005) across the world. Second, to determine
which factors are less explored in measuring the entrepreneurial intentions of stu-
dents and thus can be explored more in future studies. A clearer perspective regard-
ing various factors affecting EI of university students used by various scholars are
analyzed in this paper. The findings from this paper can support practitioners to
implement policies and take action to promote entrepreneurship in higher education
students, as well as provide insights for further research in the future. Following this brief introduction, the rest of the paper is organized as fol-
lows. Section 2 displays the review of literature on the development of various 1 3 3 1906 G. Maheshwari et al. entrepreneurial models, Sect. 3 explains the research methodology, while Sect. 4
provides the results of the review, the next section corresponds to the discussion of
the paper and the concluding remarks are provided in the last section of the paper. 2.1 The entrepreneurial event model (EEM) The entrepreneurial event model (EEM) proposed by Shapero and Sokol (1982) is
the first model to shed light on entrepreneurial intention theory. According to the
model, the three main determinants that affect an individual’s intention in entre-
preneurship are perceived desirability, perceived feasibility and propensity to act. The perception of desirability towards entrepreneurship implies the engagingness of
entrepreneurial conduct that a person can perceive. Perceived feasibility signifies the
extent that an individual believes they can perform entrepreneurial behavior; and
propensity to act indicates the possibility to become an entrepreneur. The proposed
model also highlights that the “entrepreneurial event” acts as a trigger to determine
behavior towards entrepreneurship of an individual, which can help them to make
the best decision among a range of choices. 2 Review of development of entrepreneurial models There are various models developed and used by various scholars to determine the
entrepreneurial intentions of an individual. 2.5 The entrepreneurial intention model (EIM) The entrepreneurial intention model (EIM) by Boyd and Vozikis (1994) is fur-
ther developed from Bird’s (1988) original model of entrepreneurial intentionality. According to Bird’s model (1988), a person establishes intentions towards entre-
preneurship based on both contextual and personal characteristics. Specifically, the
contextual factors include political, social and economic elements that can shape an
individual’s rational thinking. While personality, ability and background can affect
one’s intuitiveness about starting a business. Based on the original model, EIM of
Boyd and Vozikis (1994) added the self-efficacy factor as an application from social
cognitive theory (Bandura 1977a, b, 1986) to demonstrate its importance in impact-
ing entrepreneurial intentions and behaviors of individuals as well as being inter-
mediary between one’s thoughts regarding entrepreneurship and intentions towards
venture creation. 2.3 The theory of planned behavior model (TPB) The theory of planned behavior (TPB) is advanced from the theory of reasoned
action (TRA) by Ajzen and Fishbein (1980). TRA implies that intentions, which
are shaped by personal attitudes and subjective norms, will govern the actions of
an individual. Regarding the TPB model by Ajzen (1991), the behavior of a person 1 3 Factors affecting students’ entrepreneurial intentions:… 1907 is based on voluntary control and specific planning. TPB defines the three anteced-
ents that shape an individual’s intention, namely attitudes towards behavior (ATB),
social norms (SN), and perceived behavioral control (PBC). ATB implies the posi-
tive or negative perceptions of individuals regarding behavior. SN refers to how
social pressures can influence the performance of a certain behavior. PBC represents
the person’s aspects towards difficulty level of conducting the behavior. Similar to
TRA model, TPB also emphasizes that intention is the direct antecedent of behavior,
and the greater intention will more likely cause behavior to be performed (Ajzen
1991). The study by Barba-Sánchez et al. (2022), considered TPB components in
their study and found that PA and PBC have a direct influence on the EI of students,
while SN does not influence the EI of students directly, but mediate the relationship
between environmental awareness and PA; Environmental awareness and EI. 2.4 The theory of planned behavior entrepreneurial model (TPBEM) Based on TPB, the theory of planned behavior entrepreneurial model (TPBEM) by
Krueger and Carsrud (1993) explains the three factors that impact individuals’ inten-
tions to start a business: attitude towards venture creation, subjective norms and per-
ceived control for entrepreneurial demeanor. 2.6 The social cognitive career theory model (SCCT) Social learning theory (SLT) was first introduced through the Bobo Doll Experiment
in the 1960s by Bandura et al. (1961, 1963), indicating that learning can happen by
observing, imitating, practicing behaviors and encountering consequences of behav-
iors in a social context (Bandura 1977a). The learning procedure is determined by
the association between individuals and the degree of elevating emotional and prac-
tical expertise, defining self-perception and others’ perceptions (Bandura 1977a). Bandura (1977b) adopted the concept of self-efficacy to SLT to demonstrate the cor-
relation between perceived self-efficacy and changes in behavior. Self-efficacy refers 1 3 3 3 1908 G. Maheshwari et al. to a person’s belief in successfully performing a task in a certain situation. The pro-
posed model presents four primary antecedents that develop the personal efficacy
expectations, including performance achievements, vicarious experience, social per-
suasion, and physiological conditions, such as anxiety and arousal. to a person’s belief in successfully performing a task in a certain situation. The pro-
posed model presents four primary antecedents that develop the personal efficacy
expectations, including performance achievements, vicarious experience, social per-
suasion, and physiological conditions, such as anxiety and arousal. Social cognitive theory (SCT) is further developed from the SLT of Bandura,
strengthening the vital role of cognitive components in the social learning process. The self-efficacy factor from his previous studies is also included in SCT as one of
an individual’s behavioral determinants. Moreover, the model presents that human
behavior is shaped by the reciprocal causation among environmental, behavioral,
and cognitive attributes (Bandura 1986). Lent et al. (1994) expanded the social cognitive career theory (SCCT) from the
original SCT of Bandura, studying the decision-making demeanor that involves
career matters. It is denoted by SCCT that career success is affected by cognitive-
related elements, which consists of self-efficacy, expectancy of outcomes and inten-
tions; and a career decision-making procedure is regulated by both personal and
contextual factors. 2.7 Lüthje and Franke model (LFM) Lüthje and Franke Model (LFM) developed by Lüthje and Franke (2003) indicates
the crucial significance of personality traits in explaining self-employment attitudes
and entrepreneurial behaviors besides personal attitudes, social norms, contextual
and economic determinants. Specifically, in the study concerning university’s influ-
ence on the entrepreneurial intentions of students, Lüthje and Franke indicated that
the components of personality traits, including risk-taking propensity and internal
locus of control, have a significant influence on attitude towards entrepreneurship,
hence indirectly impacting the intention to establish a new business. The model also
incorporates perceived support and perceived barriers as contextual factors to deter-
mine the importance in reinforcing an individual’s intentions towards entrepreneur-
ship (Lüthje and Franke 2003). By integrating personal and environmental factors,
this model presents an extended approach to investigate the broad range of anteced-
ents that affect entrepreneurial intentions (Nabi et al. 2010). Factors affecting students’ entrepreneurial intentions:… 1909 Table 1 Sources of database
for all 290 papers (including
influential and non-influential
papers) Sources
Number
of studies
Scopus
267
ProQuest
19
Google Scholar
4 to include relevant articles. The search began using the key words such as “Entrepre-
neurial Intention” AND “determinants” OR “factors” AND “university students”,
which resulted in 387 results (including articles, books, conference papers) in which
342 articles were in English. The third step focused on extracting the articles of our
interest based on the research objective regarding factors influencing EI of students. After reading the abstract of 342 articles, 52 articles did not match our research
objective and hence those were discarded leaving us with 290 final articles relevant
to this study, most of which were found from the Scopus database (Table 1). During
the last step, the articles were analyzed using the average citations received per year
to identify the most influential papers based on the average citation per year of 10 or
above. This resulted in 36 papers (Table 2) and the thematic analysis was conducted
to identify the various themes from these papers regarding factors affecting EI of
students. The rest of the papers (n = 254) (Table in Appendix) were analyzed further
based on the identified themes. The research methodology for this paper is shown in
Fig. 1. 4.1 Region of research The first criteria to analyze the paper was based on the region of research and is
presented in Fig. 2. The analysis of the articles showed that most of the studies
were conducted in Asia (52%), followed by 19% of studies conducted in Europe/UK
region, 12% studies in both America and Africa region and the remaining 6% of the
studies were multi-country studies. 3 Research methodology A systematic review has been conducted for this study and the papers which meas-
ured the entrepreneurial intentions of the university students published from 2005
until June 2022 were analyzed. A four-step review was adopted for this study
(Maheshwari et al. 2021). The first step focused on reviewing articles measuring
the entrepreneurial intentions (EI) of students (from 2005 to June 2022). During the
next step, the data extraction was conducted using various databases such as Scopus,
Emerald, Springer, Taylor and Francis, ProQuest and JSTOR as many highly ranked
journals of educational studies are indexed in these prominent databases and aligned
with publication standards. An additional search was conducted on Google Scholar 1 3 1909
Factors affecting students’ entrepreneurial intentions:…
Table 1 Sources of database
for all 290 papers (including
influential and non-influential
papers)
Sources
Number
of studies
Scopus
267
ProQuest
19
Google Scholar
4 Table 1 Sources of database
for all 290 papers (including
influential and non-influential
papers) 4.4 Citation analysis The next stage of the analysis included the citation analysis to identify the most
influential papers from the total 290 papers identified in the study. The authors ana-
lyzed all 290 final articles and calculated the average citations received per year for
each paper and identified a total of 36 articles as the most influential paper based
on the average citation of 10 or above received per year. These 36 most cited papers
were further analyzed to identify the themes based on content similarities regarding
the factors affecting the entrepreneurial intentions of university students. After read-
ing 36 papers, the authors identified seven main themes (factors) which affect the EI
of the students. The seven themes of this paper were identified based on our holistic
understanding of two criteria (1) the core factors (variables) considered in all the
influential papers (2) the common core factors used by these influential papers to
measure the EI of students. These seven factors are cognitive factors, personality
factors, environmental factors, social factors, educational factors, contextual (situ-
ational) factors and demographic factors. These factors are classified from the 36
papers identified and Table 4 shows these seven themes (factors). i
The Education and Training Journal has published the highest number of papers
(five) identified in this study, followed by four published in International Journal
of Entrepreneurial Behaviour and Research, three in International Entrepreneurship
and Management Journal, two each respectively in Journal of Asian Finance, Eco-
nomics and Business and the Studies in Higher Education. There was one article
each in the rest of the journals. There are 15 studies from Asia, 10 studies conducted
in Europe, six were a collaboration between multiple countries, four in America and
one from Africa. 19 out of 36 studies have used the TPB model whereas seven stud-
ies have combined the TPB model with other models (as per Table 2). 4.2 Year of research Next, the articles were analyzed to determine the number of studies conducted in
different years. It was found that very few studies had been conducted until 2016
(n = 50). Most of the studies were conducted after 2016 (n = 240). This indicates the
growing interest of scholars in this area since 2017 (as in Fig. 3). 1 3 1910 G. Maheshwari et al. 4.3 Research methods used in the studies Out of the 290 studies conducted between 2005 and June 2022, most of the studies
(n = 285) used quantitative research methodology as seen in Table 3 and three stud-
ies used qualitative methodology. One study used mixed methodology while another
study was a synthesis of literature. This clearly indicates that quantitative research
methodology dominates this field of research and there is a need to use qualitative or
mixed methodology approaches in this research field (Table 3). 4.5 Themes (categories) identification from influential papers The 36 most cited papers were further analyzed to identify the themes used in these
papers following Fitz-Koch et al. (2018) research and it was found that most of these
studies revolved around seven categories (themes). The summary of the factors
under each category (theme) are summarized in Table 4. These seven identified
categories (themes) used in various influential papers are discussed next in detail. 1 3 Factors affecting students’ entrepreneurial intentions:… 1911 Table 2 List of most influential papers (n = 36)
Region
Average citation per year
Name of authors
Name of journals
Database
Model used
America
80
(Zhao et al. 2005)
Journal of Applied
Psychology
Scopus
SCT
Mix regions
54
(Iakovleva et al. 2011)
Education and Training
Scopus
TPB
Europe
51
(Nowiński et al. 2019)
Studies in Higher
Education
Scopus
TPB; EEM; SCT
Europe
44
(Hockerts 2017)
Entrepreneurship: Theory
and Practice
Scopus
TPB
Europe
38
(Maresch et al. 2016)
Technological forecasting
& social change
Scopus
TPB
Mix regions
35
(Gurel et al. 2010)
Annals of Tourism Research
Scopus
Studies of Learned (1992)
and TPBEM
Asia
34
(Zhang et al. 2014)
International
Entrepreneurship and Man-
agement Journal
Scopus
TPB; EEM
Europe
26
(Kuckertz and Wagner
2010)
Journal of Business Venturing
Scopus
Sustainable entrepreneur-
ship
Asia
26
(Turker and Selcuk 2009)
Journal of European Industrial
Training
Scopus
Entrepreneurial support
model (ESM)
Asia
26
(Al-Jubari et al. 2019)
International
Entrepreneurship and Man-
agement Journal
Scopus
TPB; SDT (self-determina-
tion theory)
Europe
20
(Iglesias-Sánchez et al. 2016)
Education and Training
Scopus
TPB
Mix regions
18
(Pruett et al. 2009)
International Journal of
Entrepreneurial
Behaviour & Research
Scopus
None
Europe
18
(Mirjana et al. 2018)
Economic Research-Ekonomska
Istrazivanja
Scopus
TPB 3 1912 G. Maheshwari et al. 1 3
(
)
Region
Average citation per year
Name of authors
Name of journals
Database
Model used
Mix regions
15
(Fragoso et al. 2020)
Journal of Small Business and
Entrepreneurship
Scopus
TPB
Asia
15
(Wu and Wu 2008)
Journal of Small Business and
Enterprise Development
Scopus
TPB
Europe
15
(Solesvik 2013)
Education and Training
Scopus
TPB
Asia
14
(Nguyen et al. 2019)
Children and youth services
review
Scopus
TPB
Asia
13
(Karimi et al. 2017)
International Journal of Psy-
chology
Scopus
TPB
Europe
12
(Maes et al. 2014)
European Management Journal
Scopus
TPB
Asia
12
(Zhang et al. 4.5 Themes (categories) identification from influential papers 1914 Research
Methodlogy
Step 1
Review of Existing
literature from 2005
- 2022
Step 2
Data extraction
based on key
words
Step 3
Extraction of
relevant studies
Step 4
Thematic
analysis and
further analysis Research
Methodlogy
Step 1
Review of Existing
literature from 2005
- 2022
Step 2
Data extraction
based on key
words
Step 3
Extraction of
relevant studies
Step 4
Thematic
analysis and
further analysis
Fig. 1 Research Methodology (adapted from Maheshwari et al. (2021)) Step 2
Data extraction
based on key
words Step 1
Review of Existing
literature from 2005
- 2022 Step 3
Extraction of
relevant studies Research
Methodlogy Fig. 1 Research Methodology (adapted from Maheshwari et al. (2021)) 52%
12%
12%
19%
6%
Region of research
Asia
Africa
America
Europe/UK
Mix regions including Middle East
and Australia/New Zealand
Fig. 2 Region of research Region of research Fig. 2 Region of research 4.5 Themes (categories) identification from influential papers 2020)
Journal of Hospitality, Leisure,
Sport and Tourism Education
Scopus
Social learning theory;
Positive psychology
theory
Africa
12
(Hattab 2014)
Journal of Entrepreneurship
Scopus
TPB; EEM
Europe
12
(Arranz et al. 2017)
Studies in Higher Education
Scopus
TPB
Asia
11
(Mustafa et al. 2016)
Journal of Entrepreneurship in
Emerging Economies
Scopus
LFM
Asia
11
(Sharahiley 2020)
Global Journal of Flexible
Systems Management
Scopus
TPB; EEM
Asia
11
(Tung et al. 2020)
The international journal of
management education
Scopus
Theory of reasoned action,
TPB, EEM and model of
entrepreneurial potential
Asia
11
(Sesen 2013)
Education and Training
Scopus
LFM
Europe
11
(Solesvik et al. 2014)
Education and Training
Scopus
Entrepreneurial event
theory, cultural values
theory and human capital
theory Factors affecting students’ entrepreneurial intentions:… 1913 f
g
p
(
)
Region
Average citation per year
Name of authors
Name of journals
Database
Model used
Mix regions
11
(Gieure et al. 2019)
International Journal of Entre-
preneurial Behaviour and
Research
Scopus
TPB
Asia
11
(Liu et al. 2019)
Frontiers in Psychology
Scopus
TPB
Asia
10
(Akhter et al. 2020)
Journal of Asian Finance, Eco-
nomics and Business
Scopus
TPB
Mix regions
10
(Giacomin et al. 2011)
International Entrepreneurship
and Management Journal
Scopus
None
America
10
(Martins and Perez 2020)
International Journal of Entre-
preneurial Behaviour and
Research
Scopus
TPB; EEM
America
10
(Zhang and Cain 2017)
International Journal of Entre-
preneurial Behaviour and
Research
Scopus
TPB
America
10
(Zhang et al. 2015)
Entrepreneurship Research
Journal
Scopus
TPB
Asia
10
(Luc 2018)
Journal of Asian Finance, Eco-
nomics and Business
Scopus
TPB
Asia
10
(Utami 2017)
European Research Studies
Journal
Scopus
TPB 1 3 1914
G. Maheshwari et al. 4.5.1 Cognitive factors
4.5.1.1 TPB factors The effect of TPB antecedents—attitudes, social norms and per-
Research
Methodlogy
Step 1
Review of Existing
literature from 2005
- 2022
Step 2
Data extraction
based on key
words
Step 3
Extraction of
relevant studies
Step 4
Thematic
analysis and
further analysis
Fig. 1 Research Methodology (adapted from Maheshwari et al. (2021))
52%
12%
12%
19%
6%
Region of research
Asia
Africa
America
Europe/UK
Mix regions including Middle East
and Australia/New Zealand
Fig. 2 Region of research G. Maheshwari et al. 4.5.1 Cognitive factors 4.5.1.1 TPB factors The effect of TPB antecedents—attitudes, social norms and per-
ceived behavioral control—on entrepreneurial intentions is discussed in many stud-
ies, including Solesvik (2013), Zhang et al. (2015), Iglesias-Sánchez et al. (2016),
Karimi et al. (2017), Mirjana et al. (2018), Al-Jubari et al. (2019). In research by Wu
and Wu (2008), personal attitude is the critical influence of entrepreneurial intentions,
regardless of the educational backgrounds of students. The three antecedents were
proven to have a direct and significant impact on entrepreneurial intentions in the 1 3 1 Factors affecting students’ entrepreneurial intentions:… 1915 1
1
2
3
3
6
3
14
7
10
30
17
50
58
52
33
0
10
20
30
40
50
60
2005 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022
Number of studies
Year
Year of study
Fig. 3 Year of study
Table 3 Research methods used in the studies
Research methods used
Number of studies
Quantitative
285
Qualitative
3
Mixed methodology/Literature review
2 study of Solesvik (2013) and Utami (2017). Furthermore, perceived entrepreneurial
motivation, which was enhanced among students who joined an enterprise education
program, stimulated entrepreneurial intentions through the mediating effect of the
three TPB factors (Solesvik, 2013). (
)
Zhang et al. (2015) proved that social norms and perceived behavior control
relate significantly to entrepreneurial intentions of students, and controlled behavior
generates greater impact on the intentions than social norms do. However, no rela-
tionship is found between attitudes and the desire to start a business, considering the
lack of experience towards entrepreneurship among undergraduate students. In the
research of Iglesias-Sánchez et al. (2016), PA and PBC have a significant impact on
students’ intentions to start ventures, while SN is not a determining component but
only demonstrates a decision-making role. In line with other studies, Karimi et al. (2017) confirmed the significant impact of TPB antecedents on Iranian students’
EI. Among the three predictors, PBC shows the strongest relationship with inten-
tions to start a business while SN demonstrates the weakest. Maresch et al. (2016)
highlighted a negative relationship between subjective norms and entrepreneurial
intentions for science and engineering students. By applying TPB to investigate
entrepreneurial intentions of students between developing and developed countries,
Iakovleva et al. (2011) showed stronger entrepreneurial intentions with higher atti-
tudes, SN and PBC in developing countries. 4.5.2 Personality factors According to Zhao et al. (2005), people having higher risk propensity will be more
confident confronting risky situations and viewing uncertain circumstances as less
risky than other individuals. Considering that, they might feel less anxious to take
on entrepreneurial occupation, fulfill the position and complete the tasks more com-
fortably, thus having higher entrepreneurial self-efficacy. This impact is also justi-
fied by the findings of the study, stating that self-efficacy plays a mediating role in
the relationship between risk propensity and intentions towards entrepreneurship of
students.fi Many studies found that self-efficacy and intentions towards entrepreneurship
of students have a positively significant relationship, such as Guzmán-Alfonso and
Guzmán-Cuevas (2012), Sesen (2013), Utami (2017), Zhang and Cain (2017). The
research of Gurel et al. (2010) indicates that innovativeness and risk-taking propen-
sity play a significant role as a predictor for entrepreneurial intentions of tourism
students in the UK and Turkey, while tolerance of ambiguity and locus of control do
not relate to the intentions towards entrepreneurship. Additionally, education does
not demonstrate the moderating impact in the relationship between those entrepre-
neurial traits and intentions of tourism students. On the other hand, locus of con-
trol shows significant influence on entrepreneurial intentions in the study of Sesen
(2013). The study also indicates that personality factors are more significant towards
the entrepreneurial intentions of students than environmental predictors such as
access to capital and social networks. Zhang et al. (2015) also stated that short-term risk-taking preference and factors
of psychological well-being positively influence the intentions towards entrepre-
neurship of an individual with the existence of TPB antecedents, namely personal
attitudes, social norms and perceived behavioral control. The study of Zhang and
Cain (2017) pointed out that there is an absence of a direct effect of risk aversion
on intentions to start a business in dental school students. Risk aversion only dimin-
ishes entrepreneurial intentions indirectly via antecedents of TPB. The impact stated
that risk aversion might not be a fixed dispositional factor, but an adjustable trait
that can be altered over time. The study of Mustafa et al. (2016) found that entre-
preneurial intentions are positively impacted by a proactive personality and the per-
ceived university support of Malaysian students. Among the two drivers, proactive
personalities have a stronger influence on intentions towards self-employment than
from the perceived concept of student development support from students. Karimi
et al. 4.5.1 Cognitive factors The result of the study indicated that
SN significantly impacted entrepreneurial intentions of students, which is different 1 3 G. Maheshwari et al. 1916 from some past papers that found no significance in the relationship between SN and
Table 4 Themes from influential papers
Themes (in italics) and relevant factors in particular theme (non-
italicized)
Cognitive factors
Cognitive styles
Self-efficacy
Personal attitude (PA)
Perceived desirability
Subjective norm (SN)
Perceived feasibility
Perceived behavioral control (PBC)
Personality factors
Risk propensity
Risk aversion
Internal locus of control
Intrinsic motivation
Creativity
Extrinsic motivation
Tolerance of ambiguity
Creativity
Leadership styles
Environmental factors
Government support
Physical infrastructure
Access to capital
Culture
Human resources
Institutional infrastructure
Social factors
Prior experience
Entrepreneurial family
Role models
Culture/Country
Life experiences
Educational factors
Entrepreneurial environment of a university
Curriculum
Entrepreneurial courses
Extracurricular activities
Contextual (situational) factors
Unemployment
Social status
Expected value of starting a business
Perception of support and barriers
Job dissatisfaction
Need for satisfaction
Opportunities
Desire for independence
Demographic factors
Gender
Age
Nationality
Educational level
Major Age from some past papers that found no significance in the relationship between SN and
intentions towards entrepreneurship (Chen et al. 1998; Wu and Wu 2008). from some past papers that found no significance in the relationship between SN and
intentions towards entrepreneurship (Chen et al. 1998; Wu and Wu 2008). 4.5.1.2 Other factors The study of Zhang et al. (2014) identified that entrepreneurial
intentions are significantly influenced by perceived desirability, but not by perceived
feasibility. This is explainable by negative environmental elements of perceived
behavioral control, uncertain locus of control and environmental controls due to lack
of prior experiences. Solesvik et al. (2014) stated that in a transitional economy con-
text, students that have perceived desirability and perceived feasibility have greater 1 3 Factors affecting students’ entrepreneurial intentions:… 1917 entrepreneurial intentions. Moreover, the study found that students who take initiative
have greater intentions to start a business, while students with low capability beliefs
yield lower entrepreneurial intentions. The research of Mirjana et al. (2018) also
highlighted the crucial relationship between innovative cognitive style and students’
intentions to become entrepreneurs. However, innovative cognitive style inconsider-
ably influences entrepreneurial intentions when the factor is considered as a solely
explanatory component. 4.5.3 Environmental factors In the study, Pruett et al. (2009) indicated that the participants’ country and cultures,
exposure to acquaintance entrepreneurs in family, family support, and the strength
of beliefs towards motives for entrepreneurship positively impact entrepreneurial
intentions. However, the significance of those variables is smaller than the effect of
entrepreneurial disposition, implying that factors related to personal characteristics
are more influential on the intentions towards self-employment. In addition, entre-
preneurial intentions are negatively affected by social barriers, which involve operat-
ing risks, or lack of knowledge and capital. But the impact is also relatively small as
compared to the disposition factor.i According to Turker and Selcuk (2009), structural support shows significant
impact on entrepreneurial intentions, stating that a greater comprehensive support
from all social sectors is required to stimulate entrepreneurship in young people. In
addition, the effect of self-confidence as a moderating component is more consid-
erable in the connection between structural support and entrepreneurial intentions. However, the study demonstrates that perceived relational support, such as monetary
and sentimental assistance from family and friends, does not influence the intention
to establish a business for students. Access capital is proven to have a negative and
significant correlation to entrepreneurial intentions of students in the study of Sesen
(2013). 4.5.2 Personality factors (2017) stated that personality factors, including the need for achievement, risk 1 3 1918 G. Maheshwari et al. taking and locus of control, indirectly affect the intentions towards entrepreneurship
of Iranian students through the entrepreneurial attitudes and PBC. taking and locus of control, indirectly affect the intentions towards entrepreneurship
of Iranian students through the entrepreneurial attitudes and PBC. In the paper of Al-Jubari et al. (2019), intrinsic and extrinsic motivations, which
are generated from psychological needs of autonomy, relatedness and competence,
are indicated to have a positive indirect effect on entrepreneurial intentions in Malay-
sian students via the three components of TPB. More importantly, the motivation
types play important roles in the entrepreneurial process, in which each kind yields
different impacts. Entrepreneurs with intrinsic motivations will demonstrate more
effective performances, more persistence and greater autonomy, leading to dynamic
entrepreneurs. Meanwhile, individuals with extrinsic motivations will be less persis-
tent when confronting challenges, more likely to discontinue nascent behaviors, and
concentrate on external achievements. In addition, the need for satisfaction and the
need for frustration are also proven to be negatively correlated. 4.5.4 Social factors Prior entrepreneurial experience, which provides students a role model and enac-
tive proficiency from empirical exposure, is proven to be strongly mediated by self-
efficacy, thus further affecting entrepreneurial intentions (Zhao et al. 2005). How-
ever, prior entrepreneurial exposure shows a notable negative impact on intentions
towards entrepreneurship in the research of Zhang et al. (2014). The reason for
this unexpected result is that the participants in the study mostly underwent nega-
tive entrepreneurial experiences, thus raising the fears and insecurities towards self-
employment. The proposed model of Kuckertz and Wagner (2010) indicated the 1 3 Factors affecting students’ entrepreneurial intentions:… 1919 orientation of an individuals’ sustainability is meaningful in the relationship with
their entrepreneurial intentions. Nevertheless, while significant potential is found
among students for entrepreneurship opportunities with sustainable orientation, it
declines as business experience is achieved. Gurel et al. (2010) included social factors into the research model to explain the
effect on entrepreneurial intentions of tourism students. Among those elements, stu-
dents with entrepreneurial families tend to have higher intentions towards entrepre-
neurship. Conversely, cultural factors are considered when there is concern in the
probability of self-employment, instead of entrepreneurial intention. The findings of
Hockerts (2017) showed that prior experience with social organizations can increase
social entrepreneurial intentions of an individual. The relationship is mediated by
factors including empathy, self-efficacy, moral obligation, and perceived social sup-
port. The self-efficacy variable holds the greatest effect. Furthermore, the study pro-
vided compelling evidence that the number of social entrepreneurship electives by
students is predicted by entrepreneurial intentions. 4.5.5 Educational factors Moreover, the research demonstrates different influences of curricular activi-
ties on the entrepreneurial competences of students in two institutions, suggesting
the development in educational methodology and strategies to enhance their compe-
tences. There are a substantial number of studies indicating the positive connection
between entrepreneurship education and entrepreneurial intention of students (Hat-
tab 2014; Zhang et al. 2014; Maresch et al. 2016; Nowiński et al. 2019). Research
from Zhang et al. (2014) shows a direct relationship between entrepreneurship
education and entrepreneurial intentions. Also, gender, study majors and univer-
sity types have considerable positive interactive impacts on the correlation between
entrepreneurship education and intentions to run a business in the context of Chi-
nese university students. The findings of Hattab (2014) and Maresch et al. (2016)
are also in line regarding the positive impact of entrepreneurship education towards
entrepreneurial intentions of students. It is noteworthy that students in business
majors may benefit more from entrepreneurship education than those in science and
engineering programs. In the study of Hattab (2014), education is indicated to posi-
tively influence perceived desirability towards entrepreneurship of students, while
the effect is less significant on perceived feasibility and inconsiderable on students’
self-efficacy. The positive significant relationship between entrepreneurship educa-
tion and entrepreneurial intentions, mediated by self-efficacy, is also confirmed in
the study of Nowiński et al. (2019). Among Visegrad countries, the correlation is
considerable only in Poland where entrepreneurship education is introduced in high
school. Another important finding is that despite lower levels of self-efficacy and
intentions towards entrepreneurship in female students, they may benefit more from
entrepreneurship education than males do. However, some studies found no rela-
tionship between university environment and entrepreneurial intentions of students,
including the work of Sesen (2013), Chen et al. (2015).fi According to Chen et al. (2015), entrepreneurship courses increase learning effi-
cacy and satisfaction of technical undergraduate students, but do not enhance the
intentions of students to start a business. The study implies that entrepreneurship
courses provided by universities would help students to recognize that entrepreneur-
ial occupations might not be suitable for them and rather to implement what they
learned to future jobs rather than pursue entrepreneurship. Solesvik et al. 4.5.5 Educational factors The perceptions of formal learning from students does not directly influence entre-
preneurial intentions but demonstrates a strong indirect impact on the decision of
an individual to start a business via a mediating factor—self-efficacy. Supporting
the idea that students’ intentions towards entrepreneurial venture creation can be
shaped by formal academic courses. The study of Zhao et al. (2005) suggests educa-
tional institutions integrate distinct types of learning ways to improve entrepreneur-
ial self-efficacy in students. Entrepreneurial intentions of students are proven to be
impacted at educational level through personal attitude effects and academic majors
through both attitudes and the PBC of individuals. However, no relationship is found
between academic accomplishment and PBC; and entrepreneurship education cur-
riculums have little to no impact on the entrepreneurial desires of students (Wu and
Wu 2008). One highly influential paper by Walter and Block (2016) identified the
effects of entrepreneurship education is higher on the EI of students in entrepre-
neurship-hostile institutional environments than in entrepreneurship-friendly institu-
tional environments. Although the paper was highly cited, we could not include it in
this list because it was based on macrolevel educational factors, whereas this study
is based on microlevel educational factors. The result from the study of Turker and Selcuk (2009) indicates that educational
support significantly impacts the entrepreneurial intentions of students in Turkey. As specified by the paper, an educational institution delivering sufficient knowledge
and motivation towards entrepreneurship to students will enhance the likelihood
of young people being involved in venture creation, thus suggesting universities
to develop educational policies and structures to effectively inspire entrepreneurs. Nevertheless, self-confidence, which is a moderator in the model, was proven to
not strengthen the relationship between educational support and intentions towards
entrepreneurship. Moreover, according to the article, the educational factor has
greater beta co-efficiency than structural support factor, the former variable is a
more influential predictor to entrepreneurial intentions than the latter. This can be 1 3 3 1920 G. Maheshwari et al. explained that students might perceive educational support as an immediate factor,
thus having more awareness towards this support (Turker and Selcuk 2009). According to the study of Arranz et al. (2017), although curricular and extra-
curricular activities have a positive impact on attitudes towards entrepreneurship,
they might reduce capacity and intention to engage in the start-up activities of stu-
dents. 4.5.6 Contextual (situational) factors According to Guzmán-Alfonso and Guzmán-Cuevas (2012) perceived social value
and entrepreneurial intentions have a significantly negative relationship for people
aged 18–64 in Latin America, which is not in line with Ajzen’s model. The study of
Karimi et al. (2017) indicated that perceived contextual support and barriers have
an indirect impact on entrepreneurial intentions of students via PBC; and perceived
barriers are also found to have a direct, negative association with the intentions
towards start-up. The entrepreneurial intentions were found to be positively related
to perception of motives such as creativity and desire for independence while per-
ceptions of barriers were negatively impacting the entrepreneurial intentions of the
students as mentioned by Pruett et al. (2009) in their study. 4.5.5 Educational factors (2014)
proved that although the relationship between students joining in entrepreneurship-
specific education and high intensity of entrepreneurial intentions is positive, the
interactions of entrepreneurship-specific education with perceived desirability, per-
ceived feasibility, and perceived cultural elements are not related to greater entrepre-
neurial intentions. 1 3 1 3 Factors affecting students’ entrepreneurial intentions:… 1921 Fig. 4 Mind map showing most common factors in (n = 36) most influential papers. Note: Mind map
reading; for example: EI affected by cognitive factors (3 studies) (on left of EI box); EI affected by cogni-
tive factors and personality factors (3 studies) (on left of EI box); EI affected by cognitive, personality
and environmental factors (1 study) (on left of EI box); EI affected by cognitive factors, environmental
factors, demographic factors (1 study) (on left of EI box); EI affected by only education factors and con-
textual factors (1 study) (on right of EI box); EI affected by personality factors and educational factor (1
study) (on right of EI box) Fig. 4 Mind map showing most common factors in (n = 36) most influential papers. Note: Mind map
reading; for example: EI affected by cognitive factors (3 studies) (on left of EI box); EI affected by cogni-
tive factors and personality factors (3 studies) (on left of EI box); EI affected by cognitive, personality
and environmental factors (1 study) (on left of EI box); EI affected by cognitive factors, environmental
factors, demographic factors (1 study) (on left of EI box); EI affected by only education factors and con-
textual factors (1 study) (on right of EI box); EI affected by personality factors and educational factor (1
study) (on right of EI box) Fig. 4 Mind map showing most common factors in (n = 36) most influential papers. Note: Mind map
reading; for example: EI affected by cognitive factors (3 studies) (on left of EI box); EI affected by cogni-
tive factors and personality factors (3 studies) (on left of EI box); EI affected by cognitive, personality
and environmental factors (1 study) (on left of EI box); EI affected by cognitive factors, environmental
factors, demographic factors (1 study) (on left of EI box); EI affected by only education factors and con-
textual factors (1 study) (on right of EI box); EI affected by personality factors and educational factor (1
study) (on right of EI box) 4.5.7 Demographic factors 4.5.7.1 Gender differences Gender was proven to have a direct relationship with
entrepreneurial intentions in the study of Zhao et al. (2005), in which females show
lower intentions to start a business than males. However, the research indicates that
there is no difference between the two genders regarding entrepreneurial self-effi-
cacy, suggesting that the connection between sexuality and entrepreneurial intentions
is shaped by theoretical mechanisms such as perceived social supports and barriers,
and outcome expectations, rather than self-efficacy. Empirical results from Zhang
et al. (2014) state that women and people from universities and backgrounds, other
than technology, have lower entrepreneurial intentions than men and people from
technological ones, suggesting eliminating the traditional entrepreneurial stereotypes
among females and increase the need for additional women role models. Further-
more, the research of Maes et al. (2014) investigated the differences between females 1 3 3 3 1922 G. Maheshwari et al. Fig. 5 Mind map showing most common factors in n = 254 papers (non-influential papers) Fig. 5 Mind map showing most common factors in n = 254 papers (non-influential papers) and males in the elements that predict entrepreneurial intentions of graduate stu-
dents in Belgium. Females tend to be driven to self-employment by the motivation
of getting organized and in consideration of their own personal abilities, while males
will base their entrepreneurial intentions on financial restraints and creativity while
perceiving entrepreneurship as a means of getting ahead. In addition, females prefer
to comply with normative role models than males; however, the gender impact on
entrepreneurial intentions is not mediated by social norms. 4.5.7.2 Nationality differences The study of Giacomin et al. (2011) determined the
national differences in entrepreneurial intentions, motivations, and perceived barriers
towards venture creation dispositions of students in American, Asian, and European
countries. Specifically, the strongest entrepreneurial intentions are found in Spanish
students, while interests towards public administration occupations are identified in
Chinese students. Furthermore, despite similarly perceived motivations and barriers
to self-employment, students in those countries show different sensitivity levels and
significant extents to each motivator and barrier, which can be explained by the socio-
economic factors of each nation. Regarding students in Beira Interior region (Portugal) and Extremadura region
(Spain), there is evidence that some differences exist in perceived desirability, per-
ceived feasibility, and entrepreneurial intentions among the countries according to
the study of Díaz-Casero et al. (2012). 1 3 4.5.7 Demographic factors University students in the two nations have
positive perceptions towards the desirability of entrepreneurship. Concerning per-
ceived feasibility, Spanish students found it easier to start a business in the present
than in the past, while this was not true for Portuguese students. Moreover, students
in the Extremadura region have higher intentions to start a business than those in
the Beira Interior region; nevertheless, the seriousness of entrepreneurial intentions 1 3 Factors affecting students’ entrepreneurial intentions:… 1923 is higher in Portuguese students compared to individuals in Spain. Additionally,
the influences of gender and entrepreneurial family members on the perceptions of
students in the two countries are also investigated, in which sexuality impacts the
perceived intentions of students. Yet no influence is found between gender, family
backgrounds and perceived feasibility in both groups. 4.6 Further analysis of themes from influential papers To visually present the themes clearly, the mind map has been used (as in Fig. 4) to
summarize as what factors are most commonly considered in these 36 most influ-
ential papers and the analysis suggests that cognitive factors are most commonly
used (three studies used them as only factors affecting entrepreneurial intentions) or
cognitive factors used with personality factors (in three studies) or cognitive factors
with educational factors (used in five studies) or cognitive factors used with envi-
ronmental factors, demographic factors and contextual factors respectively in one,
one and two studies. This shows that most of the papers (15 out of 36) have used
cognitive factors as only factors or combined with other factors affecting EI of stu-
dents. Personality factors, environmental factors or social factors are never consid-
ered alone in these most cited papers but used in combination with other factors and
that too are not very widely used. Other factors, such as social factors, educational
factors, contextual factors, and environmental factors, are rarely used as single fac-
tors to measure the EI of students.i Based on these identified seven-factors themes, the rest of the 254 papers were
also grouped in similar way as seen in Fig. 5. This analysis also indicates that the
cognitive factors are used as a single factor in 61 studies, followed by a combina-
tion of cognitive and personality factors by 20 studies, cognitive and educational
factors by 22 studies, cognitive and contextual factors by 11 studies and cognitive
factors with social, environmental, demographic factors considered by respectively
seven, three and two studies. Overall, 161 studies used cognitive factors (single or in
combination with other six factors), followed by 43 studies which used personality
factors alone or in combination with the rest of the five factors. The pattern found in
the rest of the papers are like the ones used by most cited papers except that in these
papers, the personality factors are explored in more detail. 5 Discussion Entrepreneurship studies have arisen rapidly since the published works of Shapero
40 years ago (Shapero and Sokol 1982; Shapero 1984), many research papers have
focused on EI, which is considered to have the greatest influence on entrepreneur-
ship activities. This paper systematically analyzed various publications regarding
the EI of students during the period 2005 till June 2022 and discusses the most and
common studied factors in past research and how future studies can further explore
this research area. The paper adopted citation analysis, which is a prominent method, that recog-
nizes the 36 most impactful studies in this research area over the period considered
in this study. From the analysis, seven main themes (categories) of EI determinants
were identified and implemented further in the paper’s analysis framework to ana-
lyze the rest of the articles (n = 254) used in this study. The analysis on several fac-
tors from 254 studies is briefly included in this review paper and indicated similar
results as found in most influential papers, which suggests that the cognitive factor is
most used factor in many papers which influences the EI of students. 4.7 Conceptual model variables such as gender, university type, university locality, nationality, major, age,
work experience, and exposure to entrepreneurial activities, family income, marital
status, and entrepreneurial family background. 4.7 Conceptual model Based on the synthesis of literature, we propose the integrated conceptual model of
the variables that affects the entrepreneurial intentions of the students according to
the extensive coding of 290 papers. The first important finding is that the independ-
ent variables used to measure the entrepreneurial intentions of university students
are captured in the literature at seven levels: cognitive factors, personality factors,
environmental factors, social factors, educational factors, contextual factors and
demographic factors. Most of the papers have used cognitive factors (TPB model,
EEM, EIM) as an independent variable and TPB components are used as mediators/
moderators to measure the EI of students. 1 3 G. Maheshwari et al. 1924 Fig. 6 Conceptual model (Factors influencing EI) Fig. 6 Conceptual model (Factors influencing EI) The second important finding that the model depicts is that there are a wide range
of mediators and moderators used to measure the EI of students. The mediators and
moderators are also measured at the same seven levels as mentioned for independ-
ent variables. In total, 130 studies used mediators and 61 scholars used moderators
in their study. Most of the studies have used TPB components as mediator, while
moderators used are mostly related to demographic factors, contextual factors, and
personality factors. Other than TPB factors, examples of mediators used as cognitive factors from
entrepreneurial event model are perceived desirability, perceived feasibility, and
entrepreneurial self-efficacy. Other mediators used are risk propensity, need for
achievement, and locus of control which are derived from Bandura’s social cogni-
tive theory and are related to personality factors. The next level of mediators used
are at a social level such as the family influence, social exposure, country culture,
and prior experience. The next majorly used mediators are related to situational
factors as shown in conceptual model, followed by educational factors, and demo-
graphic factors. Only 21% of scholars in their studies have used moderators. The
moderators also consist of seven identified factors (see Fig. 6). Some of the schol-
ars have also used control variables in their study which are mostly demographic 1 3 Factors affecting students’ entrepreneurial intentions:… 1925 variables such as gender, university type, university locality, nationality, major, age,
work experience, and exposure to entrepreneurial activities, family income, marital
status, and entrepreneurial family background. 5.1 Theoretical implications and suggestions on future research From a theoretical perspective, the literature review aims to identify the factors most
studied by past scholars that have an impact on the EI of university students. The
study contributes to the educational entrepreneurship research literature, which will
help higher educational institutions to understand which significant factors stimu-
late students’ intentions to start a business. The analysis of the paper clearly dem-
onstrated that TPB model and cognitive factors dominate this area of research, and
most studies are found to be conducted in Asian countries. Hence, based on the
analysis of papers, this study discusses further steps for entrepreneurship research
to better understand EI of university students and offers the following suggestions as
below and as summarized in the conceptual model for future research (Fig. 6).f It is clear that to improve the understanding of factors affecting entrepreneurial
intentions, future research should consider moving from cognitive factors using the
TPB model. It is important to access the role of social factors including the family
background and culture, contextual (situational) factors as to what forces an indi-
vidual to become an entrepreneur, and this may shed further light on the EI of stu-
dents. These combined factors will help in bringing out a holistic overview of fac-
tors affecting students EI. Cognitive factors have been extensively used in literature
and there is a need to identify other important factors which affect the EI of students. Next, the culture of the country can have a great influence on the intentions of the
students which has been less explored in the studies and hence conducting research 1 3 3 1926 G. Maheshwari et al. considering the socio-cultural factors in different countries might help in providing
multi-faceted views in terms of entrepreneurial intentions. It can also be of interest
for the scholars to further explore the demographic factors as very few studies have
considered them to determine the EI of students. The outbreak of COVID-19 has
affected entrepreneurship (Yu et al. 2021), and hence future research may consider
the impact of the pandemic, such as online education, macroeconomic factors, etc.,
on EI of university students. In the digital transformation economy, technological entrepreneurship started to
grab the attention of many countries, especially developing countries (Nathani and
Dwivedi 2019). The perception towards technology growth can influence students’
intentions to start new ventures. 5.1 Theoretical implications and suggestions on future research Future research can study the technological entre-
preneurship by investigating how combined factors such as environmental factors
(e.g., access to technology), contextual factors (e.g., perception of recent economy/
market, perception of government support, opportunities), and social factors (e.g.,
prior experiences, role models) affect the EI of university students, from which many
implications can be undertaken to benefit the young people’s entrepreneurial activi-
ties. Most of the studies provided comparisons on EI considering different nation-
alities or genders. Furthermore, to enhance understanding in the EI research area,
comparison can be provided considering other demographic factors, for instance,
online versus physical educational environment, individuals with disabilities ver-
sus the ones without disabilities. Most of the studies have focused on theory of the
planned behaviour model and hence it might be important in the future to combine
different EI models to explore and broaden the scope of literature which will further
add value and contribute to the already existing literature. More than half of the studies are conducted in Asia and hence it is important to
explore other less explored regions such as Africa, America, and Europe to deter-
mine if there are any differences in factors affecting the EI of students in these
developed and developing regions. As identified by the review, the entire literature
in this field is dominated by quantitative studies and hence to provide more robust
results, mixed studies using quantitative and qualitative should be conducted in the
future. Future research may benefit from EI determinants in the context of higher
education to conduct studies on different developmental phases, such as primary and
secondary school. In the study of Brüne and Lutz (2020), entrepreneurship educa-
tion had greater positive influence on the self-efficacy and entrepreneurial desirabil-
ity of younger students than older ones. Scholars can examine the effect of early
exposure to entrepreneurship on the development of factors that will further impact
EI of students. The research of Barth and Muehlfeld (2021) found that the interven-
tions in early entrepreneurship enhanced the entrepreneurial self-efficacy of univer-
sity students. 5.2 Practical implications There are some critical implications based on the analysis from this paper for var-
ious stakeholders. The systematic review can be useful for the scholars who aim
to conduct research in EI of students in the future and this review will inspire and 1 3 Factors affecting students’ entrepreneurial intentions:… 1927 motivate the scholars to determine the novel research framework based on the
insights provided from this systematic review which can help the community in gen-
eral. Policy practitioners can implement relevant policies and provide appropriate
support to enhance the EI of individuals and to provide an environment to build
an entrepreneurship culture in their country. Educational institutions and teachers
can find ways to inspire the entrepreneurial intentions in the students by enhanc-
ing course curriculums, developing applicable skills and knowledge, encouraging
ideas, and boosting self-confidence in students and helping them to develop overall. Entrepreneurial intentions are often used as a proxy for behavior, but entrepreneurial
intentions rarely convert into entrepreneurial action, particularly among students
who have limited experience of entrepreneurship and lack experience of work alto-
gether. Therefore, focusing on entrepreneurial intentions only, or using intentions as
a proxy for action, represents a severe limitation to entrepreneurial action and future
studies should focus on measuring the entrepreneurial actions with entrepreneurial
intentions. 6 Conclusion This paper has conducted the systematic review on more than 15 years of EI
research studied in worldwide university students to provide an understanding of the
most common factors that affect students’ intentions to become entrepreneurs used
in the literature and suggest novel lines of research in the future. Various papers
published between 2005 and June 2022 measuring the EI of the students were ana-
lyzed and the findings clearly suggest that there is an increasing trend of these stud-
ies from 2017 onwards. Most of the research focused on Asia with more than half
of the studies conducted in this region. The results highlighted that the most schol-
ars used TPB model to measure EI of students, and the factors used by most papers
are related to cognitive factors. It is believed that using a range of factors can pro-
vide a better understanding to measure the EI of students. Hence, future research
can be extended in various areas as identified by the gaps provided in the discussion
section above. Based on the above findings, it is clear that cognitive factors should
not only be accounted for to understand the intentions of students and more studies
should focus on other factors which are equally important in their influence on the
EI of the students. Like every other study, this review also has some limitations that can be
addressed in future research. First, although the authors tried their best to include
the studies between 2005 and June 2022, some studies might have been overlooked
due to the variety of databases available. Next, the comparative review between dif-
ferent regions might have helped to understand the region-specific factors affecting
the EI of students which was not explored in this study. Next, specific review can be
conducted in Africa to better understand the EI of students as there are fewer stud-
ies currently in this region and such review can motivate the scholars to explore this
region further. Finally, the comparative review between Asia and Africa might pro-
vide valuable insight on the factors affecting the EI of students, as both the regions
mostly consist of developing countries. 1 3 1928 G. Maheshwari et al. Appendix List of n = 254 papers (non-influential papers)
Title of the paper
Name of authors
Name of Journals
Database
Agro-Entrepreneurial
Intention among Uni-
versity Students: a study
under the premises
of Theory of Planned
Behavior
(Che Nawi et al. 2022)
SAGE Open
Scopus
Attitudes, subjective
norms, and perceived
control versus contex-
tual factors influencing
the entrepreneurial
intentions of students
from Poland
(Kobylińska 2022)
WSEAS Transactions on Business
and Economics
Scopus
COVID-19 pandemic and
entrepreneurial intention
among university stu-
dents: a contextualisation
of the Igbo Traditional
Business School
(Godswill Agu et al. 2022)
African Journal of Economic and
Management Studies
Scopus
Determinant factors in
entrepreneurial intention
among Social Work
degree students: the mod-
erating effect of entrepre-
neurship education
(García-Uceda et al. 2022) Social Enterprise Journal
Scopus
Determinants of Entre-
preneurial Intentions
of Youth: the Role of
Access to Finance
(Rusu et al. 2022)
Engineering Economics
Scopus
Drivers of Green Entre-
preneurial Intention:
Why Does Sustainabil-
ity Awareness Matter
Among University
Students? (Prabowo et al. 2022)
Frontiers in Psychology
Scopus
Effects of Entrepre-
neurial Competence and
Planning Guidance on
the Relation Between
University Students’
Attitude and Entrepre-
neurial Intention
(Ferreira et al. 2022)
Journal of Entrepreneurship
Scopus
Factors That Can Promote
the Green Entrepreneur-
ial Intention of College
Students: A Fuzzy Set
Qualitative Comparative
Analysis
(Cai et al. 2022)
Frontiers in Psychology
Scopus 1 3 Factors affecting students’ entrepreneurial intentions:… Factors affecting students’ entrepreneurial intentions:… 1929 f
g
p
Title of the paper
Name of authors
Name of Journals
Database
Factors that influence
the entrepreneurial
intention of psychology
students of the virtual
modality [Factores que
influyen en la intención
emprendedora de estudi-
antes de psicología de la
modalidad virtual]
(Valencia-Arias et al. 2022)
Retos(Ecuador)
Scopus
Positive psychological
capital and university
students’ entrepreneurial
intentions: does gender
make a difference? (Maslakçı et al. 2022b)
International Journal for Educa-
tional and Vocational Guidance
Scopus
Relationship between
Positive Psychological
Capital and Entrepre-
neurial Intentions of
University Students:
The Mediating Role of
Entrepreneurial Self-
efficacy
(Maslakçı et al. 2022a)
World Journal of Entrepreneurship,
Management and Sustainable
Development
Scopus
Sustainable Economic
Development Through
Entrepreneurship: A
Study on Attitude,
Opportunity Recogni-
tion, and Entrepre-
neurial Intention Among
University Students in
Malaysia
(Wiramihardja et al. Appendix 2022) Frontiers in Psychology
Scopus
Sustainable entrepreneur-
ial intentions: Explora-
tion of a model based
on the theory of planned
behaviour among
university students in
north-east Colombia
(Romero-Colmenares and
Reyes-Rodríguez 2022)
International Journal of Manage-
ment Education
Scopus
The entrepreneurial
intention of university
students: An environ-
mental perspective
(Barba-Sánchez et al. 2022)
European Research on Manage-
ment and Business Economics
Scopus
The impact of entrepre-
neurship education
and cultural context
on entrepreneurial
intentions of Ukrainian
students: the mediating
role of attitudes and
perceived control
(Mykolenko et al. 2022)
Higher Education, Skills and
Work-based Learning
Scopus (Romero-Colmenares and
Reyes-Rodríguez 2022)
International Journal of Manage-
ment Education
Scopus 3 1930 G. Maheshwari et al. Title of the paper
Name of authors
Name of Journals
Database
UPPS impulsivity,
entrepreneurial self-
efficacy and entrepre-
neurial intentions among
university students:
ADHD symptoms as a
moderator
(Tran et al. 2022)
Journal of Applied Research in
Higher Education
Scopus
The Effect of Entrepre-
neurial Education and
Culture on Entrepre-
neurial Intention
(Kayed et al. 2022)
Organizacija
ProQuest
Assessing the effective-
ness of entrepreneur-
ship education in the
universities of Tehran
province based on an
entrepreneurial intention
model
(Sherkat and Chenari
2022)
STUDIES IN HIGHER EDUCA-
TION
Taylor and
Francis
The influence of entre-
preneurship education
on entrepreneurial
intentions: Perception of
higher business educa-
tion graduates
(Magasi 2022)
International Journal of Research
in Business and Social Science
ProQuest
Determinants of Somali
Student’s Entrepreneur-
ial Intentions: The Case
Study of University
Students in Mogadishu 1
(Alin and Dil 2022)
Eskisehir Osmangazi Universitesi
Sosyal Bilimler Dergisi
ProQuest
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ProQuest 3 1936 G. Maheshwari et al. Title of the paper
Name of authors
Name of Journals
Database
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Name of authors
Name of Journals
Database
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Name of authors
Name of Journals
Database
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Scopus 1 3 1 3 Factors affecting students’ entrepreneurial intentions:… 1939 f
g
p
Title of the paper
Name of authors
Name of Journals
Database
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Name of authors
Name of Journals
Database
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Name of Journals
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g
p
Title of the paper
Name of authors
Name of Journals
Database
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programas de aprendi-
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intención emprendedora
de estudiantes universi-
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istraživanje odrednica
studentske poduzetničke
namjere]
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Name of authors
Name of Journals
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Factors affecting students entrepreneurial intentions:…
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Name of authors
Name of Journals
Database
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Name of authors
Name of Journals
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Name of authors
Name of Journals
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Database
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2020) Appendix 2017)
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Name of authors
Name of Journals
Database
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universitários]
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University
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2020)
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journal
Scopus universitários]
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University
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Technology
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journal
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journal
Scopus 3 1952 G. Maheshwari et al. Appendix Title of the paper
Name of authors
Name of Journals
Database
A critical comparison
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science and technology
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intention: a tale of two
genders
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parsert 2019)
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Research
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Scholar
Factors affecting entrepre-
neurial intention among
UniSZA students
(Ghazali et al. 2012)
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Effect of demographic fac-
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University
(Nsahlai et al. 2020) 1 3 Factors affecting students’ entrepreneurial intentions:… Factors affecting students’ entrepreneurial intentions:… 1953 Title of the paper
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(Ali et al. Appendix 2019)
International journal of gender and
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Students’ Attitudes toward
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emerging countries
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in the entrepreneurial
education practices
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pitowati 2017)
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Name of authors
Name of Journals
Database
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Students
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comparative study of
two countries university
students
(Díaz-Casero et al. 2012)
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effects on the entre-
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university students in
South Africa
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university students in
Oman
(Uddin et al. 2016)
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their effects on future
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Scopus 1 3 Factors affecting students’ entrepreneurial intentions:… Factors affecting students’ entrepreneurial intentions:… 1955 1955
Factors affecting students entrepreneurial intentions:…
Title of the paper
Name of authors
Name of Journals
Database
Factors influencing
entrepreneurial intention
and the moderating role
of entrepreneurship
education: A conceptual
model
(Shamsudin et al. 2017)
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University Students’
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university business
students in Kenya:
Lessons from Kenyatta
University
(Kilonzo and Nyambegera
2014)
International Journal of Entrepre-
neurship and Small Business
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entrepreneurial intention
of private universities
students in Malaysia
(Ismail et al. 2012)
International Journal of Entrepre-
neurship and Small Business
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Descriptive research study
on factors influencing
entrepreneurial intention
among engineering stu-
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District
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Journal of Advanced Research in
Dynamical and Control Systems
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An examination of univer-
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(Carey et al. 2010)
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Heliyon
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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/licen
ses/by/4.0/. References Abdullahi MS, Khalid N, Ahmed U et al (2021) Effect of entrepreneurship education on entrepreneurial
intention among university students. J Tech Educ Train. https://doi.org/10.30880/jtet.2021.13.03.
005l Adeyonu AG, Balogun OL, Obaniyi KS (2019) Factors influencing entrepreneurial intentions of under-
graduate agricultural students in Nigeria|Nijerya’da ziraat fakültesi öğrencilerinin girişimciliklerini
etkileyen faktörler. Yuz Yil Univ J Agric Sci. https://doi.org/10.29133/yyutbd.623910 Aggarwal V (2019) A conceptual study on factors of entrepreneurial potentiality and their impact on
entrepreneurial intention with the moderating role of entrepreneurship education. Prabandhan
Indian J Manag. https://doi.org/10.17010/pijom/2019/vl2i6/144932 p
g
p
Indian J Manag. https://doi.org/10.17010/pijom/2019/vl2i6/144932 Agu AG, Kalu OO, Esi-Ubani CO, Agu PC (2021) Drivers of sustainable entrepreneurial intentions
among university students: an integrated model from a developing world context. Int J Sustain
High Educ. https://doi.org/10.1108/IJSHE-07-2020-0277 Ahamed F, Rokhman W (2015) The role of social and psychological factors on entrepreneurial inten-
tion among islamic college students in Indonesia. Entrep Bus Econ Rev 29–41. https://doi.org/10.
15678/EBER.2015.030103 Ahmed T, Klobas JE, Ramayah T (2021) Personality traits, demographic factors and entrepreneurial
intentions: improved understanding from a moderated mediation study. Entrep Res J. https://doi.
org/10.1515/erj-2017-0062 Ajzen I (1991) The theory of planned behavior. Organ Behav Hum Decis Process 50:179–211. https://
doi.org/10.1016/0749-5978(91)90020-T g
Ajzen I, Fishbein M (1980) Understanding attitudes and predicting social behaviour. Prentice-Hall,
Hobokenfi Akadiri S, Bekun FV, Rasul O, Victor Bekun F (2017) The impact of self-efficacy on international stu-
dent entrepreneur intention. Int Rev Manag Mark 7(1):169–174 Akhtar S, Albarrak MS, Ahmad A et al (2022) Drivers of student entrepreneurial intention and the mod-
erating role of entrepreneurship education: evidence from an Indian university. Discrete Dyn Nat
Soc. https://doi.org/10.1155/2022/6767580l p
g
Akhter A, Hossain MU, Al Asheq A (2020) Influential factors of social entrepreneurial intention in Bang-
ladesh. J Asian Financ Econ Bus. https://doi.org/10.13106/JAFEB.2020.VOL7.NO8.645l Akhter A, Hossain MU, Al Asheq A (2020) Influential factors of social entrepreneurial intention in Bang-
l d
h J A i
Fi
E
B
h
//d i
/10 13106/JAFEB 2020 VOL7 NO8 645 p
g
Al Bakri A, Mehrez A (2017) Factors influencing entrepreneurial intentions among arab students. Int J
Entrep 21(3). http://hdl.handle.net/10576/15723 Al Bakri A, Mehrez A (2017) Factors influencing entrepreneurial intentions among arab students. Int Al Bakri A, Mehrez A (2017) Factors influencing entrepreneurial i
Entrep 21(3). http://hdl.handle.net/10576/15723 l
Entrep 21(3). References http://hdl.handle.net/10576/15723 Ali MSY, Abou EAEE (2020) Determinants of entrepreneurial intention among Sudanese university st Ali MSY, Abou EAEE (2020) Determinants of entrepreneurial intention among Sudanese university stu-
dents. Manag Sci Lett. https://doi.org/10.5267/j.msl.2020.4.023f Ali MSY, Abou EAEE (2020) Determinants of entrepreneurial intention
dents. Manag Sci Lett. https://doi.org/10.5267/j.msl.2020.4.023f dents. Manag Sci Lett. https://doi.org/10.5267/j.msl.2020.4.023f Ali YM, Negasi RD (2021) The differential impact of the experiential-entrepreneurial learning method
on the entrepreneurial intentions of higher education students. Int J Learn Teach Educ Res. https://
doi.org/10.26803/IJLTER.20.9.14 g
(
)f
p
p
p
g
on the entrepreneurial intentions of higher education students. Int J Learn Teach Educ Res. https://
doi.org/10.26803/IJLTER.20.9.14 g
Ali J, Zakaria N, Jaganathan M et al (2017) Determinants of entrepreneurial intention: empirical insights
from Malaysian undergraduate business students. Int J Econ Res 14(19):159–169 Ali I, Ali M, Badghish S (2019) Symmetric and asymmetric modeling of entrepreneurial ecosystem in
developing entrepreneurial intentions among female university students in Saudi Arabia. Int J Gend
Entrep. https://doi.org/10.1108/IJGE-02-2019-0039 Alin LD, Dil E (2022) Determinants of somali student’s entrepreneurial intentions: the case study of uni-
versity students in Mogadishu 1. Eskişehir Osmangazi Üniversitesi Sos Bilim Derg 23:130–142. https://doi.org/10.17494/ogusbd.1092867 Alin LD, Dil E (2022) Determinants of somali student’s entrepreneurial intentions: the case study of uni-
versity students in Mogadishu 1. Eskişehir Osmangazi Üniversitesi Sos Bilim Derg 23:130–142. https://doi.org/10.17494/ogusbd.1092867 1 1 3 3 Factors affecting students’ entrepreneurial intentions:… 1957 Al-Jubari I, Hassan A, Hashim J (2017) The role of autonomy as a predictor of entrepreneurial intention
among university students in Yemen. Int J Entrep Small Bus. https://doi.org/10.1504/IJESB.2017.
081950 Al-Jubari I, Hassan A, Hashim J (2017) The role of autonomy as a predictor of entrepreneurial intention
among university students in Yemen. Int J Entrep Small Bus. https://doi.org/10.1504/IJESB.2017.
081950 Al-Jubari I, Hassan A, Liñán F (2019) Entrepreneurial intention among University students in Malay-
sia: integrating self-determination theory and the theory of planned behavior. Int Entrep Manag J. h
//d i
/10 1007/ 11365 018 0529 0 Al-Jubari I, Hassan A, Liñán F (2019) Entrepreneurial intention among University students in Malay-
sia: integrating self-determination theory and the theory of planned behavior. Int Entrep Manag J. https://doi org/10 1007/s11365 018 0529 0 https://doi.org/10.1007/s11365-018-0529-0 p
g
Al-Jubari I, Mosbah A, Anor Salim FAB (2021) Motivational and attitudinal determinants of entrepre-
neurial intention: hospitality and tourism students’ perspectives. J Hosp Tour Educ. References https://doi.org/
10.1080/10963758.2021.1963747 Al-Jubari I, Mosbah A, Anor Salim FAB (2021) Motivational and attitudinal determinants of entrepre-
neurial intention: hospitality and tourism students’ perspectives. J Hosp Tour Educ. https://doi.org/
10.1080/10963758.2021.1963747 Al-Mamary YHS, Abdulrab M, Alwaheeb MA, Alshammari NGM (2020) Factors impacting entrepre-
neurial intentions among university students in Saudi Arabia: testing an integrated model of TPB
and EO. Educ Train. https://doi.org/10.1108/ET-04-2020-0096 Educ Train. https://doi.org/10.1108/ET-04-2020-009 Al-Shammari M, Waleed R (2018) Entrepreneurial intentions of private university students in the king-
dom of Bahrain. Int J Innov Sci. https://doi.org/10.1108/IJIS-06-2017-0058 Alshebami AS, Seraj AHA (2021) The Antecedents of saving behavior and entrepreneurial intention of
Saudi Arabia University Students. Educ Sci Theory Pract. https://doi.org/10.12738/jestp.2021.2.
005l Alzamel S, Nazri M, Omar S (2020) Factors influencing e-entrepreneurial intention among female stu-
dents in Saudi Arabia. Int J Criminol Sociol. https://doi.org/10.6000/1929-4409.2020.09.234 Amanamah RB, Acheampong A, Owusu EK (2018) An exploratory study of entrepreneurial intentio
among university students in Ghana. Int J Sci Technol Res 7(1):140–148 Amofah K, Saladrigues R (2022) Impact of attitude towards entrepreneurship education and role mode
on entrepreneurial intention. J Innov Entrep. https://doi.org/10.1186/s13731-022-00197-5 Anjum T, Farrukh M, Heidler P, Tautiva JAD (2021a) Entrepreneurial intention: creativity, entrepreneur-
ship, and university support. J Open Innov Technol Mark Complex. https://doi.org/10.3390/joitm
c7010011 Anjum T, Heidler P, Amoozegar A, Anees RT (2021b) The impact of entrepreneurial passion on the
entrepreneurial intention; moderating impact of perception of university support. Adm Sci. https://
doi.org/10.3390/admsci11020045 g
Anwar I, Saleem I, Islam KMB et al (2020) Entrepreneurial intention among female university students:
examining the moderating role of entrepreneurial education. J Int Bus Entrep Dev. https://doi.org/
10.1504/JIBED.2020.110254 Anwar I, Thoudam P, Saleem I (2022) Role of entrepreneurial education in shaping entrepreneurial inten-
tion among university students: testing the hypotheses using mediation and moderation approach. J
Educ Bus. https://doi.org/10.1080/08832323.2021.1883502 p
g
Ao J, Liu Z (2014) What impact entrepreneurial intention? Cultural, environmental, and educational fa
tors. J Manag Anal. https://doi.org/10.1080/23270012.2014.994232 Arango-Botero D, Arias MLB, Montoya MH, Valencia-Arias A (2020) Determinants of entrepreneurial
intention among engineering students based on structural equation modeling. Pertanika J Soc Sci
Humanit. https://doi.org/10.47836/PJSSH.28.4.08f Arasti Z, Pasvishe FA, Motavaseli M (2012) Normative institutional factors affecting entrepreneurial
intention in iranian information technology sector. J Manag Strategy. https://doi.org/10.5430/jms.
v3n2p16 Arasti Z, Pasvishe FA, Motavaseli M (2012) Normative institutional factors affecting entrepreneurial
intention in iranian information technology sector. J Manag Strategy. References https://doi.org/10.5430/jms.
v3n2p16f p
Arranz N, Ubierna F, Arroyabe MF et al (2017) The effect of curricular and extracurricular activities on
university students’ entrepreneurial intention and competences. Stud High Educ. https://doi.org/10.
1080/03075079.2015.1130030 p
Arranz N, Ubierna F, Arroyabe MF et al (2017) The effect of curricular and extracurricular activities on
university students’ entrepreneurial intention and competences. Stud High Educ. https://doi.org/10.
1080/03075079.2015.1130030 Arranz N, Arroyabe MF, Fdez. de Arroyabe JC (2019) Entrepreneurial intention and obstacles of under-
graduate students: the case of the universities of Andalusia. Stud High Educ. https://doi.org/10.
1080/03075079.2018.1486812 Arranz N, Arroyabe MF, Fdez. de Arroyabe JC (2019) Entrepreneurial intention and obstacles of under-
graduate students: the case of the universities of Andalusia. Stud High Educ. https://doi.org/10.
1080/03075079.2018.1486812 Arshad M, Farooq O, Sultana N, Farooq M (2016) Determinants of individuals’ entrepreneurial inte
tions: a gender-comparative study. Career Dev Int. https://doi.org/10.1108/CDI-10-2015-0135f Arshad M, Farooq O, Farooq M (2019) The effect of intrinsic and extrinsic factors on entrepreneurial
intentions: the moderating role of collectivist orientation. Manag Decis. https://doi.org/10.1108/
MD-04-2016-0248 Ashari H, Abbas I, Abdul-talib AN, Mohd Zamani SN (2022) Entrepreneurship and sustainable develop-
ment goals: a multigroup analysis of the moderating effects of entrepreneurship education on entre-
preneurial intention. Sustainability. https://doi.org/10.3390/su14010431 1 3 1958 G. Maheshwari et al. Ashokan N, Priya K, Rajeswari PS (2019) Determinants of personal attitudes dimensions on entrepr
neurial intentions. Int J Eng Adv Technol 8(5):488–490 Ashraf MA (2021) “Is Old Gold?” the role of prior experience in exploring the determinants of Islamic
social entrepreneurial intentions: evidence from Bangladesh. J Soc Entrep. https://doi.org/10.1080/
19420676.2019.1702580 Asma PX, Hassan S et al (2019) Determinants of social entrepreneurial intentions for educational pr
grams. J Public Aff. https://doi.org/10.1002/pa.1925 Asma PX, Hassan S et al (2019) Determinants of social entrepreneurial intentions for educational pro-
grams. J Public Aff. https://doi.org/10.1002/pa.1925 f
Atitsogbe KA, Mama NP, Sovet L et al (2019) Perceived employability and entrepreneurial intentions
across university students and job seekers in Togo: the effect of career adaptability and self-effi-
cacy. Front Psychol. https://doi.org/10.3389/fpsyg.2019.00180 f
Atitsogbe KA, Mama NP, Sovet L et al (2019) Perceived employability and entrepreneurial intentions
across university students and job seekers in Togo: the effect of career adaptability and self-effi-
cacy. Front Psychol. https://doi.org/10.3389/fpsyg.2019.00180 Autio E, KeeleyKlofsten RHM et al (2001) Entrepreneurial intent among students in Scandinavia and in
the USA. Enterp Innov Manag Stud. References https://doi.org/10.1080/14632440110094632 Autio E, KeeleyKlofsten RHM et al (2001) Entrepreneurial intent among students in Scandinavia and in
the USA. Enterp Innov Manag Stud. https://doi.org/10.1080/14632440110094632 Ayodele TO, Ekemode BG, Kajimo-Shakantu K (2021) Bridging the gap in real estate enterprise: the
impact of mentoring on entrepreneurial intentions of real estate students in Nigeria. Prop Manag. https://doi.org/10.1108/PM-02-2021-0015 Ayodele TO, Ekemode BG, Kajimo-Shakantu K (2021) Bridging the gap in real estate enterprise: the
impact of mentoring on entrepreneurial intentions of real estate students in Nigeria. Prop Manag. https://doi.org/10.1108/PM-02-2021-0015 p
g
Bae TJ, Qian S, Miao C, Fiet JO (2014) The relationship between entrepreneurship education and entre-
preneurial intentions: a meta-analytic review. Entrep Theory Pract. https://doi.org/10.1111/etap.
12095 Bae TJ, Qian S, Miao C, Fiet JO (2014) The relationship between entrepreneurship education and entre-
preneurial intentions: a meta-analytic review. Entrep Theory Pract. https://doi.org/10.1111/etap.
12095 Bandura A (1977a) Social learning theory. Prentice Hall, Hobokenfi Bandura A (1977a) Social learning theory. Prentice Hall, Hobokenfi Bandura A (1977a) Social learning theory. Prentice Hall, Hobokenfi andura A (1977b) Self-efficacy: toward a unifying theory of behavioral change. Psychol Rev 84:191–
215. https://doi.org/10.1037/0033-295X.84.2.191 Bandura A (1986) Social foundations of thought and action: a social cognitive theory. Prentice-Ha
Englewood Cliffs f
Bandura A, Ross D, Ross SA (1961) Transmission of aggression through imitation of aggressive models. J Abnorm Soc Psychol. https://doi.org/10.1037/h0045925i f
Bandura A, Ross D, Ross SA (1961) Transmission of aggression through imitation of aggressive models. J Abnorm Soc Psychol. https://doi.org/10.1037/h0045925i y
p
g
Bandura A, Ross D, Ross SA (1963) Imitation of film-mediated aggressive models. J Abnorm Soc Psy-
chol. https://doi.org/10.1037/h0048687 Bandura A, Ross D, Ross SA (1963) Imitation of film-mediated aggressive models. J Abnorm Soc Psy-
chol. https://doi.org/10.1037/h0048687 Bapoo MA, Tehseen S, Haider SA et al (2022) Sustainability orientation and sustainable entrepreneurship
intention: the mediating role of entrepreneurial opportunity recognition. Acad Entrep J 28:1–23 Bapoo MA, Tehseen S, Haider SA et al (2022) Sustainability orientation and sustainable entrepreneurship
intention: the mediating role of entrepreneurial opportunity recognition. Acad Entrep J 28:1–23 Barba-Sánchez V, Atienza-Sahuquillo C (2017) Entrepreneurial motivation and self-employment: evi-
dence from expectancy theory. Int Entrep Manag J. https://doi.org/10.1007/s11365-017-0441-z Barba-Sánchez V, Atienza-Sahuquillo C (2017) Entrepreneurial motivation and self-employment: evi-
dence from expectancy theory. Int Entrep Manag J. https://doi.org/10.1007/s11365-017-0441-z p
y
y
p
g
p
g
Barba-Sánchez V, Atienza-Sahuquillo C (2018) Entrepreneurial intention among engineering students:
the role of entrepreneurship education. Eur Res Manag Bus Econ. References https://doi.org/10.1111/cwe.12194 Bickenbach F, Dohse D, Liu WH (2017) University students’ entrepreneurial intentions: a comparative
analysis of Hong Kong and Guangzhou. China World Econ. https://doi.org/10.1111/cwe.12194 Bird B (1988) Implementing entrepreneurial ideas: the case for intention. Acad Manag Rev. https://do
org/10.5465/amr.1988.4306970 Biswas A, Verma RK (2021) Attitude and alertness in personality traits: a pathway to building entrepre-
neurial intentions among university students. J Entrep. https://doi.org/10.1177/097135572110256
56 Boyd NG, Vozikis GS (1994) The influence of self-efficacy on the development of entrepreneurial inten-
tions and actions. Entrep Theory Pract. https://doi.org/10.1177/104225879401800404f Brüne N, Lutz E (2020) The effect of entrepreneurship education in schools on entrepreneurial outcome
a systematic review. Manag Rev Q. https://doi.org/10.1007/s11301-019-00168-3l Bui THV, Nguyen TLT, Tran MD, Nguyen TAT (2020) Determinants influencing entrepreneurial inten-
tion among undergraduates in universities of Vietnam. J Asian Financ Econ Bus. https://doi.org/10.
13106/jafeb.2020.vol7.no7.369 Buli BM, Yesuf WM (2015) Determinants of entrepreneurial intentions: technical-vocational educatio
and training students in Ethiopia. Educ Train. https://doi.org/10.1108/ET-10-2014-0129l Buschow C, Laugemann R (2020) What makes a media entrepreneur? Factors influencing entrepreneurial
intention of mass communication students. J Mass Commun Educ. https://doi.org/10.1177/10776
95820912146 Cai X, Hussain S, Zhang Y (2022) Factors that can promote the green entrepreneurial intention of college
students: a fuzzy set qualitative comparative analysis. Front Psychol. https://doi.org/10.3389/fpsyg.
2021.776886 Canever MD, Barral MRM, Ribeiro FG (2017) How does the public and private university environme
affect students’ entrepreneurial intention? Educ Train. https://doi.org/10.1108/ET-12-2016-0187 f
Cano JA, Tabares A (2017) Determinants of university students’ entrepreneurial intention: GUESSS
Colombia study. Espacios 38(45):22 Cao VQ, Ngo TTT (2019) Linking entrepreneurial intentions and mindset models: a comparative study of
public and private universities in Vietnam. Gadjah Mada Int J Bus 21(2):115–133. https://doi.org/
10.22146/gamaijb.34753 g
j
Carey TA, Flanagan DJ, Palmer TB (2010) An examination of university student entrepreneurial inte
tions by type of venture. J Dev Entrep. https://doi.org/10.1142/S1084946710001622l Caro-González FJ, Romero-Benabent H, Sánchez-Torné I (2017) The influence of gender on the entrepre-
neurial intentions of journalism students. Intang Cap. https://doi.org/10.3926/ic.927 Castelló-Sirvent F, Pinazo-Dallenbach P (2021) Corruption shock in Mexico: FsQCA analysis of entre-
preneurial intention in university students. Mathematics. https://doi.org/10.3390/math9141702 Çelik AK, Yıldız T, Aykanat Z, Kazemzadeh S (2021) The impact of narrow personality traits on entre-
preneurial intention in developing countries: a comparison of Turkish and Iranian undergraduate
students using ordered discrete choice models. Eur Res Manag Bus Econ. References https://doi.org/10.1016/j.iedeen.
2017.04.001 p
y
y
p
g
p
g
Barba-Sánchez V, Atienza-Sahuquillo C (2018) Entrepreneurial intention among engineering students:
the role of entrepreneurship education. Eur Res Manag Bus Econ. https://doi.org/10.1016/j.iedeen.
2017.04.001 Barba-Sánchez V, Mitre-Aranda M, del Brío-González J (2022) The entrepreneurial intention of univer-
sity students: an environmental perspective. Eur Res Manag Bus Econ. https://doi.org/10.1016/j.
iedeen.2021.100184 Barth J, Muehlfeld K (2021) Thinking out of the box—by thinking in other boxes: a systematic review
of interventions in early entrepreneurship vs. STEM education research. Manag Rev Q. https://doi.
org/10.1007/s11301-021-00248-3 g
Bazan C, Shaikh A, Frederick S et al (2019) Effect of memorial university’s environment & support sy
tem in shaping entrepreneurial intention of students. J Entrep Educ 22(1):1–35i Bazkiaei HA, Heng LH, Khan NU et al (2020) Do entrepreneurial education and big-five personality
traits predict entrepreneurial intention among universities students? Cogent Bus Manag. https://doi.
org/10.1080/23311975.2020.1801217 g
Bazkiaei HA, Khan NU, Irshad A-R, Ahmed A (2021) Pathways toward entrepreneurial intention among
Malaysian universities’ students. Bus Process Manag J 27:1009–1032. https://doi.org/10.1108/
BPMJ-01-2021-0021 Belas J, Gavurova B, Schonfeld J et al (2017) Social and economic factors affecting the entrepreneuri
intention of university students. Transform Bus Econ 16(3):220–239 Bell R (2019) Predicting entrepreneurial intention across the university. Educ Train. https://doi.org/10.
1108/ET-05-2018-0117 Bello MI, Danjuma IM (2017) Relationship between psychological factors and entrepreneurial intentions
of university undergraduates in north east, Nigeria. J Tech Educ Train 9(2):81–93 Benedictohanu I, Shodipe TO, Chukwu DU, Chukwuma JNW (2020) Impact of behavioural factors as
related to available resources on entrepreneurial intentions of electrical installation and mainte-
nance works students. Int J Eng Educ 36(1):142–154 1 1 3 3 Factors affecting students’ entrepreneurial intentions:… 1959 Bernhofer L, Han Z (2014) Contextual factors and their effects on future entrepreneurs in China: a com-
parative study of entrepreneurial intentions. Int J Technol Manag. https://doi.org/10.1504/IJTM.
2014.060955 Bernhofer L, Han Z (2014) Contextual factors and their effects on future entrepreneurs in China: a com-
parative study of entrepreneurial intentions. Int J Technol Manag. https://doi.org/10.1504/IJTM.
2014.060955 Bhat IH, Singh S (2018) Analyzing the moderating effect of entrepreneurship education on the anteced-
ents of entrepreneurial intention. J Entrep Educ 21(1):1–10 Bhat IH, Singh S (2018) Analyzing the moderating effect of entrepreneurship education on the anteced-
ents of entrepreneurial intention. J Entrep Educ 21(1):1–10 Bickenbach F, Dohse D, Liu WH (2017) University students’ entrepreneurial intentions: a comparative
analysis of Hong Kong and Guangzhou. China World Econ. References https://doi.org/10.7341/20211715 Doanh DC (2021a) The moderating role of self-efficacy on the cognitive process of entrepreneurship: an
empirical study in Vietnam. J Entrep Manag Innov. https://doi.org/10.7341/20211715 p
y
p
g
p
g
Doanh DC (2021b) The role of contextual factors on predicting entrepreneurial intention among Viet-
namese students. Entrep Bus Econ Rev. https://doi.org/10.15678/EBER.2021b.090111fi Doanh DC (2021b) The role of contextual factors on predicting entrepreneurial intention among Viet-
namese students. Entrep Bus Econ Rev. https://doi.org/10.15678/EBER.2021b.090111fi Doanh DC, Bernat T (2019) Entrepreneurial self-efficacy and intention among Vietnamese students:
a meta-analytic path analysis based on the theory of planned behavior. Procedia Comput Sci
159:2447–2460. https://doi.org/10.1016/j.procs.2019.09.420l Doanh DC, Bernat T (2019) Entrepreneurial self-efficacy and intention among Vietnamese students:
a meta-analytic path analysis based on the theory of planned behavior. Procedia Comput Sci
159:2447–2460. https://doi.org/10.1016/j.procs.2019.09.420l p
g
j p
Dong Y, Pang L, Fu L (2019) Research on the influencing factors of entrepreneurial intentions based on
mediating effect of self-actualization. Int J Innov Sci. https://doi.org/10.1108/IJIS-03-2018-0027 p
g
j p
Dong Y, Pang L, Fu L (2019) Research on the influencing factors of entrepreneurial intentions based on
mediating effect of self-actualization. Int J Innov Sci. https://doi.org/10.1108/IJIS-03-2018-0027 f
Dragin AS, Mijatov MB, Munitlak Ivanović O et al (2022) Entrepreneurial intention of students (man-
agers in training): personal and family characteristics. Sustainability 14:7345. https://doi.org/10.
3390/su14127345 f
Dragin AS, Mijatov MB, Munitlak Ivanović O et al (2022) Entrepreneurial intention of students (man-
agers in training): personal and family characteristics. Sustainability 14:7345. https://doi.org/10.
3390/su14127345 Duong CD (2021) Exploring the link between entrepreneurship education and entrepreneurial intentions:
the moderating role of educational fields. Educ Train. https://doi.org/10.1108/ET-05-2021-0173 Duong CD (2021) Exploring the link between entrepreneurship education and entrepreneurial intention
the moderating role of educational fields. Educ Train. https://doi.org/10.1108/ET-05-2021-0173 i
Duong CD, Nguyen HX, Ngo TVN et al (2020) The impact of individual and environmental character-
istics on students’ entrepreneurial intention. Manag Sci Lett. https://doi.org/10.5267/j.msl.2019.9.
020 i
Duong CD, Nguyen HX, Ngo TVN et al (2020) The impact of individual and environmental character-
istics on students’ entrepreneurial intention. Manag Sci Lett. https://doi.org/10.5267/j.msl.2019.9.
020 Duque Aldaz FJ, Pazan Gómez EG, Álvarez Vasco WA (2018) Factors affecting entrepreneurial intentio
of Senior University Students. Espacios 39(9)f Dzomonda O, Fatoki O, Oni O (2015) The effect of psychological and contextual factors on the entrepre-
neurial intention of university students in South Africa. Corp Ownersh Control. References https://doi.org/10.
1016/j heliyon 2021 e06381 1016/j.heliyon.2021.e06381
Datta A, Bazan C, Arnold K (2021) Effect of gender role identity on the entrepreneurial intention of uni-
versity students. J Small Bus Entrep. https://doi.org/10.1080/08276331.2021.1981729l Datta A, Bazan C, Arnold K (2021) Effect of gender role identity on the entrepreneurial intention of uni-
versity students. J Small Bus Entrep. https://doi.org/10.1080/08276331.2021.1981729l Díaz-Casero JC, Ferreira JJM, Mogollón RH, Raposo MLB (2012) Influence of institutional environment
on entrepreneurial intention: a comparative study of two countries university students. Int Entrep
Manag J. https://doi.org/10.1007/s11365-009-0134-3 Díaz Casero JC, Ferreira JJM, Mogollón RH, Raposo MLB (2012) Influence of institutional environment
on entrepreneurial intention: a comparative study of two countries university students. Int Entrep
Manag J. https://doi.org/10.1007/s11365-009-0134-3 g
p
g
Diegoli RB, Gutierrez HSM, Los Salmones MDMG De (2018) Teachers as entrepreneurial role models:
the impact of a teacher’s entrepreneurial experience and student learning styles in entrepreneurial
intentions. J Entrep Educ 21(1)l g
p
g
Diegoli RB, Gutierrez HSM, Los Salmones MDMG De (2018) Teachers as entrepreneurial role models:
the impact of a teacher’s entrepreneurial experience and student learning styles in entrepreneurial
intentions. J Entrep Educ 21(1)l Díez-Echavarría L, Valencia-Arias A, Bermúdez-Hernández J (2020) Tool for measuring the influence of
the field of knowledge on entrepreneurial intention among university students. Period Polytech Soc
Manag Sci. https://doi.org/10.3311/PPso.12873 Díez-Echavarría L, Valencia-Arias A, Bermúdez-Hernández J (2020) Tool for measuring the influence of
the field of knowledge on entrepreneurial intention among university students. Period Polytech Soc
Manag Sci. https://doi.org/10.3311/PPso.12873l g
p
g
Do BR, Dadvari A (2017) The influence of the dark triad on the relationship between entrepreneurial atti-
tude orientation and entrepreneurial intention: a study among students in Taiwan University. Asia
Pacific Manag Rev https://doi org/10 1016/j apmrv 2017 07 011 g
p
g
Do BR, Dadvari A (2017) The influence of the dark triad on the relationship between entrepreneurial atti-
tude orientation and entrepreneurial intention: a study among students in Taiwan University. Asia
Pacific Manag Rev. https://doi.org/10.1016/j.apmrv.2017.07.011 Pacific Manag Rev. https://doi.org/10.1016/j.apmrv.2017.07.011 i
g
p
g
j p
Doan XT, Phan TTH (2020) The impact of entrepreneurial education on entrepreneurial intention: the
case of Vietnamese. Manag Sci Lett. https://doi.org/10.5267/j.msl.2019.12.040fi i
Doan XT, Phan TTH (2020) The impact of entrepreneurial education on entrepreneurial intention: the
case of Vietnamese. Manag Sci Lett. https://doi.org/10.5267/j.msl.2019.12.040fi Doanh DC (2021a) The moderating role of self-efficacy on the cognitive process of entrepreneurship: an
empirical study in Vietnam. J Entrep Manag Innov. References https://doi.org/10.1016/j.
iedeen.2020.100138 Chang YY, Wannamakok W, Kao CP (2021) Entrepreneurship education, academic major, and university
students’ social entrepreneurial intention: the perspective of Planned Behavior Theory. Stud High
Educ https://doi org/10 1080/03075079 2021 2021875 Chang YY, Wannamakok W, Kao CP (2021) Entrepreneurship education, academic major, and university
students’ social entrepreneurial intention: the perspective of Planned Behavior Theory. Stud High
Educ. https://doi.org/10.1080/03075079.2021.2021875 Che Nawi N, Al MA, Hassan AA et al (2022) Agro-entrepreneurial Intention among University Students:
a study under the premises of theory of planned behavior. SAGE Open. https://doi.org/10.1177/
21582440211069144 Che Nawi N, Al MA, Hassan AA et al (2022) Agro-entrepreneurial Intention among University Students:
a study under the premises of theory of planned behavior. SAGE Open. https://doi.org/10.1177/
21582440211069144fi Chen CC, Greene PG, Crick A (1998) Does entrepreneurial self-efficacy distinguish entrepreneurs from
managers? J Bus Ventur. https://doi.org/10.1016/S0883-9026(97)00029-3 Chen CC, Greene PG, Crick A (1998) Does entrepreneurial self-efficacy distinguish entrepreneurs from
managers? J Bus Ventur. https://doi.org/10.1016/S0883-9026(97)00029-3 Chen SC, Hsiao HC, Chang JC et al (2015) Can the entrepreneurship course improve the entrepreneurial
intentions of students? Int Entrep Manag J. https://doi.org/10.1007/s11365-013-0293-0l Chen SC, Hsiao HC, Chang JC et al (2015) Can the entrepreneurship course improve the entrepreneurial
intentions of students? Int Entrep Manag J. https://doi.org/10.1007/s11365-013-0293-0l Chinaire TI, Chimucheka T, Khayundi HA (2021) Factors influencing social entrepreneurial intentions
students at a University in South Africa. Acad Entrep J 27:1–16l y
p
Chukwuma-Nwuba EO (2018) The influence of culture on entrepreneurial intentions: a Nigerian univer-
sity graduates’ perspective. Transnatl Corp Rev. https://doi.org/10.1080/19186444.2018.1507877 Chukwuma-Nwuba EO (2018) The influence of culture on entrepreneurial intentions: a Nigerian unive
sity graduates’ perspective. Transnatl Corp Rev. https://doi.org/10.1080/19186444.2018.1507877 1 3 1960 G. Maheshwari et al. Contreras-Barraza N, Espinosa-Cristia JF, Salazar-Sepulveda G, Vega-Muñoz A (2021) Entrepreneurial
intention: a gender study in business and economics students from Chile. Sustainability. https://doi.
org/10.3390/su13094693 Contreras-Barraza N, Espinosa-Cristia JF, Salazar-Sepulveda G, Vega-Muñoz A (2021) Entrepreneurial
intention: a gender study in business and economics students from Chile. Sustainability. https://doi.
org/10.3390/su13094693 g
Dao TK, Bui AT, Doan TTT et al (2021) Impact of academic majors on entrepreneurial intentions of
Vietnamese students: an extension of the theory of planned behavior. Heliyon. https://doi.org/10.
1016/j h li
2021 06381 g
Dao TK, Bui AT, Doan TTT et al (2021) Impact of academic majors on entrepreneurial intentions of
Vietnamese students: an extension of the theory of planned behavior. Heliyon. References https://doi.org/10.1177/09713557211069261 Ferreira ADSM, Loiola E, Gondim SMG, Pereira CR (2022) Effects of entrepreneurial competence and
planning guidance on the relation between university students’ attitude and entrepreneurial inten-
tion. J Entrep. https://doi.org/10.1177/09713557211069261 p
p
g
Ferri L, Ginesti G, Spano R, Zampella A (2019) Exploring factors motivating entrepreneurial intentions:
the case of Italian university students. Int J Train Dev. https://doi.org/10.1111/ijtd.12158 Fitz-Koch S, Nordqvist M, Carter S, Hunter E (2018) Entrepreneurship in the agricultural sector: a litera-
ture review and future research opportunities. Entrep Theory Pract. https://doi.org/10.1177/10422
58717732958 Fragoso R, Rocha-Junior W, Xavier A (2020) Determinant factors of entrepreneurial intention among
university students in Brazil and Portugal. J Small Bus Entrep. https://doi.org/10.1080/08276331.
2018.1551459 Fukuda K (2014) An empirical study on entrepreneurial intentions among Japanese university student
Int J Entrep Small Bus. https://doi.org/10.1504/IJESB.2014.059474 Galloway L, Brown W (2002) Entrepreneurship education at university: a driver in the creation of hig
growth firms? Educ Train. https://doi.org/10.1108/00400910210449231l i
Gangwani S, Ballout S (2020) Factors influencing the entrepreneurial intention of business graduate stu-
dents of Saudiarabia University. Acad Entrep J 26(4):1–7f Garcez A, Franco M (2021) Effects of psychological and cognitive factors on the relation between
entrepreneurial intention and academic hazing: case of the new students in the faculty of social
and human sciences at the University of Beira - Portugal. Entrep Res J. https://doi.org/10.1515/
erj-2020-0351 j
García-Uceda E, Murillo-Luna JL, Lafuente JA (2022) Determinant factors in entrepreneurial inten-
tion among social work degree students: the moderating effect of entrepreneurship education. Soc
Enterp J. https://doi.org/10.1108/SEJ-07-2021-0061 p
p
g
Ghazali Z, Ibrahim NA, Zainol FA (2012) Factors affecting entrepreneurial intention among UniSZA
students. Asian Soc Sci. https://doi.org/10.5539/ass.v9n1p85f Giacomin O, Janssen F, Pruett M et al (2011) Entrepreneurial intentions, motivations and barriers: differ-
ences among American, Asian and European students. Int Entrep Manag J. https://doi.org/10.1007/
s11365-010-0155-y Gieure C, del Benavides-Espinosa M, M, Roig-Dobón S, (2019) Entrepreneurial intentions in
an international university environment. Int J Entrep Behav Res. https://doi.org/10.1108/
IJEBR-12-2018-0810 Gismera Tierno L, Pérez-Macías N, Medina-Molina C (2021) Visual thinking boosting Spanish
higher education students’ entrepreneurial intentions. J Knowl Econ. https://doi.org/10.1007/
s13132-020-00700-x Godswill Agu A, Okwara OO, Okocha ER, Madichie NO (2022) COVID-19 pandemic and entrepre-
neurial intention among university students: a contextualisation of the Igbo Traditional Business
School. Afr J Econ Manag Stud. https://doi.org/10.1108/AJEMS-05-2021-0227 González-Serrano MH, Valantine I, Hervás JC et al (2018) Sports university education and entrepre-
neurial intentions: a comparison between Spain and Lithuania. Educ Train. References https://doi.org/10.
22495/cocv13i1c11p2 p
Echchabi A, Omar MMS, Ayedh AM (2020) Entrepreneurial intention among female university students
in Oman. J Int Bus Entrep Dev. https://doi.org/10.1504/JIBED.2020.110251 Eid R, Badewi A, Selim H, El-Gohary H (2019) Integrating and extending competing intention models to
understand the entrepreneurial intention of senior university students. Educ Train. https://doi.org/
10.1108/ET-02-2018-0030fi Elnadi M, Gheith MH (2021) Entrepreneurial ecosystem, entrepreneurial self-efficacy, and entrepreneur-
ial intention in higher education: evidence from Saudi Arabia. Int J Manag Educ. https://doi.org/10.
1016/j.ijme.2021.100458f j j
Elnadi M, Gheith MH, Farag T (2020) How does the perception of entrepreneurial ecosystem affect
entrepreneurial intention among university students in Saudi Arabia? Int J Entrep 24 Entrialgo M, Iglesias V (2020) Entrepreneurial intentions among university students: the moderating ro
of creativity. Eur Manag Rev. https://doi.org/10.1111/emre.12386 Ezeh PC, Nkamnebe AD, Omodafe UP (2020) Determinants of entrepreneurial intention among unde
graduates in a Muslim community. Manag Res Rev. https://doi.org/10.1108/MRR-09-2018-0348 1 3 Factors affecting students’ entrepreneurial intentions:… 1961 Fanea-Ivanovici M, Baber H (2022) Sustainability at universities as a determinant of entrepreneurship for
sustainability. Sustainability. https://doi.org/10.3390/su14010454f Fanea-Ivanovici M, Baber H (2022) Sustainability at universities as a determinant of entrepreneurship for
sustainability. Sustainability. https://doi.org/10.3390/su14010454f Fatoki O (2014) Parental and gender effects on the entrepreneurial intention of university students in
South Africa. Mediterr J Soc Sci. https://doi.org/10.5901/mjss.2014.v5n7p157 Fatoki O (2014) Parental and gender effects on the entrepreneurial intention of university students in
South Africa. Mediterr J Soc Sci. https://doi.org/10.5901/mjss.2014.v5n7p157 Fatoki O (2014) Parental and gender effects on the entrepreneurial intention of university students in
South Africa. Mediterr J Soc Sci. https://doi.org/10.5901/mjss.2014.v5n7p157 Fatoki O (2019) Sustainability orientation and sustainable entrepreneurial intentions of University stu-
dents in South Africa. Entrep Sustain Issues. https://doi.org/10.9770/jesi.2019.7.2(14) Fatoki O (2019) Sustainability orientation and sustainable entrepreneurial intentions of University stu-
dents in South Africa. Entrep Sustain Issues. https://doi.org/10.9770/jesi.2019.7.2(14) Fatoki O (2019) Sustainability orientation and sustainable entrepreneurial intentions of University stu-
dents in South Africa. Entrep Sustain Issues. https://doi.org/10.9770/jesi.2019.7.2(14) Fernandes C, Ferreira JJ, Raposo M et al (2018) Determinants of entrepreneurial intentions: an intern
tional cross-border study. Int J Innov Sci. https://doi.org/10.1108/IJIS-02-2017-0017f Fernandes C, Ferreira JJ, Raposo M et al (2018) Determinants of entrepreneurial intentions: an interna-
tional cross-border study. Int J Innov Sci. https://doi.org/10.1108/IJIS-02-2017-0017f Ferreira ADSM, Loiola E, Gondim SMG, Pereira CR (2022) Effects of entrepreneurial competence and
planning guidance on the relation between university students’ attitude and entrepreneurial inten-
tion. J Entrep. p
doi.org/10.1108/IJEBR-01-2017-0031 Hien DTT, Cho SE (2018) Relationship between entrepreneurship education and innovative start-up
intentions among university students. Int J Entrep 22(3):1–16 Hoang G, Le TTT, Tran AKT, Du T (2021) Entrepreneurship education and entrepreneurial intentions of
university students in Vietnam: the mediating roles of self-efficacy and learning orientation. Educ
Train. https://doi.org/10.1108/ET-05-2020-0142 Hockerts K (2017) Determinants of social entrepreneurial intentions. Entrep Theory Pract. https://doi.
org/10.1111/etap.12171 Hoda N, Ahmad N, Ahmad M et al (2020) Validating the entrepreneurial intention model on the Univer-
sity Students in Saudi Arabia. J Asian Financ Econ Bus. https://doi.org/10.13106/jafeb.2020.vol7.
no11.469 Hossain MU, Al Asheq A, Arifuzzaman SM (2019) Entrepreneurial intention of Bangladeshi students:
impact of individual and contextual factors. Probl Perspect Manag. https://doi.org/10.21511/ppm.
17(4).2019.40 Hossain MU, Al Asheq A, Arifuzzaman SM (2019) Entrepreneurial intention of Bangladeshi students:
impact of individual and contextual factors. Probl Perspect Manag. https://doi.org/10.21511/ppm.
17(4).2019.40 Hou F, Su Y, Lu M, Qi M (2019) Model of the entrepreneurial intention of university students in the
Pearl River Delta of China. Front Psychol. https://doi.org/10.3389/fpsyg.2019.00916l Hou F, Su Y, Lu M, Qi M (2019) Model of the entrepreneurial intention of university students in the
Pearl River Delta of China. Front Psychol. https://doi.org/10.3389/fpsyg.2019.00916l Hsu CY, Wang SM (2019) Social entrepreneurial intentions and its influential factors: a comparison of
students in Taiwan and Hong Kong. Innov Educ Teach Int. https://doi.org/10.1080/14703297.2018.
1427611 Hsu CY, Wang SM (2019) Social entrepreneurial intentions and its influential factors: a comparison of
students in Taiwan and Hong Kong. Innov Educ Teach Int. https://doi.org/10.1080/14703297.2018.
1427611 Hudea OS, Toma SG, Burcea M (2021) A non-parametric analysis of the relationship between business
experience and entrepreneurial intention of final-year university students. Mathematics. https://doi.
org/10.3390/math9161955 Hudea OS, Toma SG, Burcea M (2021) A non-parametric analysis of the relationship between business
experience and entrepreneurial intention of final-year university students. Mathematics. https://doi.
org/10 3390/math9161955 p
p
org/10.3390/math9161955 g
Huston SA (2018) Factors associated with entrepreneurial intentions in doctor of pharmacy students. A
J Pharm Educ 82:6355. https://doi.org/10.5688/ajpe6355 p
g
jp
Iakovleva T, Kolvereid L, Stephan U (2011) Entrepreneurial intentions in developing and developed
countries. Educ Train. https://doi.org/10.1108/00400911111147686 p
g
jp
Iakovleva T, Kolvereid L, Stephan U (2011) Entrepreneurial intentions in developing and develope
countries. Educ Train. https://doi.org/10.1108/00400911111147686 Ibidunni AS, Mozie D, Ayeni AWAA (2021) Entrepreneurial characteristics amongst university students:
insights for understanding entrepreneurial intentions amongst youths in a developing economy. Educ Train. https://doi.org/10.1108/ET-09-2019-0204 Ibidunni AS, Mozie D, Ayeni AWAA (2021) Entrepreneurial characteristics amongst university students:
insights for understanding entrepreneurial intentions amongst youths in a developing economy. Educ Train. https://doi.org/10.1108/ET-09-2019-0204 p
g
lesias-Sánchez PP, Jambrino-Maldonado C, Velasco AP, Kokash H (2016) Impact of entrepreneurship
programmes on university students. Educ Train. https://doi.org/10.1108/ET-01-2015-0004 p
g
Iglesias-Sánchez PP, Jambrino-Maldonado C, Velasco AP, Kokash H (2016) Impact of entrepreneursh
programmes on university students. Educ Train. https://doi.org/10.1108/ET-01-2015-0004 p
g
y
p
g
İlerisoy ZY, Aycı A, Aycı H, Kınacı EB (2021) Impacts of architectural education on entrepreneurial
intention: a case study of senior architects from six universities in Turkey. Archnet-IJAR. https://
doi.org/10.1108/ARCH-11-2020-0269 İlerisoy ZY, Aycı A, Aycı H, Kınacı EB (2021) Impacts of architectural education on entrepreneurial
intention: a case study of senior architects from six universities in Turkey. Archnet-IJAR. https://
doi.org/10.1108/ARCH-11-2020-0269 g
Ismail N, Jaffar N, Khan S, Leng TS (2012) Tracking the cyber entrepreneurial intention of private uni-
versities students in Malaysia. Int J Entrep Small Bus. https://doi.org/10.1504/IJESB.2012.050169 Ismail N, Jaffar N, Khan S, Leng TS (2012) Tracking the cyber entrepreneurial intention of private uni-
versities students in Malaysia. Int J Entrep Small Bus. https://doi.org/10.1504/IJESB.2012.050169 Ismail N, Jaffar N, Khan S, Leng TS (2012) Tracking the cyber entrepreneurial intention of private uni-
versities students in Malaysia. Int J Entrep Small Bus. https://doi.org/10.1504/IJESB.2012.050169
J
Y H dl
B S
J S
C (2019) D
i
f
d
’
i l i
i ng Y, Hadley B, Son J, Song C (2019) Determinants of students’ entrepreneurial intention to compete
in a fast-pitch competition. J Educ Bus. https://doi.org/10.1080/08832323.2018.1540389 Jang Y, Hadley B, Son J, Song C (2019) Determinants of students’ entrepreneurial intention to compe
in a fast-pitch competition. J Educ Bus. https://doi.org/10.1080/08832323.2018.1540389 ang X, Sun Y (2015) Study on constructing an education platform for innovation and entrepreneurship
of university student. Res J Appl Sci Eng Technol. References https://doi.org/10.1108/
ET-12-2017-0205f Goyanes M (2015) Factors affecting the entrepreneurial intention of students pursuing journalism and
media studies: evidence from Spain. JMM Int J Media Manag. https://doi.org/10.1080/14241277.
2015.1055748 Gulzar F, Fayaz A (2021) Youth entrepreneurial intentions: an integrated model of individual and contex-
tual factors. Int J Organ Anal. https://doi.org/10.1108/IJOA-08-2021-2928 Gurel E, Altinay L, Daniele R (2010) Tourism students’ entrepreneurial intentions. Ann Tour Res. https
doi.org/10.1016/j.annals.2009.12.003 urel E, Madanoglu M, Altinay L (2021) Gender, risk-taking and entrepreneurial intentions: assessing the
impact of higher education longitudinally. Educ Train. https://doi.org/10.1108/ET-08-2019-0190 1 3 1962 G. Maheshwari et al. Guzmán-Alfonso C, Guzmán-Cuevas J (2012) Entrepreneurial intention models as applied to Latin
America. J Organ Chang Manag. https://doi.org/10.1108/09534811211254608 Hassan A, Saleem I, Anwar I, Hussain SA (2020) Entrepreneurial intention of Indian university students:
the role of opportunity recognition and entrepreneurship education. Educ Train. https://doi.org/10.
1108/ET-02-2020-0033 Hattab HW (2014) Impact of entrepreneurship education on entrepreneurial intentions of university st
dents in Egypt. J Entrep. https://doi.org/10.1177/0971355713513346 Hendieh J, Aoun D, Osta A (2019) Students’ attitudes toward entrepreneurship at the Arab Open Unive
sity-Lebanon. J Entrep Educ 22(2):1–13 Henley A, Contreras F, Espinosa JC, Barbosa D (2017) Entrepreneurial intentions of Colombian business
students: planned behaviour, leadership skills and social capital. Int J Entrep Behav Res. https://
doi.org/10.1108/IJEBR-01-2017-0031 p
p
org/10.3390/math9161955 https://doi.org/10.2478/orga-2022-0002 Kayed H, Al-Madadha A, Abualbasal A (2022) The effect of entrepreneurial education and culture on
entrepreneurial intention. Organizacija. https://doi.org/10.2478/orga-2022-0002 Kilonzo PM, Nyambegera SM (2014) Determinants of entrepreneurial intention among university busi-
ness students in Kenya: lessons from Kenyatta University. Int J Entrep Small Bus. https://doi.org/
10.1504/IJESB.2014.062503 Kilonzo PM, Nyambegera SM (2014) Determinants of entrepreneurial intention among university busi-
ness students in Kenya: lessons from Kenyatta University. Int J Entrep Small Bus. https://doi.org/
10.1504/IJESB.2014.062503 Kim J-R (2022) People-centered entrepreneurship: the impact of empathy and social entrepreneurial self-
efficacy for social entrepreneurial intention. Glob Bus Financ Rev 27:108–118. https://doi.org/10.
17549/gbfr.2022.27.1.108 Kim CNT, Van HP, Van HT, Kim TP (2020) The relationship between higher education and entrepreneu
ial intention among Vietnamese students. Manag Sci Lett. https://doi.org/10.5267/j.msl.2020.1.00 Kobylińska U (2022) Attitudes, subjective norms, and perceived control versus contextual factors influ-
encing the entrepreneurial intentions of students from Poland. WSEAS Trans Bus Econ. https://doi.
org/10.37394/23207.2022.19.10l Kör B, Wakkee I, Mutlutürk M (2020) An investigation of factors influencing entrepreneurial intention
amongst university students. J High Educ Theory Pract. https://doi.org/10.33423/jhetp.v20i1.2777 Kowang TO, Apandi SZBA, Hee OC et al (2021) Undergraduates entrepreneurial intention: holist
determinants matter. Int J Eval Res Educ. https://doi.org/10.11591/ijere.v10i1.20733 Krueger N (2009) Entrepreneurial intentions are dead: long live entrepreneurial intentions. Understan
ing the entrepreneurial mind. Springer, Cham, pp 13–34 Krueger NF, Carsrud AL (1993) Entrepreneurial intentions: applying the theory of planned behaviour. Entrep Reg Dev. https://doi.org/10.1080/08985629300000020 p
g
p
g
Krueger NF, Reilly MD, Carsrud AL (2000) Competing models of entrepreneurial intentions. J Bus Ve
tur. https://doi.org/10.1016/S0883-9026(98)00033-0 Kuckertz A, Block J (2021) Reviewing systematic literature reviews: ten key questions and criteria f
reviewers. Manag Rev Q 71(3):519–524l Kuckertz A, Wagner M (2010) The influence of sustainability orientation on entrepreneurial intentions—
investigating the role of business experience. J Bus Ventur. https://doi.org/10.1016/j.jbusvent.2009.
09.001 Kurniawan MEH, Yudoko G, Basri MH, Umbara AN (2019) Do entrepreneurship students have an inten-
tion to become an entrepreneur? J Entrep Educ 22(2):1–14 Laguía A, Moriano JA, Gorgievski MJ (2019) A psychosocial study of self-perceived creativity and
entrepreneurial intentions in a sample of university students. Think Ski Creat. https://doi.org/10.
1016/j.tsc.2018.11.004 j
Law KMY, Jakubiak M (2020) Determinants of students’ entrepreneurial intention: a comparative study
in Poland and Hong Kong. Int J Innov Learn. https://doi.org/10.1504/IJIL.2020.108459 Le Trung T, Xuan HD, Ngoc HN et al (2020) A comparative analysis of entrepreneurial intention and
migration attitudes of students in Vietnam and Poland. Manag Sci Lett. p
p
org/10.3390/math9161955 https://doi.org/10.19026/rjaset.9.2630 Jiang X, Sun Y (2015) Study on constructing an education platform for innovation and entrepreneursh
of university student. Res J Appl Sci Eng Technol. https://doi.org/10.19026/rjaset.9.2630 y
pp
g
p
g
j
Jwara N, Hoque M (2018) Entrepreneurial intentions among university students: a case study of Durban
University of Technology. Acad Entrep J 24(3):1–19 Jwara N, Hoque M (2018) Entrepreneurial intentions among university students: a case study of Durban
University of Technology. Acad Entrep J 24(3):1–19 Jwara N, Hoque M (2018) Entrepreneurial intentions among
University of Technology. Acad Entrep J 24(3):1–19 Kalitanyi V, Bbenkele E (2017) Assessing the role of socio-economic values on entrepreneurial inten-
tions among university students in Cape Town. S Afr J Econ Manag Sci. https://doi.org/10.4102/
sajems.v20i1.1768 Kalitanyi V, Bbenkele E (2017) Assessing the role of socio-economic values on entrepreneurial inten-
tions among university students in Cape Town. S Afr J Econ Manag Sci. https://doi.org/10.4102/
sajems.v20i1.1768 1 3 Factors affecting students’ entrepreneurial intentions:… 1963 Kalitanyi V, Bbenkele E (2018) Cultural values as determinants of entrepreneurial intentions among
university students in Cape Town-South Africa. J Enterp Communities. https://doi.org/10.1108/
JEC-01-2017-0017 Kalitanyi V, Bbenkele E (2018) Cultural values as determinants of entrepreneurial intentions among
university students in Cape Town-South Africa. J Enterp Communities. https://doi.org/10.1108/
JEC-01-2017-0017 Kalitanyi V, Visser DJ (2016) Social values as determinants of entrepreneurial intentions among uni-
versity students in Cape Town-South Africa. Probl Perspect Manag. https://doi.org/10.21511/ppm.
14(3-1).2016.05 Kalitanyi V, Visser DJ (2016) Social values as determinants of entrepreneurial intentions among uni-
versity students in Cape Town-South Africa. Probl Perspect Manag. https://doi.org/10.21511/ppm.
14(3-1).2016.05 (
)
Karimi S, Makreet AS (2020) The role of personal values in forming students’ entrepreneurial intentions
in developing countries. Front Psychol. https://doi.org/10.3389/fpsyg.2020.525844 (
)
Karimi S, Makreet AS (2020) The role of personal values in forming students’ entrepreneurial intentions
in developing countries. Front Psychol. https://doi.org/10.3389/fpsyg.2020.525844 Karimi S, Biemans HJA, Naderi Mahdei K et al (2017) Testing the relationship between personality char-
acteristics, contextual factors and entrepreneurial intentions in a developing country. Int J Psychol. https://doi.org/10.1002/ijop.12209f Karimi S, Biemans HJA, Naderi Mahdei K et al (2017) Testing the relationship between personality char-
acteristics, contextual factors and entrepreneurial intentions in a developing country. Int J Psychol. https://doi.org/10.1002/ijop.12209f p
g
j p
Kayed H, Al-Madadha A, Abualbasal A (2022) The effect of entrepreneurial education and culture on
entrepreneurial intention. Organizacija. p
p
org/10.3390/math9161955 https://doi.org/10.1177/0971355719893500 p
p
g
Lopes J, Teixeira SJ, Ferreira JJM et al (2020) University entrepreneurial intentions: mainland and insular
regions—are they different? Educ Train. https://doi.org/10.1108/ET-03-2019-0055 Lopes J, Teixeira SJ, Ferreira JJM et al (2020) University entrepreneurial intentions: mainland and insular
regions—are they different? Educ Train. https://doi.org/10.1108/ET-03-2019-0055l f
Lopez T, Alvarez C (2019) Influence of university-related factors on students’ entrepreneurial intentions. Int J Entrep Ventur. https://doi.org/10.1504/IJEV.2019.103751 f
Lopez T, Alvarez C (2019) Influence of university-related factors on s
Int J Entrep Ventur. https://doi.org/10.1504/IJEV.2019.103751 f
Lopez T, Alvarez C (2019) Influence of university-related factors on students’ entrepreneurial intentions. Int J Entrep Ventur. https://doi.org/10.1504/IJEV.2019.103751 l
rep Ventur. https://doi.org/10.1504/IJEV.2019.10375 López-Delgado P, Iglesias-Sánchez PP, Jambrino-Maldonado C (2019) Gender and university degree:
a new analysis of entrepreneurial intention. Educ Train. https://doi.org/10.1108/ET-04-2018-0085f Lu G, Song Y, Pan B (2021) How university entrepreneurship support affects college students’ entrepre-
neurial intentions: an empirical analysis from China. Sustainability. https://doi.org/10.3390/su130
63224 Luc PT (2018) The relationship between perceived access to finance and social entrepreneurship inten-
tions among university students in Vietnam. J Asian Financ Econ Bus. https://doi.org/10.13106/
jafeb.2018.vol5.no1.63 j
Lüthje C, Franke N (2003) The “making” of an entrepreneur: testing a model of entrepreneurial intent
among engineering students at MIT. R D Manag. https://doi.org/10.1111/1467-9310.00288f j
Lüthje C, Franke N (2003) The “making” of an entrepreneur: testing a model of entrepreneurial inte
among engineering students at MIT. R D Manag. https://doi.org/10.1111/1467-9310.00288f Maes J, Leroy H, Sels L (2014) Gender differences in entrepreneurial intentions: a TPB multi-group ana
ysis at factor and indicator level. Eur Manag J. https://doi.org/10.1016/j.emj.2014.01.001l Maes J, Leroy H, Sels L (2014) Gender differences in entrepreneurial intentions: a TPB multi-group anal-
ysis at factor and indicator level. Eur Manag J. https://doi.org/10.1016/j.emj.2014.01.001l Magasi C (2022) The influence of entrepreneurship education on entrepreneurial intentions. Int J Res Bus
Soc Sci 11:371–380. https://doi.org/10.20525/ijrbs.v11i2.1701l Magasi C (2022) The influence of entrepreneurship education on entre
Soc Sci 11:371–380. https://doi.org/10.20525/ijrbs.v11i2.1701l Magasi C (2022) The influence of entrepreneurship education on entrepreneurial intentions. Int J Res Bus
Soc Sci 11:371–380. https://doi.org/10.20525/ijrbs.v11i2.1701l Maheshwari G (2021) Factors influencing entrepreneurial intentions the most for university students in
Vietnam: educational support, personality traits or TPB components? Educ Train. https://doi.org/
10.1108/ET-02-2021-0074 Maheshwari G (2021) Factors influencing entrepreneurial intentions the most for university students in
Vietnam: educational support, personality traits or TPB components? Educ Train. p
p
org/10.3390/math9161955 https://doi.org/10.5267/j.
msl.2019.8.022 Lee-Ross D (2017) An examination of the entrepreneurial intent of MBA students in Australia using the
entrepreneurial intention questionnaire. J Manag Dev. https://doi.org/10.1108/jmd-10-2016-0200 Leiva JC, Mora-Esquivel R, Krauss-Delorme C et al (2021) Entrepreneurial intention among Latin Amer-
ican university students [Intención emprendedora entre estudiantes universitarios en Latinoamé-
rica]. Acad Rev Latinoam Adm 34(3):399–418. https://doi.org/10.1108/ARLA-05-2020-0106 Lent RW, Brown SD, Hackett G (1994) Toward a unifying social cognitive theory of career and academic
interest, choice, and performance. J Vocat Behav 45(1):79–122. https://doi.org/10.1006/jvbe.1994 1 3 1964 G. Maheshwari et al. Lim WL, Yusof M, Nor LM (2017) The impact of entrepreneurial alertness on entrepreneurial intention
among university students in Malaysia: theory of planned behaviour. J Eng Appl Sci. https://doi.
org/10.3923/jeasci.2017.1409.1418 Lim WL, Yusof M, Nor LM (2017) The impact of entrepreneurial alertness on entrepreneurial intention
among university students in Malaysia: theory of planned behaviour. J Eng Appl Sci. https://doi.
org/10.3923/jeasci.2017.1409.1418 g
j
Lin S, De-Pablos-Heredero C, Montes Botella JL, Lin-Lian C (2022) Entrepreneurial intention of Chi-
nese students studying at universities in the community of Madrid. Sustainability 14:5475. https://
doi.org/10.3390/su14095475 g
Liñán F, Fayolle A (2015) A systematic literature review on entrepreneurial intentions: citation, thematic
analyses, and research agenda. Int Entrep Manag J. https://doi.org/10.1007/s11365-015-0356-5l Lingappa AK, Shah A, Mathew AO (2020) Academic, family, and peer influence on entrepreneuri
intention of engineering students. SAGE Open. https://doi.org/10.1177/2158244020933877 Liu X, Zhao WGW (2021) Family education? Unpacking parental factors for tourism and hospitality
students’ entrepreneurial intention. J Hosp Leis Sport Tour Educ. https://doi.org/10.1016/j.jhlste.
2020.100284f Liu X, Lin C, Zhao G, Zhao D (2019) Research on the effects of entrepreneurial education and entrepre-
neurial self-efficacy on college students’ entrepreneurial intention. Front Psychol. https://doi.org/
10.3389/fpsyg.2019.00869 p yg
Lo C, Sun H, Law K (2017) Comparing the entrepreneurial intention between female and male engineer-
ing students. J Women’s Entrep Educ 1–2:28–51 g
p
Locke E, Baum R (2007) Entrepreneurial motivation. In: Baum JR, Frese M, Baron RA (eds) The psy- Locke E, Baum R (2007) Entrepreneurial motivation. In: Baum JR, Frese M, Baron RA (eds) The psy- Locke E, Baum R (2007) Entrepreneurial motivation. In: Baum JR, Frese M, Baron RA (eds) The ps
chology of entrepreneurship. Lawrence Erlbaum Associates Publishers, pp 93–112 gy
p
p
, pp
Looi KH (2020) Contextual motivations for undergraduates’ entrepreneurial intentions in emerging Asia
economies. J Entrep. https://doi.org/10.1177/0971355719893500 (
)
g
p
economies. J Entrep. p
p
org/10.3390/math9161955 W ld J E t
M
S
t i D
18 305 319 Maslakçı A, Sürücü L, Şeşen H (2022a) Relationship between positive psychological capital and entr
preneurial intentions of university students: the mediating role of entrepreneurial self-efficac Maslakçı A, Sürücü L, Şeşen H (2022a) Relationship between positive psychological capital and entre-
preneurial intentions of university students: the mediating role of entrepreneurial self-efficacy. World J Entrep Manag Sustain Dev 18:305–319 World J Entrep Manag Sustain Dev 18:305–319 Maslakçı A, Sürücü L, Şeşen H (2022b) Positive psychological capital and university students’ entrepre-
neurial intentions: does gender make a difference? Int J Educ Vocat Guid. https://doi.org/10.1007/
s10775-022-09545-z Maziriri ET, Maramura TC, Nzewi OI (2019) Determinants of entrepreneurial intention among genera-
tion y students within the Johannesburg Metropolitan Area of South Africa. African J Bus Econ
Res. https://doi.org/10.31920/1750-4562/2019/14n3a6 Mirjana PB, Ana A, Marjana MS (2018) Examining determinants of entrepreneurial intentions in Slove-
nia: applying the theory of planned behaviour and an innovative cognitive style. Econ Res Istraz. https://doi.org/10.1080/1331677X.2018.1478321 Mohammadi P, Kamarudin S, Omar R (2020) Do Islamic values impact social entrepreneurial intention
of university students in Malaysia? An empirical investigation into the mediating role of empathy. Int J Econ Manag 14(3):365–378l Mónico L, Carvalho C, Nejati S et al (2021) Entrepreneurship education and its influence on higher edu-
cation students’ entrepreneurial intentions and motivation in Portugal. BAR Braz Adm Rev. https://
doi.org/10.1590/1807-7692BAR2021190088 Morales-Alonso G, Pablo-LerchundiNúñez-Del-Río IMC (2016) Entrepreneurial intention of engineering
students and associated influence of contextual factors/Intención emprendedora de los estudiantes
de ingeniería e influencia de factores contextuales. Rev Psicol Soc. https://doi.org/10.1080/02134
748.2015.1101314 Mustafa MJ, Hernandez E, Mahon C, Chee LK (2016) Entrepreneurial intentions of university students
in an emerging economy: the influence of university support and proactive personality on students’
entrepreneurial intention. J Entrep Emerg Econ. https://doi.org/10.1108/JEEE-10-2015-0058 Mykolenko O, Ippolitova I, Doroshenko H, Strapchuk S (2022) The impact of entrepreneurship edu-
cation and cultural context on entrepreneurial intentions of Ukrainian students: the mediat-
ing role of attitudes and perceived control. High Educ Ski Work Learn. https://doi.org/10.1108/
HESWBL-08-2020-0190 Nabi G, Holden R, Walmsley A (2010) Entrepreneurial intentions among students: towards a re-focused
research agenda. J Small Bus Enterp Dev 17:537–551. https://doi.org/10.1108/146260010110887
14 Nabi G, LiñáN F, Fayolle A et al (2017) The impact of entrepreneurship education in higher education:
a systematic review and research agenda. Acad Manag Learn Educ 16(2):277–299. p
p
org/10.3390/math9161955 https://doi.org/
10.1108/ET-02-2021-0074 Maheshwari G, Kha KL (2021) Investigating the relationship between educational support and entrepre-
neurial intention in Vietnam: the mediating role of entrepreneurial self-efficacy in the theory of
planned behavior. Int J Manag Educ. https://doi.org/10.1016/j.ijme.2021.100553 Maheshwari G, Kha KL (2021) Investigating the relationship between educational support and entrepre-
neurial intention in Vietnam: the mediating role of entrepreneurial self-efficacy in the theory of
planned behavior. Int J Manag Educ. https://doi.org/10.1016/j.ijme.2021.100553 Maheshwari G, Nayak R, Ngyyen T (2021) Review of research for two decades for women leadership
in higher education around the world and in Vietnam: a comparative analysis. Gend Manag Int J
36(5):640–658 Maheshwari G, Nayak R, Ngyyen T (2021) Review of research for two decades for women leadership
in higher education around the world and in Vietnam: a comparative analysis. Gend Manag Int J
36(5):640–658 Mahola S, Aderibigbe JK, Chimucheka T (2019) South African university students’ entrepreneurial
intention as a correlate of entrepreneurship risk perceptions and aversion. J Hum Ecol. https://doi.
org/10.31901/24566608.2019/67.1-3.3153 g
Maresch D, Harms R, Kailer N, Wimmer-Wurm B (2016) The impact of entrepreneurship education on
the entrepreneurial intention of students in science and engineering versus business studies univer-
sity programs. Technol Forecast Soc Change. https://doi.org/10.1016/j.techfore.2015.11.006 y p
g
g
p
g
j
Margaça C, Hernández-Sánchez B, Sánchez-García JC, Cardella GM (2021) The roles of psychological
capital and gender in university students’ entrepreneurial intentions. Front Psychol. https://doi.org/
10.3389/fpsyg.2020.615910 1 3 Factors affecting students’ entrepreneurial intentions:… 1965 Martins I, Perez JP (2020) Testing mediating effects of individual entrepreneurial orientation on the rela-
tion between close environmental factors and entrepreneurial intention. Int J Entrep Behav Res. https://doi.org/10.1108/IJEBR-08-2019-0505 Martins I, Perez JP (2020) Testing mediating effects of individual entrepreneurial orientation on the rela-
tion between close environmental factors and entrepreneurial intention. Int J Entrep Behav Res. https://doi.org/10.1108/IJEBR-08-2019-0505 p
g
Maslakçı A, Sürücü L, Şeşen H (2022a) Relationship between positive psychological capital and entre-
preneurial intentions of university students: the mediating role of entrepreneurial self-efficacy. ld
S
i
18 305 319 p
g
Maslakçı A, Sürücü L, Şeşen H (2022a) Relationship between positive psychological capital and entre-
preneurial intentions of university students: the mediating role of entrepreneurial self-efficacy. p
p
org/10.3390/math9161955 https://doi.org/10.1080/03075079.2017.1365359l g
p
g
Nsahlai VK, Zogli LJ, Lawa E, Dlamini BI (2020) Factors influencing entrepreneurial intention: a case of
students in a South African University. Acad Entrep J 26(1) Nsahlai VK, Zogli LJ, Lawa E, Dlamini BI (2020) Factors influencing entrepreneurial intention: a case of
students in a South African University. Acad Entrep J 26(1) Nuan W, Xin G (2012) Rational thinking upon entrepreneurship education of college students. High Edu
Soc Sci 3:41–44. https://doi.org/10.3968/j.hess.1927024020120303.1121 Nuringsih K, Puspitowati I (2017) Determinants of eco entrepreneurial intention among students: stud
in the entrepreneurial education practices. Adv Sci Lett. https://doi.org/10.1166/asl.2017.9351l hanu IB, Shodipe TO (2021) Influence of the link between resources and behavioural factors on the
entrepreneurial intentions of electrical installation and maintenance work students. J Innov Entrep. Ohanu IB, Shodipe TO (2021) Influence of the link between resources and behavioural factors on the
entrepreneurial intentions of electrical installation and maintenance work students. J Innov Entrep. https://doi.org/10.1186/s13731-021-00154-8 Ojewumi KA, Fagbenro DA, Babatunde SI (2020) Psycho-demographic factors and entrepreneurial
intention among university students. Open J Psychol Res. https://doi.org/10.32591/coas.ojpr.0401.
03031o Ozcan NA, Sahin S, Cankir B (2021) The validity and reliability of thriving scale in academic context:
mindfulness, GPA, and entrepreneurial intention among university students. Curr Psychol. https://
doi.org/10.1007/s12144-021-01590-1 g
Paiva LEB, Sousa ES, Lima TCB, Da Silva D (2020) Planned behavior and religious beliefs as anteced-
ents to entrepreneurial intention: a study with university students. Rev Adm Mackenzie. https://doi.
org/10.1590/1678-6971/eRAMG200022 g
Palalić R, Ramadani V, Ðilović A et al (2017) Entrepreneurial intentions of university students: a cas
based study. J Enterp Communities. https://doi.org/10.1108/JEC-12-2016-0046 Paul J, Hermel P, Srivatava A (2017) Entrepreneurial intentions—theory and evidence from Asia, Amer-
ica, and Europe. J Int Entrep. https://doi.org/10.1007/s10843-017-0208-1 Paul J, Hermel P, Srivatava A (2017) Entrepreneurial intentions—theory and evidence from Asia, Amer-
ica, and Europe. J Int Entrep. https://doi.org/10.1007/s10843-017-0208-1 p
p
p
g
Pérez-Macías N, Fernández-Fernández JL, Rúa Vieites A (2020) The impact of network ties, shared lan-
guages and shared visions on entrepreneurial intentions of online university students. Stud High
Educ. https://doi.org/10.1080/03075079.2019.1619682 Pérez-Macías N, Fernández-Fernández JL, Rúa Vieites A (2020) The impact of network ties, shared lan-
guages and shared visions on entrepreneurial intentions of online university students. Stud High
Educ. https://doi.org/10.1080/03075079.2019.1619682 Pérez-Macías N, Fernández-Fernández JL, Rúa-Vieites A (2021) Entrepreneurial intention among online
and face-to-face university students: the influence of structural and cognitive social capital dimen-
sions. J Int Entrep. p
p
org/10.3390/math9161955 https://doi.org/
10.5465/amle.2015.0026 Nasar A, Kamarudin S, Rizal AM et al (2019) Short-term and long-term entrepreneurial intention com-
parison between Pakistan and Vietnam. Sustainability. https://doi.org/10.3390/su11236529 Nasip S, Amirul SR, Sondoh SL, Tanakinjal GH (2017) Psychological characteristics and entrepreneurial
intention: a study among university students in North Borneo, Malaysia. Educ Train. https://doi.
org/10.1108/ET-10-2015-0092l g
Nathani N, Dwivedi G (2019) Influence of technology entrepreneurship on entrepreneurial intentions: a
cross country analysis. SSRN Electron J. https://doi.org/10.2139/ssrn.3319889 Neneh BN (2014) An assessment of entrepreneurial intention among university students
Mediterr J Soc Sci 5:542–552. https://doi.org/10.5901/mjss.2014.v5n20p542 Neneh BN (2014) An assessment of entrepreneurial intention among university stude
Mediterr J Soc Sci 5:542–552. https://doi.org/10.5901/mjss.2014.v5n20p542 p
g
y
Mediterr J Soc Sci 5:542–552. https://doi.org/10.5901/mjss.2014.v5n20p542 Nguyen C (2017) Entrepreneurial intention of international business students in Viet Nam: a survey
of the country joining the Trans-Pacific Partnership. J Innov Entrep. https://doi.org/10.1186/
s13731-017-0066-z Nguyen TT (2020) The impact of access to finance and environmental factors on entrepreneurial inten-
tion: the mediator role of entrepreneurial behavioural control. Entrep Bus Econ Rev. https://doi.
org/10.15678/EBER.2020.080207f Nguyen TT (2020) The impact of access to finance and environmental factors on entrepreneurial inten-
tion: the mediator role of entrepreneurial behavioural control. Entrep Bus Econ Rev. https://doi.
org/10.15678/EBER.2020.080207f g
Nguyen AT, Do THH, Vu TBT et al (2019) Factors affecting entrepreneurial intentions among youths in
Vietnam. Child Youth Serv Rev. https://doi.org/10.1016/j.childyouth.2019.01.039 g
Nguyen AT, Do THH, Vu TBT et al (2019) Factors affecting entrepreneurial intentions among youths in
Vietnam. Child Youth Serv Rev. https://doi.org/10.1016/j.childyouth.2019.01.039 Nguyen PM, Dinh VT, Luu TMN, Choo Y (2020) Sociological and theory of planned behaviour approach
to understanding entrepreneurship: comparison of Vietnam and South Korea. Cogent Bus Manag. https://doi.org/10.1080/23311975.2020.1815288 Nguyen PM, Dinh VT, Luu TMN, Choo Y (2020) Sociological and theory of planned behaviour approach
to understanding entrepreneurship: comparison of Vietnam and South Korea. Cogent Bus Manag. https://doi.org/10.1080/23311975.2020.1815288 1 3 1966 G. Maheshwari et al. Nowiński W, Haddoud MY, Lančarič D et al (2019) The impact of entrepreneurship education, entrepre-
neurial self-efficacy and gender on entrepreneurial intentions of university students in the Visegrad
countries. Stud High Educ 44:361–379. https://doi.org/10.1080/03075079.2017.1365359l Nowiński W, Haddoud MY, Lančarič D et al (2019) The impact of entrepreneurship education, entrepre-
neurial self-efficacy and gender on entrepreneurial intentions of university students in the Visegrad
countries. Stud High Educ 44:361–379. p
p
org/10.3390/math9161955 https://doi.org/10.1007/s10843-020-00280-6 Pérez-Macías N, Fernández-Fernández JL, Rúa-Vieites A (2021) Entrepreneurial intention among online
and face-to-face university students: the influence of structural and cognitive social capital dimen-
sions. J Int Entrep. https://doi.org/10.1007/s10843-020-00280-6 p
p
g
Pérez-Pérez C, González-Torres T, Nájera-Sánchez JJ (2021) Boosting entrepreneurial intention of uni-
versity students: is a serious business game the key? Int J Manag Educ. https://doi.org/10.1016/j.
ijme.2021.100506 p
p
g
Pérez-Pérez C, González-Torres T, Nájera-Sánchez JJ (2021) Boosting entrepreneurial intention of uni-
versity students: is a serious business game the key? Int J Manag Educ. https://doi.org/10.1016/j.
ijme.2021.100506f j
Phuc PT, Vinh NQ, Do QH (2020) Factors affecting entrepreneurial intention among tourism undergra
uate students in Vietnam. Manag Sci Lett. https://doi.org/10.5267/j.msl.2020.6.026 Phuong NTM, Quoc TH, Van CL, Lien LTK (2021) The students’ attitudes and entrepreneurial intentio
evidence from Vietnam universities. Manag Sci Lett. https://doi.org/10.5267/j.msl.2020.10.028 Phuong NTM, Quoc TH, Van CL, Lien LTK (2021) The students’ attitudes and entrepreneurial intention:
evidence from Vietnam universities. Manag Sci Lett. https://doi.org/10.5267/j.msl.2020.10.028 Pittaway L, Cope J (2007) Entrepreneurship education: a systematic rev
Bus J 25(5):479–510. https://doi.org/10.1177/0266242607080656 Pittaway L, Cope J (2007) Entrepreneurship education: a systematic review of the evidence. Int Small
Bus J 25(5):479–510. https://doi.org/10.1177/0266242607080656 Pittaway L, Cope J (2007) Entrepreneurship education: a systematic review of the evidence. Int Small
Bus J 25(5):479–510. https://doi.org/10.1177/0266242607080656 p
g
Popescu CC, Bostan I, Robu IB et al (2016) An analysis of the determinants of entrepreneurial intention
among students: a Romanian case study. Sustainability. https://doi.org/10.3390/su8080771 Pouratashi M (2015) Entrepreneurial intentions of agricultural students: levels and determinants. J Agr
Educ Ext. https://doi.org/10.1080/1389224X.2014.960528 Prabowo H, Ikhsan RB, Yuniarty Y (2022) Drivers of green entrepreneurial intention: why does sustain-
ability awareness matter among university students? Front Psychol. https://doi.org/10.3389/fpsyg.
2022.873140 Pradana M, Wardhana A, Wijayangka C et al (2020) Indonesian university students’ entrepreneuri
intention: a conceptual study. J Crit Rev. https://doi.org/10.31838/jcr.07.07.101 Pruett M, Shinnar R, Toney B et al (2009) Explaining entrepreneurial intentions of university students:
cross-cultural study. Int J Entrep Behav Res. https://doi.org/10.1108/13552550910995443 y
p
p
g
Qudus A, Mazhar M, Tabassum MF, Farhan M (2022) The role of psychological factors on entrepre-
neurial intentions among business students 32(1) Qudus A, Mazhar M, Tabassum MF, Farhan M (2022) The role of psychological factors on entrepre-
neurial intentions among business students 32(1) Rahaman MA, Ali MJ, Mamoon ZR, Al Asheq A (2020) Understanding the entrepreneurial intention
in the light of contextual factors: gender analysis. J Asian Finance Econ Bus. p
p
org/10.3390/math9161955 https://doi.org/10.
13106/JAFEB.2020.VOL7.NO9.639 Rahaman MA, Ali MJ, Mamoon ZR, Al Asheq A (2020) Understanding the entrepreneurial intention
in the light of contextual factors: gender analysis. J Asian Finance Econ Bus. https://doi.org/10.
13106/JAFEB.2020.VOL7.NO9.639 Rakib M, Tawe A, Azis M et al (2020) Determinants of entrepreneurial intention: empirical study of stu-
dent entrepreneurs. Acad Entrep J 26(3):1–12 Rakib M, Tawe A, Azis M et al (2020) Determinants of entrepreneurial intention: empirical study of stu-
dent entrepreneurs. Acad Entrep J 26(3):1–12 1 3 Factors affecting students’ entrepreneurial intentions:… 1967 Ramayah T, Rahman SA, Taghizadeh SK (2019) Modelling green entrepreneurial intention among uni-
versity students using the entrepreneurial event and cultural values theory. Int J Entrep Ventur. https://doi.org/10.1504/IJEV.2019.101629 Ramayah T, Rahman SA, Taghizadeh SK (2019) Modelling green entrepreneurial intention among uni-
versity students using the entrepreneurial event and cultural values theory. Int J Entrep Ventur. https://doi.org/10.1504/IJEV.2019.101629 p
g
Reuel Johnmark D, Munene JC, Balunywa W (2016) Robustness of personal initiative in moderating
entrepreneurial intentions and actions of disabled students. Cogent Bus Manag. https://doi.org/10.
1080/23311975.2016.1169575 p
g
Reuel Johnmark D, Munene JC, Balunywa W (2016) Robustness of personal initiative in moderating
entrepreneurial intentions and actions of disabled students. Cogent Bus Manag. https://doi.org/10.
1080/23311975.2016.1169575 Rodriguez-Gutierrez P, Cabeza-Ramírez LJ, Muñoz-Fernández GA (2020) University students’ behav-
iour towards entrepreneurial intention in Ecuador: testing for the influence of gender. Int J Environ
Res Public Health. https://doi.org/10.3390/ijerph17228475 Romero-Colmenares LM, Reyes-Rodríguez JF (2022) Sustainable entrepreneurial intentions: explora-
tion of a model based on the theory of planned behaviour among university students in north-east
Colombia. Int J Manag Educ. https://doi.org/10.1016/j.ijme.2022.100627f Roxas B (2014) Effects of entrepreneurial knowledge on entrepreneurial intentions: a longitudinal study
of selected South-east Asian business students. J Educ Work. https://doi.org/10.1080/13639080.
2012.760191f Roy R, Das N (2020) A critical comparison of factors affecting science and technology students’
entrepreneurial intention: a tale of two genders. Int J Educ Vocat Guid. https://doi.org/10.1007/
s10775-019-09393-4 Rueda Barrios GE, Rodriguez JFR, Plaza AV et al (2022) Entrepreneurial intentions of university stu-
dents in Colombia: exploration based on the theory of planned behavior. J Educ Bus. https://doi.
org/10.1080/08832323.2021.1918615 Rungsrisawat S, Sutduean C (2019) Entrepreneurial education and entrepreneurial intention: the mediat-
ing role of creativity disposition among University Students in Thailand. Int J Innov Creat Change
6(10):213–232 (
)
Rusu VD, Roman A, Tudose MB (2022) Determinants of entrepreneurial intentions of youth: the role of
access to finance. Eng Econ. p
p
org/10.3390/math9161955 https://doi.org/10.5755/j01.ee.33.1.28716f i
Salhieh SM, Al-Abdallat Y (2022) Technopreneurial intentions: the effect of innate innovativeness an
academic self-efficacy. Sustainability. https://doi.org/10.3390/su14010238 Salhieh SM, Al-Abdallat Y (2022) Technopreneurial intentions: the effect of innate innovativeness
academic self-efficacy. Sustainability. https://doi.org/10.3390/su14010238 fi
Sánchez AC (2021) Personal values as predictors of entrepreneurial intentions of university students. Evol Stud Bus. https://doi.org/10.1344/jesb2021.2.j096 Sankar P, Irin Sudha A (2016) Determinants of entrepreneurial intention of pharmacy students in Chen-
nai. Int J Pharm Sci Rev Res 41(1):150–154 ( )
Saptono A, Purwana D, Wibowo A et al (2019) Assessing the university students’ entrepreneurial inten-
tion: entrepreneurial education and creativity. Humanit Soc Sci Rev. https://doi.org/10.18510/hssr.
2019.7158 Schlaegel C, Engle RL, Richter NF, Taureck PC (2021) Personal factors, entrepreneurial intention, and
entrepreneurial status: a multinational study in three institutional environments. J Int Entrep. https://doi.org/10.1007/s10843-021-00287-7 p
g
Sesen H (2013) Personality or environment? A comprehensive study on the entrepreneurial intentions of
university students. Educ Train. https://doi.org/10.1108/ET-05-2012-0059 Shah N, Soomro BA (2017) Investigating entrepreneurial intention among public sector university st
dents of Pakistan. Educ Train. https://doi.org/10.1108/ET-11-2016-0168 Shahid MS, Ahsen SR (2021) Linking entrepreneurship education and entrepreneurial intentions: an
interactive effect of social and personal factors. Int J Learn Change. https://doi.org/10.1504/IJLC.
2021.111670 Shahid MS, Imran Y, Shehryar H (2018) Determinants of entrepreneurial intentions: an institutional
embeddedness perspective. J Small Bus Entrep. https://doi.org/10.1080/08276331.2017.1389053 Shahneaz MA, Bin Amin M, Eni LN (2020) The interplay between the psychological factors and entre-
preneurial intention: an empirical investigation. J Asian Financ Econ Bus. https://doi.org/10.13106/
JAFEB.2020.VOL7.NO12.139f Shahzad MF, Khan KI, Saleem S, Rashid T (2021) What factors affect the entrepreneurial intention to
start-ups? The role of entrepreneurial skills, propensity to take risks, and innovativeness in open
business models. J Open Innov Technol Mark Complex. https://doi.org/10.3390/JOITMC7030173l Shamsudin SFFB, Al MA, Nawi NBC et al (2017) Factors influencing entrepreneurial intention and the
moderating role of entrepreneurship education: a conceptual model. Adv Sci Lett. https://doi.org/
10.1166/asl.2017.7635 1 3 1968 G. Maheshwari et al. Shamsudin MF, Alias MN, Majid ZA et al (2021) Does the support system mediate the relationship
between university roles and entrepreneurial intentions among university students? J Inf Technol
Manag. https://doi.org/10.22059/JITM.2021.83112 Shapero A (1984) The entrepreneurial event. College of Administrative Science, Ohio State University,
Columbus Shapero A, Sokol L (1982) The social dimensions of entrepreneurship. In: The encyclopaedia
entrepreneurship. Prentice-Hall, pp 72–90 Sharahiley SM (2020) Examining entrepreneurial intention of the Saudi Arabia’s university students: ana-
lyzing alternative integrated research model of TPB and EEM. org/10.1007/s40171-019-00231-8 Sherkat A, Chenari A (2022) Assessing the effectiveness of entrepreneurship education in the universities
of Tehran province based on an entrepreneurial intention model. Stud High Educ. https://doi.org/
10.1080/03075079.2020.1732906fi Shi L, Yao X, Wu W (2020) Perceived university support, entrepreneurial self-efficacy, heterogeneous
entrepreneurial intentions in entrepreneurship education: the moderating role of the Chinese sense
of face. J Entrep Emerg Econ. https://doi.org/10.1108/JEEE-04-2019-0040l Silva N, Fernández-Robin C, Yáñez D, Romaní G (2021) Influence of educational programs oriented
toward entrepreneurship on the entrepreneurial intention of university students: the case of Chile. Acad Rev Latinoam Adm. https://doi.org/10.1108/ARLA-06-2020-0146 Solesvik MZ (2013) Entrepreneurial motivations and intentions: investigating the role of education majo
Educ Train. https://doi.org/10.1108/00400911311309314 Solesvik MZ (2013) Entrepreneurial motivations and intentions: i
Educ Train. https://doi.org/10.1108/00400911311309314 olesvik M, Westhead P, Matlay H (2014) Cultural factors and entrepreneurial intention: The role of
entrepreneurship education. Educ Train. https://doi.org/10.1108/ET-07-2014-0075 Solesvik M, Westhead P, Matlay H (2014) Cultural factors and entrepreneurial intention: The role
entrepreneurship education. Educ Train. https://doi.org/10.1108/ET-07-2014-0075 Soria K, Honores G, Gutiérrez J (2016) Gender and social legitimacy of entrepreneurship: contribution to
entrepreneurial intention in university students from Chile and Colombia. J Technol Manag Innov. https://doi.org/10.4067/S0718-27242016000300008 Sriyakul T, Jermsittiparsert K (2019) The mediating role of entrepreneurial passion in the relationship
between entrepreneur education and entrepreneurial intention among university students in Thai-
land. Int J Innov Creat Chang 6(10):193–212 St-Jean É, Nafa A, Tremblay M et al (2014) Entrepreneurial intentions of university students: an interna-
tional comparison between African, European and Canadian students. Int J Entrep Innov Manag. https://doi.org/10.1504/IJEIM.2014.062878l p
g
Su Y, Zhu Z, Chen J et al (2021) Factors influencing entrepreneurial intention of university students in
China: integrating the perceived university support and theory of planned behavior. Sustainability. https://doi.org/10.3390/su13084519l p
g
Sumathi P, Ahamed SBI, Karthikeyan M (2018) Descriptive research study on factors influencing entre-
preneurial intention among engineering students in Virudhunagar District. J Adv Res Dyn Control
Syst 10(1):605–611 y
( )
Suratno E, Kusmana A (2019) The analysis of the effect of entrepreneurship education, perceived desir-
ability, and entrepreneurial self-efficacy on university students’ entrepreneurial intention. Univers J
Educ Res. https://doi.org/10.13189/ujer.2019.071131 Tamprateep P, Tanaboriboon C, Rathprasert B (2019) Factors affecting the entrepreneurial intentions
among university students of Thailand. J Comput Theor Nanosci. https://doi.org/10.1166/jctn.
2019.8389 Tassawa C (2019) A structural model for predicting entrepreneurial intention of university students. Humanit Arts Soc Sci Stud. p
p
org/10.3390/math9161955 Glob J Flex Syst Manag. https://doi.
org/10.1007/s40171-019-00231-8 org/10.1007/s40171-019-00231-8 Int J Sci Technol Res 8(11):3737–3743 Utami CW (2017) Attitude, subjective norms, perceived behavior, entrepreneurship education and sel
efficacy toward entrepreneurial intention university student in Indonesia. Eur Res Stud J 20l fi
Valencia-Arias A, Rodríguez Correa PA, Cárdenas-Ruiz JA, Gómez-Molina S (2022) Factors that influ-
ence the entrepreneurial intention of psychology students of the virtual modality [Factores que
influyen en la intención emprendedora de estudiantes de psicología de la modalidad virtual]. Retos
12:5–24. https://doi.org/10.17163/ret.n23.2022.01 Van Ewijk AR, Belghiti-Mahut S (2019) Context, gender and entrepreneurial intentions: how entre-
preneurship education changes the equation. Int J Gend Entrep. https://doi.org/10.1108/
IJGE-05-2018-0054 Van Trang T, Doanh DC (2019) The role of structural support in predicting entrepreneurial intention:
insights from Vietnam. Manag Sci Lett. https://doi.org/10.5267/j.msl.2019.6.012 Van Trang T, Do QH, Loan NTB (2020) Relationships between students’ work values and
intention among Vietnamese students. Acad Entrep J 26:1–13 Velásquez JAT, Arias AV, Hernández JB et al (2018) Characterization of entrepreneurial intention in
university students as from systemic entrepreneurship intention Model: a case study. Cuad Gest. https://doi.org/10.5295/cdg.160670jt p
g
g
j
Virginia Guadalupe LT, Yesenia ST, Luis Ramón MM, Diego Alfredo PR (2021) Determinantes de la
intención emprendedora en estudiantes universitarios mexicanos. Rev Ciencias Soc. https://doi.org/
10.31876/rcs.v27i.36998 Vroom V (1964) Work and motivation. Wiley, New York Vroom V (1964) Work and motivation. Wiley, New Yorkf Vuong BN, Phuong NND, Huan DD, Quan TN (2020) A model of factors affecting entrepreneurial inten-
tion among information technology students in Vietnam. J Asian Financ Econ Bus. https://doi.org/
10.13106/JAFEB.2020.VOL7.NO8.461 Walter SG, Block JH (2016) Outcomes of entrepreneurship education: an institutional perspective. J Bus
Ventur. https://doi.org/10.1016/j.jbusvent.2015.10.003ifi Wang W, Tang Y, Liu Y et al (2019) Can sense of opportunity identification efficacy play a mediating
role? Relationship between network embeddedness and social entrepreneurial intention of univer-
sity students. Front Psychol. https://doi.org/10.3389/fpsyg.2019.01342 Wang W, Tang Y, Liu Y et al (2019) Can sense of opportunity identification efficacy play a mediating
role? Relationship between network embeddedness and social entrepreneurial intention of univer-
sity students. Front Psychol. https://doi.org/10.3389/fpsyg.2019.01342 Wang Q, Sun Z, Wu C (2021) The impact of university innovation and entrepreneurship education on
entrepreneurial intention from the perspective of educational psychology. Front Psychol. https://
doi.org/10.3389/fpsyg.2021.745976f Wang Q, Sun Z, Wu C (2021) The impact of university innovation and entrepreneurship education on
entrepreneurial intention from the perspective of educational psychology. Front Psychol. org/10.1007/s40171-019-00231-8 https://doi.org/10.14456/hasss.2019.11f p
g
Thanh LT, Hau DX, Huyen NN et al (2020) The effects of internal and external barriers on Vietnamese
students’ entrepreneurial intention. Manag Sci Lett. https://doi.org/10.5267/j.msl.2019.8.032 Tiwari P, Bhat AK, Tikoria J, Saha K (2020) Exploring the factors responsible in predicting entrepre-
neurial intention among nascent entrepreneurs: a field research. South Asian J Bus Stud. https://
doi.org/10.1108/SAJBS-05-2018-0054 Tomal M, Szromnik A (2021) Determinants of entrepreneurial intentions of university students in
selected post-communist countries in Europe: investigating cross-cultural differences. J Bus Econ
Manag. https://doi.org/10.3846/jbem.2021.15546 g
p
g
j
orres FC, Méndez JCE, Barreto KS et al (2017) Exploring entrepreneurial intentions in Latin American
university students. Int J Psychol Res. https://doi.org/10.21500/20112084.2794 Torres FC, Méndez JCE, Barreto KS et al (2017) Exploring entrepreneurial intentions in Latin American
university students. Int J Psychol Res. https://doi.org/10.21500/20112084.2794 1 3 Factors affecting students’ entrepreneurial intentions:… 1969 Tran TVH, Duong CD, Nguyen TH et al (2022) UPPS impulsivity, entrepreneurial self-efficacy and
entrepreneurial intentions among university students: ADHD symptoms as a moderator. J Appl Res
High Educ. https://doi.org/10.1108/JARHE-12-2021-0464 Tran TVH, Duong CD, Nguyen TH et al (2022) UPPS impulsivity, entrepreneurial self-efficacy and
entrepreneurial intentions among university students: ADHD symptoms as a moderator. J Appl Res
High Educ. https://doi.org/10.1108/JARHE-12-2021-0464 g
p
g
Tung DT, Hung NT, Phuong NTC et al (2020) Enterprise development from students: the case of uni-
versities in Vietnam and the Philippines. Int J Manag Educ. https://doi.org/10.1016/j.ijme.2019.
100333f Tung DT, Hung NT, Phuong NTC et al (2020) Enterprise development from students: the case of uni-
versities in Vietnam and the Philippines. Int J Manag Educ. https://doi.org/10.1016/j.ijme.2019.
100333f Turker D, Selcuk SS (2009) Which factors affect entrepreneurial intention of university students? J Eur
Ind Train. https://doi.org/10.1108/03090590910939049 Turker D, Selcuk SS (2009) Which factors affect entrepreneurial intention of university students? J E
Ind Train. https://doi.org/10.1108/03090590910939049 Ubierna F, Arranz N, de Arroyabe JCF (2014) Entrepreneurial intentions of university students: a stud
of design undergraduates in Spain. Ind High Educ. https://doi.org/10.5367/ihe.2014.0191l Uddin MA, Mohammad S, Hammami S (2016) Influence of demographic factors on the entrepreneurial
intentions of university students in Oman. Investig Manag Financ Innov. https://doi.org/10.21511/
imfi.13(1-1).2016.08f ,
,
(
)l
g p
p
intentions of university students in Oman. Investig Manag Financ Innov. https://doi.org/10.21511/
imfi.13(1-1).2016.08f i
Uike DD (2019) Effect of demographic factors on entrepreneurial intention of management students
Nagpur university, India. org/10.1007/s40171-019-00231-8 https://doi.org/10.1108/14626000810917843i Wu YCJ, Wu T (2017) A decade of entrepreneurship education in the Asia Pacific for future directions
theory and practice. Manag Decis 55. https://doi.org/10.1108/MD-05-2017-0518 Wu WH, Wei CW, Yu MC, Kao HY (2020) Exploring factors surrounding students’ entrepreneurial
intentions in medical informatics: the theory of planning behavior perspective. Front Psychol. https://doi.org/10.3389/fpsyg.2020.544887lfi Wu WH, Wei CW, Yu MC, Kao HY (2020) Exploring factors surrounding students’ entrepreneurial
intentions in medical informatics: the theory of planning behavior perspective. Front Psychol. https://doi.org/10.3389/fpsyg.2020.544887lfi p
g
p yg
Wu J, Alshaabani A, Rudnák I (2022) Testing the influence of self-efficacy and demographic characteris-
tics among international students on entrepreneurial intention in the context of Hungary. Sustain-
ability. https://doi.org/10.3390/su14031069 Wu J, Alshaabani A, Rudnák I (2022) Testing the influence of self-efficacy and demographic characteris-
tics among international students on entrepreneurial intention in the context of Hungary. Sustain-
ability. https://doi.org/10.3390/su14031069 y
p
g
Xuan HD, Le Trung T, Ngoc HN et al (2020) The effect of educational background on entrepreneurial
intention. Manag Sci Lett. https://doi.org/10.5267/j.msl.2019.8.013l y
p
g
Xuan HD, Le Trung T, Ngoc HN et al (2020) The effect of educational background on entrepreneurial
intention. Manag Sci Lett. https://doi.org/10.5267/j.msl.2019.8.013l Yaghmaei O, Ghasemi I, Assadian S (2015) Relationship among influential factors of entrepreneurial
intention in terms of gender: case of postgraduate students in Malaysia. Mediterr J Soc Sci. https://
doi.org/10.5901/mjss.2015.v6n3p195 Yaghmaei O, Ghasemi I, Assadian S (2015) Relationship among influential factors of entrepreneurial
intention in terms of gender: case of postgraduate students in Malaysia. Mediterr J Soc Sci. https://
doi.org/10.5901/mjss.2015.v6n3p195 g
j
p
Yasir N, Liren A, Mehmood N, Arfat Y (2019) Impact of personality traits on entrepreneurial intention
and demographic factors as moderator. Int J Entrep 23:1–20 Yasir N, Liren A, Mehmood N, Arfat Y (2019) Impact of personality traits on entrepreneurial intention
and demographic factors as moderator. Int J Entrep 23:1–20 Yasir N, Xie R, Zhang J (2022) The impact of personal values and attitude toward sustainable entre-
preneurship on entrepreneurial intention to enhance sustainable development: empirical evidence
from Pakistan. Sustainability 14:6792. https://doi.org/10.3390/su14116792l Yasir N, Xie R, Zhang J (2022) The impact of personal values and attitude toward sustainable entre-
preneurship on entrepreneurial intention to enhance sustainable development: empirical evidence
from Pakistan. Sustainability 14:6792. https://doi.org/10.3390/su14116792l y
p
g
Yu T, Khalid N, Ahmed U (2021) Factors influencing entrepreneurial intention among foreigners in
Kazakhstan. Sustainability. org/10.1007/s40171-019-00231-8 https://doi.org/10.3390/su13137066 Yu T, Khalid N, Ahmed U (2021) Factors influencing entrepreneurial intention among foreigners in
Kazakhstan. Sustainability. https://doi.org/10.3390/su13137066 Zhang P, Cain KW (2017) Reassessing the link between risk aversion and entrepreneurial intention: the
mediating role of the determinants of planned behavior. Int J Entrep Behav Res. https://doi.org/10.
1108/IJEBR-08-2016-0248fi Zhang P, Cain KW (2017) Reassessing the link between risk aversion and entrepreneurial intention: the
mediating role of the determinants of planned behavior. Int J Entrep Behav Res. https://doi.org/10.
1108/IJEBR-08-2016-0248fi Zhang J, Huang J (2021) Entrepreneurial self-efficacy mediates the impact of the post-pandemic entrepre-
neurship environment on college students’ entrepreneurial intention. Front Psychol. https://doi.org/
10.3389/fpsyg.2021.643184 Zhang J, Huang J (2021) Entrepreneurial self-efficacy mediates the impact of the post-pandemic entrepre-
neurship environment on college students’ entrepreneurial intention. Front Psychol. https://doi.org/
10.3389/fpsyg.2021.643184 p yg
Zhang Y, Duysters G, Cloodt M (2014) The role of entrepreneurship education as a predictor of universi
students’ entrepreneurial intention. Int Entrep Manag J. https://doi.org/10.1007/s11365-012-0246 p yg
hang Y, Duysters G, Cloodt M (2014) The role of entrepreneurship education as a predictor of university
students’ entrepreneurial intention. Int Entrep Manag J. https://doi.org/10.1007/s11365-012-0246-z Zhang P, Wang DD, Owen CL (2015) A study of entrepreneurial intention of university students. Entre
Res J. https://doi.org/10.1515/erj-2014-0004i Zhang SN, Li YQ, Liu CH, Ruan WQ (2020) Critical factors identification and prediction of tourism
and hospitality students’ entrepreneurial intention. J Hosp Leis Sport Tour Educ. https://doi.org/10.
1016/j.jhlste.2019.100234 Zhang SN, Li YQ, Liu CH, Ruan WQ (2020) Critical factors identification and prediction of tourism
and hospitality students’ entrepreneurial intention. J Hosp Leis Sport Tour Educ. https://doi.org/10.
1016/j.jhlste.2019.100234fi j j
Zhao H, Hills GE, Seibert SE (2005) The mediating role of self-efficacy in the development of entrepr
neurial intentions. J Appl Psychol. https://doi.org/10.1037/0021-9010.90.6.1265 Zhao J, Wei G, Chen KH, Yien JM (2020) Psychological capital and university students’ entrepreneurial
intention in China: mediation effect of entrepreneurial capitals. Front Psychol. https://doi.org/10.
3389/fpsyg.2019.02984 Zhao J, Wei G, Chen KH, Yien JM (2020) Psychological capital and university students’ entrepreneurial
intention in China: mediation effect of entrepreneurial capitals. Front Psychol. https://doi.org/10.
3389/fpsyg.2019.02984 p yg
hu R, Zhao G, Long Z et al (2022) Entrepreneurship or employment? A survey of college students’
sustainable entrepreneurial intentions. Sustainability 14:5466. https://doi.org/10.3390/su14095466
ovko L, Dulčić Ž, Bilić I (2020) Determinants of students’ entrepreneurial intention: an empirical
research. Management 25:25–44 p yg
Zhu R, Zhao G, Long Z et al (2022) Entrepreneurship or employment? org/10.1007/s40171-019-00231-8 https://
doi.org/10.3389/fpsyg.2021.745976f g
p yg
Ward A, Hernández-Sánchez BR, Sánchez-García JC (2019) Entrepreneurial potential and gender effects:
the role of personality traits in university students’ entrepreneurial intentions. Front Psychol. https://doi.org/10.3389/fpsyg.2019.02700 Ward A, Hernández-Sánchez BR, Sánchez-García JC (2019) Entrepreneurial potential and gender effects:
the role of personality traits in university students’ entrepreneurial intentions. Front Psychol. https://doi.org/10.3389/fpsyg.2019.02700 p
g
p yg
Waris I, Barkat W, Ahmed A, Hameed I (2021) Fostering sustainable businesses: understanding sustain-
ability-driven entrepreneurial intention among university students in Pakistan. Soc Responsib J. https://doi.org/10.1108/SRJ-10-2020-0399 p
g
p yg
Waris I, Barkat W, Ahmed A, Hameed I (2021) Fostering sustainable businesses: understanding sustain-
ability-driven entrepreneurial intention among university students in Pakistan. Soc Responsib J. https://doi.org/10.1108/SRJ-10-2020-0399 p
g
Wegner D, Thomas E, Teixeira EK, Maehler AE (2020) University entrepreneurial push strat-
egy and students’ entrepreneurial intention. Int J Entrep Behav Res. https://doi.org/10.1108/
IJEBR-10-2018-0648 p
g
Wegner D, Thomas E, Teixeira EK, Maehler AE (2020) University entrepreneurial push strat-
egy and students’ entrepreneurial intention. Int J Entrep Behav Res. https://doi.org/10.1108/
IJEBR-10-2018-0648 Wibowo SF, Purwana D, Wibowo A, Saptono A (2019) Determinants of entrepreneurial intention among
millennial generation in emerging countries. Int J Entrep 23:1 Wibowo SF, Purwana D, Wibowo A, Saptono A (2019) Determinants of entrepreneurial intention among
millennial generation in emerging countries. Int J Entrep 23:1 1 3 1970 G. Maheshwari et al. Wiramihardja K, N’daryAl Mamun VA et al (2022) Sustainable economic development through entrepre-
neurship: a study on attitude, opportunity recognition, and entrepreneurial intention among univer-
sity students in Malaysia. Front Psychol. https://doi.org/10.3389/fpsyg.2022.866753 Wiramihardja K, N’daryAl Mamun VA et al (2022) Sustainable economic development through entrepre-
neurship: a study on attitude, opportunity recognition, and entrepreneurial intention among univer-
sity students in Malaysia. Front Psychol. https://doi.org/10.3389/fpsyg.2022.866753 y
y
y
p
g
p yg
Wu J, Rudnák I (2021) Exploring the impact of studying abroad in Hungary on entrepreneurial intention
among international students. Sustainability. https://doi.org/10.3390/su13179545 Wu J, Rudnák I (2021) Exploring the impact of studying abroad in Hungary on entrepreneurial intention
among international students. Sustainability. https://doi.org/10.3390/su13179545 Wu S, Wu L (2008) The impact of higher education on entrepreneurial intentions of university students in
China. J Small Bus Enterp Dev. https://doi.org/10.1108/14626000810917843i Wu S, Wu L (2008) The impact of higher education on entrepreneurial intentions of university students in
China. J Small Bus Enterp Dev. org/10.1007/s40171-019-00231-8 A survey of college students’
sustainable entrepreneurial intentions. Sustainability 14:5466. https://doi.org/10.3390/su14095466
Ž p yg
Zhu R, Zhao G, Long Z et al (2022) Entrepreneurship or employment? A survey of college students’
sustainable entrepreneurial intentions. Sustainability 14:5466. https://doi.org/10.3390/su14095466
Zovko L, Dulčić Ž, Bilić I (2020) Determinants of students’ entrepreneurial intention: an empirical
research. Management 25:25–44 sustainable entrepreneurial intentions. Sustainability 14:5466. https://doi.org/10.3390/su14095466
Zovko L, Dulčić Ž, Bilić I (2020) Determinants of students’ entrepreneurial intention: an empirical
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Games-Based Learning as an Interdisciplinary Approach to Literacy across Curriculum for Excellence
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Abstract Literacy remains an area of concern in early secondary education in
Scotland (ages 12-14), with recent research suggesting a continued
decline in attainment levels. As literacy underpins learning,
interdisciplinary and collaborative approaches to teaching literacy are
now being emphasized through the new Curriculum for Excellence that
aims to address this issue. It is not clear, however, what types of
learning activity are most appropriate for implementing this new, more
cooperative approach. One candidate is the use of educational games
and reflective writing. So, to what extent do learners demonstrate
transferable literacy skills through engaging with educational games? This paper evaluates the effectiveness of the multi-user simulation
game, Mars Colony Challenger (MCC), which portrays a scientifically
accurate Mars colonisation mission in a way that aims to facilitate both
scientific and literary development. A class of secondary school pupils
(n=28) used the game within the context of a science class on ‘The
Three States of Matter’. They then produced written narratives that
captured the experiential learning undertaken. Comparing these
narratives with the remaining pupils in the cohort, who had not used
MCC in their science class, revealed a statistically significant difference
in literacy ability. Further qualitative analysis of the narratives
themselves highlighted a high level of engagement and inspiration
evoked through the experience. Consequently, these results highlight
the efficacy of MCC as a means of literacy development, and they
suggest a means to elicit greater frequency of opportunity for pupil
engagement with, and subsequent assessment of, literacy
competencies. Games-Based Learning as an
Interdisciplinary Approach to Literacy
across Curriculum for Excellence Hugh O’Donnell
University of Edinburgh Press Start Press Start Games-based Interdisciplinary Learning in CfE O’Donnell O’Donnell Games-based Interdisciplinary Learning in CfE Keywords Literacy; Curriculum for Excellence; Interdisciplinary Learning; Science;
Game-based learning; Digital games; Serious games Press Start 2015 | Volume 2 | Issue 2
ISSN: 2055-8198
URL: http://press-start.gla.ac.uk Press Start 2015 | Volume 2 | Issue 2
ISSN: 2055-8198
URL: http://press-start.gla.ac.uk Press Start is an open access student journal that publishes the best
undergraduate and postgraduate research, essays and dissertations from across
the multidisciplinary subject of game studies. Press Start is published by HATII
at the University of Glasgow. O’Donnell Games-based Interdisciplinary Learning in CfE 13-14 years old and have been studying English as a compulsory subject
since they were 4-5 years old (Primary 1). Therefore, this project was
quasi-experimental since all pupils in S2 study English. Pupils utilised a networked, multi-user digital game in order that they
could write personal and imaginative narratives within the English and
Science curriculums, utilising all four competencies of Literacy
independent of the subject area. 1. Introduction According to the 2012 and 2014 ‘Scottish Survey of Literacy and
Numeracy’ (The Scottish Government, 2013 & 2015) levels of
attainment in writing continue to decline. As stated by the Department
of Education (2012) there is a paucity of evidence as to why writing lags
behind the other literacy competencies and research has not provided
evidence as to what interventions help secondary pupils succeed in
written competency. The importance of writing proficiency cannot be underestimated. Emig
(1977 in Thompson, 2012) contends that the teaching and learning of
writing has a foundational and underpinning role in children’s
development: Writing serves learning uniquely because writing as process-and-
product possesses a cluster of attributes that correspond
uniquely to certain powerful learning strategies (p. 122). In order to tackle this problem, a new Curriculum for Excellence (CfE)
has been introduced in Scotland. Part of the CfE strategy is an
interdisciplinary and collaborative approach to literacy education. Accordingly, it is now within the remit of all teaching professionals to
embed the overarching themes of literacy into their daily practice. Literacy – reading, writing, speaking and listening – is: Fundamental to all areas of learning, as it unlocks access to the
wider curriculum. Being literate increases opportunities for the
individual in all aspects of life, lays the foundations for lifelong
learning and work (LTScotland (2012a), p. 4). Lawless & Brown (2015) assert in their paper on digital game-based
science and literacy learning: “researchers have illustrated for decades
that leveraging interdisciplinary contexts… provide opportunities for
students to engage in real-world problem solving that can deepen
students’ understanding, flexibility in application and transfer of
knowledge to novel situations and also reduces the likelihood of inert
knowledge (e.g., Bednar, Cunningham, Duffy, & Perry, 1992; Bransford,
Vye, Adams, & Perfetto, 1989; Brown & King, 2000; Jonassen, 2009;
Koschmann, Kelson, Feltovich, & Barrows, 1996; Mergendoller,
Bellisimo, & Maxwell, 2000) (p. 4)”. This paper presents the outcomes of an action research project that was
undertaken within the context of Curriculum of Excellence, a series of
interdisciplinary lessons between the Science and English Departments
of a Scottish secondary school. The target population was a pupil cohort
in their second year (S2) of post-compulsory education. S2 pupils are 2015 | Volume 2 | Issue 2 Press Start
ISSN: 2055-8198
URL: http://press-start.gla.ac.uk 76 O’Donnell Games-based Interdisciplinary Learning in CfE in the pupils’ final written pieces while accommodating the adoption of
scientifically accurate information and an understanding of the natural
process involved. 2. Games, Play & Learning In addition to the widening responsibility for Literacy, Curriculum for
Excellence it has extended the definition of what constitutes a ‘text’. In
the document ‘Curriculum for Excellence: Literacy and English. Principles
and Practice’ (LTScotland, 2013a) under the section ‘What is meant by
‘texts’ (p. 4) the traditional “novels, short stories, plays, poems” are
now joined by the now ubiquitous multimodal and digital mediums -
“blogs and social networking sites…text messages…games” (ibid.) - of
the 21st Century. Digital games, themselves cultural texts (Buckingham & Burn, 2007;
Salen, 2007 in Altura & Curwood 2015), are composed of distinct yet
interconnected elements: graphics, sound, interface, gameplay and
story, as defined by Berens and Howard in Newman (2004), whereas
traditional narratives exhibit the “sequential nature of language (you
read or hear one word at a time)” (Greenfield, 1984, p. 101). Games
provide the opportunity for parallel processing and therefore mimic the
real world where there are “multiple interacting variables…the world is
not a simple system, but rather many complex systems of multiple
interacting factors” (Greenfield, 1984, p. 103; Blunt, 2013). Simulation games allow for ‘real’ experiences to be recorded
simultaneously, enabling pupils to provide an imaginative element to
their experiences. With the affordance of a multi-player context, these
activities may be undertaken within “a group that is bonded primarily
through shared endeavours, goals, and practices” (Gee, 2003, p. 197),
providing richer shared corroborative narratives. Wouter et al. (2013) in their meta-analysis of 39 previous studies
concluded that ‘serious games’ were effective in improving both learning
and retention. Connolly, et al. (2012) also found that during their meta-
analysis of game-based learning that the most frequent instances of
efficacy were related to understanding and acquisition of content
knowledge. Mars Colony Challenger (MCC) offered an experience analogous to the
establishment of a sustainable human colony on Mars. The physical and
scientific constraints would determine the survival of both individuals
and groups. The loose ‘scriptedness’ of the game allowed for creativity 2015 | Volume 2 | Issue 2 Press Start
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URL: http://press-start.gla.ac.uk 77 77 3. Mars Colony Challenger Mars Colony Challenger (MCC) is a 3D first-person video game,
developed by Hyperkat Games1 in the USA, that offers players the
challenge of setting up a remote base on the surface of Mars. At its
core, the game was derived from a simulator where everything is fully
integrated. The developer took a scientific approach in order to develop
and authentic experience drawing upon accurate principles and
technologies, resulting in a game that is both educational and
challenging. Figure 1. In-game footage from Mars Colony Challenger of the water
pumps installation. Figure 1. In-game footage from Mars Colony Challenger of the water
pumps installation. Using this networked, multi-user application, pupils collaborated in
separate teams consisting of no more than four players2. Of the game’s
seven phases, only ‘Phase #1: Establish Life Support’ was played by
pupils as it was supported by the Science and English-based curricular
activities and would involve pupils in learning about the processes
associated with the ‘three states of matter’ (LTScotland, 2012b) – solid,
liquid and gas. ‘Stage 1:Water Pumps’ requires that players locate,
assemble and activate two heated water pumps to melt subsurface ice,
and produce a water supply; ‘Stage 2: Separator’ requires that players 1 http://www.hyperkat.com/
2 Some groups (e.g. G and H) occasionally produced a combined
grouping of 5 players. 1 http://www.hyperkat.com/
2 Some groups (e.g. G and H) occasionally produced a combined
grouping of 5 players. 2015 | Volume 2 | Issue 2 Press Start
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79 O’Donnell Games-based Interdisciplinary Learning in CfE Games-based Interdisciplinary Learning in CfE locate, assemble and enable the Separator Unit which extracts oxygen
and hydrogen from the newly established water supply (the hydrogen is
used to fuel a utility rover vehicle); ‘Stage 3: Scrubber’ requires that
players locate, assemble and activate the Scrubber Unit which mixes the
oxygen with carbon dioxide and nitrogen from Mars’ low pressure
atmosphere to create a breathable atmosphere of 14-psi within the
base. It was important to build upon the use of MCC to support pupils in the
development of their personal narratives. Pupils began to think about
their ‘destination’ and garnered the necessary and realistic back-story
(Kane, 2004) for the first two weeks, working within their designated
groups, encountering non-fiction and fiction texts aimed at creating the
imaginative backdrop, and which posed the themes:
What is Necessary to Support Life on Earth?
How Could Mankind Colonise Mars? An existing BBC book and DVD series (BBC, 2004) was used as the
narrative backdrop (Royle, 2008; Newman 2004) against which their
virtual personas could be “stimulated or scaffolded by external
elements” (van der Meij et al., 2011) and operate in “both fictional and
non-fictional” realities” (Barab et al., 2005, p. 16). Sitzmann (2011 in
Wouters et al., 2013) suggests evidence that when “a simulation game
was supplemented with other instructional methods [it] yielded higher
levels of learning.” This was not the case when the game was used on
its own. After engaging with related Science concepts, fictional and non-fictional
pieces about Mars and an imagined manned exploration, pupils were
expected to produce a final personal-imaginative piece of writing that
captured their experiences ‘on’ Mars, and elicit an understanding of the
science behind establishing a small human colony. Press Start
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URL: http://press-start.gla.ac.uk 2015 | Volume 2 | Issue 2
79 79 79 O’Donnell Games-based Interdisciplinary Learning in CfE 3 It is not uncommon that the Guidance Department – often acting upon
a parental request – will issue directives for certain pupils to be
separated from each other within classrooms: historic instances of
bullying, etc. Separation of pupils is also undertaken by classroom
teacher in order that misbehaviour between individuals is limited. 4.2 Data Collection Qualitative and quantitative data were derived from the main corpus of
pupil work as follows:
Interim journal writing and group discussion
Final personal-imaginative writing O’Donnell O’Donnell Games-based Interdisciplinary Learning in CfE Games-based Interdisciplinary Learning in CfE Games-based Interdisciplinary Learning in CfE 4. Research Methods Within this paper, the following research question is addressed: To what extent do pupils demonstrate transferable literacy skills
through writing reflective pieces about their experience in a
game-based learning activity presented in the context of science-
themed English lessons? For the purpose of this experiment, the sampling frame consisted of
second year (S2) classes of male and female pupils aged between 12
and 14 at a Scottish school. The experiment encompassed the academic
term between January and April 2014 and was aimed at exploring the
introduction of the Mars simulation software package, Mars Colony
Challenger (MCC). Its effectiveness as a ‘mediating tool’ in satisfying a
number of curricular outcomes for English, Literacy and Science through
inquiry-based, experiential learning activities in “an open-ended journey
towards understanding” (Kane, 2004) is measured as the dependent
variable, while the use of MCC is the independent variable. The rest of
the pupils in the year group – the control group – undertook prose-
related activities involving short stories chosen by their class teacher. Because of school management restrictions random sampling could not
be achieved and it was possible only to draw a sample of 30 pupils (17
female and 13 male) from a cohort of 132. The experimental group was
one class taught by a single teacher. The control group included four
classes taught by four different teachers. The 30 pupils were distributed into 6 groups of 4 (Groups A-F); and
there were 2 groups of 3 (Groups G and H). Group construction was
undertaken with attention afforded to (where possible):
evenly-matched gender pairing;
behaviour management and/or Guidance Department
stipulations3;
attainment (Science and English assessment data from S1);
the anticipated peer-support. The pupils in the control group were similarly of mixed ability and
therefore showed varying degrees of competency across subject areas;
pupils are not set according to ability at this academic stage. 3 It is not uncommon that the Guidance Department – often acting u
a parental request – will issue directives for certain pupils to be
separated from each other within classrooms: historic instances of
bullying, etc. Separation of pupils is also undertaken by classroom
teacher in order that misbehaviour between individuals is limited. 2015 | Volume 2 | Issue 2 Press Start
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URL: http://press-start.gla.ac.uk Press Start
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URL: http://press-start.gla.ac.uk 80 2015 | Volume 2 | Issue 2
82 4.2.1 Quantitative Data The final personal-imaginative written piece contains the personal-
imaginative narratives that were a montage of the pupils’ experiences of
the ‘Mars 2050 Colonisation’. The pieces were assessed using a subset
of the Curriculum for Excellence Literacy and English Experiences and
Outcomes (LTScotland, 2012a). Each writing outcome is ascribed an ‘I Can’ statement, and these
marking descriptors range from Second (2), Third (3) and Fourth(4) in
ascending complexity in confirming competency in a particular aspect of
a piece of writing. For example, Figure 2 shows spelling competencies
(LIT *-21a) required for a piece of work to be given either a category
Second (2), Third (3) and Fourth (4). Figure 2. Literacy – Writing – Spelling: -21a. Figure 2. Literacy – Writing – Spelling: -21a. Each piece of writing was first graded under three Literacy
(orthographic) outcomes, each of which would be assigned level 2, 3 or
4. And each piece of writing was then graded under two English (form
and content) outcomes each of which would be 2, 3 or 4. Final
narratives were then assigned three levels: an aggregate level for LIT;
an aggregate level for ENG; and an overall aggregate level which was
the LIT and ENG aggregates combined. The English (‘ENG’) Outcomes (Figure 3) required further definition
during the purposes of coding and the development of a richer narrative
analysis for this project, and Fisher’s (2012) classifications (Table 1)
were used. 2015 | Volume 2 | Issue 2
81 Press Start
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URL: http://press-start.gla.ac.uk 81 Games-based Interdisciplinary Learning in CfE Games-based Interdisciplinary Learning in CfE O’Donnell O’Donnell Figure 3. Literacy – English: -30a & -31a. Figure 3. Literacy – English: -30a & -31a. 4 Introduced in 2003, The Chartered Teacher was a new qualification-
based teacher grade focusing on enhanced professional practice and
effectiveness in teaching and learning.
http://www.gov.scot/Publications/2003/07/17538/22895
5 A graduate based in a school and who is working through the process
of becoming a fully qualified teacher. 2015 | Volume 2 | Issue 2
83 6 CHAT is an augmentation of Vygotsky’s concept of ‘mediation’ known
as ‘Activity Theory’ (AT). AT conceptualizes “learning as activity and
activity as learning” (Engeström, 1999 in Barab et al., 2002, p. 5) and
describes the inextricable link between learning and acting: “activity
(sensory, mental, and physical) is a precursor to learning (Jonassen &
Rohrer-Murphy in Barab et al., 2002, p. 80). Figure 3. Literacy – English: -30a & -31a. Fisher’s definitions in Table 1 expand on characterisation, setting, as
well as personal reflection and interaction and allowed for a broader
analysis of the communal aspects of the pupils’ colonisation activities. Fisher’s definitions in Table 1 expand on characterisation, setting, as
well as personal reflection and interaction and allowed for a broader
analysis of the communal aspects of the pupils’ colonisation activities. Press Start
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82 82 82 Games-based Interdisciplinary Learning in CfE O’Donnell CfE
Fisher
(2012)
Elaborations
Literacy (LIT)
Orthographic
Spelling,
Punctuation,
Paragraphing,
Meaning. English (ENG)
Form
Word Choice,
Sentence
Construction,
Figurative
Language. English (ENG)
Content
Setting
Description,
Personal
Reflection,
Emotional
Response,
Interpersonal
Relationships. Table 1. Coding from Fisher (2012). Two English Department colleagues volunteered their support to assess
the samples of final personal-imaginative narratives: one with more Table 1. Coding from Fisher (2012). Two English Department colleagues volunteered their support to assess
the samples of final personal-imaginative narratives: one with more
than twenty years’ experience, and the first ‘Chartered Teacher4’ in the
school; the other, a Probationer Teacher5. The Science Teacher reviewed
a selection of final narratives, assessing the scientific content. 2015 | Volume 2 | Issue 2 Press Start
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URL: http://press-start.gla.ac.uk 83 83 O’Donnell Games-based Interdisciplinary Learning in CfE Games-based Interdisciplinary Learning in CfE Games-based Interdisciplinary Learning in CfE O’Donnell Games-based Interdisciplinary Learning in CfE 4.2.2 Qualitative Data In education “collaborative group activity is the key to promote student
interaction in the classroom” (Hashim & Jones, 2007) and to aid
capturing such events, Cultural Historical Activity Theory6 (CHAT) was
used. CHAT, proposed by Engeström (1999 & 2001) is used to
encompass the social contexts of learning: ‘rules’, include grammatical
and genre rules, science principles, lesson plans, game rules;
‘community’ refers to the pupils participating in the same ‘object’ or
‘outcome’ and the other organisational hierarchies related to this
system; ‘division of labour' refers to the degree of contribution provided
by activity participants, pupils and teacher. Whilst primarily a data
analysis tool used to describe systems of human activity, the collection
method was informed by this conceptual lens and assisted through the
use of a template (Hardman, 2008) that helped capture the disorganised
and complex data associated with “multi-mediational processes in
human activity” (Yagamatchi-Lynch, p. 25, 2010). Other material that allowed for a qualitative analysis of the project was
drawn from questionnaires (e.g. how they rated the game and which
features were most enjoyable; how they rated the BBC Space Odyssey
DVD clips and text; group interaction) and semi-structured interviews. Additionally, there were observations of social interaction during game
play, since players’ “reaction to game play, or debriefing, is one of the
key stones of the research programme of many recent studies on video
games” (Guillén-Nieto et al., 2012) and analysed using Cultural
Historical Activity Theory (CHAT). Data was collated pertaining to each of the interpersonal ‘activity
systems’ that were manifest during the use of the MCC game and during
other periods of collaborative group work; identification of initial codes
and subsequently-derived themes were undertaken in accordance with
the methodology presented by Strauss & Corbin (1990), Corbin (1998)
& Saldana (2012), allowing the production of the rich qualitative
narratives that were used to explore each dynamic system of activities. Such qualitative studies of the social and affective aspects of digital
game-based learning are encouraged by Connolly et al (2012). 2015 | Volume 2 | Issue 2 Press Start
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URL: http://press-start.gla.ac.uk 84 84 4.3 Data Analysis Mann-Whitney U was used to compare the MCC experimental group and
the control group because the data were ordinal and did not follow a
known distribution. In addition to the quantitative data analysis, the
qualitative analysis was driven by theory (Hamalainen et al., 2006, p
.52) and utilised the processes of Yagamatchi-Lynch (2010) and
Hardman (2008): producing CHAT diagrams for important scenes within
episodes of gameplay; coding key scenes using CHAT terminology. During the course of pupils’ engagement in the in-game activities, CHAT
was the theoretical lens used to analyse how each – MCC, science fiction
and non-fiction texts, workbooks, etc. – operated as a ‘mediating’ tool. Pupil relationships and the division of labour within each activity system
were highlighted, as were the dialogic and written artefacts produced
during the transformations occurring as subjects (pupils) engaged in
attaining their objectives and outcomes. The final versions of pupils’ personal-imaginative narratives, in addition
to the re-viewing of video evidence and questionnaire data that had
been transcribed and coded facilitated qualitative descriptions of the
“rich participant activities” (Yagamatchi-Lynch, p.33 , 2010) and the
implications and perspectives for the future use of the MCC software. Using CHAT methodology, game-based interaction and non-game based
classroom activities – traditional classroom activities and group
interviews – were analysed to “determine tensions which emerged
through the blended learning experience” (Karasavvidis, p. 198, 2009). Tensions can affect the subject’s ability to attain the object by taking a
role as an obstacle, making it difficult for the subject to attain the
object, or by taking a role as an enabling influence for the subject to
attain the object” (Yagamatchi-Lynch, p. 18, 2010). Game analysis was conducted by transcribing key events from the audio
and audio-visual recordings of ‘over the shoulder’ observed lessons as
well as group discussion interviews. Transana7 was used to facilitate the
‘coding’ process using CHAT and collate supplementary data that related
to other aspects – enjoyment, interaction, frustration, etc. – obtained
through periodic logging of performance and achievements. 5. Results
5.1 Quantitative Findings
5.1.1 Final Writing Levels Two of the 30 pupils failed to submit the final pieces of personal-
imaginative writing: absences and the loss of interim written material 2015 | Volume 2 | Issue 2
85 Press Start
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86 O’Donnell were the major factors and as such attrition should be factored into any
secondary school context. The results for the experimental group using
MCC (n=28) were then compared with the control group (n=102). Figure 4. Histogram showing the post-project performance writing levels
of pupils involved in the MCC project and the control group
In Figure 4 the graph on the left is skewed to the right, showing a Figure 4. Histogram showing the post-project performance writing levels
of pupils involved in the MCC project and the control group In Figure 4 the graph on the left is skewed to the right, showing a
significant trend in the performance of the pupils involved in the MCC
project. The majority of these pupils attained levels between 3.50 and
4.50. In contrast, the second graph is skewed to the left, showing that
the majority of the pupils independent of the MCC project attained levels
between 2.50 and 3.50. In Figure 4 the graph on the left is skewed to the right, showing a
significant trend in the performance of the pupils involved in the MCC
project. The majority of these pupils attained levels between 3.50 and
4.50. In contrast, the second graph is skewed to the left, showing that
the majority of the pupils independent of the MCC project attained levels
between 2.50 and 3.50. In Figure 4 the graph on the left is skewed to the right, showing a
significant trend in the performance of the pupils involved in the MCC
project. The majority of these pupils attained levels between 3.50 and
4.50. In contrast, the second graph is skewed to the left, showing that
the majority of the pupils independent of the MCC project attained levels
between 2.50 and 3.50. Comparatively, the control group performed better than their year group
peers. Although the lack of a true control group disallows for asserting
causal claims about the impact of MCC on writing attainment – the
corollary ‘Baseline (2013)’ levels are unavailable for the rest of the year
group – the results do support that the simulation has facilitated
development in writing. 2015 | Volume 2 | Issue 2
87 8 Grouping Variable: Cohort Press Start
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8 Grouping Variable: Cohort 9 Curriculum for Excellence: literacy and English. Experiences and
Outcomes (p.8, LTScotland, 2012a)
10 A quotation is a segment from a Primary Document (PD) that is
interesting or important to the user. The PDs in this project refer to the
pupils’ final narratives, and the depicted quotations relate to the result
of Boolean queries. The format used is: (Primary Document Number :
Quotation Creation Order). O’Donnell Games-based Interdisciplinary Learning in CfE Games-based Interdisciplinary Learning in CfE O’Donnell Press Start
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86 86 Games-based Interdisciplinary Learning in CfE O’Donnell
Games-based Interdisciplinary Learning in CfE
Ranks
Cohort
N
Mean Rank
Sum of
Ranks
Experimental group
Control Group
Total
28
102
130
77.20
62.29
2161.50
6353.50
Test Statistics8
Level
Mann-Whitney U
Wilcoxon W
Z
Asymp. Sig. (2-tailed)
1100.500
6353.500
-2.140
.032
Table 2. Results of Mann-Whitney Test comparing experimental group
and control group final writing levels
A Mann-Whitney U Test revealed a statistically significant difference
between the experimental group and the control group, as shown in
Table 2. From this data, it can be concluded that there is a difference in
distribution between those pupils who were involved in the MCC project
and those who were not, with those using MCC having a higher
probability of achieving a higher rank than their peers. Table 2. Results of Mann-Whitney Test comparing experimental group
and control group final writing levels Table 2. Results of Mann-Whitney Test comparing experimental group
and control group final writing levels A Mann-Whitney U Test revealed a statistically significant difference
between the experimental group and the control group, as shown in
Table 2. From this data, it can be concluded that there is a difference in
distribution between those pupils who were involved in the MCC project
and those who were not, with those using MCC having a higher
probability of achieving a higher rank than their peers. A Mann-Whitney U Test revealed a statistically significant difference
between the experimental group and the control group, as shown in
Table 2. From this data, it can be concluded that there is a difference in
distribution between those pupils who were involved in the MCC project
and those who were not, with those using MCC having a higher
probability of achieving a higher rank than their peers. 2015 | Volume 2 | Issue 2
87 Press Start
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URL: http://press-start.gla.ac.uk 87 O’Donnell 5.2 Qualitative Findings There remains a disparity between competency in English and Literacy
Outcomes. For instance, Pupil-11 (Group-D), despite orthographic
issues (achieving a Level 2 in LIT 2-22a9, punctuation, varied sentence
structures, and paragraphing) was still able to demonstrate effective
word choice: 33:2810 not enough resources
33:30 locate
33:32 fully installed
33:33 different objectives
33:34 connect
Table 3. Atlas.ti Quotations for Effective Word Choice use by Pupil-11
Group-D To illustrate, consistent sentence construction – punctuation, for
example – is inconsistently applied and remains an issue in the case of
Pupil-1 and Pupil-15, despite the astute form and content exemplified: “The sky looks like butter scotch and I have a dry mouth
because it’s so hot we have no water yet because we haven’t
installed the water pumps” (Pupil-1, Atlas.ti, Primary Document
P81). “…for us to take the kit of, It took a while because to install the
scrubber you need a scrubber unit compressor…” (Pupil-15,
Atlas.ti, Primary Document P65). This was a general feature across most of the groups: Pupil-8 and Pupil-
11 of Group-D similarly displayed lapses in sentence construction –
especially punctuation (full stops) despite displaying aptitude in form
and content. Further peer- and self-assessment should resolve such
frequent lapses; this supports the necessity that all curriculum teachers
provide the interventions to correct orthographic issues, when pupils can
produce writing that is engaged and purposeful. 2015 | Volume 2 | Issue 2
88 Press Start
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90 O’Donnell Games-based Interdisciplinary Learning in CfE Games-based Interdisciplinary Learning in CfE In Group-A, as with the other groups, the activities resulted in the
adoption of strong verb usage, verbs that suited the identities that were
co-constructed during the activities they were carrying out on Mars;
verbs and language that constructed the social aspect of the character
and his/her imagined character’s back-story on Earth. For example, the
first block contains language that constitutes their scientific and
engineering identity: 38:14 tightening
38:15 installing
38:18 (which had been deployed earlier)
68:18 Unpacking and setting
68:11 rendezvous
82:15 well heads
Table 4. Atlas.ti Quotations for Strong Word Choice/Verb use by
Group-A 38:14 tightening
38:15 installing
38:18 (which had been deployed earlier)
68:18 Unpacking and setting
68:11 rendezvous
82:15 well heads
Table 4. Atlas.ti Quotations for Strong Word Choice/Verb use by
Group-A The second block contains language that constitutes their personal
identity: 38:27 (my favourite horse at my riding school. 38:40 you could see a faint light
68:8 it feels like a life jacket but. 82:17 [Pupil-1] is the funniest of the group. 80:15 I am nervous about staying here. 80:16 I am missing home very much just now. 80:18 I got barely any sleep last night. Table 5. Atlas.ti Quotations for Form and Content use by Group-A Table 5. Atlas.ti Quotations for Form and Content use by Group-A As is present in the other group writing pieces, we have a mixture of
‘mission jargon’ and language that is creative and rich in delivering
personal and imaginative narration from multiple identities. Here we have the application of a real-world adjectival (‘heavy’) to what
is essentially the object of a digitally simulated activity: “We installed the water pumps yesterday. They were quite heavy
but we managed to get them fixed onto the tops of the well
heads. It took most of the day to install both of them, it was hard
work but rewarding because we can now pump water up from
under the surface of mars” (Pupil-7, Atlas.ti, Primary Document
P87). “[Pupil-7] and [Pupil-27] were doing all the heavy lifting, bringing
the equipment that we needed inside from the garage the
installing the bulky pumps. I and [Pupil-28] were the ones who 2015 | Volume 2 | Issue 2 Press Start
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URL: http://press-start.gla.ac.uk 89 O’Donnell O’Donnell Games-based Interdisciplinary Learning in CfE Apart from Pupil-7, all the pupils in Group-B attained the highest Level 4
in ENG 3-30a (Personal) and ENG 3-31a (Imaginative) as a result of
their richness and “convincing and appropriate structures
and/or…interesting and convincing characters” (Education Scotland,
2004, p. 17). Apart from Pupil-7, all the pupils in Group-B attained the highest Level 4
in ENG 3-30a (Personal) and ENG 3-31a (Imaginative) as a result of
their richness and “convincing and appropriate structures
and/or…interesting and convincing characters” (Education Scotland,
2004, p. 17). Royle (2008) suggests that players engage if: O’Donnell O’Donnell Games-based Interdisciplinary Learning in CfE 6.1 Experimental group performance The use of the digital game, Mars Colony Challenger (MCC) and a limited
set of text-based and audio-visual resources have supported 28 of the
initial 30 pupils in expressing themselves in both imaginative and
personal writing, which displayed improved attainment between 2013
and 2014. Pupils involved in the project achieved a mean literacy level of 77.20,
whereas the control group achieved a mean of 66.29. The data
supports the assertion that there is a significant statistical difference
between the two groups (one of the project limitations was having no
access to 2013 ‘baseline’ levels for the control group) and that the
adoption of a digital game has successfully operated as a ‘text’ as
specified by Curriculum for Excellence. Royle (2008) suggests that players engage if: The backstory or narrative provides the believability or
authenticity of engagement. Characterization makes the player's role in the narrative
believable so that the player can engage fully in the game. From Group-B, Pupil-29 demonstrates the hybrid nature of narratives
that originate from game-play and thinking imaginatively: “Thinking back, my favourite moment on Mars was when our first
plant broke the rocky surface: the first Martian plant ever to have
been grown by humans. I think the only moment to compete
with that moment was when I went to check on the fish eggs on
morning and our first fish had hatched; they were so small,
having seen them on Earth I would never have had thought that
they would have been the first Earth species to have been born
on Mars” (Pupil-29, Atlas.ti, Primary Document P81). Press Start
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90 90 6.2 Science Curriculum Planning & Instructional Design The final personal-imaginative narratives show overall progression in
writing for all pupils. Despite the constraint of having no formally-
timetabled Science lessons to coincide with the project timeline, pupils
remained appreciative of what they were attempting to learn. Future timetabling to complement the narrative events encountered in
Mars Colony Challenger events, and an awareness of the asymmetric
distribution of the tasks within the game and its impact on the
acquisition of learning scientific processes – and associated
nomenclature – would in enrich future collective story-telling that is
obviously at the kernel of this digital game. Mars Colony Challenger (MCC) - and similar games - should be
employed and be externally supported by lessons that maintain a
realistic and relevant scientific pedagogy, so that teachers can develop
in their pupils “reflective practice, analysis, and evaluation, all essential
high-level skills in today’s information-based society” (Royle, 2008). In
Jackson (2009) the author notes that “Prensky (2001a) ties the power of
stories to the evocation of emotion” (p. 297) and Royle (2008) asserts
that: Storytelling is a potent learning tool: Stories are a powerful communication tool because they enable
listeners to make connections between what is said and their own
experience — this helps create meaning and can trigger people
into action. (Bonner, Chartier, and Lapointe 2006, p. 4) 2015 | Volume 2 | Issue 2
91 Press Start
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URL: http://press-start.gla.ac.uk 91 91 O’Donnell Games-based Interdisciplinary Learning in CfE Science should recognise that “narratives enable students to develop
research skills, critical and creative thinking… related to managing and
utilising scientific knowledge in everyday life” (Vrasidas et al., 2015, p. 3). Steinkuehler and Duncan (2009) observed evidence of such higher-
order cognitive skills in episodes containing sophisticated ranges of
language during discussion between digital game players, which
included the use of content knowledge in constructive argumentation
and reasoning. Having pupils participate in the creation of such stories
using games like Mars Colony Challenger, when supplemented by other
methods of instruction, and which afford pupils to work in groups can be
more effective with regards to learning and content retention (Wouters,
et al., 2013; Vrasidas et al., 2015; Connolly et al., 2012) . Furthermore,
when there was an underlying “theme that is loosely related to the
learning goals [it] may improve the effect of a narrative” (p. 12). However, with regards to the Clark/Kozma debate, which continues to
question the efficacy of media in enhancing learning - “that no single
media attribute forms a unique cognitive effect” (Clark, 2007 in Becker
2010), this article suggests that the use of a computer simulation does
facilitate ‘learning by doing’ (Schank et al., 1999) but that responsibility
for such successful implementation “must be placed squarely on the
shoulders of both [digital games and instructional design]” (Becker,
2010). 6.5 Diegesis – Were they there? English Departments stand to benefit from opportunities whereby pupils
write both imaginatively and from experience of a text such as MCC: “When [Pupil-15] turned her light on her space suit you could see
a faint light down the hill!” (Atlas.ti, Primary Document P38) As part of the qualitative data gathered, there was evidence from
questionnaires and interviews that pupils enjoyed the MCC project and
the group-based activities derived from it; it is clear that the activities in
MCC “originate logically from the narrative structure of the game”
(Royle, 2008) which itself is rule-based and grounded in scientific laws:
“Opportunities to promote storytelling should be maximised in the Game
Design” (ibid). Pupils’ identity as Mars Colonisers within MCC was through what they
were actually doing and at the same time reflecting upon their current
(or imagined) personal lives – far from home, missing family and pets,
acknowledging frustration with their fellow crewmembers. Such learning
within the context of a game “involves taking on and playing with
identities… There is a tripartite play of identities as learners relate, and
reflect on, their multiple real-world identities, a virtual identity, and a
projective identity” (Gee, 2003, p. 67). This is also supported by
Vrasidas et al. (2015) and their use of the Science Fiction genre to 2015 | Volume 2 | Issue 2 Press Start
ISSN: 2055-8198
URL: http://press-start.gla.ac.uk 92 2015 | Volume 2 | Issue 2
93 O’Donnell Games-based Interdisciplinary Learning in CfE Games-based Interdisciplinary Learning in CfE Games-based Interdisciplinary Learning in CfE introduce interdisciplinary learning of Science, stressing the importance
“of narratives in involving the reader in the story emotionally” (p. 2), as
well a pointing to research by Avraamidou & Osborne (2009) that
supports the multiple cognitive benefits “the introduction of narratives in
science education significantly increases students’ memory, interest, and
understanding” (ibid): introduce interdisciplinary learning of Science, stressing the importance
“of narratives in involving the reader in the story emotionally” (p. 2), as
well a pointing to research by Avraamidou & Osborne (2009) that
supports the multiple cognitive benefits “the introduction of narratives in
science education significantly increases students’ memory, interest, and
understanding” (ibid): I’m missing my family and of course my beloved horses from
Velvet Path…I brought my bracelet I made of Juniper’s mane to
remind me of them. Even in space, horses smell the same so I
am still a bit homesick… Yesterday we did our first orbit refuelling
when we had just arrived…one of Mars’ two moons, Deimos, passed in front of us. What a majestic sight! Today we worked much better as a team than yesterday as we
completed the water pumps, scrubber (which was to make the air
breathable), and sabieter (which mixed together the right
chemicals for fuel for the rover). I then began to shutting down the machines after I made sure
everyone had their space suits on. We made sure nothing had
been left behind and slowly made our way outside. We took our final steps on Mars and boarded the lander.” (Pupil-14, Atlas.ti, Primary Document P38) (Pupil-14, Atlas.ti, Primary Document P38) From the pupil narratives the high proportion of occurrence of the
exclusive pronoun ‘we’ supports – and corroborates – the societal
aspects of the lessons. ‘We and ‘they’ allows pupils to reflect on events
from their own and the perspectives of others; they consider others,
their contributions and the social dynamics of their group – all in a safe
context, proven to have generated little if any angst amongst its
members. 2015 | Volume 2 | Issue 2
93 Press Start
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URL: http://press-start.gla.ac.uk Press Start
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93 93 93 Games-based Interdisciplinary Learning in CfE O’Donnell Figure 5. In-game footage from Mars Colony Challenger - the base at
night. Figure 5. In-game footage from Mars Colony Challenger - the base at
night. 6.6 Limitations A limitation of this study was the unavailability of baseline data for the
entire cohort of S2 pupils, which would have been used to, firstly,
control for prior experience, and secondly, provide insight into the gains
made by pupils of different levels of ability. Furthermore, due to the
lack of random allocation of students to experimental and control group,
the research was quasi-experimental in design. While the author has no
reason to believe that this class differed meaningfully from the control
group, follow up studies that confirm these results using a more rigorous
experimental design are advised. O’Donnell Games-based Interdisciplinary Learning in CfE Games-based Interdisciplinary Learning in CfE associated with pieces of writing that are drawn from the recall of
specific episodes in the digital game; the impact of group collaboration
and discussion on written lexical variety and richness should also be
considered (Charsky, 2010 in Guillén-Nieto et al., 2012). Furthermore, an exploration of the use and acquisition of scientific
language could lead to isolating game episodes that provide the richest
imaginative responses and evidence of scientific content knowledge
within the loose narrative structure, which would provide Science
teachers and English teachers with clearer evidence of areas facilitating
collaboration. 7. Conclusion Within the context of the Curriculum for Excellence, the sciences and
other subject areas must now share responsibility for pupils' literary
development. Science teachers are, therefore, afforded a new
collaborative role in helping pupils to actively engage with texts, enable
pupils to access the wealth of literary genres that the scientific
disciplines offer, and facilitate opportunities for literacy assessment. However, it is not clear how science teachers can design learning
environments that will help students develop transferrable literacy skills. This paper has explored the use of the educational simulation game,
Mars Colony Challenger, as one possible approach. The results of an
experimental trial showed a statistically significant difference in literacy
ability between those pupils using the game and those who did not. Literacy must be supported by other subject specialists; problems with
orthographic ability cannot be eradicated simply by the introduction of a
digital game. But game-based learning, such as the activities
undertaken with Mars Colony Challenger, can support writing across
many disciplines. Press Start
2015 | Volume 2 | Issue 2
ISSN: 2055-8198
95
URL: http://press-start.gla.ac.uk
References
Altura, G.J. & Curwood, J.S. (2015). Hitting Restart. Journal of Adolescent &
Adult Literacy, 59(1), 25–27. Barab, S., et al (2005). Eat Your Vegetables and Do Your Homework: A Design-
Based Investigation of Enjoyment and Meaning in Learning. Educational
Technology. January-February 2005. BBC. (2004). Space Odyssey: Voyage to the Planets (Press Release). Retrieved
rom
http://www.bbc.co.uk/pressoffice/pressreleases/stories/2004/10_october/13/sp
ace_odyssey.pdf
Becker, K. 2010. The Clark-Kozma Debate in the 21st Century. Retrieved from
http://mruir.mtroyal.ca:8080/xmlui/handle/11205/143 6.7 Future Research The MCC project activities are clearly enculturated into the written and
spoken language of the pupils involved: the MCC simulation software
provided a “flexible learning context in which students both work
independently and collaboratively as they complete game tasks” (Spires,
H., 2015, p. 5). From the qualitative results, a number of future
considerations are suggested in relation to the spoken and written
language associated with engagement with a digital simulation for
learning: an analysis of the performance of pupils in the individual
elements of ‘Eng-‘ and ‘Lit-’ based on gender and ability. The compilation of a body of personal-imaginative corpora from game
play involving MCC should continue, and support a deeper linguistic
analysis of the personal-imaginative narratives. Of interest will be an
exploration of the interplay of the ‘I’ and ‘We’ pronoun frequencies with
regards to pupil attainment and game engagement. This computation of
collocations based on pronoun use and lexical variety is of particular
interest, and may reveal correlations whereby greater lexical richness 2015 | Volume 2 | Issue 2 Press Start
ISSN: 2055-8198
URL: http://press-start.gla.ac.uk 94 References Altura, G.J. & Curwood, J.S. (2015). Hitting Restart. Journal of Adolescent &
Adult Literacy, 59(1), 25–27. Press Start
2015 | Vo
ISSN: 2055-8198
URL: http://press-start.gla.ac.uk
Barab, S., et al (2005). Eat Your Vegetables and Do Your Homework: A Design-
Based Investigation of Enjoyment and Meaning in Learning. Educational
Technology. January-February 2005. BBC. (2004). Space Odyssey: Voyage to the Planets (Press Release). Retrieved
rom
http://www.bbc.co.uk/pressoffice/pressreleases/stories/2004/10_october/13/sp
ace_odyssey.pdf
Becker, K. 2010. The Clark-Kozma Debate in the 21st Century. Retrieved from
http://mruir.mtroyal.ca:8080/xmlui/handle/11205/143 Barab, S., et al (2005). Eat Your Vegetables and Do Your Homework: A Design-
Based Investigation of Enjoyment and Meaning in Learning. Educational
Technology. January-February 2005. BBC. (2004). Space Odyssey: Voyage to the Planets (Press Release). Retrieved
rom BBC. (2004). Space Odyssey: Voyage to the Planets (Press Release). Retrieved
rom
http://www.bbc.co.uk/pressoffice/pressreleases/stories/2004/10_october/13/sp
ace_odyssey.pdf http://www.bbc.co.uk/pressoffice/pressreleases/stories/2004/10_october/13/sp
ace_odyssey.pdf Becker, K. 2010. The Clark-Kozma Debate in the 21st Century. Retrieved from
http://mruir.mtroyal.ca:8080/xmlui/handle/11205/143 Press Start
2015 | Volume 2 | Issue 2
ISSN: 2055-8198
95
URL: http://press-start.gla.ac.uk 95 Games-based Interdisciplinary Learning in CfE O’Donnell Blunt, R. Does Games-Based Learning Work? Results from Three Recent Studies. Retrieved from
http://www.johnseelybrown.com/newlearning.pdfhttp://patrickdunn.squarespac
e.com/storage/blunt_game_studies.pdf Connolly, T. M., Boyle, E. A., MacArthur, E., Hainey,T & Boyle, J. M. A systematic
literature review of empirical evidence on computer games and serious games. Computers & Education, 59( 2), 661–686, 2012. Corbin, J. 1998. The Basics of Qualitative Research. Sage. Department for Education. 2012. What is the research evidence on writing? DFE-
RR238 Education Scotland. About Interdisciplinary Learning. Retrieved from
http://www.educationscotland.gov.uk/learningteachingandassessment/learninga
crossthecurriculum/interdisciplinarylearning/about/benefits.asp Engeström,Y, Miettinen, R & Punamaki, R. (eds.) 1999. Perspectives on Activity
Theory. Cambridge University Press. Engeström, Y. 2001. Expansive Learning at Work: toward an activity theoretical
reconceptualization. Journal of Education and Work, 14(1), 133-156. Retrieved
from http://www.handover.eu/upload/library/jimaw6szeyuluh4tho6oq.pdf
Fisher, R. 2012. Teaching writing: a situated dynamic. British Educational
Research Journal 38(2) 299-317 Engeström, Y. 2001. Expansive Learning at Work: toward an activity theoretical
reconceptualization. Journal of Education and Work, 14(1), 133-156. Retrieved
from http://www.handover.eu/upload/library/jimaw6szeyuluh4tho6oq.pdf Fisher, R. 2012. Teaching writing: a situated dynamic. British Educational
Research Journal, 38(2), 299-317. Gee, J. 2003. What Video Games Have to Teach Us about Learning and Literacy. Palgrave Macmillan. Greenfield, P. 1984. Mind & Media: the Effects of Television Video Games &
Computers. In Videogames. (Fontana Paperbacks, London). Guillén-Nieto, V. & Aleson-Carbonell, M. 2012. Serious games and learning
effectiveness: The case of It's a Deal!. Computers & Education. 58(1), 435-448 Hämäläinen, R., Oksanen, K., & Häkkinen, P. (2008). Designing and analyzing
collaboration in a scripted game for vocational education. Computers in Human
Behavior, 24(6), 2496–2506. Hardman, Joanne. 2008. Researching pedagogy: an Activity Theory approach. Journal of Education, 45, 65-95. Hashim, N. H., & Jones, M. L. 2007. Activity theory : a framework for qualitative
analysis, in 4th International Qualitative Research Convention (QRC), 3-5
September, 2007, FJ Hilton, Malaysia. Jackson, J. 2009. Game-based teaching: what educators can learn from
videogames. Teaching Education, 20(3), 291-304 Kane, Pat. 2004. The Play Ethic: A Manifesto For a Different Way of Living. 35-
64. In A General Theory of Play. MacMillan, London. Karasavvidis, I., 2009. Activity Theory as a theoretical framework for the study
of blended learning: a case study. 6th International Conference on Networked Press Start
2015 | Volume 2 | Issue 2
ISSN: 2055-8198
96
URL: http://press-start.gla.ac.uk 96 96 Games-based Interdisciplinary Learning in CfE O’Donnell Learning, 195-202. Retrieved from
http://nlc.ell.aau.dk/past/nlc2008/abstracts/PDFs/Karasawidis_195-202.pdf. Lawless & Brown. 2015.. Developing scientific literacy skills through
interdisciplinary, technology-based global simulations: GlobalEd 2.In The
Curriculum Journal. LTScotland. Curriculum for Excellence: literacy and English. Experiences and
Outcomes. Retrieved from
http://www.ltscotland.org.uk/Images/literacy_english_experiences_outcomes_tc
m4-539867.pdf
LTScotland. Curriculum for Excellence: sciences. Experiences and Outcomes. Retrieved from
http://www.educationscotland.gov.uk/Images/sciences_experiences_outcomes_
tcm4-539890.pdf LTScotland. Curriculum for Excellence: sciences. Experiences and Outcomes. Retrieved from
http://www.educationscotland.gov.uk/Images/sciences_experiences_outcomes_
tcm4-539890.pdf Newman, J. 2004. What is a videogame? Rules, puzzles and simulations. In
Videogames. 9-28. Routledge, London. Royle, K. 2008. Game-Based Learning: A different perspective. Innovate 4(4). Retrieved from http://www.innovateonline.info/index.php?view=article&id=433 Saldana, J. 2012. The Coding Manual for Qualitative Researchers. (Sage). Schank, R. C., Berman, T. R., & Macpherson, K. A. 1999. Learning by Doing
Instructional-design theories and models : vol. 2, a new paradigm of
instructional theory. 161-181. Mahwah, N.J.: Lawrence Erlbaum Associates. Scottish Government, The. 2013. Scottish Survey of Literacy and Numeracy
2012 (Literacy). Retrieved from
http://www.scotland.gov.uk/Publications/2013/04/8843 Scottish Government, The. 2015. Scottish Survey of Literacy and Numeracy
2012 (Literacy). Retrieved from
http://www.gov.scot/Topics/Statistics/Browse/School-Education/SSLN Scottish Executive, The. 2004. A Curriculum for Excellence. Blackwell. Spires, H. 2015. Digital Game-Based Learning: What's Literacy Got To Do With
It? Journal of Adolescent & Adult Literacy. doi:10.1002/jaal.424 Steinkuehler, C. and Duncan, S. 2009. Informal scientific reasoning in online
virtual worlds, Journal of Science Education and Technology 1(1). Strauss, A. & Corbin, J. 1990. Basics of Qualitative Research. Sage. Strauss, A. & Corbin, J. 1990. Basics of Qualitative Research. Sage. Thompson, I. 2012. Stimulating reluctant writers: a Vygotskian approach to
teaching writing in secondary schools. English in Education, 46, 85–100. doi: 10.1111/j.1754-8845.2011.01117.x van der Meij, H. et al. 2011. Learning from games: Does collaboration help? British Journal of Educational Technology, 42(4) 2011 655–664 Press Start
ISSN: 2055-8198
URL: http://press-start.gla.ac.uk 2015 | Volume 2 | Issue 2
97 2015 | Volume 2 | Issue 2
97 97 2015 | Volume 2 | Issue 2
98 O’Donnell Games-based Interdisciplinary Learning in CfE Games-based Interdisciplinary Learning in CfE Vrasidas, C., Avraamidou, L., Theodoridou, K., Themistokleous, S. & Panaou, P. 2015. Science Fiction in Education: case studies from classroom
implementations. Educational Media International. Wouters, P., Van Nimwegen, C., Van Oostendorp, H. & Van Der Spek, E.D. 2013. A meta-analysis of the cognitive and motivational effects of serious games. Journal of Educational Psychology, 105(2) Yamagata-Lynch, L. 2010. Activity Systems Analysis Methods: Understanding
Complex Learning Environments. Springer Press Start
ISSN: 2055-8198
URL: http://press-start.gla.ac.uk 2015 | Volume 2 | Issue 2
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Glyoxal and methylglyoxal in Atlantic seawater and marine aerosol particles: method development and first application during the Polarstern cruise ANT XXVII/4
| null | 2,013
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cc-by
| 10,801
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Glyoxal and methylglyoxal in Atlantic seawater and marine aerosol
particles: method development and first application during the
Polarstern cruise ANT XXVII/4 M. van Pinxteren and H. Herrmann
Leibniz-Institut für Troposphärenforschung (TROPOS), Chemistry Department, Permoserstr. 15, 0 Correspondence to: H. Herrmann (herrmann@tropos.de) Received: 13 May 2013 – Published in Atmos. Chem. Phys. Discuss.: 11 June 2013
Revised: 6 September 2013 – Accepted: 31 October 2013 – Published: 5 December 2013 Received: 13 May 2013 – Published in Atmos. Chem. Phys. Discuss.: 11 June 2013
Revised: 6 September 2013 – Accepted: 31 October 2013 – Published: 5 December 2013 Abstract. An analytical method for the determination
of the alpha dicarbonyls glyoxal (GLY) and methylgly-
oxal (MGLY) from seawater and marine aerosol par-
ticles is presented. The method is based on derivati-
zation with o-(2,3,4,5,6-Pentafluorobenzyl)-hydroxylamine
(PFBHA) reagent, solvent extraction and GC-MS (SIM)
analysis. The method showed good precision (RSD < 10 %),
sensitivity (detection limits in the low ng L−1 range), and
accuracy (good agreement between external calibration and
standard addition). The method was applied to determine
GLY and MGLY in oceanic water sampled during the Po-
larstern cruise ANT XXVII/4 from Capetown to Bremer-
haven in spring 2011. GLY and MGLY were determined
in the sea surface microlayer (SML) of the ocean and cor-
responding bulk water (BW) with average concentrations
of 228 ng L−1 (GLY) and 196 ng L−1 (MGLY). The results
show a significant enrichment (factor of 4) of GLY and
MGLY in the SML. Furthermore, marine aerosol particles
(PM1) were sampled during the cruise and analyzed for GLY
(average concentration 0.19 ng m−3) and MGLY (average
concentration 0.15 ng m−3). On aerosol particles, both car-
bonyls show a very good correlation with oxalate, support-
ing the idea of a secondary formation of oxalic acid via GLY
and MGLY. Concentrations of GLY and MGLY in seawa-
ter and on aerosol particles were correlated to environmental
parameters such as global radiation, temperature, distance to
the coastline and biological activity. There are slight hints
for a photochemical production of GLY and MGLY in the
SML (significant enrichment in the SML, higher enrichment
at higher temperature). However, a clear connection of GLY
and MGLY to global radiation as well as to biological ac- tivity cannot be concluded from the data. A slight correlation
between GLY and MGLY in the SML and in aerosol particles
could be a hint for interactions, in particular of GLY, between
seawater and the atmosphere. 1
Introduction Oceans cover around two-thirds of the earth and most of
the manifold exchange processes between oceans and the at-
mosphere are only rarely understood (Cunliffe et al., 2013). Among these interaction processes, organic export from the
oceans to the atmosphere via gaseous emission or via bubble
bursting play an essential role as this is one pathway towards
organic compounds on marine aerosol particles (Liss and
Duce, 1997). The two α-dicarbonyls, glyoxal (CHOCHO;
GLY) and methylglyoxal (CH3COCHO; MGLY), have at-
tracted increasing attention over the past years because of
their potential role in secondary organic aerosol (SOA) for-
mation (Carlton et al., 2007; Fu et al., 2008; Ervens et al.,
2011). They can be produced via oxidation of volatile or-
ganic compounds (VOCs) and typical precursors are iso-
prene, toluene, acetylene and acetone (Fu et al., 2008; War-
neck, 2003; Shaw et al., 2010). Besides secondary formation,
direct emission of both GLY and MGLY to the atmosphere is
also reported (Fu et al., 2008; Sinreich et al., 2010). Although
the volatility of GLY and MGLY is high and these com-
pounds are found in the gaseous phase, large shifts of GLY
and MGLY in the atmosphere towards the particle phase
are reported (Ervens and Volkamer, 2010). Aerosol uptake
of highly soluble compounds like GLY is most efficient on M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater (2007) found that glyoxal uptake on aerosol par-
ticles can compete with glyoxal loss in the gas phase due
to oxidation processes. Aqueous phase chemistry of GLY
and MGLY in aerosol particles leads to further oxidized low
volatility products such as oxalic acid (via OH radical re-
action) and non-radical reaction including the formation of
high molecular weight compounds (Tilgner and Herrmann,
2010; Ervens et al., 2011; Lim et al., 2010). Recently Sin-
reich et al. (2010) attributed the open ocean as an important
source for GLY in the atmosphere. They measured concen-
trations of GLY up to 140 ppt in the atmosphere via differ-
ential optical absorption spectroscopy (DOAS) over the re-
mote tropical Pacific Ocean. Due to the short atmospheric
lifetime of GLY and MGLY of about 2 h these authors as-
sumed a local marine source. They concluded that there must
be an airborne formation mechanism of GLY in form of a
process at the air–sea interface or via bubble bursting. Shaw
et al. (2010) recently described that the oceans also emit GLY
and MGLY precursors, especially isoprene. Zhou and Mop-
per (1997) investigated carbonyls including GLY and MGLY
in the open ocean (Sargasso Sea, Atlantic) and found that
the surface of the ocean is photochemically active and pro-
duces carbonyls. These authors further suggested that besides
biological processes also photochemical processes influence
carbonyl abundance in seawater (Kieber and Mopper, 1987). In the atmosphere, photochemical activity (e.g., in the ox-
idation of isoprene) is reported to lead to the formation of
GLY and MGLY (Sinreich et al., 2010). Generally, the mech-
anism of the production regarding GLY and MGLY and the
exchange between the surface of the oceans and the atmo-
sphere are not well understood but the ocean is assumed to
play an important role (Sinreich et al., 2010). (GC-MS). There are several studies using o-(2,3,4,5,6-
Pentafluorobenzyl)-hydroxylamine (PFBHA) derivatization
and GC-MS for carbonyls in atmospheric samples (Ortiz et
al., 2006; Pang et al., 2013), though less studies exist for
these devices in high-matrix aqueous samples. One study
(Saison et al., 2009) applies PFBHA derivatization and GC-
MS for carbonyls in beer, however for relatively high con-
centrations (µg L−1 range). EPA method 556 applies PF-
BHA derivatization followed by hexane extraction and GC-
electron capture detection as reference method for carbonyls
(including GLY and MGLY) in drinking water. M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater As the open ocean and marine interactions are suggested
to be an important source for the alpha-dicarbonyls in the
atmosphere and because of the few available field measure-
ments in the marine area, the aim of this work was to ana-
lyze GLY and MGLY in marine samples from the Atlantic
Ocean. Firstly, the easy-to-use approach of PFBHA derivati-
zation combined with solvent extraction and GC-MS analy-
sis was validated for the seawater samples and aerosol parti-
cles especially regarding matrix interferences. Secondly, the
method was applied to the field samples collected during a
transatlantic Polarstern cruise in spring 2011. In seawater the
focus was on the SML, as it is the direct interface between
ocean and atmosphere. Connections of GLY/MGLY in the
SML and on aerosol particles to environmental parameters
such as global radiation, temperature, distance to the coast-
line and biological activity should help to find hints regarding
the production of these compounds and a connection between
these carbonyls in the ocean and in the atmosphere. 2.1
Cruise Figure 1 presents the Polarstern ANT XXVII/4 cruise plot
from Capetown (South Africa) to Bremerhaven (Germany)
between 20 April 2011 and 20 May 2011. Each day of
the cruise is marked with a black square including 5 days
backwards trajectories (blue lines), determined in a height
of 30 m (NOAA HYSPLIT model, http://www.arl.noaa.gov,
NOAA Air Resources Laboratory, Silver Spring, MD, USA). Aerosol particle sampling was performed continuously along
the cruise. However due to sampling problems only 11
suitable aerosol filters could be loaded (reported in detail
in section “Sampling problems and artifacts”). Water sam-
pling was performed on 15 days, marked with a cross in
Fig. 1. The green crosses present the sampling points where
oceanic water sampling and clean marine aerosol sampling
was co-located, meaning that SML samples were taken in
the midterm of aerosol sampling. p y
p
(
,
)
Analytical methods for GLY/MGLY analysis in environ-
mental samples typically include a derivatization step in
order to reduce reactivity and polarity of the compounds. The most extensively used derivatization agent for atmo-
spheric samples is 2.4-dinitrophenylhydrazine (DNPH) often
in combination with solid phase extraction (SPE) and liquid
chromatography-mass spectrometry (LC-MS) or ultraviolet
(UV) detection (Liggio and McLaren, 2003; Kampf et al.,
2011). Zhou and Mopper (1997) applied DNPH derivatiza-
tion followed by SPE-LC-UV for seawater analysis. How-
ever, especially for seawater samples problems in SPE due
to clogging of the cartridge due to matrix compounds are
reported (Mbukwa et al., 2012). Recently a very sensitive
HPLC method with fluorescence detection using dansylac-
etamidooxyamine as derivatization agent was presented for
carbonyls in environmental water (Houdier et al., 2011). Kawamura and Ikushima (1993) developed a method for al-
pha dicarbonyls and atmospheric acids using BF3/butanol
derivatization and gas chromatography-mass spectrometry M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater aqueous aerosol particles, especially marine aerosol particles
(Liggio et al., 2005), as the solubility of GYL and MGLY
in aerosol water is expected to be strongly enhanced due to
the present concentrated solutes (Lim et al., 2010). Volka-
mer et al. (2007) found that glyoxal uptake on aerosol par-
ticles can compete with glyoxal loss in the gas phase due
to oxidation processes. Aqueous phase chemistry of GLY
and MGLY in aerosol particles leads to further oxidized low
volatility products such as oxalic acid (via OH radical re-
action) and non-radical reaction including the formation of
high molecular weight compounds (Tilgner and Herrmann,
2010; Ervens et al., 2011; Lim et al., 2010). Recently Sin-
reich et al. (2010) attributed the open ocean as an important
source for GLY in the atmosphere. They measured concen-
trations of GLY up to 140 ppt in the atmosphere via differ-
ential optical absorption spectroscopy (DOAS) over the re-
mote tropical Pacific Ocean. Due to the short atmospheric
lifetime of GLY and MGLY of about 2 h these authors as-
sumed a local marine source. They concluded that there must
be an airborne formation mechanism of GLY in form of a
process at the air–sea interface or via bubble bursting. Shaw
et al. (2010) recently described that the oceans also emit GLY
and MGLY precursors, especially isoprene. Zhou and Mop-
per (1997) investigated carbonyls including GLY and MGLY
in the open ocean (Sargasso Sea, Atlantic) and found that
the surface of the ocean is photochemically active and pro-
duces carbonyls. These authors further suggested that besides
biological processes also photochemical processes influence
carbonyl abundance in seawater (Kieber and Mopper, 1987). In the atmosphere, photochemical activity (e.g., in the ox-
idation of isoprene) is reported to lead to the formation of
GLY and MGLY (Sinreich et al., 2010). Generally, the mech-
anism of the production regarding GLY and MGLY and the
exchange between the surface of the oceans and the atmo-
sphere are not well understood but the ocean is assumed to
play an important role (Sinreich et al., 2010). aqueous aerosol particles, especially marine aerosol particles
(Liggio et al., 2005), as the solubility of GYL and MGLY
in aerosol water is expected to be strongly enhanced due to
the present concentrated solutes (Lim et al., 2010). Volka-
mer et al. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 11792 M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater 2.2
Sampling details Oceanic water sampling was performed with a rubber boat
that was launched from the Polarstern ship. For SML Atmos. Chem. Phys., 13, 11791–11802, 2013 2.3
Analysis 25
GLY (40 % w/w) and MGLY (40 %) were purchased from
Sigma Chemical Co. (St Louis, MO) Stock solutions with
a concentration of 1 mg L−1 were prepared and used for
5 days (kept in the fridge at −20 ◦C). From the stock solu-
tions, five calibration standards were prepared in the range
of 0.25–2 µg L−1 in volumes between 20 mL (for parti-
cle analysis) and 100 mL (for seawater analysis). Standards
for seawater samples were always set to the same salin-
ity as real samples (35 PSU) by adding 3.5 g sea salt mix-
ture (Sigma Chemical Co.) to 100 mL milliQ water. In-
ternal standards 2-Trifluormethylbenzaldehyd and 2,2,6,6-
Cyclohexanone (Sigma Chemical Co.) were added to the
sample in a concentration of 1 µg L−1 each. For standard ad-
dition experiments, the samples were divided in 3–5 aliquots
and spiked with different amounts of standard solution. For
derivatization, freshly prepared acidic PFBHA was added to
the sample in a 10 000 fold excess corresponding to a concen-
tration of 1 µg L−1 GLY. The free form of GLY and MGLY
is in equilibrium with various forms of hydrated oligomers
varying with concentration and solution pH, however, at con-
centrations below 1 mol L−1 the monomeric form is dom-
inant (Whipple, 1970). Due to acidic derivatization condi-
tions, equilibrium is shifted towards the free form of GLY
and MGLY (Liggio et al., 2005). Therefore, the measure-
ments here represent the sum of the monomer carbonyls, its
hydrated form and reversibly formed high molecular weight
compounds (Kampf et al., 2012). To ensure complete deriva-
tization, the reaction mixture was allowed to stand overnight
at room temperature. For extraction, 0.5–1 mL of hexane
was added as extraction solvent. Formation of emulsion can
be a problem using LLE for complex samples, but not ob-
served for our seawater and particle samples. After stirring
for 1 h the hexane layer was removed, dried over sodium sul-
fate and transferred to a GC autosampler vial. In all, 2 µL
were injected in splitless mode into the GC-MS. A HP-5 col-
umn (Agilent technologies, Waldbronn, Germany) was ap-
plied (30 m length, 0.25 mm i.D. 0.25 µm film thickness) and
inlet temperature was set at 250 ◦C and the oven program
was as follows: 50 ◦C (2 min) with 3 ◦C min−1 to 130 ◦C,
with 10 ◦C min−1 to 280 ◦C. inxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater 720
Fig. 1. Cruise plot including seawater sampling (red crosses), si-
multaneous seawater and aerosol particle sampling (green crosses)
and backwards trajectories (blue lines). laxation time of 5 s. The viewing angle is 180◦and corre-
lates with cosine properties. Under a glass dome is a black
solar radiation absorbing thermopile and 64 thermocouples. Incoming short-wave radiation heats the top and the resulting
temperature gradient of thermocouples generates an electric
potential which is proportional to radiation flux density. 2.3
Analysis The transfer line was set at
280 ◦C. For GLY m/z of 181 (quantifier ion) and 448 (qual-
ifier ion), for MGLY m/z of 181 (quantifier ion) and 462
(qualifier ion) were detected. Internal calibration was per-
formed using five concentration levels and dividing the peak
areas of GLY and MGLY through the peak areas of the in-
ternal standard 2-Trifluormethylbenzaldehyd. For standard 720
Fig. 1. Cruise plot including seawater sampling (red crosses), si-
multaneous seawater and aerosol particle sampling (green crosses)
and backwards trajectories (blue lines). sampling, a glass plate (sampling area 2000 cm2) was ver-
tically put in the water and slowly drawn upwards. The film
adheres to the surface of the glass and is taken off by framed
Teflon wipers (Stolle et al., 2010; van Pinxteren et al., 2012). This sampling technique is easy to use, requires only sim-
ple equipment and provides sampling of uniform SML thick-
nesses of ca. 100 µm (thickness = sampled volume / sam-
pling area). Bulk water (BW) from 1 m depth was collected
using a self-made device consisting of a glass bottle mounted
on a telescopic rod that regulates sampling depth. After each
sampling all material was extensively cleaned with reagent
water. All water samples were stored in glass bottles and kept
at −20 ◦C until analysis. Details of the sampling points for
seawater sampling together with environmental parameters
are listed in Table 1. A high volume Digitel sampler DHA-80 (Walter Riemer
Messtechnik, Germany) was installed on top of the observa-
tion deck at a height of ca. 30 m. Aerosol particles (PM1)
were collected on preheated 150 mm quartz fiber filters
(Minktell, MK 360) at a flow rate of 500 L min−1. Sampling
time was 24 h from midnight to midnight (UTC). After sam-
pling, the filters were stored in aluminum boxes and kept at
−20 ◦C in a freezer. After the cruise they were transported in
dry ice to the TROPOS laboratories in Leipzig. Air temperature was achieved from the Scalable Auto-
matic Weather Station SCAWS. Broadband global radiation
was measured with a Kipp and Zonen CM21 pyranometer. Data are sampled with a 1 Hz resolution despite a 95 % re- Atmos. Chem. Phys., 13, 11791–11802, 2013 www.atmos-chem-phys.net/13/11791/2013/ www.atmos-chem-phys.net/13/11791/2013/ www.atmos-chem-phys.net/13/11791/2013/ M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater 11793 www.atmos-chem-phys.net/13/11791/2013/ 3.2
Method validation The limit of detection (LOD) was determined as the concen-
tration corresponding to the mean blank (n = 6) plus three
times the standard deviation (Kaiser and Specker, 1956). The
values in Table 2 correspond to a sample volume of 100 mL
oceanic water and a 20 mL aqueous extract of 25 % of the
filter sample (area: 177 cm2) for aerosol particles. LODs
can be further lowered by increasing the sample volume
or reducing the extraction solvent volume (e.g., by evapo-
ration). The LODs for seawater samples reported here are
similar to other applications using GC-MS, for example by
Kawamura (1993a) with LODs for carbonyls of 50 ng L−1
(rain and snow samples). Zhou and Mopper (1997) found
lower LODs for GLY and MGLY of 3–4 ng L−1 after en-
richment via SPE and DNPH-LC-UV analysis probably due
to the very high injection volumes of 2000 µL into the LC
system. Using highly fluorescence derivatization agents and
LC-FLD analysis, LODs of 16 ng L−1 (GLY) and 12 ng L−1
(MGLY) can be achieved without preconcentration (Houdier
et al., 2011). However due to the high matrix content of
real samples, mostly sample preparation steps for a combi-
nation of clean up and analyte enrichment are applied. The
LODs achieved for aerosol particle analysis (25 % of filter,
aqueous extract of 20 mL, and extraction solvent of 1 mL)
are 353 ng L−1 for GLY and 259 ng L−1 for MGLY. The
higher LODs for an aqueous extract of 20 mL are caused by www.atmos-chem-phys.net/13/11791/2013/ M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater 1794
M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic
able 1. Seawater sampling: Sampling dates and corresponding environmental parameters
Distance
Air
Water
Global
Sampling
Local time
Coordinates
to coast
temp.b
temp.b
radiationb
date
sampling
latitude
longitude
(km)a
(◦C)
(◦C)
(W m−2)
23.04.2011
10:05 (UTC+1)
24◦24.47′ S
9◦18.50′ E
560
19.3
20.7
402
24.04.2011
12:04 (UTC)
20◦48.90′ S
6◦4.26′ E
780
21.6
22.7
326
25.04.2011
12:03 (UTC)
17◦22.89′ S
3◦2.86′ E
920
22.9
23.9
413
29.04.2011
12:05 (UTC)
8◦42.44′ S
4◦22.05′ W
1496
26.4
27.7
538
30.04.2011
13:02 (UTC)
5◦30.22′ S
7◦3.21′ W
1056
27.8
28.5
478
01.05.2011
12:02 (UTC−1)
2◦29.84′ S
9◦33.70′ W
760
28.2
28.5
551
02.05.2011
08:05 (UTC−1)
0◦0.75′ S
11◦37.64′ W
600
27.4
27.9
–c
03.05.2011
12:01 (UTC−1)
3◦32.22′ N
14◦33.95′ W
484
27.3
29.5
541
04.05.2011
12:04 (UTC−1)
6◦45.68′ N
17◦14.39′ W
460
27.6
29.6
575
05.05.2011
12:01 (UTC−1)
9◦52.69′ N
19◦50.63′ W
430
25.2
25.9
563
08.05.2011
12:04 (UTC−1)
13◦32.49′ N
20◦31.61′ W
380
22.9
23.8
552
09.05.2011
12:03 (UTC−1)
23◦39.87′ N
20◦15.97′ W
410
20.4
21.4
374
12.05.2011
11:00 (UTC−1)
25◦9.95′ N
18◦3.28′ W
328
20.2
20.9
508
13.05.2011
12:19 (UTC)
34◦57.29′ N
12◦55.29′ W
498
18.1
18.2
489
16.05.2011
07:08 (UTC−1)
37◦34.94′ N
12◦5.52′ W
340
17.0
17.6
404
a Shortest distance to coastline estimated via google earth; b at the sampling time; c not available. 11794 Table 1. Seawater sampling: Sampling dates and corresponding environmental parameters a Shortest distance to coastline estimated via google earth; b at the sampling time; c not available. and the second peak to cis isomer (Standard Method Num-
ber 6252B, 1995), (EPA method 556, 1998). As the ratio be-
tween trans and cis GLY was constant (1 : 3) only cis GLY
was interpreted throughout this study. addition, 2–4 concentration levels were added to a real sam-
ple and compared to external calibration. Oxalic acid was
measured from aqueous particle extracts (25 % of the filter
in 20 mL) as oxalate with ion chromatography, for details
see Müller et al. (2010). Dissolved organic carbon was deter-
mined from the aqueous particle extracts using a TOC-VCPH
analyzer (Shimadzu, Japan) as described in van Pinxteren et
al. (2009). M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater To test for matrix interferences,
internal calibration was compared with standard addition and
the graphs are shown in Fig. 3. No interferences with the ma-
trix were found as the slopes of the calibration curves are
very close (same for MGLY). Therefore, external calibration
could be applied to the oceanic water samples as well as to
the aqueous aerosol extracts. 7
7
7
7
7
7
7
7
7
7
7
7
7 area/area i.S. Fig. 3. Calibration curve for internal calibration and standard ad-
dition for GLY (a) 100 mL seawater sample, (b) 20 mL aqueous
extract of aerosol particle sample. M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater 11795 M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater
11795
721
Figure 2
722
723
724
725
37.00
37.50
38.00
38.50
39.00
39.50
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
Time-->
Abundance
Ocean sample (PS SML 8)
MGLY standard (c= 1 µg L ‐1)
blank
GLY standard (c= 1 µg L ‐1)
trans GLY
cis GLY
Fig. 2. SIM chromatograms of GLY and MGLY standard, ocean sample and blank (milliQ water + PFBHA), 100 mL sample volume each,
PFBHA was pre-cleaned over a SPE C18 cartridge. g
723
724
725
Fig. 2. SIM chromatograms of GLY and MGLY standard, ocean sample and blank (milliQ water + PFBHA), 100 mL sample volume each,
PFBHA was pre-cleaned over a SPE C18 cartridge. 727
728
729
Table 2. Validation data for GLY and MGLY; relative standard de-
viation (RSD), extraction yields and limits of detection (LOD). 26
(a)
(b)
y = 0.5148x + 0.0678
R² = 0.9868
y = 0.4664x ‐ 0.0239
R² = 0.9643
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0
0.2
0.4
0.6
0.8
1
1.2
standard addition
internal calibration
area/area i.S. concentration (µg L‐1)
y = 0.3644x + 0.222
R² = 0.9857
y = 0.3305x ‐ 0.0472
R² = 0.9994
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
standard addition
internal calibration
area/area i.S. concentration (µg L‐1)
Fig. 3. Calibration curve for internal calibration and standard ad-
dition for GLY (a) 100 mL seawater sample, (b) 20 mL aqueous
extract of aerosol particle sample. 727
728
729
730
731
732
733
734
735
736
737
738
Table 2. Validation data for GLY and MGLY; relative standard de-
viation (RSD), extraction yields and limits of detection (LOD). GLY
MGLY
RSD, (%) n = 6
8.9
5.4
extraction yield (%)
80
70
LOD seawater(ng L−1) a
54
50
LOD aerosolextract(ng L−1) b
353
259
LOD aerosolparticle(ng m−3) c
0.05
0.04
a For 100 mL saltwater; b for 20 mL aqueous extract; c for
545 m3 air volume. (a)
y = 0.5148x + 0.0678
R² = 0.9868
y = 0.4664x ‐ 0.0239
R² = 0.9643
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0
0.2
0.4
0.6
0.8
1
1.2
standard addition
internal calibration
area/area i.S. 3.1
Method development: extraction and derivatization For seawater analysis, 100 mL of oceanic water samples
(SML and BW samples) were derivatized and for aerosol
analysis up to 20 mL aqueous extract of 25 % of the fil-
ter sample were derivatized with PFBHA as described in
the experimental part. Repetitive extraction of one and the
same filter piece showed complete extraction in the first step. Blank problems due to the derivatization agent (especially
DNPH) for carbonyls are often reported and can be over-
come by re-crystallization and further cleaning of the deriva-
tization reagent with a SPE column (Liggio and McLaren,
2003; Kampf et al., 2011). Using freshly prepared PFBHA
solution blank values in the chromatograms were low, still
a purification step by passing PFBHA over a SPE column
(C18) led to a further reduction of the blank values. Figure 2
shows typical chromatograms for standards and samples us-
ing pre-cleaned PFBHA. While derivatized MGLY gave one
peak, for derivatized GLY two isomers were found in the
chromatograms. The two GLY isomers were assigned based
on prior literature, the first eluting peak is referred to trans Atmos. Chem. Phys., 13, 11791–11802, 2013 www.atmos-chem-phys.net/13/11791/2013/ M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater concentration (µg L‐1) y = 0.5148x + 0.0678
R² = 0.9868 26
(b)
y = 0.3644x + 0.222
R² = 0.9857
y = 0.3305x ‐ 0.0472
R² = 0.9994
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
standard addition
internal calibration
area/area i.S. concentration (µg L‐1) higher blank values from the filter material. The LODs for
the aerosol particle analysis reported here are slightly lower
compared to Kampf et al. (2011) applying DNPH-LC-MS
analysis (510 ng L−1 for GLY, 620 ng L−1 for MGLY) and
similar to Kawamura (1993a). Solvent extraction yields in
this work were estimated by three successive extraction steps
with hexane regarding the sum of them as 100 % value. The
extraction yields in the first step were 80 % for GLY (76 %
for 100 mL; 82 % for 20 mL) and 70 % for MGLY (65 %
for 100 mL; 75 % for 20 mL). Repeatability, determined by
6 fold measurements of aqueous standards with a concentra-
tion of 1 µg L−1, was very good with relative standard de-
viations (RSD) below 10 %. To test for matrix interferences,
internal calibration was compared with standard addition and
the graphs are shown in Fig. 3. No interferences with the ma-
trix were found as the slopes of the calibration curves are
very close (same for MGLY). Therefore, external calibration
could be applied to the oceanic water samples as well as to
the aqueous aerosol extracts. 7
7
7
7
7
7
7
7
7
7
7
7
7 higher blank values from the filter material. The LODs for
the aerosol particle analysis reported here are slightly lower
compared to Kampf et al. (2011) applying DNPH-LC-MS
analysis (510 ng L−1 for GLY, 620 ng L−1 for MGLY) and
similar to Kawamura (1993a). Solvent extraction yields in
this work were estimated by three successive extraction steps
with hexane regarding the sum of them as 100 % value. The
extraction yields in the first step were 80 % for GLY (76 %
for 100 mL; 82 % for 20 mL) and 70 % for MGLY (65 %
for 100 mL; 75 % for 20 mL). Repeatability, determined by
6 fold measurements of aqueous standards with a concentra-
tion of 1 µg L−1, was very good with relative standard de-
viations (RSD) below 10 %. 3.3.1
GLY and MGLY in Atlantic seawater Air temperature
is strongly correlated to water temperature (R2 = 0.98, val-
ues in Table 1). A correlation to SML concentrations of
GLY and MGLY to air temperature was not found (Figs. S7–
S8). However, enrichment of the two carbonyls seems to be
connected to air temperature, indicated by a positive cor-
relation between EF and temperature (R2 = 0.4, Figs. S9–
S10). A connection of GLY and MGLY to biological activity
was evaluated by correlating GLY and MGLY concentrations
to Chlorophyll a (Chl a) as an indicator for biological ac-
tivity. Chl a satellite data – provided by the Ocean Color
Web from NASA (http://oceancolor.gsfc.nasa.gov/), radius In summary the enrichment factor > 1 implies photochem-
ical production of the carbonyls that is also supported by the
trend of a higher enrichment at higher temperatures. How-
ever, a direct correlation to global radiation was less pro-
nounced. No indications of biological carbonyl production
or depletion could be concluded from our data. Furthermore,
only small dependencies regarding carbonyls in seawater and
the distance to coastline were found. 3.3.1
GLY and MGLY in Atlantic seawater Kieber and Mopper (1987) sug-
gested microbial activity to be rather a sink than a source
in well-oxygenated conditions such as the SML. The enrich-
ment of GLY and MGLY found in this work might be an
indicator for photochemical production of the carbonyls in
the SML, as the SML is more exposed to sunlight. However,
only weak correlations were found between global radiation
and concentration of GLY and MGLY in the SML as well as
enrichment of GLY/MGLY (R2 ∼0.2, Figs. S1–S4). Global
radiation is slightly correlated to air temperature within the
SML sampling time (R2 = 0.39 , Fig. S5). Air temperature
is strongly correlated to water temperature (R2 = 0.98, val-
ues in Table 1). A correlation to SML concentrations of
GLY and MGLY to air temperature was not found (Figs. S7–
S8). However, enrichment of the two carbonyls seems to be
connected to air temperature, indicated by a positive cor-
relation between EF and temperature (R2 = 0.4, Figs. S9–
S10). A connection of GLY and MGLY to biological activity of 78 km, n = 10 – showed no uniform trend regarding Chl
a and GLY/MGLY concentration or enrichment in the wa-
ter samples (Figs. S11–S14). However, it must be mentioned
that Chl a is a very broad indicator for biological activity and
does not reflect carbonyl specific microorganisms. Moreover,
satellite measurements are sensitive to cloud cover. There-
fore, an important issue in further sampling campaigns is the
development of analytical analysis of biologic pigments such
as Chl a and others. Zhou and Mopper (1997) reported that
concentrations of carbonyls are higher in coastal areas com-
pared to the open ocean but enrichment in the SML showed
the opposite trend (higher enrichment in open ocean, lower
enrichment in coastal waters). This is in agreement to Carl-
son (1983), who reported the same trend for dissolved or-
ganic carbon (DOC) in general. Regarding GLY and MGLY
in the present work, the trend for decreasing concentrations
in the SML towards the open ocean is weak (R2 = 0.14
for GLY, R2 = 0.05 for MGLY, Figs. S15–S16). Higher en-
richment towards the ocean was observed as very slight for
MGLY (R2 = 0.2, Fig. S18). concentration for MGLY were on average slightly lower
(261 ng L−1) with a higher fluctuation (77–760 ng L−1). In
BW, average concentrations were 121 ng L−1 (GLY) and
94 ng L−1 (MGLY). 3.3.1
GLY and MGLY in Atlantic seawater MGLY also showed higher fluctuation
and in six BW samples MGLY concentrations were be-
low LOD. There was a weak correlation between GLY and
MGLY concentrations in the SML (R2 = 0.28), while no cor-
relation was found for these compounds in BW. The enrich-
ment factor (EF = cSML/cBW) for GLY and MGLY in the
SML varied strongly but was mostly > 1. The average EF
for both compounds was ca. 4 and EF GLY and EF MGLY are
slightly correlated (R2 = 0.30). The actual EF is in fact even
higher considering that for several BW samples GLY and es-
pecially MGLY concentrations were below LOD, whereas
SML concentrations were detectable. Presently, there are
few measured GLY and MGYL concentrations in real sea-
water samples available from literature. In one application
from Zhou and Mopper (1997) GLY and MGLY were mea-
sured (besides other aldehydes) in the Sargasso Sea (Atlantic
Ocean, near Bahamas). The concentrations are in the same
range (lower nM range) compared to the data presented here. The authors also observed enrichment of GLY and MGYL in
the SML with higher average enrichment factors than found
in the present study but also with higher variability (EF: 1–
21 for GLY; EF: 2–13 for MGLY). Regarding the forma-
tion and enrichment of carbonyls in seawater, photochem-
ical production has been reported to be a major source of
carbonyls in surface water (Kieber et al., 1990). Zhou and
Mopper (1997) found higher photoproduction rates of car-
bonyls in SML compared to BW. Moreover, microbial activ-
ity is discussed to be an important factor influencing carbonyl
abundance in seawater (Zhou and Mopper, 1997; Kieber and
Mopper, 1987). Microorganisms may either take up carbonyl
compounds or release them. Kieber and Mopper (1987) sug-
gested microbial activity to be rather a sink than a source
in well-oxygenated conditions such as the SML. The enrich-
ment of GLY and MGLY found in this work might be an
indicator for photochemical production of the carbonyls in
the SML, as the SML is more exposed to sunlight. However,
only weak correlations were found between global radiation
and concentration of GLY and MGLY in the SML as well as
enrichment of GLY/MGLY (R2 ∼0.2, Figs. S1–S4). Global
radiation is slightly correlated to air temperature within the
SML sampling time (R2 = 0.39 , Fig. S5). 3.3.1
GLY and MGLY in Atlantic seawater The results for GLY and MGLY concentrations in SML and
bulk water are listed in Table 3. Average concentration of
GLY in the SML was 330 ng L−1 (123–616 ng L−1). SML www.atmos-chem-phys.net/13/11791/2013/ Atmos. Chem. Phys., 13, 11791–11802, 2013 M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater 11796 concentration for MGLY were on average slightly lower
(261 ng L−1) with a higher fluctuation (77–760 ng L−1). In
BW, average concentrations were 121 ng L−1 (GLY) and
94 ng L−1 (MGLY). MGLY also showed higher fluctuation
and in six BW samples MGLY concentrations were be-
low LOD. There was a weak correlation between GLY and
MGLY concentrations in the SML (R2 = 0.28), while no cor-
relation was found for these compounds in BW. The enrich-
ment factor (EF = cSML/cBW) for GLY and MGLY in the
SML varied strongly but was mostly > 1. The average EF
for both compounds was ca. 4 and EF GLY and EF MGLY are
slightly correlated (R2 = 0.30). The actual EF is in fact even
higher considering that for several BW samples GLY and es-
pecially MGLY concentrations were below LOD, whereas
SML concentrations were detectable. Presently, there are
few measured GLY and MGYL concentrations in real sea-
water samples available from literature. In one application
from Zhou and Mopper (1997) GLY and MGLY were mea-
sured (besides other aldehydes) in the Sargasso Sea (Atlantic
Ocean, near Bahamas). The concentrations are in the same
range (lower nM range) compared to the data presented here. The authors also observed enrichment of GLY and MGYL in
the SML with higher average enrichment factors than found
in the present study but also with higher variability (EF: 1–
21 for GLY; EF: 2–13 for MGLY). Regarding the forma-
tion and enrichment of carbonyls in seawater, photochem-
ical production has been reported to be a major source of
carbonyls in surface water (Kieber et al., 1990). Zhou and
Mopper (1997) found higher photoproduction rates of car-
bonyls in SML compared to BW. Moreover, microbial activ-
ity is discussed to be an important factor influencing carbonyl
abundance in seawater (Zhou and Mopper, 1997; Kieber and
Mopper, 1987). Microorganisms may either take up carbonyl
compounds or release them. Sampling problems and artifacts As reported in Sect. 2.1, PM1 aerosol particles were collected
along the entire cruise. For several sampling days and at cer-
tain wind directions, the exhaust plume affected the filter
sampling, resulting in a contamination of the filters. There-
fore, all filters with elemental carbon values above 60 ng m−3
were excluded. Furthermore, some filters needed to be ex-
cluded due to water entrance into the Digitel sampler inlet at
high wind speeds resulting in wet filters. Overall we achieved
11 filter samples (24 h sampling each) which were suggested
to be of marine origin due to their low values of elemental
carbon and air masses from the open ocean. At the moment it is not possible to assess positive and
negative artifacts during aerosol sampling and the GLY and
MGLY concentrations reported here may be affected by un-
certainties such as adsorption of organic vapors, volatiliza-
tion or further reactions of the analytes. However, care was
taken that directly after sampling and during transport to the
laboratories, filters were cooled throughout at −20 ◦C un-
til analysis. Quartz filters are employed in similar applica-
tions for the sampling of GLY and MGLY (Kawamura et al.,
2010; Pavuluri et al., 2010; Li and Yu, 2005). Miyazaki et
al. (2010) reported that levels of field blanks were generally Atmos. Chem. Phys., 13, 11791–11802, 2013 www.atmos-chem-phys.net/13/11791/2013/ M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater
Table 3. Surface microlayer thickness, GLY and MGLY concentrations in surface microlayer (SML) and bulk water (B
enrichment factor (EF). . van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater 11797 less than 17 % of mass concentrations for diacids and related
compounds (such as GLY and MGLY) for quartz filters. (average 0.31 ng m−3) on Arctic aerosol particles showing
a seasonal variation with a peak in April being produced
during polar sunrise events. Over the western North Pacific
the concentration of MGLY concentrations were on aver-
age 0.5 ng m−3 while GLY concentrations were much lower
(0.06–0.12 ng m−3) (Miyazaki et al., 2010). In tropical India
aerosol particles GLY and MGLY were found in concentra-
tions between 4 and 23 ng m−3 (Pavuluri et al., 2010). An
explanation for the higher concentrations could be that the
air was mainly coming from the Arabian Sea, the Indian
Ocean but also partly continentally influenced. Generally
higher concentrations of GLY and MGLY on aerosol parti-
cles are reported in anthropogenic areas compared to remote
areas. In megacities, GLY and MGLY can be up to hundreds
of ng m−3 (for example up to 218 ng m−3 in Hong Kong
(Li and Yu, 2005; Ho et al., 2007; Kawamura and Yasui,
2005). Liggio and McLaren (2003) reported the alpha dicar-
bonyls in the lower ng m−3 range at urban and rural sites in
Canada (GLY: 0.43–3.3 ng m−3; MGLY: 0.03–1.2 ng m−3). At a background measurement site in the Rhine–Main area in
Germany, concentration were in average 1 ng m−3 for GLY
and 1.4 ng m−3 for MGLY (Kampf et al., 2011). In a cor-
relation of GLY and MGLY to NOx the authors showed
an anthropogenic influence on the SOA formation poten-
tial of GLY and MGLY. In another study the authors found
0.81 ng m−3 for GLY and 0.31 ng m−3 for MGLY in a bo-
real forest in Finland (Kampf et al., 2012). In a very recent
study, Kawamura et al. (2013) presented GLY and MGLY
aerosol concentrations with a maximum of 70 ng m−3 and
120 ng m−3 at Mt. Tai (North China). The corresponding GLY/MGLY concentrations on aerosol particles The concentrations of GLY and MGLY in the PM1 samples
(Fig. 4, Table S1) ranged between 0.11–0.41 ng m−3 (GLY)
and 0.06–0.29 ng m−3 (MGLY). Due to strong acidic condi-
tions during derivatization, GLY and MGLY values represent
the sum of the monomer carbonyls, its hydrated form and
reversibly formed high molecular weight compounds (Lig-
gio et al., 2003). The very good correlation between GLY
and MGLY (R2 = 0.82 ) suggest a similar transport and/or
formation mechanism for these two compounds in the atmo-
sphere. GLY concentrations are always slightly higher than
MGLY (factor 1.3) which might be due to the increased
solubility of GLY KH = 3.6 × 105 M atm−1; MGLY: KH =
3.7 × 103 M atm−1 data from Fu et al. (2008) which might
lead to a stronger uptake to the aerosol phase. As GLY and
MGLY are short-living compounds in the atmosphere with
an atmospheric lifetime of ca. 2 h in an marine environment
(Sinreich et al., 2010), GLY and MGLY are not expected to
originate from continental sources but rather from the open
sea. The concentrations for GLY and MGLY reported here
are in the same order of magnitude as reported for marine
aerosol particles by other authors in different places. No mea-
sured GLY and MGLY data for aerosol particles over the
Atlantic Ocean were found in the literature. Kawamura et
al. (2010) observed GLY (average: 0.55 ng m−3) and MGLY Atmos. Chem. Phys., 13, 11791–11802, 2013 www.atmos-chem-phys.net/13/11791/2013/ Sampling problems and artifacts Sampling
SML thickness
date
(µm)
GLY
MGLY
SML
BW
EF
SML
BW
EF
23.04.2011
132
257
< LOD
> 5.8
234
< LOD
> 5.9
24.04.2011
122
239
71.1
3.4
111
< LOD
> 3.4
25.04.2011
132
123
83.8
1.5
130
< LOD
> 1.5
29.04.2011
–
312
134
2.3
172
50.9
3.4
30.04.2011
142
419
59.4
7.1
325
< LOD
> 8.1
01.05.2011
146
284
75.2
3.8
145
51.4
2.8
02.05.2011
128
453
75.1
6.0
760
246
3.1
03.05.2011
127
276
69.7
4.0
305
68.1
4.5
04.05.2011
93
492
93.7
5.3
650
< LOD
> 16
05.05.2011
93
389
101
3.9
179
213
0.8
08.05.2011
108
360
93.9
3.8
253
150
1.7
09.05.2011
134
208
114
1.8
225
143
1.6
12.05.2011
111
204
216
0.9
77.2
< LOD
> 1.9
13.05.2011
–
616
257
2.4
199
63.8
3.1
16.05.2011
113
313
322
1.0
143
186
0.8
average
–
330
121
3.5
94.2
4.0
–
∗For average values concentrations < LOD were set at LOD (GLY: 54 ng L−1; MGLY: 50 ng L−1). M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater www.atmos-chem-phys.net/13/11791/2013/ Besides pho-
tochemical activity, Ervens and Volkamer (2010) revealed
further important parameters that strongly affect GLY on
aerosol particles such as particle constitution (liquid water
content, pH, chemical composition). Therefore, a combina-
tion of environmental factors might affect GLY and MGLY
abundance on aerosol particles. For oxalate concentrations
determined in this work, correlation to global radiation was
quite strongly pronounced (R2 = 0.60, n = 10, Fig. S23) in-
dicating photochemical production to be a dominant source. This might indicate that GLY and MGLY production could
result from the photo-oxidation of a VOC precursor in the
gas phase. Fig. 4. Concentration of GLY, MGLY and oxalate together with
DOC and environmental parameters. gas phase concentrations were by a factor of 10–20 higher. They found a strong correlation to levoglucosan, suggesting
biomass burning to be an important source for alpha dicar-
bonyls. For the data presented here, a very good correlation be-
tween the two carbonyls and oxalate was found (Fig. 5),
regarding the absolute concentrations as well as their DOC
fraction on the aerosol particles. The strong correlation is
partly caused by the last sampling point close to Europe
(highest GLY, MGLY and oxalate concentrations) but still
pronounced without this data point (R2 = 0.4 for GLY and
MGLY). The correlation between GLY and MGLY to ox-
alate might show a linkage towards a secondary formation
of oxalic acid via these alpha dicarbonyls, that is a detailed
described pathway in the literature (Tilgner and Herrmann,
2010; Ervens et al., 2011). Tilgner and Herrmann (2010)
showed that oxalic acid is formed preferably in the atmo-
spheric aqueous phase due to aldehyde precursors such as
GLY and MGLY. Rinaldi et al. (2011) suggested that cloud-
mediated oxidation of gaseous GLY might be an important
source for oxalate on aerosol particles. Oxalate concentra-
tions in this work are by a factor of 29 higher than the sum of
GLY and MGLY concentrations. This might be due to mul-
tiple potential precursors for oxalic acid or higher accumu-
lation of oxalic acid in the aerosol particles due to its much
lower volatility compared to GLY and MGLY. Positive corre-
lations between alpha dicarbonyls and oxalic acid were also
reported by Kawamura et al. (2013) regarding aerosol par-
ticles from Mt. www.atmos-chem-phys.net/13/11791/2013/ M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater 11798 0
0.1
0.2
0.3
0.4
0
100
200
300
400
500
solar radiation
CHL‐A
0
0.1
0.2
0.3
0.4
0.5
0
5
10
15
20
25
30
35
40
temp. DOC
south‐west Africa
north‐west Africa
Europe
oxalate (ng m-3 )
GLY/MGLY (ng m-3 )
Chl-a (µg L-1 )
global rad. .(W m-2 )
temp. ( C)
DOC (µg m-3)
0
5
10
15
20
25
30
35
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
0.450
GLY
MGLY
Ox
Chl‐a
oxalate
golbal radiation
Fig. 4. Concentration of GLY, MGLY and oxalate together with
DOC and environmental parameters. factor of 19 higher than GLY and MGLY together (Kawa-
mura et al., 2010). factor of 19 higher than GLY and MGLY together (Kawa-
mura et al., 2010). factor of 19 higher than GLY and MGLY together (Kawa-
mura et al., 2010). 28
Figure 4
Regarding the data presented here, the last sampling point
(17 May 2011) was very close to European mainland (Fig. 1)
measurements. Although air masses were marine and EC
concentrations below 60 ng m−3, the higher concentration
of GLY and MGLY (and also oxalate) might be caused
by stronger anthropogenic influences such as enhanced ship
traffic in this region. Different authors (Liggio and McLaren,
2003; Kampf et al., 2011) report stronger GLY and MGLY
abundance in higher anthropogenic influenced areas prob-
ably due to an increased concentration of aromatic precur-
sors. Also for oxalic acid, higher concentrations are reported
in areas where a continental influence becomes noticeable
(Warneck, 2003; Kawamura and Usukura, 1993b). Probably
other than ocean driven mechanisms and different precursors
are important in this area close to Europe. Disregarding the
last sampling point, a positive correlation between GLY and
MGLY concentration to temperature (R2 = 0.42, R2 = 0.30,
respectively, n = 10, Figs. S19–S20) was found. Global radi-
ation correlated to MGLY (R2 = 0.42; n = 10, Fig. S21) but
weakly to GLY (R2 = 0.10, n = 10, Fig. S22). Photochem-
ical production from precursors such as isoprene is reported
to be the dominant source for GLY and MGLY, but photol-
ysis and oxidation with OH also represent sinks for these
carbonyls in the atmosphere (Fu et al., 2008). M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater R² = 0,3254
0
100
200
300
400
500
600
0,05
0,1
0,15
0,2
0,25
concentration GLY aer (ng m-3)
concentration GLY SML (ng L-1)
(a) Figure 5
R² = 0,116
0
100
200
300
400
500
600
700
0,05
0,1
0,15
0,2
concentration MGLY aer (ng m-3)
c oncentration MGLY SML (ng L-1) (a) (b) Fig. 6. Correlation of (a) GLY and (b) MGLY in SML and in aerosol particle. Figure 6
(1) gaseous transfer from the ocean into the atmosphere and
gas-to-particle conversion, (2) incorporation of material from
the organic rich SML during wind driven aerosol production,
and (3) accumulation of organics to the surface of bubbles
and its subsequent injection to the atmosphere (bubble burst-
ing). Although the last process is mostly expected for sur-
face active material, also non-surface active organics can be
associated with the surface active agent (Duce, 1976). Gen-
erally, all these processes are possible for GLY and MGLY –
as well as for their precursors – including subsequent depo-
sition back to the ocean. Due to the high solubility of GLY
and MGLY their net flow is expected from air to sea (Zhou
and Mopper, 1990). Sinreich et al. (2010) observed high con-
centrations of gaseous GLY over the Pacific Ocean and sug-
gested an airborne source of GLY in the form of a process at
the air–sea interface or by a bubble bursting process. due to the limited data set and the lack of gas phase measure-
ments clearly more research is needed on this subject. GLY/MGLY in the SML and aerosol Within the Polarstern cruise there were six sampling dates
when seawater and clean marine aerosol particles sampling
was co-located, meaning that SML sampling was in the
midterm of the 24 h aerosol sampling time. These sampling
dates are illustrated with a green cross in Fig. 1. A posi-
tive correlation between the carbonyls in the SML and in
aerosol particles was found for GLY (R2 = 0.33; less pro-
nounced for MGLY: R2 = 0.12) presented in Fig. 6. There
are multiple ways of exchange processes between oceans
and the atmosphere for the carbonyls that could be the rea-
son for a positive correlation. Turekian et al. (2003) summa-
rized three main processes for sea–air transfer of organics: Atmos. Chem. Phys., 13, 11791–11802, 2013 www.atmos-chem-phys.net/13/11791/2013/ Tai in China as well as for Arctic aerosol
particles where oxalate concentrations were on average by a In contrast to seawater, where no connection to Chl a was
found, a negative correlation of GLY and MGLY to Chl a was
observed for GLY (R2 = 0.54, Fig. S24) and less pronounced
for MGLY (R2 = 0.28, n = 6, Fig. S25). Also oxalic acid
on aerosol particles showed a slightly negative correlation to
Chl a (R2 = 0.28, n = 6, Fig. S26). Miyazaki et al. (2010)
also observed increased concentrations for GLY on marine
aerosol particles in times of lower biological activity, while
MGLY concentrations were hardly affected. One possible
explanation for the diminished concentrations of GLY and
MGLY in the aerosol particles without changes in seawater
concentrations could be changes in the gas phase chemistry
depending on biological activity. Biological activity could
affect the concentrations of the carbonyls and their precur-
sors in the gas phase leading to a reduced production and/or
stronger degradation of the alpha dicarbonyls and affecting
their partitioning between gas and aerosol phase. However Atmos. Chem. Phys., 13, 11791–11802, 2013 www.atmos-chem-phys.net/13/11791/2013/ M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater 11799 M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater
11799
R² = 0,8028
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0,4
0,45
0
0,01
0,02
0,03
0,04
concentration GLY aer (ng m-3)
concentration oxalate aer (µg m-3)
R² = 0,7702
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0
0,01
0,02
0,03
0,04
concentration MGLY aer (ng m-3)
concentration oxalate aer (µg m-3)
R² = 0,6647
0
0,01
0,02
0,03
0,04
0,05
0,06
0,07
0,00
0,50
1,00
1,50
2,00
2,50
3,00
DOC fraction oxalate (%)
DOC fraction GLY ( %)
R² = 0,5644
0
0,05
0,1
0,15
0,2
0,25
0,00
1,00
2,00
3,00
DOC fraction oxalate (%)
DOC fraction MGLY (%)
(a)
(b)
(c)
(d)
Fig. 5. Correlation of GLY/MGLY and oxalate: (a) and (b) concentration, (c) and (d) DOC fraction. ∗DOC fraction was calculated from
GLY, MGLY and oxalate concentrations and their contribution to DOC concentrations on a carbon basis. R² = 0,8028
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0,4
0,45
0
0,01
0,02
0,03
0,04
concentration GLY aer (ng m-3)
concentration oxalate aer (µg m-3)
(a) R² = 0,7702
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0
0,01
0,02
0,03
0,04
concentration MGLY aer (ng m-3)
concentration oxalate aer (µg m-3) (a) R² = 0,5644
0
0,05
0,1
0,15
0,2
0,25
0,00
1,00
2,00
3,00
DOC fraction oxalate (%)
DOC fraction MGLY (%)
(b)
(d) (b) R² = 0,6647
0
0,01
0,02
0,03
0,04
0,05
0,06
0,07
0,00
0,50
1,00
1,50
2,00
2,50
3,00
DOC fraction oxalate (%)
DOC fraction GLY ( %)
(c) (c) (d) Fig. 5. Correlation of GLY/MGLY and oxalate: (a) and (b) concentration, (c) and (d) DOC fraction. ∗DOC fraction was calculated from
GLY, MGLY and oxalate concentrations and their contribution to DOC concentrations on a carbon basis. Figure 5
R² = 0,3254
0
100
200
300
400
500
600
0,05
0,1
0,15
0,2
0,25
concentration GLY aer (ng m-3)
concentration GLY SML (ng L-1)
R² = 0,116
0
100
200
300
400
500
600
700
0,05
0,1
0,15
0,2
concentration MGLY aer (ng m-3)
c oncentration MGLY SML (ng L-1)
(a)
(b)
Fig. 6. Correlation of (a) GLY and (b) MGLY in SML and in aerosol particle. M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater 11800 In summary, the slight correlation of the alpha dicarbonyls
in the SML and in the aerosol particles reported here could
be a hint for interactions of these compounds – especially
GLY – in seawater and in the atmosphere although specific
mechanisms cannot be concluded from this work. Carlton, A. G., Turpin, B. J., Altieri, K. E., Seitzinger, S.,
Reff, A., Lim, H. J., and Ervens, B.: Atmospheric oxalic
acid and SOA production from glyoxal, Results of aqueous
photooxidation experiments, Atmos. Environ. 41, 7588–7602,
doi:10.1016/j.atmosenv.2007.05.035, 2007. Cunliffe, M., Engel, A., Frka, S., Gasparovic, B., Guitart, C., Mur-
rell, J. C., Salter, M., Stolle, C., Upstill-Goddard, R., and Wurl,
O: Sea surface microlayers, A unified physicochemical and bi-
ological perspective of the air-ocean interface, Prog. Oceanogr.,
109, 104–116, doi:10.1016/j.pocean.2012.08.004, 2013. 4
Conclusions Further investigations of important precursors of
GLY and MGLY (especially isoprene), gas phase sampling
of the carbonyls and a larger data set are necessary for a bet-
ter understanding of GLY and MGLY interactions between
air and sea. Fu, T. M., Jacob, D. J., Wittrock, F., Burrows, J. P., Vrekous-
sis, M., and Henze, D. K.: Global budgets of atmospheric gly-
oxal and methylglyoxal, and implications for formation of sec-
ondary organic aerosols, J. Geophys. Res.-Atmos., 113, D15303,
doi:10.1029/2007jd009505, 2008. Ho, K. F., Cao, J. J., Lee, S. C., Kawamura, K., Zhang, R. J., Chow,
J. C., and Watson, J. G.: Dicarboxylic acids, ketocarboxylic
acids, and dicarbonyls in the urban atmosphere of China, J. Geo-
phys. Res.-Atmos., 112, D22s27, doi:10.1029/2006jd008011,
2007. Supplementary material related to this article is
available online at http://www.atmos-chem-phys.net/13/
11791/2013/acp-13-11791-2013-supplement.pdf. Houdier, S., Barret, M., Domine, F., Charbouillot, T., Deguillaume,
L., and Voisin, D.: Sensitive determination of glyoxal, methyl-
glyoxal and hydroxyacetaldehyde in environmental water sam-
ples by using dansylacetamidooxyamine derivatization and liq-
uid chromatography/fluorescence, Anal. Chim. Ac., 704, 162–
173, doi:10.1016/j.aca.2011.08.002, 2011. Kaiser, H. and Specker, H.: Bewertung und Vergleich von Analy-
senverfahren, Fresenius Zeitschrift für Analytische Chemie, 149,
46–66, doi:10.1007/bf00454145, 1956. Acknowledgements. The authors acknowledge the team from the
Polarstern ANT XXVII/4 cruise, M. Brückner for global radiation
measurements, S. Fuchs for sampling, M. Voyevoda for technical
assistance and the Bundesministerium fuer Bildung und Forschung
(BMBF) for funding within the SOPRAN project (FKZ03F0662-J). Kampf, C. J., Bonn, B., and Hoffmann, T.: Development and
validation of a selective HPLC-ESI-MS/MS method for the
quantification of glyoxal and methylglyoxal in atmospheric
aerosols (PM2.5), Anal. Bioanal. Chem., 401, 3115–3124,
doi:10.1007/s00216-011-5192-z, 2011. Edited by: R. McLaren Kampf, C. J., Corrigan, A. L., Johnson, A. M., Song, W., Kero-
nen, P., Königstedt, R., Williams, J., Russell, L. M., Petäjä, T.,
Fischer, H., and Hoffmann, T.: First measurements of reactive
α-dicarbonyl concentrations on PM2.5 aerosol over the Boreal
forest in Finland during HUMPPA-COPEC 2010 – source ap-
portionment and links to aerosol aging, Atmos. Chem. Phys., 12,
6145–6155, doi:10.5194/acp-12-6145-2012, 2012. www.atmos-chem-phys.net/13/11791/2013/ M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater References Carlson, D. J.: Dissolved Organic Materials in Surface Microlay-
ers – Temporal and Spatial Variability and Relation to Sea State,
Limnol. Oceanogr., 28, 415–431, 1983. Atmos. Chem. Phys., 13, 11791–11802, 2013 4
Conclusions This work presents a suitable and easy-to-use method for the
analysis of GLY and MGLY in seawater and aerosol parti-
cles. To our knowledge, these data present the first measured
GLY and MGLY concentrations on Atlantic aerosol particles
and the corresponding seawater samples. In seawater, the en-
richment in the SML implies a photochemical production of
GLY and MGLY though a clear connection to global radia-
tion was not observed. GLY and MGLY on marine aerosol
particles are strongly connected to each other, suggesting
similar formation mechanisms. Moreover they are both cor-
related to oxalate. A negative correlation of GLY and MGLY
on aerosol particles to biological activity was found. As no
connection between SML concentrations of the carbonyls
and Chl a was observed, the reason might be due to changes
in gas phase chemistry of the carbonyls and their precursors. The results of GLY and MGLY in marine aerosol particles
and in the ocean water give a first insight towards the inter-
action processes between ocean and atmosphere, though de-
tailed mechanisms and net fluxes cannot be concluded from
our data. Further investigations of important precursors of
GLY and MGLY (especially isoprene), gas phase sampling
of the carbonyls and a larger data set are necessary for a bet-
ter understanding of GLY and MGLY interactions between
air and sea. Duce, R. and Hoffmann, E. J.: Chemical fractionation at the air/sea
interface, Annu. Rev. Earth Planet Sci., 4, 187–228, 1976. EPA method 556: Determination of carbonyl compounds in drink-
ing water by pentafluorobenzylhydroxylamine derivatization and
capillary gas chromatography with electron capture detection,
National exposure research laboratory office of research and
development US Environmental Protection Agency Cincinnati,
Ohio 45268, 1998. Ervens, B. and Volkamer, R.: Glyoxal processing by aerosol multi-
phase chemistry: towards a kinetic modeling framework of sec-
ondary organic aerosol formation in aqueous particles, Atmos. Chem. Phys., 10, 8219–8244, doi:10.5194/acp-10-8219-2010,
2010. Ervens, B., Turpin, B. J., and Weber, R. J.: Secondary or-
ganic aerosol formation in cloud droplets and aqueous parti-
cles (aqSOA), a review of laboratory, field and model stud-
ies, Atmos. Chem. Phys., 11, 11069–11102, doi:10.5194/acp-11-
11069-2011, 2011. g
p
y
y
p
The results of GLY and MGLY in marine aerosol particles
and in the ocean water give a first insight towards the inter-
action processes between ocean and atmosphere, though de-
tailed mechanisms and net fluxes cannot be concluded from
our data. M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater 11801 over the western North Pacific in summer, J. Geophys. Res.-
Atmos., 115, D23210, doi:10.1029/2010jd014439, 2010. Kawamura, K.: Identification of C-2-C-10 omega-oxocarboxylic
acids, pyruvic-acid, and C-2-C-3 alpha-dicarbonyls in wet pre-
cipitation and aerosol samples by capillary GC and GC / MS,
Anal. Chem., 65, 3505–3511, doi:10.1021/ac00071a030, 1993a. Müller, K., Lehmann, S., van Pinxteren, D., Gnauk, T., Nieder-
meier, N., Wiedensohler, A., and Herrmann, H.: Particle charac-
terization at the Cape Verde atmospheric observatory during the
2007 RHaMBLe intensive, Atmos. Chem. Phys., 10, 2709–2721,
doi:10.5194/acp-10-2709-2010, 2010. Kawamura, K. and Ikushima, K.: Seasonal-changes in the distribu-
tion of dicarboxylic-acids in the urban atmosphere, Environ. Sci. Technol., 27, 2227–2235, doi:10.1021/es00047a033, 1993. Ortiz,
R.,
Hagino,
H.,
Sekiguchi,
K.,
Wang,
Q. Y.,
and
Sakamoto, K.: Ambient air measurements of six bifunctional
carbonyls in a suburban area, Atmos. Res., 82, 709–718,
doi:10.1016/j.atmosres.2006.02.025, 2006. Kawamura, K. and Usukura, K.,: Distribution of low molecular
weight dicarboxylic acids in the north pacific aerosol samples,
J. Oceanogr., 49, 271–283, 1993b Kawamura, K. and Yasui, O.: Diurnal changes in the distribution
of dicarboxylic acids, ketocarboxylic acids and dicarbonyls in
the urban Tokyo atmosphere, Atmos. Environ., 391, 1945–1960,
doi:10.1016/j.atmosenv.2004.12.014, 2005. j
Pang, X., Lewis, A. C., and Ródenas-Garcia M.: Microfluidic lab-
on-a-chip derivatization for gaseous carbonyl analysis, J. Chro-
matogr. A, 1296, 93–103, 2013. Pavuluri, C. M., Kawamura, K., and Swaminathan, T.: Water-
soluble organic carbon, dicarboxylic acids, ketoacids, and alpha-
dicarbonyls in the tropical Indian aerosols, J. Geophys. Res.-
Atmos., 115, D11302, doi:10.1029/2009jd012661, 2010. Kawamura, K., Kasukabe, H., and Barrie, L. A.: Secondary for-
mation of water-soluble organic acids and alpha-dicarbonyls
and their contributions to total carbon and water-soluble or-
ganic carbon, Photochemical aging of organic aerosols in
the Arctic spring, J. Geophys. Res.-Atmos., 115, D21306,
doi:10.1029/2010jd014299, 2010. Rinaldi, M., Decesari, S., Carbone, C., Finessi, E., Fuzzi, S., Ce-
burnis, D., O’Dowd, C. D., Sciare, J., Burrows, J. P., Vrekous-
sis, M., Ervens, B., Tsigaridis, K., and Facchini, M. C.: Ev-
idence of a natural marine source of oxalic acid and a pos-
sible link to glyoxal, J. Geophys. Res.-Atmos., 116, D16204,
doi:10.1029/2011jd015659, 2011. Kawamura, K., Okuzawa, K., Aggarwal, S. G., Irie, H., Kanaya,
Y., and Wang, Z.: Determination of gaseous and particulate
carbonyls (glycolaldehyde, hydroxyacetone, glyoxal, methylgly-
oxal, nonanal and decanal) in the atmosphere at Mt. Tai, At-
mos. Chem. Phys., 13, 5369–5380, doi:10.5194/acp-13-5369-
2013, 2013. Saison, D., De Schutter, D. P., Delvaux, F., and Delvaux, F. M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater R.: De-
termination of carbonyl compounds in beer by derivatisation and
headspace solid-phase microextraction in combination with gas
chromatography and mass spectrometry, J. Chromatogr. A, 26,
5061–5068, doi:10.1016/j.chroma.2009.04.077, 2009. Kieber, D. J. and Mopper, K.: Photochemical fromation of gly-
oxylic and pyruvic acid in seawater, Mar. Chem., 21, 135–149,
doi:10.1016/0304-4203(87)90034-x, 1987. Kieber, R. J., Zhou, X. L., and Mopper, K.: Formation of carbonyl-
compounds from UV-induced photodegradation of humic sub-
stances in natural-waters – fate of riverine carbon in the sea, Lim-
nol. Oceanogr., 35, 1503–1515, 1990. Shaw, S. L., Gantt, B., and Meskhidze, N.: Production and Emis-
sions of Marine Isoprene and Monoterpenes, A Review, Adv. Meteorol., 24, 408696, doi:10.1155/2010/408696, 2010. Sinreich, R., Coburn, S., Dix, B., and Volkamer, R.: Ship-based
detection of glyoxal over the remote tropical Pacific Ocean,
Atmos. Chem. Phys., 10, 11359–11371, doi:10.5194/acp-10-
11359-2010, 2010. Li, Y. C. and Yu, J. Z.: Simultaneous determination of mono- and
dicarboxylic acids, omega-oxo-carboxylic acids, midchain keto-
carboxylic acids, and aldehydes in atmospheric aerosol samples,
Environ. Sci. Technol., 39, 7616–7624, doi:10.1021/es050896d,
2005. Standard Method Number 6252B: “PFBHA Liquid-Liquid Extrac-
tion Gas Chromatographic Method”, Standard Methods for the
Examination of Water and Wastewater, 6–83, American Public
Health Assoc., Washington, DC, 1995. Liggio, J., Li, S. M., and McLaren, R.: Reactive uptake of glyoxal
by particulate matter, J. Geophys. Res.-Atmos., 110, D10304,
doi:10.1029/2004jd005113, 2005. Stolle, C., Nagel, K., Labrenz, M., and Jürgens, K.: Succession of
the sea-surface microlayer in the coastal Baltic Sea under nat-
ural and experimentally induced low-wind conditions, Biogeo-
sciences, 7, 2975–2988, doi:10.5194/bg-7-2975-2010, 2010. Liggio, J. and McLaren, R.: An optimized method for the deter-
mination of volatile and semi-volatile aldehydes and ketones in
ambient particulate matter, Int. J. Environ. An. Ch., 83, 819–835,
doi:10.1080/03067310310001597653, 2003. Tilgner, A. and Herrmann, H.: Radical-driven carbonyl-to-acid
conversion and acid degradation in tropospheric aqueous sys-
tems studied by CAPRAM, Atmos. Environ., 44, 5415–5422,
doi:10.1016/j.atmosenv.2010.07.050, 2010. Lim, Y. B., Tan, Y., Perri, M. J., Seitzinger, S. P., and Turpin,
B. J.: Aqueous chemistry and its role in secondary organic
aerosol (SOA) formation, Atmos. Chem. Phys., 10, 10521–
10539, doi:10.5194/acp-10-10521-2010, 2010. Turekian, V. C., Macko, S. A., and Keene, W. C.: Concentra-
tions, isotopic compositions, and sources of size-resolved, par-
ticulate organic carbon and oxalate in near-surface marine air
at Bermuda during spring, J. Geophys. Res.-Atmos., 108, 4157,
doi:10.1029/2002jd002053, 2003. Liss., P. S. and Duce, R. Atmos. Chem. Phys., 13, 11791–11802, 2013 www.atmos-chem-phys.net/13/11791/2013/ M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater A.: The Sea Surface and Global Change,
Cambridge University Press, vCambridge, UK, 1997 RAD, The
Sea Surface and Global Change, 1997. Mbukwa, E. A., Msagati, T. A. M., and Mamba, B. B.: Sup-
ported liquid membrane-liquid chromatography-mass spectrom-
etry analysis of cyanobacterial toxins in fresh water systems,
Phys. Chem. Earth, 50, 84–91, doi:10.1016/j.pce.2012.09.005,
2012. van Pinxteren, D., Bruggemann, E., Gnauk, T., Iinuma, Y., Muller,
K., Nowak, A., Achtert, P., Wiedensohler, A., and Herrmann,
H.: Size- and time-resolved chemical particle characteriza-
tion during CAREBeijing-2006, Different pollution regimes
and diurnal profiles, J. Geophys. Res.-Atmos., 114, D00g09,
doi:10.1029/2008jd010890, 2009. Miyazaki, Y., Kawamura, K., and Sawano, M.: Size distributions
and chemical characterization of water-soluble organic aerosols Atmos. Chem. Phys., 13, 11791–11802, 2013 www.atmos-chem-phys.net/13/11791/2013/ www.atmos-chem-phys.net/13/11791/2013/ Atmos. Chem. Phys., 13, 11791–11802, 2013 M. van Pinxteren and H. Herrmann: Glyoxal and methylglyoxal in Atlantic seawater 11802 Whipple, E. B.: Structure of glyoxal in water, J. Am. Chem. Soc.,
92, 7183–7186, doi:10.1021/ja00727a027, 1970. van Pinxteren, M., Müller, C., Iinuma, Y., Stolle, C., and Her-
rmann, H.: Chemical Characterization of Dissolved Organic
Compounds from Coastal Sea Surface Micro layers (Baltic
Sea, Germany), Environ. Sci. Technol., 46, 10455–10462,
doi:10.1021/es204492b, 2012. Zhou, X. L. and Mopper, K.: Apparent partition-coefficients of 15
carbonyl-compounds between air and seawater and between air
and fresh-water – implications for air sea exchange, Environ. Sci. Technol., 24, 1864–1869, doi:10.1021/es00082a013, 1990. Volkamer, R., Martini, F. S., Molina, L. T., Salcedo, D., Jimenez, J. L., and Molina, M. J.: A missing sink for gas-phase glyoxal in
Mexico City, Formation of secondary organic aerosol, Geophys. Res. Lett., 34, L19807, doi:10.1029/2007gl030752, 2007. Zhou, X. L. and Mopper, K.: Photochemical production of low-
molecular-weight carbonyl compounds in seawater and surface
microlayer and their air-sea exchange, Mar. Chem., 56, 201–213,
doi:10.1016/s0304-4203(96)00076-x, 1997. Warneck, P.: In-cloud chemistry opens pathway to the formation
of oxalic acid in the marine atmosphere, Atmos. Environ., 37,
2423–2427, doi:10.1016/s1352-2310(03)00136-5, 2003. Atmos. Chem. Phys., 13, 11791–11802, 2013 www.atmos-chem-phys.net/13/11791/2013/ www.atmos-chem-phys.net/13/11791/2013/
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https://opus.lib.uts.edu.au/bitstream/10453/124651/1/Integrated%20solutions%20for%20sustainable%20fall%20prevention%20in%20primary%20care%2c%20the%20iSOLVE%20project%3a%20a%20type%202%20hybrid%20effectiveness-implementation%20design.pdf
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Integrated solutions for sustainable fall prevention in primary care, the iSOLVE project: a type 2 hybrid effectiveness-implementation design
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Implementation science
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STUDY PROTOCOL STUDY PROTOCOL © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Integrated solutions for sustainable fall
prevention in primary care, the iSOLVE
project: a type 2 hybrid effectiveness-
implementation design Lindy Clemson1,2*
, Lynette Mackenzie1, Chris Roberts3, Roslyn Poulos4, Amy Tan1, Meryl Lovarini1,
Cathie Sherrington5, Judy M. Simpson6, Karen Willis7, Mary Lam1, Anne Tiedemann5, Dimity Pond8, David Peiris5,
Sarah Hilmer3,9, Sabrina Winona Pit10, Kirsten Howard6, Lorraine Lovitt11 and Fiona White1 * Correspondence: lindy.clemson@sydney.edu.au
1Faculty of Health Sciences, The University of Sydney, Lidcombe, Australia
2Centre of Excellence in Population Ageing Research, Sydney, Australia
Full list of author information is available at the end of the article Clemson et al. Implementation Science (2017) 12:12
DOI 10.1186/s13012-016-0529-9 Clemson et al. Implementation Science (2017) 12:12
DOI 10.1186/s13012-016-0529-9 Open Access Background the way advice is delivered and by whom). A systematic
review of interventions conducting training and dissemin-
ation of evidence to healthcare professionals [20] provides
evidence that training can improve implementation, but
conclusions were based on six studies of which only one
was a controlled trial. There was mixed support for
effectiveness of changes to primary care management
of falls, peer or lay-volunteer interventions, and com-
munity awareness campaigns. g
There is strong evidence that falls in community-
residing older people can be prevented with clear guid-
ance for effective interventions [1–3]. Primary care,
which can include general practice, allied health services,
community health, and community pharmacy, is gener-
ally the first point of contact people have with a health
system. General practitioners (GP), in particular are re-
lied on to manage the needs of older patients who are
falling [4, 5]. However, there is currently no clear model
for engaging GPs in fall prevention, and few older people
are asked by their GP about falls or are offered interven-
tions to prevent falls [6]. This leaves a huge gap of
missed opportunity [7, 8]. Among those GPs that do ad-
dress falls, few base their falls prevention practice on
recognized clinical guidelines [9]. Further, the develop-
ment of care plans and routine use of allied health pro-
fessionals in fall prevention has been slow. This has been
attributed to organizational barriers, difficulty for GPs in
initiating referral and/or reimbursement processes [10],
time constraints, a lack of educational materials [9], and
limited understanding of what allied health professionals
can offer in fall prevention [11]. While there has been
research about the acceptability and uptake of care
plans, there is limited research on the efficacy of multi-
disciplinary team approaches as an intervention to im-
prove clinical outcomes [12]. Identified factors for uptake and implementation of fall
prevention in primary care include accessible pathways
for referral which identify people most at risk, engage-
ment
in
collaborative
partnerships
and
developing
opportunities for knowledge transfer and up-skilling,
thereby building capacity and ensuring better outcomes
and broader population reach [9, 13]. For example, Ganz
et al.’s implementation of fall prevention into a veteran’s
organization [21, 22] found that the key to cultural
change was collaboration and links between older people
and health service providers and the development and
support of healthcare organizations/systems to better
deliver falls prevention interventions. Mahoney et al. Abstract Implementation Science (2017) 12:12 Page 2 of 12 Page 2 of 12 Abstract Background: Despite strong evidence giving guidance for effective fall prevention interventions in community-
residing older people, there is currently no clear model for engaging general medical practitioners in fall prevention
and routine use of allied health professionals in fall prevention has been slow, limiting widespread dissemination. This protocol paper outlines an implementation-effectiveness study of the Integrated Solutions for Sustainable Fall
Prevention (iSOLVE) intervention which has developed integrated processes and pathways to identify older people
at risk of falls and engage a whole of primary care approach to fall prevention. Methods/design: This protocol paper presents the iSOLVE implementation processes and change strategies and
outlines the study design of a blended type 2 hybrid design. The study consists of a two-arm cluster randomized
controlled trial in 28 general practices and recruiting 560 patients in Sydney, Australia, to evaluate effectiveness of
the iSOLVE intervention in changing general practitioner fall management practices and reducing patient falls and
the cost effectiveness from a healthcare funder perspective. Secondary outcomes include change in medications
known to increase fall risk. We will simultaneously conduct a multi-methodology evaluation to investigate the
workability and utility of the implementation intervention. The implementation evaluation includes in-depth
interviews and surveys with general practitioners and allied health professionals to explore acceptability and
uptake of the intervention, the coherence of the proposed changes for those in the work setting, and how to
facilitate the collective action needed to implement changes in practice; social network mapping will explore
professional relationships and influences on referral patterns; and, a survey of GPs in the geographical intervention
zone will test diffusion of evidence-based fall prevention practices. The project works in partnership with a primary
care health network, state fall prevention leaders, and a community of practice of fall prevention advocates. Discussion: The design is aimed at providing clear direction for sustainability and informing decisions about
generalization of the iSOLVE intervention processes and change strategies. While challenges exist in hybrid designs,
there is a potential for significant outcomes as the iSOLVE pathways project brings together practice and research
to collectively solve a major national problem with implications for policy service delivery. Trial registration: Australian New Zealand Clinial Trials Registry ACTRN12615000401550 * Correspondence: lindy.clemson@sydney.edu.au
1Faculty of Health Sciences, The University of Sydney, Lidcombe, Australia
2Centre of Excellence in Population Ageing Research, Sydney, Australia
Full list of author information is available at the end of the article Clemson et al. Background [23], when implementing a community-based fall pre-
vention program for widespread dissemination in the
USA, demonstrated how evidence-based training needs
to be supported by implementation guidelines for part-
ner organizations to ensure successful adoption. Another major problem for evidence uptake is that
successful trials of fall interventions have more often
been provided within a research context. Several studies
have failed to find fall prevention effects from interven-
tions that identify older people at risk of falls in general
practice followed by referral to usual care [7, 13–16]. It
appears that usual care may not reflect evidence-based
fall prevention. To determine why multi-factorial pro-
grams are successful or not, careful consideration needs
to be given to the content of the fall prevention inter-
vention, the process in which the intervention content
is delivered and if the intervention is targeted to the ap-
propriate group [17]. Lovarini et al.’s systematic review
[18] demonstrated a lack of fall prevention research
empirically testing sustainability of intervention imple-
mentation. We need to find ways of implementation in
real-life contexts that are evidence-based, generalizable,
and are sustainable. In order to test a model where general practice and al-
lied health services are integrated and evidence-based,
we conducted a small feasibility trial [24] involving two
GP practices consisting of eight GPs and two private
practices of physiotherapists and occupational therapists,
with fall prevention interventions delivered under a
Medicare rebate system (Enhanced Primary Care for
Allied Health Professionals) providing partial service
payment. Individual measures showed clinically import-
ant changes with excellent adherence. Research questions Research questions The iSOLVE intervention
Theoretical framework Methods/design Our study uses a type 2 blended design which places equal
importance on evaluating both intervention effectiveness
and implementation [25]. This design is aimed at providing
clear direction for sustainability and informing decisions
about generalization of the iSOLVE strategies [25]. We will
test the effectiveness of the iSOLVE pathways model
through a cluster randomized controlled trial to determine
effectiveness for individual patients and change in GP refer-
ral behaviours. The implementation evaluation is utilizing a
multi-methodology process including in-depth interviews
and surveys, social network mapping, and surveying of GPs
in the geographical intervention zone. Components of the iSOLVE pathway model of intervention
Table 1 provides a description of the four components
of the iSOLVE intervention and summarizes the planned
active ingredients of these components. Component one:
Identifying and managing fall risk in general practice
comprises individual face-to-face training and a compre-
hensive set of tools to support GP’s in knowledge trans-
lation, education, and in identifying and managing fall
risk. Component two: Knowledge translation, education,
and up-skilling the allied health local workforce com-
prises evidence-based fall prevention workshops, sharing
implementation strategies and opportunities for linking
services to GP practices. Component three: Referral
pathways in primary care outlines the role of GPs, allied
health, and the SNPHN in fall prevention and the strat-
egies used to facilitate patient pathways to fall preven-
tion
assessment
and
to
the
different
intervention
options. In Component 4: Diffusion and dissemination: a
guiding strategy document will be developed to document
the iSOLVE approach as a model of care for supporting
regional health networks, GP practices, ambulance ser-
vices, allied health, and community pharmacists to engage
in integrated pathways and evidence-based effective prac-
tices to protect older people from falling. The iSOLVE intervention
Theoretical framework 1. The primary effectiveness research questions are to
evaluate if the iSOLVE model will be effective in (i)
reducing falls in patients of participating practices
compared to control practices and (ii) increasing
general practitioners’ engagement in fall prevention
management and referral practices compared to the
control GPs. Our theoretically informed iSOLVE pathways model will
be integrated within one region, the Sydney North Pri-
mary Health Network (SNPHN) using the GP practice
as the core focus to develop and implement effective
clinical pathways by re-shaping the professional connec-
tions between potential fall service providers and the GP
practice. Building decision support tools, pathways, rou-
tine referral processes, and active networks will be a way
of levering evidence into practice [28]. The development
has been guided by the knowledge-to-action framework
(KAT) [29] which uses practical steps to implement and
sustain evidence in practice: adaptation of knowledge to
the local context, assessing barriers to implementation,
tailoring the intervention, and evaluating outcomes. Michie et al.’s [30] behaviour change wheel provides a
resource for determining key elements of behaviour
change within iSOLVE such as understanding what as-
pects of incentives, enabling and training are key to ef-
fective functioning of our emergent model. Further, the
normalization process theory (NPT) [31], considered a
major theory to guide the sustainability of healthcare in-
novations, will frame our understanding of, and our ap-
proaches to, how new practices can be routinely used by
organizations as it focuses on processes to enable new
practices to become embedded. And finally, Lau et al.'s
review [5] provides a useful and practical example of fac-
tors which influence change in practice in primary care. 2. The primary implementation research questions will
(i) investigate how the iSOLVE strategies are
adopted (or not) at individual (GP and allied health
professional (AHP)) practice, and primary care
network levels; and (ii) identify the factors that will
facilitate the embedding of the intervention in usual
care, and thus its sustainability. Aim and research questions q
To better inform authentic sustainable population
approaches to fall prevention, we have developed the
Integrated Solutions for Sustainable Fall Prevention
(iSOLVE) project. The aim of iSOLVE is to establish
integrated processes and pathways to identify older
people at risk of falls and engage a whole of primary
care approach to fall prevention. We will achieve this
by improving access to appropriate fall prevention
interventions
for
older
people,
ensuring
ongoing
knowledge acquisition and sustainable action by med-
ical practitioners and allied healthcare professionals;
implementing decision-making support within general
practice; and generating pathways to facilitate imple-
mentation through collaboration within a regional
health network. A systematic review of qualitative studies investigated
factors influencing the implementation of falls preven-
tion programs. [19] This review included studies that
collected information on the views of older people and
health professionals. Barriers and facilitators include
practical considerations (economic, access, time), adapt-
ability of the program for the community served (social
and cultural factors), and psychosocial factors (being
identified as a faller, impact of falls, client preferences, Page 3 of 12 Page 3 of 12 Clemson et al. Implementation Science (2017) 12:12 Component 1. Identifying and managing fall risk in general practice: knowledge translation and synthesis (Note that these
approaches differ from the recruitment strategy used for patients to the trial which was adapted to ensure
blinding of research assistants.)
1.7 Medication reviews
Medication reviews. Drawing on successful methods for reducing falls by medication review conducted by GPs
in the trial conducted by Pit et al. [62]. GPs are provided with information regarding both the evidence base
for reduction or ceasing medications to prevent falls and detailed lists of medications with specific fall risks. Medication reviews may also be requested using the Medicare funded Home Medicines Review by accredited
pharmacists and this option is included in the Tailoring Interventions to Fall Risk Chart. Component 2. Knowledge translation, education and up-skilling the allied health local workforce
2.1 Evidence-based interactive fall
prevention workshops
Educational approaches are effective in facilitating knowledge translation by AHPs [56], but active training and
planning for change are needed for effective implementation and sustainability [18, 20]. Evidence-based fall
prevention education workshops are offered to allied health professionals and service providers within the SNHN. These have been developed by experts within their fields and include Home Hazard and Environment interventions
(LC, LM) [3]; Exercise interventions (ATi, CS) [63] and the LiFE exercise program (LC) [64], Medication Management
(SH), and Foot and Ankle interventions [65]. 2.2 Active planning for fall
prevention implementation
and sustainability
These interactive workshops comprise knowledge and skill development as well as a planning session for
implementation and sustainability. Planning strategies documented by participants in each workshop form
part of a developing working document shared to all workshop participants Decision support tools and fall management tailoring introduced in the GP training session. These decision making
tools enable fall risk assessment and management and are part of a practice resource package for the GP. The
decision tool and resources are adapted from a primary care resource for falls prevention, developed by the
Centers for Disease Control in the USA [60]. A working group who were experts in fall prevention research, (LC,
CS, ATi) drawing from the US resource and using evidence from systematic review meta-analyses of interventions
[1] and of risk factors [61], developed the iSOLVE risk assessment/fall management algorithm and decision tools. The US Stay Independent Patient Check List and the GP Fall Risk Assessment chart were updated based on the
iSOLVE algorithm. 1.2 Decision support tools and fall
management tailoring. GP
resources (e.g., background
information/evidence, case
studies, Medicare
reimbursement options) 2.2 Active planning for fall
prevention implementation
and sustainability Component 1. Identifying and managing fall risk in general practice: knowledge translation and synthesis A new chart, Tailoring Interventions to Fall Risk, was developed which maps risk factors and risk
factor profiles to appropriate interventions. This was based on the intervention evidence (e.g., medication review;
balance, and strength training) and additionally, where intervention evidence did not exist, was based on modifiable
risk factor evidence which strongly supported a guideline for practice (e.g., postural dizziness). Other iSOLVE resources
include background information supporting the evidence for interventions; five case studies which each illustrate
the algorithm and tailoring options and were validated by a local expert group; a detailed summary of known
medications to be a risk of falling; Medicare reimbursement options for GPs; and, examples of “how to talk with
patients about falls.” A summary of local allied health professionals who offer fall prevention services and who
attended the workshops is provided for each GP with contact details. GP computer systems supports: The decision tools can be embedded into the GP systems and software by a fall
prevention add-on developed for the practice software supported by the SNHN. Embedded decision tools in
practice software recognize the barriers to GPs adoption of new practices and the need for speed and efficiency. The software add-on automatically creates the iSOLVE decision support tools. Once the GP completes the Fall Risk
Assessment, the program produces the recommended, individualized, and tailored interventions that match their
fall risk. Sample referral forms are provided. There are hard copies of all these documents, so that the whole
process can be manually done if the GP chooses to or if the practice is not computerized. Fall or fall risk alert to GP: People who report a fall in the past year or report “yes” to one of the risk questions on
the Stay Independent Fall Check list will indicate an alert to the GP who then starts the process of assessment and
management. Where practices agree a tablet device will be given to people 65 years and over by the practice
nurse in the waiting room and the fall screen completed to assist in determining risk factors. If the tablet is used,
this automatically sends the fall risk information to the GP’s software to speed up the process. GP managing patient fall risk: The GP uses the patient check list, conducts a risk assessment, and determines a
tailored management plan. The management plan is generated automatically if the computer system is used. 2.1 Evidence-based interactive fall
prevention workshops 1.6 Identifying eligible older
people 1.3 GP computer systems 1.7 Medication reviews Component 1. Identifying and managing fall risk in general practice: knowledge translation and synthesis An individual face-to-face training session is used to educate GPs in the various components of the iSOLVE
intervention including decision support tools, evidence-based interventions, potential referral pathways and fall
prevention strategies. The training is based on academic detailing which is characterized by principles such as
involvement of a “peer” to enable rapport and credibility, concise graphic print materials and applies social
marketing principles to facilitate behavior change [58, 59]. 1.1 Individual face-to-face training
sessions 1.2 Decision support tools and fall
management tailoring. GP
resources (e.g., background
information/evidence, case
studies, Medicare
reimbursement options)
Decision support tools and fall management tailoring introduced in the GP training session. These decision making
tools enable fall risk assessment and management and are part of a practice resource package for the GP. The
decision tool and resources are adapted from a primary care resource for falls prevention, developed by the
Centers for Disease Control in the USA [60]. A working group who were experts in fall prevention research, (LC,
CS, ATi) drawing from the US resource and using evidence from systematic review meta-analyses of interventions
[1] and of risk factors [61], developed the iSOLVE risk assessment/fall management algorithm and decision tools. The US Stay Independent Patient Check List and the GP Fall Risk Assessment chart were updated based on the
iSOLVE algorithm. A new chart, Tailoring Interventions to Fall Risk, was developed which maps risk factors and risk
factor profiles to appropriate interventions. This was based on the intervention evidence (e.g., medication review;
balance, and strength training) and additionally, where intervention evidence did not exist, was based on modifiable
risk factor evidence which strongly supported a guideline for practice (e.g., postural dizziness). Other iSOLVE resources
include background information supporting the evidence for interventions; five case studies which each illustrate
the algorithm and tailoring options and were validated by a local expert group; a detailed summary of known
medications to be a risk of falling; Medicare reimbursement options for GPs; and, examples of “how to talk with
patients about falls.” A summary of local allied health professionals who offer fall prevention services and who
attended the workshops is provided for each GP with contact details. 1.3 GP computer systems
GP computer systems supports: The decision tools can be embedded into the GP systems and software by a fall
prevention add-on developed for the practice software supported by the SNHN. Component 1. Identifying and managing fall risk in general practice: knowledge translation and synthesis Embedded decision tools in
practice software recognize the barriers to GPs adoption of new practices and the need for speed and efficiency. The software add-on automatically creates the iSOLVE decision support tools. Once the GP completes the Fall Risk
Assessment, the program produces the recommended, individualized, and tailored interventions that match their
fall risk. Sample referral forms are provided. There are hard copies of all these documents, so that the whole
process can be manually done if the GP chooses to or if the practice is not computerized. 1.4 Fall or fall risk alert to GP
Fall or fall risk alert to GP: People who report a fall in the past year or report “yes” to one of the risk questions on
the Stay Independent Fall Check list will indicate an alert to the GP who then starts the process of assessment and
management. Where practices agree a tablet device will be given to people 65 years and over by the practice
nurse in the waiting room and the fall screen completed to assist in determining risk factors. If the tablet is used,
this automatically sends the fall risk information to the GP’s software to speed up the process. 1.5 GP managing patient fall risk
GP managing patient fall risk: The GP uses the patient check list, conducts a risk assessment, and determines a
tailored management plan. The management plan is generated automatically if the computer system is used. The GP may review medications and check cataracts or postural hypotension where clinically indicated. The GP
also initiates appropriate referrals to local fall services (e.g., allied health and/or community exercise and/or
medication review) which specify “fall prevention”. 1.6 Identifying eligible older
people
Identifying eligible older people: People aged 65 years and over who have had a fall and or have a fall risk are
identified by several processes: opportunistic presentation to the GP practice and complete Stay Independent
Patient check list (or tablet) in the waiting room and/or are asked about falls by their GP; or, identify a fall during
a 75+ annual health checks. Additionally, the falls prevention computer program initiates an annual review of fall
status. These strategies are intended as routine and ongoing identification of older people who have fallen or
are at risk of falling. Marketing poster and brochures are also provided for the waiting room. Study setting
h Partnerships are crucial in all phases of the project: develop-
ment, implementation and in longer term plans for dissem-
ination and sustainability [26]. [27] We are conducting the
project in a metropolitan area and partnering initially with
the Northern Sydney Medicare Local which, due to change
in Federal Government policy, was replaced in 2016 by the
Sydney North Primary Health Network (SNPHN), effect-
ively doubling its geographic area. The networks have a role
in educating and engaging primary care and allied health
professions to enable access to effective health services. An-
other partner is the New South Wales (NSW) state Clinical
Excellence Commission’s Fall Prevention Program which
provides state-wide leadership in implementing NSW
Health Falls Policy and sharing of knowledge and resources. Our final partnership is with the community of practice of
fall prevention advocates. An advisory committee consisting
of 14 members represents the range of allied health, com-
munity pharmacy, and primary care physicians within the
district, along with consumer representation. The commit-
tee members are a highly skilled group with a broad range
of perspectives on implementing fall prevention, providing
support, advocacy, and informal guidance to the project. Page 4 of 12 Clemson et al. Implementation Science (2017) 12:12 Effectiveness trial methodology status questionnaire). Inclusion criteria for general prac-
tices are that they are within the SNPHN area. A two-arm cluster randomized controlled parallel trial is
being conducted with the general practice as the unit of
randomization and outcomes measured at both practice
change and patient outcome levels. Cluster randomization
is used in order to avoid contamination among patients of
the same GP because the intervention is delivered by the
GP. Randomizing GPs within the same practice to the same
arm likewise avoids contamination among GPs. The trial is
being conducted according to CONSORT guidelines [32]. Figure 1 provides a flow chart of participants through the
cluster trial, and Table 2 summarizes the research questions
and measurements. Recruitment commenced in June 2015. 3.5 Network communication
strategies Component 4. Diffusion and dissemination of the iSOLVE model Component 4. Diffusion and dissemination of the iSOLVE 4.1 Development of a guiding
strategy document “The aim is to facilitate sustained implementation of evidence-based fall prevention interventions by GPs and allied
health workforce. Theoretically, informed models of sustainable education and support (such as the potential for train
the trainer) will be developed drawing on data gained from workshops, interviews, and observations. The Conditions
for Sustainability Theory [69] and Behaviour Change Wheel Framework [30] will be used to guide this process. A guiding strategy document will be developed which outlines the Integrated Solutions for Sustainable Fall
Prevention (iSOLVE) approach as a model of care for supporting regional health networks, GP practices, ambulance
services, allied health, and community pharmacists to engage in integrated pathways and evidence-based effective
practices to protect older people from falling. This will be developed by the investigators in collaboration
with the partner representatives. It will be subject to extensive consultation with the key stakeholders and the
Project Advisory Group. Outcomes The primary outcome is the number of falls per person in
12 months. Secondary outcomes are Drug Burden Index
at the patient level and GP engagement in fall prevention
management and GP referral practices. Secondary
outcomes are measured using surveys at baseline and
12 months for patients and baseline, 3 and 12 months
for GPs in order to compare change from baseline be-
tween groups. Table 1 The iSOLVE intervention: planned active ingredients of iSOLVE components (Continued) 2.3 Linking AHPs with GPs to
facilitate referrals
There is also the opportunity for AHPs to opt to be linked to GPs, thus further enhancing pathways and
implementation. Component 3. Establishing referral pathways in primary care 3.1 Decision support tools and fall
management plans 3.1 Decision support tools and fall
management plans The decision support tools and fall management plans assist in determining the best option/s for the older person. Depending on risk assessment, this can include one or more approaches: medication review, postural
hypotension assessment, referral for cataract removal, home safety assessment, community exercise
programs, home-based exercise programs for higher risk patients, group-based fall exercise programs, tai
chi, a community-based multifaceted fall prevention program Stepping On [66], or referral to a falls clinic. Pathway facilitation. We use a range of options to facilitate the pathway to local fall service providers, including
but not limited to, the use of the Enhanced Primary Care service to encourage referral to private therapists and
facilitating evidence-based fall-specific services to be provided in health and other potential care services. The
aim is to provide education and knowledge translation that will up-skill and increase the fall prevention work
force across the SNPHN. 3.3 Referrals to fall prevention
services
3.4 Links with ambulance services Referrals to fall prevention services: The health network (SNPHN) and the workshops have been central to mapping
local health professionals who can engage in specific interventions. Ambulance services. Fifty eight percent of older people seen by the ambulance service for a fall and not
transported to hospital will fall again in the next 6 months [67]. During the development phase of the project,
we will determine a process to initiate referral to GPs for fall management and follow-up. Barriers to engaging
ambulance services have been investigated [68], and we will explore local options through consultation. 3.4 Links with ambulance services Communication. A website provides information about the project, education options, and links to state fall
prevention initiatives such as Stepping On and the Active & Healthy website (a state directory of evidence-based
fall prevention exercise options suitable for older people). Component 1. Identifying and managing fall risk in general practice: knowledge translation and synthesis Diffusion and dissemination of the iSOLVE model
4.1 Development of a guiding
strategy document
“The aim is to facilitate sustained implementation of evidence-based fall prevention interventions by GPs and allied
health workforce. Theoretically, informed models of sustainable education and support (such as the potential for train
the trainer) will be developed drawing on data gained from workshops, interviews, and observations. The Conditions
for Sustainability Theory [69] and Behaviour Change Wheel Framework [30] will be used to guide this process. A guiding strategy document will be developed which outlines the Integrated Solutions for Sustainable Fall
Prevention (iSOLVE) approach as a model of care for supporting regional health networks, GP practices, ambulance
services, allied health, and community pharmacists to engage in integrated pathways and evidence-based effective
practices to protect older people from falling. This will be developed by the investigators in collaboration
with the partner representatives. It will be subject to extensive consultation with the key stakeholders and the
Project Advisory Group. Component 1. Identifying and managing fall risk in general practice: knowledge translation and synthesis The GP may review medications and check cataracts or postural hypotension where clinically indicated. The GP
also initiates appropriate referrals to local fall services (e.g., allied health and/or community exercise and/or
medication review) which specify “fall prevention”. Identifying eligible older people: People aged 65 years and over who have had a fall and or have a fall risk are
identified by several processes: opportunistic presentation to the GP practice and complete Stay Independent
Patient check list (or tablet) in the waiting room and/or are asked about falls by their GP; or, identify a fall during
a 75+ annual health checks. Additionally, the falls prevention computer program initiates an annual review of fall
status. These strategies are intended as routine and ongoing identification of older people who have fallen or
are at risk of falling. Marketing poster and brochures are also provided for the waiting room. (Note that these
approaches differ from the recruitment strategy used for patients to the trial which was adapted to ensure
blinding of research assistants.) 1.7 Medication reviews Medication reviews. Drawing on successful methods for reducing falls by medication review conducted by GPs
in the trial conducted by Pit et al. [62]. GPs are provided with information regarding both the evidence base
for reduction or ceasing medications to prevent falls and detailed lists of medications with specific fall risks. Medication reviews may also be requested using the Medicare funded Home Medicines Review by accredited
pharmacists and this option is included in the Tailoring Interventions to Fall Risk Chart. Component 2. Knowledge translation, education and up-skilling the allied health local workforce Educational approaches are effective in facilitating knowledge translation by AHPs [56], but active training and
planning for change are needed for effective implementation and sustainability [18, 20]. Evidence-based fall
prevention education workshops are offered to allied health professionals and service providers within the SNHN. These have been developed by experts within their fields and include Home Hazard and Environment interventions
(LC, LM) [3]; Exercise interventions (ATi, CS) [63] and the LiFE exercise program (LC) [64], Medication Management
(SH), and Foot and Ankle interventions [65]. These interactive workshops comprise knowledge and skill development as well as a planning session for
implementation and sustainability. Planning strategies documented by participants in each workshop form
part of a developing working document shared to all workshop participants. Clemson et al. Component 1. Identifying and managing fall risk in general practice: knowledge translation and synthesis Implementation Science (2017) 12:12 Page 5 of 12 Page 5 of 12 Table 1 The iSOLVE intervention: planned active ingredients of iSOLVE components (Continued)
2.3 Linking AHPs with GPs to
facilitate referrals
There is also the opportunity for AHPs to opt to be linked to GPs, thus further enhancing pathways and
implementation. Component 3. Establishing referral pathways in primary care
3.1 Decision support tools and fall
management plans
The decision support tools and fall management plans assist in determining the best option/s for the older person. Depending on risk assessment, this can include one or more approaches: medication review, postural
hypotension assessment, referral for cataract removal, home safety assessment, community exercise
programs, home-based exercise programs for higher risk patients, group-based fall exercise programs, tai
chi, a community-based multifaceted fall prevention program Stepping On [66], or referral to a falls clinic. 3.2 Referral pathway facilitation
Pathway facilitation. We use a range of options to facilitate the pathway to local fall service providers, including
but not limited to, the use of the Enhanced Primary Care service to encourage referral to private therapists and
facilitating evidence-based fall-specific services to be provided in health and other potential care services. The
aim is to provide education and knowledge translation that will up-skill and increase the fall prevention work
force across the SNPHN. 3.3 Referrals to fall prevention
services
Referrals to fall prevention services: The health network (SNPHN) and the workshops have been central to mapping
local health professionals who can engage in specific interventions. 3.4 Links with ambulance services
Ambulance services. Fifty eight percent of older people seen by the ambulance service for a fall and not
transported to hospital will fall again in the next 6 months [67]. During the development phase of the project,
we will determine a process to initiate referral to GPs for fall management and follow-up. Barriers to engaging
ambulance services have been investigated [68], and we will explore local options through consultation. 3.5 Network communication
strategies
Communication. A website provides information about the project, education options, and links to state fall
prevention initiatives such as Stepping On and the Active & Healthy website (a state directory of evidence-based
fall prevention exercise options suitable for older people). Component 4. Recruitment of patients Letters are sent from the GP to people 65 years and over
from their patient database. The GPs scan the list to en-
sure the identified patients are not living in high-care resi-
dential or hostel accommodation, do not have an unstable
medical condition, are not in palliative care and are
current patients. The letter invites patients to contact the
project RA by phone, email, or mail if they have had a fall
in the past 12 months or are worried about falling. Eligibility criteria This cluster randomized trial of 560 patients from 28
GP practices is designed to have 80% power to detect as
significant at the two-sided 5% level a 15% between
group differences in the proportion of participants falling,
from 50% in the control group to 35% in the intervention
group (30% relative reduction). We have assumed 50%
will fall in the control group, based on previous trials. A reduction to 35% is feasible, as a reduction of this size Inclusion criteria for patients are being identified as having
a fall in the past year or being worried about falling (veri-
fied using the short form of the Falls Efficacy Scale) and
residing in the community. Exclusion criteria are being un-
able to understand the study information, an unstable
medical condition, severe physical disability, or moderate
to severe dementia (measured by the short portable mental Clemson et al. Implementation Science (2017) 12:12 Page 6 of 12 Fig. 1 Flow of participants though cluster randomized trial Fig. 1 Flow of participants though cluster randomized trial primary care officers, attached to the iSOLVE annual GP
area survey and opportunistic such as word of mouth. For
practices with multiple GPs, once one has agreed to be part
of the study, others are provided with information and in-
vited to participate. was found in a previous meta-analysis [33]. Assuming a
cluster size of 20 participants per GP practice, an intra-
cluster correlation coefficient (ICC = 0.01) and a 15%
loss of patients, we require 26 clusters with a total of
520 patients. In all, 28 GP practices (560 patients) will
be recruited to allow for possible loss of whole practices
during the 12-month follow-up. Recruitment of GP practices Geographical survey of GPs in SNPHN Geographical survey of GPs in SNPHN Intervention The randomization schedule was computer-generated
by a researcher at a distant site and not involved in
group allocation or data collection (JS) using random
permuted blocks of size 6, 4, or 2, stratified by the
number of eligible patients attending the GP practice
(≤80; >80). Allocation is concealed using sequentially
numbered opaque envelopes. Patient lists of eligible
patients are compiled by the GP practice. The alloca-
tion of practice to group occurs after the GP baseline
survey, and patient lists are generated and allocation
is done by a researcher not involved in practice re-
cruitment or data collection (LC). Practices are re-
cruited by the project manager (ATa) who also advises
them of their allocation. Another researcher who is
blinded to the group allocation of the GP practices
contacts individual participants to explain the study,
obtains signed consent, and conducts the assessments. Baseline assessments are conducted on a home visit. Patients attending the control group practices are
asked not to disclose their participation in the study
to their GP. The practices randomized to the intervention will receive
the iSOLVE intervention, and the practices allocated to
control will continue to offer usual care. Control practices
will be offered the iSOLVE academic detailing, resources
and software when all their patients complete the 12-
month-follow-up period. Recruitment of GP practices Will iSOLVE be effective in reducing burden of drug risk and
polypharmacy in use of drugs associated with falls in patients of
participating practices compared to control practices? Will iSOLVE be cost effective from a healthcare funder perspective,
and expressed as an incremental cost per fall avoided? Will patients in the intervention group report significantly higher
interaction with their GP about fall prevention and engagement in
fall prevention activities compared to the control group? Implementation
How are the iSOLVE strategies adopted (or not) by general
practitioners (GPs), by Allied health professional’s (AHP)), and
by the primary care network? How does this change practice in fall prevention? How does this influence the nature of pathways for identifying
older people at risk and managing fall risk within primary care? Drug Burden Index; Falls Risk Increasing Drugs (FRID);
Changes in polypharmacy and in use of drugs associated
with falls (e.g., psychotropics). Falls and Health care utilization—monthly calendars
intervention costs—staff, training, capital costs, consumables. Pre-post patient survey. In-depth interviews with a sample of the participating trial
GPs, AHPs participating in the workshops and with key
coordinating stakeholders, such as Primary Care Network staff In-depth interviews as above
Meeting minutes, observations, and field notes. AHP planning strategies document from the workshops. In-depth interviews of GPs and AHPs as above
Pre- and post surveys of AHPs who participate in the workshops
AHP planning strategies document from the workshops
Relational network (who and influence) questions in survey
for trial GPs. Network influence question in AHPs survey and
in the ecological survey of GPs in geographical area of
SNPHN In-depth interviews as above
Meeting minutes, observations, and field notes. AHP planning strategies document from the workshop What are the factors that facilitate embedding of the intervention
in usual care? In-depth interviews of GPs and AHPs as above
Pre- and post surveys of AHPs who participate in the workshops
AHP planning strategies document from the workshops What factors will influence sustainability of training and knowledge
translation for GPs, AHPs and community pharmacist? What is the GPs professional network in relation to fall prevention
and who is influential within their network? Relational network (who and influence) questions in survey
for trial GPs. Network influence question in AHPs survey and
in the ecological survey of GPs in geographical area of
SNPHN How effective is iSOLVE dissemination across the SNPHN network? How effective is iSOLVE dissemination across the SNPHN network? Recruitment of GP practices GPs are invited to participate in the study by a number
of approaches: promotional flyers and information distrib-
uted via the SNHN database and a database developed by
the iSOLVE team by mapping GP practices using available
web resources (e.g., Google™health engines and business
directories), during SNPHN events, distributed by SNPHN Page 7 of 12 Clemson et al. Implementation Science (2017) 12:12 Table 2 Research questions and evaluation
Study outcomes Research questions
Measurement
Effectiveness
Will iSOLVE be effective in reducing falls in patients of participating
practices compared to control practices? Falls over 12 months—recorded daily on monthly calendars
Will iSOLVE increase general practitioners’ engagement in fall
Pre-post survey of GPs in trial—management of fall Table 2 Research questions and evaluation
Study outcomes Research questions
Measurement
Effectiveness
Will iSOLVE be effective in reducing falls in patients of participating
practices compared to control practices? Falls over 12 months—recorded daily on monthly calendars
Will iSOLVE increase general practitioners’ engagement in fall
prevention management and referral practices compared to the
control GPs? Pre-post survey of GPs in trial—management of fall
prevention and any changes in practice (Q11: frequency of
risk factor assessment, medication review, advice); referral Pre-post survey of GPs in trial—management of fall
prevention and any changes in practice (Q11: frequency of
risk factor assessment, medication review, advice); referral
patterns (Q12: frequency of referral and to whom); knowledge
of fall prevention services. Will iSOLVE increase general practitioners’ engagement in fall
prevention management and referral practices compared to the
control GPs? Will iSOLVE be effective in reducing burden of drug risk and
polypharmacy in use of drugs associated with falls in patients of
participating practices compared to control practices? Will iSOLVE be cost effective from a healthcare funder perspective,
and expressed as an incremental cost per fall avoided? Will patients in the intervention group report significantly higher
interaction with their GP about fall prevention and engagement in
fall prevention activities compared to the control group? Implementation
How are the iSOLVE strategies adopted (or not) by general
practitioners (GPs), by Allied health professional’s (AHP)), and
by the primary care network? How does this change practice in fall prevention? How does this influence the nature of pathways for identifying
older people at risk and managing fall risk within primary care? Implementation Implementation Measures and data collection Patient baseline data for control and intervention pa-
tients will include history of falls and fall injuries in the
past 12 months, age, hospitalization in the past year,
current medications, medication dosage and frequency,
and comorbidities using the Functional Comorbidity
Index [34]. Patients are asked to record falls using a pro-
spective falls calendar to be completed on a daily basis
and returned by mail monthly. Fall calendars also
prompt patients to record the circumstances of a fall,
whether any injuries occurred and whether any medical
help was sought as a result of the fall. If a person does
not return their fall calendar, they are telephoned to de-
termine whether they have fallen. A 12-month follow-up Clemson et al. Implementation Science (2017) 12:12 Clemson et al. Implementation Science (2017) 12:12 Page 8 of 12 Page 8 of 12 Mean costs and the mean health outcomes in each trial
arm will be calculated. The incremental cost-effectiveness
ratio of the intervention compared to control group will
be calculated as the cost per fall prevented; results will be
plotted on a cost-effectiveness plane. Bootstrapping will
be used to estimate a distribution around costs and health
outcomes and to calculate the CIs around the incremental
cost-effectiveness ratios. One-way sensitivity analysis will
be conducted around key variables and assumptions; a
probabilistic sensitivity analysis will be used to examine
joint uncertainty in all parameters. A cost-effectiveness
acceptability curve will be plotted to provide information
about the probability that the intervention is cost-
effective, given decision makers’ willingness to pay for
each fall prevented. survey ascertains patient engagement in fall prevention
interventions or activities over the past year, interaction
with GP about fall prevention, current medications in-
cluding dosage and frequency. Medication survey ques-
tions will also generate data to determine prevalence
and change over 12 months in medication exposures
that have been associated with increased risk of falls
with Drug Burden Index [35] as the major secondary
measure. Other medication data will include polyphar-
macy, falls risk-increasing drugs [36, 37], and use of psy-
chotropics (antipsychotics, antidepressants, and other
mood stabilizers, benzodiazepines, and z-drugs) and other
drug classes of interest. The GPs complete a survey at baseline, 3 and 12 months,
comprising a combination of open-ended and Likert scale
response questions. Measures and data collection The questions cover beliefs about fall
prevention, knowledge of local fall prevention services,
management of fall prevention and any changes in
practice, and referral patterns. Multi-methodology implementation evaluation
While intervention researchers ask “does the proposed
intervention” work, process evaluation is required to
understand “how” it works [38]. This pragmatic ap-
proach using multi-methodology aims to understand fall
prevention within the “real-world” setting [39], in this
case, medical and allied health practice. The evaluation
will also enable a deeper understanding of the coordinat-
ing role that the Primary Care Network can play in this
area. In developing an intervention within an existing set
of processes, referrals, and relationships (the GP and
allied health settings), Normalization Process Theory
(NPT) [31] can enable identification of facilitation fac-
tors. NPT focuses on the “work” of the interventio-
n—and how changes in the work that is required to
identify and refer people at risk of falls can become
routinized into the everyday practice of the GP. NPT
provides a framework for designing the inquiry and
framing the interpretation of data. In-depth interviews and surveys The process of understanding implementation of the
trial is iterative, seeking explanatory data at all layers,
and over the entire time, of the intervention. Initial
process evaluation will involve gathering data synchro-
nistically with the intended intervention. Data will in-
clude surveys, in-depth interviews, observations and
field notes, and documentation of meetings. Guided by
NPT, we will examine the “coherence” of the proposed
change for those in the work setting and how to facili-
tate the “collective action” needed to implement changes
in practice, as follows: Data analysis plan Data analysis will be on an intention-to-treat basis. For
the primary analysis of falls, negative binomial regression
will be used to compare the total number of falls per
person in the two groups, using an offset for exposure
time and accounting for clustering. GP engagement in
fall prevention management and GP referral practices
will be assessed using questions 11 and 12, respectively,
of the 12-month GP survey and corresponding questions
on the baseline survey. For management, a score from 0
to 3 will be allocated to each of the first three items and
summed to give a total out of 9. The total score for each
GP at 12 months will be compared between groups
using regression analysis with baseline score as a covari-
ate and adjusting for clustering by GP practice. For re-
ferral practice, the number of types of practitioners (out
of 13 possible) to whom a GP sometimes or often refers
older patients at risk of falling will be counted. The
change from baseline for each GP will be compared be-
tween groups using Poisson regression, adjusting for
clustering by GP practice. Change in Drug Burden Index
will be calculated for each patient and compared be-
tween groups using regression analysis, controlling for
any baseline differences in risk factors (age, gender,
comorbidities, hospitalization in past year, and use of
walking aid) and accounting for clustering. Social network mapping for their patients; the barriers and facilitators to falls
identification and to implementing fall management
and referral practices; and how the iSOLVE resources
are used (or not) and the perceived value of the
education package and resources to changing practice. Relational data relating to the GPs professional network
is being collected within the GP surveys as part of the
cluster randomized trial. A question about who is influ-
ential is also included in the AHP survey and the area
GP survey. Social network analysis is emerging as a po-
tential tool to evaluate networks in healthcare settings
[42, 43]. The network of roles from whom GPs develop
their knowledge base in falls prevention will be visual-
ized using an alluvial map generator. We will explore
who in their social or professional networks have influ-
enced their knowledge and learning of fall prevention
and the depth of such influence, the people who they see
as important in providing fall prevention services and if
there are associations between these relationships and
GP fall management and referral practices. An alluvial
map can show how the impact of differing professional
roles can change between, before, and after the academic
detailing intervention [44]. (b)Allied Health Professionals (AHPs). AHPs are
recruited to the workshops primarily thorough the
SNHN website and newsletter. Other strategies have
included professional association websites and
interest groups, a Google™search of service
directories and service providers identified from
GPs. AHPs are eligible if they are located in the
geographical area of the SNHN, provide community
services, provide or intend to provide fall prevention
services to older people, and are able to receive GP
referrals. A pre-, 3-, and 12-month post-workshop
survey is administered following AHP education
sessions and in-depth interviews conducted with a
sample of AHPs participating in the workshops. The
AHP implementation planning strategies compiled
from data collected at the workshops will be used
as an additional data source. The surveys include a
combination of open-ended and Likert scale response
questions; practice change, frequency of assessment
and interventions related to fall prevention, referral
patterns (to/from), type of services offered, and level
of confidence in related skills. Demographic questions
record profession, service sector, and nature of
employment status. The interviews will ascertain
the views of AHPs on any changes (or not) in the
referral process; how effective the referral process is,
and the factors affecting adoption and implementation
of the intervention. Survey across geographical area y
g
g
p
A survey will be used to evaluate GP referral patterns
and practices as markers of uptake and implementation
across the intervention region, that is, the SHPHN. The
survey comprises the same questions as in the trial sur-
vey for participating GPs Trends in program perceived
efficacy, adoption, and implementation [40, 41] will be
monitored over the 5 years of the program. The survey
is being distributed annually to all GPs via the SNPHN
mail lists in electronic format. As response rates are
usually low for GPs [4], we are also randomly selecting
10% of all GPs in the region and mailing them a hard
copy of the survey with pen. The random sample was
selected using a computer-generated random permuta-
tion of integers. Cost-effectiveness study A healthcare funder perspective will be used for the eco-
nomic evaluation. Health outcomes will be measured in
terms of falls prevented. Costs will include healthcare
utilization collected by monthly calendars in interven-
tion and control participants and intervention costs (in-
cluding staff, training, capital costs, and consumables). (a)General practices. Data will comprise documentation
of the academic detailing process and in-depth
interviews with a sample of GPs and practice nurses
participating in the intervention cluster. The aim is to
elucidate how they perceive fall prevention as an issue Clemson et al. Implementation Science (2017) 12:12 Page 9 of 12 Social network mapping The social network analysis of personal relationships
with professionals who provide a clinical service to falls
patients will first describe the structure of each network
of referral relations. Second, it will explore the roles of
other professionals relevant to their contribution to the
GPs management of patients who had or are at risk of
falls. In the second stage, we will quantify the contribu-
tion of relational processes that may have contributed to
the structure of referral networks that we observed using
exponential random graph models (ERGMs) and model
the probability of a referral relationship formation. ERGMs provide statistical tests to assess if these effects
are significantly different from zero [45]. Analogous to
odds ratio in logistic regression models, model coeffi-
cients are interpreted as additive factors on the log odds
of forming a relationship around referral. Significant and
positive coefficients indicate higher log odds of forming
a relation in accordance with the underlying process;
negative coefficients make it less likely. The goodness of
fit of each of these models will be assessed, following the
procedures in Hunter et al. [46]. Interviews will also be conducted with key coordinating
stakeholders, such as Primary Care Network staff. The
views of these stakeholders will provide additional insights
into the factors that may affect uptake of the intervention. Discussion The fundamental goal of this research project is to
achieve dissemination and planned sustainability of an
applied model of delivery integrated processes and path-
ways to identify older people at risk of falls and engage a
whole of primary care approach to fall prevention. The
longer term aim is a model that could be used for wide-
spread dissemination. Historically, there have been a
number of challenges in implementing prevention pro-
grams into primary health. Over the past decade, there
have been calls for improved detection of early fall risk,
awareness and access to services, and strengthening
pathways between referrers and service providers [50]. As an implementation project, the context of one geo-
graphical area may well be different from others, such as
rural versus metropolitan, thus introducing an effect
modification. Willis et al. [55] warn how intentions for
embedded strategies into existing services can be hin-
dered by a reliance on material resources for continu-
ation rather than embedding the processes based on key
features for sustainability. Further, we know that training
is a crucial and necessary first step in the process of
change [56] but much more needs to be understood
about how change occurs and what planning is needed
[57]. Our expectation is that the qualitative analysis
approach using Normalization Process Theory will give
the necessary depth of understanding to the contextual
issues related to implementation and sustainability. This should enable us to place appropriate emphasis on
the lessons we will learn, what the key drivers for
change are and what can be generalized, or what will
need further research. The advantages of a blended effectiveness-implementation
design is that it uses mixed-methods which are designed to
evaluate both content and context as a way of accelerating
research into practice, in particular by avoiding lengthy step-
wise approaches [51, 52]. It also offers a way of optimizing
our knowledge and understanding as there is a blending and
interaction of both intervention and implementation, and
it provides opportunities for triangulation and richly vali-
dated findings [53]. At the same time, this approach raises a number of
methodological challenges [25]. While core features of
design rigor in cluster randomized trials are being
adhered to [32], this is a pragmatic trial in which the
intervention is being delivered in a real-world setting. Implementation data analysis: surveys, observation data,
and interviews The information gathered will be used as indicative and
descriptive information to inform further dissemination. Responses to survey data will be coded and compared
within group and over time. Data will be explored using
descriptive and graphical methods and, where possible,
measure change in knowledge or practice using inde-
pendent sample t tests and multinomial logistic regres-
sion. Documentation relating to the intervention (e.g.,
minutes of meetings, discussions with stakeholders) will
be examined using content analysis. Analysis will focus
on the “work of the intervention” [47]—how doing the Page 10 of 12 Page 10 of 12 Clemson et al. Implementation Science (2017) 12:12 who have fallen or at risk of falls, combined with their
new skills to implement fall prevention. There are the
usual generalization issues related to cluster randomized
trials in defining how included practices (and patients)
might differ from those not included [54]. Initial recruit-
ment via newsletters may have included those most
keen, and ongoing recruitment has been expanded to
include multiple methods. Despite this, so far, we have
experienced a high response rate and interest by GP
practice indicating fall prevention is a topic of interest to
many GPs. While threats to external validity may be
heightened in this blended design, we are applying
robust trial rigour, will review self-report of patient
engagement in fall prevention over the follow-up period,
and active dissemination of the GP resources and pro-
cesses throughout the SNHN will not occur until com-
pletion of the cluster randomized trial. work, concerns raised about the work, and how respon-
sibility for falls prevention strategies is distributed across
different groups. All interviews will be audio-recorded
and transcribed verbatim. Thematic analysis (developing
codes) will identify patterns within and across the study
groups [48]. A combination of inductive and deductive
coding will be used—coding will commence with examin-
ation of barriers and facilitators, but will be open to unex-
pected findings that may contribute to these. Data analysis
will examine similarities and differences within and be-
tween stakeholder and participant groups and changes
evident at different time points in the intervention [49]. Discussion We therefore have less control both of intervention fi-
delity for GPs and also of whether and how the recruited
patients follow through with any GP-advised strategies,
recommendations, or referrals. Given resources in a
hybrid design need to cover a variety of expertise and
personnel [25] and the fact that we minimized patient
secondary assessment compared to what would be usual
in an effectiveness/explanatory trial, there is a limited
opportunity to capture, for example, quantitative ad-
herence data and validated measures of participant en-
gagement. Contamination may occur as allied health
professionals who attended workshops could see control
patients referred to them for other reasons or from other
sources, as they have been encouraged to identify people While challenges exist, there is potential for significant
outcomes as the iSOLVE pathways project brings to-
gether practice and research to collectively solve a major
national problem with implications for policy service de-
livery. The project outcomes will be a multi-disciplinary
pathway model for fall prevention that will have been
tested empirically in an urban area. It will provide
evidence of resource need and allocation, and feasible
processes and guidelines for fall identification and
prevention in general practice and community-based
healthcare practices. Practical outcomes include training
modules
and
processes
as
well
as
a
model
for
organizational and system support needs for implemen-
tation and sustainability of GP and allied health evi-
dence-based practice in falls prevention. Partnerships
with
regional,
state,
and professional organizations
provide opportunities for widespread dissemination,
leadership, and diffusion. Page 11 of 12 Page 11 of 12 Clemson et al. Implementation Science (2017) 12:12 Clemson et al. Implementation Science (2017) 12:12 1.
Gillespie LD, Robertson MC, Gillespie WJ, Sherrington C, Gates S, Clemson
LM, et al. Interventions for preventing falls in older people living in the
community. Cochrane Database Syst Rev. 2012;9(9):CD007146. doi:10.1002/
14651858.CD007146.pub3. The Cochrane Collaboration [Internet].
2.
Sherrington C, Whitney JC, Lord SR, Herbert RD, Cumming RG, Close JCT.
Effective exercise for the prevention of falls: a systematic review and
meta-analysis. J Am Geriatr Soc. 2008;56(12):2234–43.
3.
Clemson L, Mackenzie L, Ballinger C, Close JCT, Cumming RG. Environmental
interventions to prevent falls in community-dwelling older people a meta-
analysis of randomized trials. J Aging Health. 2008;20(8):954–71.
4.
Keilich K, Mackenzie L, Lovarini M, Clemson L. Urban Australian general
practitioners’ perceptions of falls risk screening, falls risk assessment, and
referral practices for falls prevention: an exploratory cross-sectional survey
study. Australian Health Review. 2016. published online early 21 April.
5.
Lau R, Stevenson F, Ong BN, Dziedzic K, Treweek S, Eldridge S, et al.
Achieving change in primary care-effectiveness of strategies for improving
implementation of complex interventions: systematic review of reviews.
BMJ Open. 2015;5(12):e009993. Funding
h 9. Jones TS, Ghosh TS, Horn K, Smith J, Vogt RL. Primary care physicians
perceptions and practices regarding fall prevention in adult’s 65 years
and over. Accid Anal Prev. 2011;43(5):1605–9. 9. Jones TS, Ghosh TS, Horn K, Smith J, Vogt RL. Primary care physicians
perceptions and practices regarding fall prevention in adult’s 65 years
and over. Accid Anal Prev. 2011;43(5):1605–9. g
The project is supported by a National Health and Medical Research Council
((NHMRC) Partnership grant 1072790). The project is supported by a National Health and Medical Research Council
((NHMRC) Partnership grant 1072790). 10. Preen D, Bailey B, Wright A. Willingness of general practitioners to participate
in enhanced primary care discharge care planning. Med J Aus. 2006;184:90–1. Availability of data and materials The datasets used and/or analyzed during the current study will be available
from the corresponding author on reasonable request. 11. Wilson S, Marks R, Donohoe S, Chapman M, Zwar N. General practitioner
multidisciplinary skills for enhanced primary care. Aust Fam Physician. 2004;33:479–80. Ethics approval and consent to participate The trial was registered prospectively with the Australian New Zealand
Clincial Trial Registry (ACTRN12615000401550) and approved by the Human
Ethics committee of The University of Sydney (2014/848). All participating
GPs and patients in the cluster trial sign consent forms as per ethical approval. Clincial Trial Registry (ACTRN12615000401550) and approved by the Human
Ethics committee of The University of Sydney (2014/848). All participating
GPs and patients in the cluster trial sign consent forms as per ethical approval. 16. Clemson L. Prevention of falls in the community: is successful in trial
settings, but translation into practice remains a challenge. Br Med J. 2010;340(15 May):C2244. Acknowledgements 6. Wenger NS, Solomon DH, Roth CP, MacLean CH, Saliba D, Kamberg CJ, et
al. The quality of medical care provided to vulnerable community-
dwelling older patients. Ann Intern Med. 2003;139(9):740–7. We wish to acknowledge the engagement and support from our partners,
initially Di Sfiligoi from the Northern Sydney Medicare Local and, later,
Cynthia Stanton and staff from the Sydney North Primary Health Network,
Margaret Armstrong, Northern Sydney Local Health District Coordinator for
NSW Fall Policy, and all the members of our Advisory Board. 7. Tinetti ME. Multifactorial fall-prevention strategies: time to retreat or advance. J Am Geriatr Soc. 2008;56(8):1563–5. 8. Fried TR, Tinetti ME, Iannone L. Primary care clinicians’ experiences with
treatment decision making for older persons with multiple conditions. Arch Intern Med. 2011;171(1):75–80. Consent for publication
No individual personal data is included in the manuscript. Consent for publication
No individual personal data is included in the manuscript. 15. Russell MA, Hill KD, Day LM, Blackberry I, Schwartz J, Giummarra MJ, et al. A
randomized controlled trial of a multifactorial falls prevention intervention
for older fallers presenting to emergency departments. J Am Geriatr Soc. 2010;58(12):2265–74. Received: 9 November 2016 Accepted: 1 December 2016 23. Mahoney J, Clemson L, Lovarini M. Stepping On, a community-based fall
prevention program. In: Malone ML, Capezuti E, Palmer RM, editors. Geriatrics models of care bringing ‘best practice’ to an aging America. Switzerland: Springer International Publishing; 2015. p. 193–8. Competing interests p
g
The authors declare that they have no competing interests. The authors declare that they have no competing interests. 14. Elley CR, Robertson MC, Garrett S, Kerse NM, McKinlay E, Lawton B, et al. Effectiveness of a falls-and-fracture nurse coordinator to reduce falls: a
randomized, controlled trial of at-risk older adults. J Am Geriatr Soc. 2008;56(8):1383–9. Authors’ contributions 12. Zwar N, Hermiz O, Comino E, Shortus T, Burns J, Harris M. Do multidisciplinary
care plan result in better care for patients with type 2 diabetes? Aust Fam
Physician. 2007;36:85–9. All authors have contributed to the conception, design or acquisition of
data, have been involved in drafting or revising the manuscript and all
authors have given approval for the final version of this paper submitted. 13. Michael YL, Whitlock EP, Lin JS, Fu R, O’Connor EA, Gold R. Primary care-
relevant interventions to prevent falling in older adults: a systematic
evidence review for the U.S. Preventive Services Task Force. Ann Intern Med. 2010;153(12):815–25. Author details
1 17. Mahoney JE. Why multifactorial fall-prevention interventions may not work. Arch Intern Med. 2010;170(13):1117–9. 1Faculty of Health Sciences, The University of Sydney, Lidcombe, Australia. 2Centre of Excellence in Population Ageing Research, Sydney, Australia. 3Sydney Medical School - Northern, The University of Sydney, Sydney,
Australia. 4School of Public Health & Community Medicine, University of New
South Wales, Sydney, Australia. 5The George Institute for Global Health,
Sydney Medical School, The University of Sydney, Sydney, Australia. 6Sydney
School of Public Health, The University of Sydney, Sydney, Australia. 7Faculty
of Health Sciences, Australian Catholic University, Melbourne, Australia. 8School of Medicine & Public Health, University of Newcastle, Newcastle,
Australia. 9Kolling Institute of Medical Research, The University of Sydney,
Sydney, Australia. 10University Centre for Rural Health, Sydney Medical
School, The University of Sydney, Sydney, Australia. 11Clinical Excellence
Commission, Sydney, Australia. 18. Lovarini M, Clemson LM, Dean C. Sustainability of community-based fall
prevention programs: a systematic review. J Safety Res. 2013;47:9–17. 19. Child S, Goodwin V, Garside R, Jones-Hughes T, Boddy K, Stein K. Factors
influencing the implementation of fall prevention programmes: a systematic
review and synthesis of qualitative studies. Implement Sci. 2012;7:91. 20. Goodwin V, Jones-Hughes T, Thompson-Coon J, Boddy K, Stein K. Implementing the evidence for prevention falls among community-
dwelling older people: a systematic review. J Safety Res. 2011;42(6):443–51. 21. Ganz D, Alkema GE, Wu S. It takes a village to prevent falls: reconceptualizing
fall prevention and management for older adults. Injury Prevention. 2008;14:266–71. 22. Ganz D, Yano EM, Salibal D, Shekelle P. Design of a continuous quality
improvement program to prevent falls among community-dwelling older
adults in an integrated healthcare system. BMC Health Serv Res. 2009;9:206. available online: http://www.biomedcentral.com/1472-6963/9/206. Received: 9 November 2016 Accepted: 1 December 2016 Consent for publication Consent for publication
No individual personal data is included in the manuscript. References The effect of education with workplace supports on
practitioners’ evidence-based practice knowledge and implementation
behaviors. Aust Occup Ther J. 2010;57(6):386–93. 33. Robertson MC, Campbell AJ, Gardner MM, Devlin N. Preventing injuries in
older people by preventing falls: a meta-analysis of individual-level data. J Am Geriatr Soc. 2002;50(5):905–11. 58. Cherry CO, Arford N, Schuster R. Implementing physician guidelines to
prevent falls: using academic detailing to induce change in physician
practices. Georgia: Center for Global Health, The University of Georgia; 2011. 34. Groll DL, To T, Bombardier C, Wright JG. The development of a
comorbidity index with physical function as the outcome. J Clin Epidemiol. 2005;58(6):595–602. 59. Soumerai SB, Avorn J. Principles of educational outreach (‘academic detailing’)
to improve clinical decision making. J Am Med Assoc. 1990;263(4):549–56. 35. Hilmer SN, Mager DE, Simonsick EM, Cao Y, Ling SM, Windham BG, et al. A
drug burden index to define the functional burden of medications in older
people. Arch Intern Med. 2007;167(8):781–7. 60. Stevens J, Phelan E. Development of STEADI: a fall prevention resource for
health care providers. Health Promot Pract. 2013;14(5):706–14. 61. Deandrea S, Lucenteforte E, Bravi F, Foschi R, La Vecchia C, Negri E. Risk
factors for falls in community-dwelling older people: a systematic review
and meta-analysis. Epidemiology. 2010;21(5):658–68. 36. van der Velde N, Stricker BH, Pols HA, van der Cammen TJ. Risk of falls after
withdrawal of fall-risk-increasing drugs: a prospective cohort study. Br J Clin
Pharmacol. 2007;63(2):232–7. 62. Pit SW, Byles JE, Henry DA, Holt L, Hansen V, Bowman DA. A quality use of
medicines program for general practitioners and older people: a cluster
randomized controlled trial. Med J Aust. 2007;187(1):23–30. 37. Laflamme L, Monarrez-Espino J, Johnell K, Elling B, Moller J. Type, number
or both? A population-based matched case-control study on the risk of
fall injuries among older people and number of medications beyond fall-
inducing drugs. PLoS One [Electron Resour]. 2015;10(3):e0123390. 63. Sherrington C, Michaleff Z, Fairhall N, Paul S, Tiedemann A, Whitney J, et al. Exercise to prevent falls in older adults: an updated systematic review and
meta-analysis. Br J Sports Med. 2016;22(4):78–83. Online first 4 October. 38. Oakley A, Strange V, Bonnell C, Allen E, Stephenson J. Process evaluation
in randomized controlled trials of complex interventions. Br Med J. 2006;332:413–6. 64. Clemson L, Fiatarone Singh MA, Bundy A, Cumming RG, Manollaras K,
O’Loughlin P, et al. References 1. Gillespie LD, Robertson MC, Gillespie WJ, Sherrington C, Gates S, Clemson
LM, et al. Interventions for preventing falls in older people living in the
community. Cochrane Database Syst Rev. 2012;9(9):CD007146. doi:10.1002/
14651858.CD007146.pub3. The Cochrane Collaboration [Internet]. 24. Mackenzie L, Clemson L. Can chronic disease management plans including
occupational therapy and physiotherapy services contribute to reducing
falls risk in older people? Aust Fam Physician. 2014;43(4):211–5. 2. Sherrington C, Whitney JC, Lord SR, Herbert RD, Cumming RG, Close JCT. Effective exercise for the prevention of falls: a systematic review and
meta-analysis. J Am Geriatr Soc. 2008;56(12):2234–43. 25. Curran GM, Bauer M, Mittman B, Pyne JM, Stetler C. Effectiveness-
implementation hybrid designs: combining elements of clinical effectiveness
and implementation research to enhance public health impact. Med Care. 2012;50(3):217–26. 26. Durlak JA, DuPre EP. Implementation matters: a review of research on the
influence of implementation on program outcomes and the factors
affecting implementation. Am J Community Psychol. 2008;41(3-4):327–50. 27. Israel BA, Schulz AJ, Parker EA, Becker AB. Review of community-based
research: assessing partnership approaches to improve public health. Annu Rev Public Health. 1998;19:173–202. 28. Hunter B, Segrott J. Re-mapping client journeys and professional identities: a
review of the literature on clinical pathways. Int J Nurs Stud. 2008;45(4):
608–25. Page 12 of 12 Page 12 of 12 Clemson et al. Implementation Science (2017) 12:12 29. Graham I, Logan J, Harrison M, Straus S, Tetroe J, Caswell W, et al. Lost
in knowledge translation: time for a map? J Contin Educ Health Prof. 2006;26(1):13–24. 54. Eldridge S, Kerry S, Torgerson DJ. Bias in identifying and recruiting
participants in cluster randomized trials: what can be done? Br Med J. 2009;339:b4006. 55. Willis K, Small R, Brown S. Using documents to investigate links between
implementation and sustainability in a complex community intervention:
the PRISM study. SocSciMed. 2012;12:1222–9. 30. Michie S, van Stralen MM, West R. The behavior change wheel: a new
method for characterising and designing behavior change interventions. Implement Sci. 2011;6:42. 31. May C, Finch T. Implementing, embedding and integrating practices: an
outline of Normalization Process Theory. Sociology. 2009;43(3):535–54. 56. Scott SD, Albrecht L, O’Leary K, Ball GD, Hartling L, Hofmeyer A, et al. Systematic review of knowledge translation strategies in the allied health
professions. Implement Sci. 2012;7:70. 32. Campbell MK, Piaggio G, Elbourne DR, Altman DG, Group C. Consort 2010
statement: extension to cluster randomized trials. BMJ. 2012;345:e5661. 57. Novak I, McIntyre S. References Integration of balance and strength training into
daily life activity to reduce rate of falls in older people (the LiFE study):
randomized parallel trial. Br Med J. 2012;345:e4547. pp1-15. 39. Glasgow RE, Steiner JF. Comparative effectiveness research to accelerate
translation: recommendations for an emerging field of science. In:
Brownson RC, Colditz GA, Procotor EK, editors. Dissemination and
implementation research translating science to practice. New York:
Oxford; 2012. p. 72–93. 65. Menz HB, Lord SR, Fitzpatrick RC. A structural equation model relating
impaired sensorimotor function, fear of falling and gait patterns in older
people. Gait Posture. 2007;25(2):243–9. 40. Glasgow RE, Vogt TM, Boles SM. Evaluating the public health impact of
health promotion interventions: the RE-AIM framework. Am J Public Health. 1999;89(9):1322–7. 66. Clemson L, Cumming RG, Kendig H, Swann M, Heard R, Taylor K. The
effectiveness of a community-based program for reducing the incidence
of falls among the elderly: a randomized trial. J Am Geriatr Soc. 2004;
52(9):1487–94. 41. Poulos RG, Donaldson A. Is sports safety policy being translated into practice:
what can be learnt from the Australian rugby union Mayday procedure? Br J
Sports Med. 2012;46(8):585–90. 67. Tiedemann A, Mikolaizak AS, Sherrington C, Segin K, Lord SR, Close JCT. Older fallers attended to by an ambulance but not transported to hospital:
a vulnerable population at high risk of future falls. Aust N Z J Public Health. 2013;37(2):179–85. 42. Chambers D, Wilson P, Thompson C, Harden M. Social network analysis in
healthcare settings: a systematic scoping review. PLoS One [Electron Resour]. 2012;7(8):e41911. 68. Comans TA, Currin ML, Quin J, Tippett V, Rogers A, Haines TP. Problems
with a great idea: referral by prehospital emergency services to a
community-based falls-prevention service. Inj Prev. 2013;19:134–8. 43. Hossain L, Chung KSK. Towards a social network model for understanding
information and communication technology use for general practitioners in
rural Australia. Comput Hum Behav. 2010;26:562–71. 43. Hossain L, Chung KSK. Towards a social network model for understanding
information and communication technology use for general practitioners in
rural Australia. Comput Hum Behav. 2010;26:562–71. 69. Lovarini MP. Sustainability of a community-based falls prevention program:
A grounded theory: The University of Sydney; 2012. 44. Rosvall M, Bergstrom CT. Mapping change in large networks. PLoS One
[Electron Resour]. 2010;5(1):e8694. 44. Rosvall M, Bergstrom CT. Mapping change in large networks. PLoS One
[Electron Resour]. 2010;5(1):e8694. 45. Robins G, Pattison P, Kalish Y, Lusher D. Clemson et al. Implementation Science (2017) 12:12 References An introduction to exponential
random graph (p*) models for social networks. Soc Netw. 2007;29(2):173–91. 45. Robins G, Pattison P, Kalish Y, Lusher D. An introduction to exponential
random graph (p*) models for social networks. Soc Netw. 2007;29(2):173–91. 46. Hunter D, Goodreau S, Handcock M. Goodness of fit of social network models. J Am Stat Assoc. 2008;103:248–58. 46. Hunter D, Goodreau S, Handcock M. Goodness of fit of social network models. J Am Stat Assoc. 2008;103:248–58. 47. May C.Towards a general theory of implementation. Implement Sci. 2013;8(18). doi:10.1186/1748-5908-8-18. 47. May C.Towards a general theory of impleme
2013;8(18). doi:10.1186/1748-5908-8-18. 48. Braun V, Clarke V. Using thematic analysis in psychology. Qual Res Psychol. 2006;2:77–101. 48. Braun V, Clarke V. Using thematic analysis in psychology. Qual Res Psychol. 2006;2:77–101. 49. Green J, Willis K, Hughes E, Small R, Gibbs L, Daly J. Generating best
evidence from qualitative research: the role of data analysis. Aust N Z J
Public Health. 2007;31(6):545–50. 49. Green J, Willis K, Hughes E, Small R, Gibbs L, Daly J. Generating best
evidence from qualitative research: the role of data analysis. Aust N Z J
Public Health. 2007;31(6):545–50. Submit your next manuscript to BioMed Central
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and we will help you at every step: 50. Clemson L, Finch C, Hill K, Lewin G. Fall prevention in Australia: policies and
activities. Clin Geriatr Med. 2010;26(4):733–49. 50. Clemson L, Finch C, Hill K, Lewin G. Fall prevention in Australia: policies and
activities. Clin Geriatr Med. 2010;26(4):733–49. • We accept pre-submission inquiries 51. Glasgow RE, Chambers D. Developing robust, sustainable, implementation
systems using rigorous, rapid and relevant science. Clin Transl Sci. 2012;5(1):48–55. 51. Glasgow RE, Chambers D. Developing robust, sustainable, implementation
systems using rigorous, rapid and relevant science. Clin Transl Sci. 2012;5(1):48–55. 52. Landsverk J, Brown CH, Chamberlain P, Palinkas L, Ogihara M, Czaja S, et
al. Design and analysis in disseminatin and implementation research. In:
Brownson RC, Colditz GA, Proctor EK, editors. Dissemination and
Implementation Research in Health. New York: Oxford University Press;
2012. p. 212–60. 53. Fielding N. Triangulation and mixed methods designs data integration with
new research technologies. J Mixed Methods Res. 2012;6(2):124–36.
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Covid-19'un Uluslararası Pay Piyasalarına Etkisi
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Ekonomi, Politika & Finans Araştırmaları Dergisi, 2020, 5(Özel Sayı):59-75
Journal of Research in Economics, Politics & Finance, 2020, 5(Special Issue): 59-75
Araştırma Makalesi, DOI: 10.30784/epfad.808308 Ekonomi, Politika & Finans Araştırmaları Dergisi, 2020, 5(Özel Sayı):59-75
Journal of Research in Economics, Politics & Finance, 2020, 5(Special Issue): 59-75
Araştırma Makalesi, DOI: 10.30784/epfad.808308 * Arş. Gör., Anadolu Üniversitesi İktisadi ve İdari Bilimler Fakültesi, denizsevinc@anadolu.edu.tr,
ORCID: 0000-0002-6223-9450 Özet Pay piyasaları, ekonominin bir göstergesi olarak sayılmakta, bunun yanında
sosyal ve ekonomik sorunlar da bu piyasalarda volatiliteye neden olmaktadır. 2019’un sonlarında Çin’de ortaya çıkan ve epidemiden pandemiye dönüşen
Covid-19, tüm dünyada sosyal hayatı, ekonomiyi ve finansal sistemi
etkilemiştir. Özellikle salgının yayılmasıyla beraber yaşanan belirsizlik,
uluslararası pay piyasalarında aşırı fiyat düşüşlerine ve volatiliteye neden
olmuştur. Bu çalışmanın amacı, Covid-19’un uluslararası pay piyasalarına
etkisini incelemek, yeni geliştirilen ve pandeminin toplumsal etkisini sayısal
olarak gösteren ―Salgın Hastalıklar Piyasa Volatilitesi Takipçisi‖ (Infectious
Infectious Disease Equity Market Volatility Tracker – EMV-ID) endeksinin bu
piyasalardaki volatiliteyi nasıl etkilediğini tespit etmektir. Çalışmada, WHO
verilerine göre en fazla vaka ve ölüm sayısının olduğu 13 ülkenin pay piyasası
endeksleri ele alınmıştır. Covid-19’un etkisinin daha net bir şekilde görülmesi
için her ülkede ilk vakanın görüldüğü tarih ile 30.09.2020 arası dönemdeki
günlük getiriler incelenmiştir. Yapılan EGARCH(1,1) modelinin sonucunda
farklı piyasaların farklı tepkiler verdiği, ancak EMV-ID endeksinin analizde
yer alan piyasaların birçoğunda volatiliteyi artırıcı etkiye sahip olduğu
sonucuna ulaşılmıştır. Anahtar Kelimeler:
Covid-19, EMV-ID,
Pay Piyasası, Volatilite JEL Kodları:
D53, G15, N20 COVID-19’UN ULUSLARARASI PAY PİYASALARINA ETKİSİ
The Impact of Covid-19 on International Stock Markets Deniz SEVİNÇ* Makale Geliş Tarihi (Received Date): 9.10.2020 Makale Kabul Tarihi (Accepted Date): 2.12.2020 Keywords:
Covid-19, EMV-ID,
Stock Market,
Volatility * Arş. Gör., Anadolu Üniversitesi İktisadi ve İdari Bilimler Fakültesi, denizsevinc@anadolu.edu.tr,
ORCID: 0000-0002-6223-9450
Makale Geliş Tarihi (Received Date): 9 10 2020
Makale Kabul Tarihi (Accepted Date): 2 12 2020 Makale Geliş Tarihi (Received Date): 9.10.2020 1. Giriş Aralık 2019’da Çin’de Covid-19 virüsünün ortaya çıkması ve 11 Mart 2020’de Dünya
Sağlık Örgütü’nün (World Health Organization-WHO) pandemi ilan etmesi ile birlikte bu
salgın küresel çapta sosyal, politik, ekonomik ve finans alanında büyük etki yaratmıştır. Ülkelerarası sınırların kapatılması ve ülke içinde de ağır tedbirlerinin alınması salgının
yayılmasını önlemeye yardımcı olmuş ancak kısa dönemde hem küresel hem de ulusal ticarette
ve reel sektörde büyük sıkıntılara yol açmıştır. Uzun dönemde ise işsizlik sayısında aşırı artışa,
iş dünyasında çöküşlere ve küresel ekonomide resesyona neden olacağı beklenmektedir. Covid-19 pandemisi Çin’de ortaya çıkması ve 170’den fazla ülkeye yayılmasına rağmen
Amerika ve Avrupa ülkelerini ekonomik ve finansal açıdan daha fazla etkilemiştir. Salgının ve
salgınla birlikte uluslararası politik olayların yaşanması sistematik riski artırmış, bunun yanında
oluşan panik ortamı ve yatırımcı davranışlarını etkilemesi ile birlikte finansal piyasalarda
bugüne kadar gerçekleşen en yüksek dalgalanmalar ortaya çıkmıştır. Mart 2020’de S&P 500 ve
Dow Jones %20’den fazla düşüş yaşanmış, Avrupa ve Asya piyasaları da arkasından gelmiştir. İngiltere’nin en büyük piyasa endeksi olan FTSE’de %10’dan fazla azalırken Japonya’da pay
piyasası %20’den fazla kayıp ortaya çıkmıştır. Piyasalardaki endişenin derecesini gösteren VIX
(Volatility Index) tüm zamanların en yüksek seviyesine ulaşmış (82.69 - 16 Mart 2020), 2008
Küresel Finansal Krizi’ndeki değerin üzerine çıkmıştır. IMF (International Monetary Fund),
gelişmekte olan piyasalardan 83 milyar $’a yakın sermaye çıkışının olduğunu ve bunun bugüne
kadar yaşanan en büyük sermaye çıkışı olduğunun kaydetmiştir (Gopinath, 2020; Zhang, Hu ve
Ji, 2020). Covid-19’un ortaya çıkması ve tedbirlerin alınmasından itibaren literatürde ekonomiye ve
finansal sisteme olan etkisi araştırılmaya başlanmıştır (Adıgüzel, 2020; Albulescu, 2020; Altig
vd., 2020; Baker vd., 2020; Campello, Kankanhalli ve Muthukrishnan, 2020; Corbet, Larkin ve
Lucey, 2020; Demir, Bilgin, Karabulut ve Doker, 2020; Duran ve Acar, 2020; Fernandes, 2020;
Haroon ve Rizvi, 2020; Kargar vd., 2020; Kwan ve Mertens, 2020; Sharif, Aloui ve Yarovaya,
2020; Soylu, 2020; Zhang vd., 2020). Ancak Covid-19 salgının henüz yeni olması ve devam
etmesi nedeniyle literatürde finansal piyasalara olan etkisini inceleyen ampirik çalışmalar
sınırlıdır. Bu çalışma Covid-19 ve finansal piyasalar arasındaki ilişkisini incelemek için, Baker
vd. (2020) tarafından oluşturulan ―Salgın Hastalıklar Piyasa Volatilitesi Takipçisi‖ (Infectious
Infectious Disease Equity Market Volatility Tracker- EMV-ID) endeksinin piyasalardaki
volatiliteye olan etkisini ortaya koymayı amaçlamaktadır. EMV-ID endeksi Ocak 1985’ten
gümünüze kadar olan bulaşıcı hastalık pandemilerinin kamuoyuna etkisini nicel olarak
ölçülmesini sağlamaktadır. Abstract Stock markets are considered as an indicator of the economy, and social and
economic problems also cause volatility in these markets. Covid-19, which
emerged in China in late 2019 and turned from an epidemic to a pandemic, has
affected social life, economy and financial system all over the world. The
uncertainty, especially with the spread of the epidemic, caused excessive price
declines and volatility in international stock markets. The purpose of this study
is to examine the impact of Covid-19 on international stock markets and detect
how Infectious Disease Equity Market Volatility Tracker (EMV-ID) index, the
newly developed, which numerically shows the social impact of the pandemic,
affects the volatility in international stock markets. In the study, it was
considered 13 countries’ the stock market indices which they have the highest
number of cases and deaths according to WHO data. In order to see the effect
of Covid-19 more clearly, daily returns were examined between the date of the
first case of each country and 30.09.2020 period. As a result of the EGARCH
(1,1) model, it is concluded that different markets give different reactions, but
the EMV-ID index has an increasing effect on volatility in most of the markets
included in the analysis. 59 D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ 11929 Krizi ―Büyük Buhran‖ (The Great Depression), 2008 Krizi ―Büyük Resesyon‖ (The Great
Recession) olarak adlandırılmaktadır. 1. Giriş Literatürde EMV-ID endeksinin etkisini ölçen çok az çalışma
bulunmaktadır (Bai, Wei, Wei, Li ve Zhang, 2020; Baker vd., 2020; Capelle-Blancard ve
Desroziers, 2020; Gupta, Subramaniam, Bouri ve Ji, 2021; Li, Liang, Ma ve Wang, 2020). Bu
nedenle bu çalışmanın literatüre katkı sağlayacağı düşünülmektedir. Çalışmada WHO tarafından onaylanan verilerle en fazla vaka ve ölüm sayısının olduğu
13 ülkenin piyasa endeksleri ele alınmıştır. Salgının her ülkede farklı tarihlerde başlaması
nedeniyle bu ülkelerde görülen ilk vakanın olduğu tarihten itibaren olan dönem analiz
edilmiştir. Çalışmanın devamında Covid-19 ve diğer küresel salgınların finansal piyasalara olan
etkisi ile ilgili literatür taraması yer almakta, üçüncü ve dördüncü kısımda ise kullanılan veriler
ve yöntem ile yapılan ampirik analiz ortaya konmaktadır. Son bölümde ise analizin sonuçları 60 Ekonomi, Politika & Finans Araştırmaları Dergisi, 2020, 5(Özel Sayı):59-75
Journal of Research in Economics, Politics & Finance, 2020, 5(Special Issue): 59-75 değerlendirilmiştir. Etik kurul izni ve/veya yasal/özel izin alınmasına gerek olmayan bu
çalışmada araştırma ve yayın etiğine uyulmuştur. D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ Ashraf (2020), yaptığı çalışmada 64 ülkedeki Covid-19 günlük vaka ve ölüm sayıları ile
piyasa endeksi getirileri arasındaki ilişkiyi araştırmış ve getirilerin günlük vaka ve ölüm
sayılarından negatif olarak etkilendiği sonucuna ulaşmıştır. Haroon ve Rizvi (2020), Covid-19 ile ilgili basında oluşan paniğin ABD piyasalarına
etkisini incelemişler ve pandeminin basında paniği arttırdığı ve bu paniğin de yüksek
volatiliteye neden olduğunu gözlemlemişlerdir. Baker vd. (2020), günlük EMV-ID endeksini ortaya koydukları ve salgın hastalık
haberlerinin üzerine yaptıkları araştırmada Covid-19’un bugüne kadarki en büyük piyasa
volatilitesine neden olduğunu bulmuşlardır. Bai vd. (2020), Baker vd. (2020) tarafından
geliştirilen EMV-ID endeksini aylık seriye dönüştürerek salgın hastalık pandemisinin
uluslararası piyasalara olan etkisini incelemişlerdir. Çalışmada kuş gribi, domuz gribi (H1N1),
Ebola, Çocuk Felci, Zika virüsü ve Covid-19’un ortaya çıkması ve yayılmasını içeren 4 Ocak
2005-30 Nisan 2020 dönemi analiz edilmiştir. Bai vd. (2020) yaptıkları GARCH-MIDAS
analizinde salgın hastalık pandemilerinin ABD, İngiltere ve Japonya piyasalarındaki volatiliteyi
artırdığı, ancak Çin piyasasının bu salgınlar sırasında daha istikrarlı olduğu sonucuna
ulaşılmıştır. Li vd. (2020), EMV-ID ve VIX endekslerinin Fransa, İngiltere ve Almanya’da
piyasa getirilerine etkisini analiz etmişlerdir. EMV-ID İngiltere ve Fransa piyasalarındaki
volatiliteyi tahminde güçlü etki gösterirken VIX üç piyasadaki volatiliteyi de tahminde büyük
güce sahiptir. Capelle-Blancard ve Desroziers (2020) EMV-ID endeksinin 74 ülkenin piyasa
getirilerine etkisini analiz etmişler, yapılan panel modelinde endeksin piyasa getirilerine
herhangi bir etkisinin olmadığı sonucuna ulaşmışlar ancak Covid-19’un her ülkeye farklı etkide
bulunduğunu belirtmişlerdir. Gupta vd. (2021) ise EMV-ID endeksinin ABD devlet tahvillerine
etkisini incelemişler, EMV-ID ve devlet tahvilleri arasında negatif bir ilişki olduğunu; salgın
hastalıklar diğer piyasalarda volatiliteyi artırdığı için devlet tahvillerinin riski hedge ettiğini
tespit etmişlerdir. Türkiye’de yer alan literatürde ise Covid-19 ve pay piyasaları arasındaki ilişkiyi
inceleyen çalışmalar kısıtlıdır. Hacıevliyagil ve Gümüş (2020) Türkiye’nin de yer aldığı en fazla
vaka görülen on ülkenin borsa endekslerini incelemiş ve Covid-19’un farklı etkilerinin
olduğunu gözlemişlerdir. Barut ve Yerdelen Kaygın (2020), Zeren ve Hızarcı (2020) ise
salgının erken döneminde Covid-19 ve çeşitli ülkelerin pay piyasaları arasındaki ilişkiyi
incelemiş ve bazı ülkelerde eşbütünleşme ilişkisi mevcutken diğerlerinin eşbütünleşik
olmadığını tespit etmişlerdir. Keleş (2020) ve Çetin (2020), Covid-19’un Türkiye’de pay
piyasalarına etkisini araştırmışlar ve alınan tedbirlerin salgının etkisini azalttığını bulmuşlardır. Bunun yanında Tayar, Gümüştekin, Dayan ve Mandi (2020) salgının BIST sektör endekslerine
etkisini incelmişlerdir. Yapılan çalışmada Covid-19’un elektrik, ulaşım, mali ve teknoloji
endekslerine güçlü negatif etkisi oluğunu belirtmişlerdir. Yapılan literatür taraması sonucunda, çalışmalarda yer alan farklı piyasaların salgına
farklı tepkiler verdiği görülmektedir. 2. Literatür Taraması Finansal piyasalar ulusal ve küresel çapta yaşanan olaylara büyük tepkiler vermektedir. Literatürde bu olayların piyasa getirilerini ve volatilitesini nasıl etkilediğine dair birçok çalışma
bulunmaktadır. Bazı araştırmacılar politik olayların etkisini (Breinlich, Leromain, Novy,
Sampson ve Usman, 2018; Burggraf, Fendel ve Huynh, 2020; Kenourgios, Dadinakis ve
Tsakalos, 2020; Wagner, Zeckhauser ve Ziegler, 2017), bazıları ise salgın hastalıklarla ilişkisini
(Alfaro, Chari, Greenland ve Schott, 2020; Ichev ve Marinč, 2018; Nippani ve Washer, 2004)
incelemişlerdir. İçinde bulunduğumuz dönemde Covid-19 pandemisinin de ekonomiye ve
finansal piyasalara büyük zararlar verdiği literatürde tartışılmaktadır (Albulescu, 2020;
Albuquerque, Koskinen, Yang ve Zhang, 2020; Altig vd., 2020; Baker vd., 2020; Sharif vd.,
2020). Harvey (2020), Covid-19’u 2008’deki Küresel Finansal Kriz ile karşılaştırmış ve
pandemiyi ―Büyük Baskı‖ (The Great Compression) olarak isimlendirmiştir1. Finans basınında
ve literatüründe Covid-19 salgını diğer salgınlarla ve büyük krizlerle karşılaştırılmakta; gelecek
yıllarda etkisinin daha fazla olacağı ve bugüne kadar olan salgın ve krizlerden daha fazla yıkıcı
etkisi olacağı düşünülmektedir. Goodell (2020), Covid-19’un finansal piyasalara geniş çapta ve uzun dönemde etki
edeceğini savunmuş; pandeminin ekonomiyi, politikaları ve finansal sistemi nasıl
etkileyebileceğini tartışmıştır. Sharif vd. (2020) ise Goodell'in (2020) tartıştığı başlıklar altında
yaptıkları çalışmada Covid-19’un ABD’de ekonomik istikrara ve piyasa getirilerine negatif etki
ettiğini ancak jeopolitik riski daha fazla etkilediği sonucuna ulaşmışlardır. Albulescu (2020), Covid-19’un finansal piyasalara etkisini inceleyen ilk çalışmalardan
birini gerçekleştirmiş ve Çin dışında ortaya çıkan yeni vakaların VIX’in yükselmesine neden
olduğunu bulmuşlardır. Mishra, Rath ve Dash (2020) Covid-19’un Hindistan’da BSE Sensex, BSE, BSE 100 ve
alt sektör endekslerinin getirilerine etkisini incelemişler ve hastalığın yayılmasından beri tüm
getirilerin negatif olduğu ve volatilitelerinin önceki döneme göre yükseldiği sonucuna
ulaşmışlardır. Ali, Alam ve Rizvi (2020), Covid-19’un finansal piyasalara etkisini üç evreye
ayırmışlardır: 1. sadece Çin’de yayılması (epidemi); 2. evre, Avrupa’ya yayılması ve 3. evre,
Kuzey Amerika’ya yayılması (pandemi). Çalışmada yapılan analiz, Çin piyasalarının epidemi
ve pandemi döneminde diğer bölgelere göre daha istikrarlı olduğu, Avrupa ve ABD
piyasalarının ise pandemi döneminde aşırı dalgalandığını göstermektedir. Zhang vd. (2020), 12 ülke için yaptığı analizde Çin dışında diğer tüm piyasalarda günlük
vaka sayısının volatiliteyi artırdığı sonucuna ulaşmışlar, pandeminin gelecekteki belirsizliğinin
küresel piyasa riski yükselttiğini belirtmişlerdir. 61 ,
,
,
,
,
,
}(
,
;
,
)
3 Çalışmada kullanılan veriler için etik kurul izni ve yasal izin alınması gerekmemektedir. D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ Bazı piyasalarda negatif etkiye rastlanırken, bazı
piyasalarda piyasaların Covid-19 ile herhangi bir ilişkiye sahip olmadığı gözlemlenmiştir. Bu
çalışmada da benzer bir sonuç ortaya çıkması beklenmektedir. 62 Ekonomi, Politika & Finans Araştırmaları Dergisi, 2020, 5(Özel Sayı):59-75
Journal of Research in Economics, Politics & Finance, 2020, 5(Special Issue): 59-75 Ekonomi, Politika & Finans Araştırmaları Dergisi, 2020, 5(Özel Sayı):59-75
Journal of Research in Economics, Politics & Finance, 2020, 5(Special Issue): 59-75 2 1) E: {economic, economy, financial}; 2) M: {stock market, equity, equities, Standard and Poors}; 3) V:
{volatility, volatile, uncertain, uncertainty, risk, risky} and 4) ID: {epidemic, pandemic, virus, flu,
disease, coronavirus, MERS, SARS, EBOLA, H5N1, H1N1}(Bai vd., 2020; Baker vd., 2020). 3. Veri ve Metodoloji Bu çalışmanın amacı, Covid-19’un uluslararası piyasalardaki volatiliteye olan etkisini
analiz etmektir. Çalışmada WHO (2020) tarafından açıklanan en çok vaka ve ölüm sayısına
sahip ülkelerin piyasa endeksleri yer almaktadır. ABD, Hindistan, Brezilya, Rusya, Kolombiya,
Peru, Meksika, Arjantin, İspanya ve Güney Afrika vaka sayısının en çok olduğu ülkeler iken,
İtalya, Fransa ve İngiltere ölü sayısının en fazla olduğu on ülke arasında görülmektedir. Bu
nedenle analiz için Covid-19’dan en çok etkilenen bu 13 ülkenin piyasaları ele alınmıştır. Her
ülke için WHO tarafından onaylanan ilk vakanın görüldüğü tarih ile 30.09.2020 arası
dönemdeki iş günleri analiz edilmiştir (Tablo 1). Çalışmada Covid-19’un etkisini sayısal olarak ölçen EMV-ID endeksi kullanılmıştır. Baker vd. (2020), EMV-ID endeksini oluşturmak için öncelikle dört farklı kümede terimleri (ve
terim uyarlamalarını) belirlemişlerdir: 1) E: (ekonomik, ekonomi, finansal); 2) M: (pay senedi,
pay senetleri, öz sermaye, Standard & Poors); 3) V: (volatilite, volatil, belirsiz, belirsizlik, risk,
riskli) ve 4) ID: (epidemik, pandemik, virüs, grip, hastalık, koronavirüs, MERS, SARS,
EBOLA, H5N1, H1N1)2. Daha sonra E, M,V ve ID kümelerinden en az bir terim içeren günlük
haberleri yaklaşık 3000 gazetede araştırmışlar ve günlük haber sayısını elde etmişlerdir. Bir
sonraki adımda EMV-ID içeren haber sayısını günlük tüm haber sayısı ile oranlamışlardır. En
son olarak da genel bir EMV-ID takibi için seriyi çarpımsal olarak yeniden ölçeklendirmişler;
oluşturulan endeksi VIX ile karşılaştırarak EMV-ID haberlerinin toplam EMV haberlerine
oranını yansıtacak bir hale getirmişlerdir. Tablo 1. Analizde Kullanılan Veri Seti
Ülke
Borsa Endeksi
İlk Vakanın Görüldüğü
Tarih
Gözlem Sayısı
ABD
S&P 500
24.01.2020
174
Hindistan
BSE Sensex 30
30.01.2020
168
Brezilya
BOVESPA
26.02.2020
151
Rusya
RTSI
31.01.2020
167
Kolombiya
COLCAP
06.03.2020
138
Peru
S&P Lima General
07.03.2020
143
Meksika
S&P/BMV IPC
28.02.2020
149
Arjantin
S&P Merval
03.03.2020
141
İspanya
IBEX 35
02.02.2020
170
Güney Afrika
South Africa Top 40
05.03.2020
143
İngiltere
FTSE 100
01.02.2020
168
İtalya
FTSE MIB
29.01.2020
173
Fransa
CAC 40
24.01.2020
176
Kaynak: WHO (2020), investing.com (2020) Günlük piyasa endeksi getirileri investing.com üzerinden, Baker vd. (2020) tarafından
geliştirilen EMV-ID endeksi ise policyunceartinty.com sitesinden elde edilmiştir3. Finansal zaman serilerinde hata terimlerinin varyansı zamana bağlı olarak değişmektedir
(heteroskedasticity) ancak geleneksel zaman serisi modelleri varyansın sabit olduğunu 63 D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ varsaymaktadır (Özden, 2008). 3. Veri ve Metodoloji 64 Ekonomi, Politika & Finans Araştırmaları Dergisi, 2020, 5(Özel Sayı):59-75
Journal of Research in Economics, Politics & Finance, 2020, 5(Special Issue): 59-75 Ekonomi, Politika & Finans Araştırmaları Dergisi, 2020, 5(Özel Sayı):59-75
Journal of Research in Economics, Politics & Finance, 2020, 5(Special Issue): 59-75 3. Veri ve Metodoloji İlk olarak Engle (1982) tarafından ortaya koyulan Otoregresif
Koşullu Değişen Varyans (ARCH - Autoregresif Conditional Heteroskedasticity) modeli
zamana bağlı olarak değişen varyansın tahmin edilebilmesini, zaman serilerinde karşılaşılan
otokorelasyonun modellenmesini sağlamıştır ancak ARCH modelinde çok fazla sayıda
parametre tahmin edilmesi gerekmektedir. Bollerslev (1986) bu sorunu gidermek için
Genelleştirilmiş Otoregresif Koşullu Değişen Varyans (GARCH – General ARCH) modelini
geliştirmiştir. Fakat ARCH ve GARCH modelleri olumlu ve olumsuz tüm şokların volatilite
üzerinde simetrik etkisi olduğunu varsaymakta ve sadece volatilitenin derecesini ortaya
koymaktadır. Bunun yanında GARCH modelinde tahmin edilen parametrelerin pozitif olması
gerekliliği bulunmaktadır. Özellikle finansal zaman serilerinde olumlu ve olumsuz şokların
volatilite üzerinde farklı etkileri bulunmaktadır. Nelson (1991), GARCH modelindeki bu
eksiklikleri gidermek amacıyla, koşullu varyans parametrelerinin pozitif olma zorunluluğunu
logaritmik dönüşüm ile ortadan kaldıran ve şokların asimetrik etkisini (kaldıraç etkisi) gösteren
EGARCH (Exponential GARCH) modelini geliştirmiştir. Çalışmada kullanılan EGARCH(1,1)
modeli şu şekilde ifade edilebilir: ∑
(1)
√
(2)
(
√
√
)
√
t=1,...,n (3) (2) (3) Denklem (1) ortalama eşitliği (mean equation), Denklem (3) ve (4) ise koşullu varyans
eşitliğini (conditional variance equation) göstermektedir. Denklem (1) ortalama eşitliği (mean equation), Denklem (3) ve (4) ise koşullu varyans
eşitliğini (conditional variance equation) göstermektedir. , piyasa endekslerinin getirilerini ifade etmektedir. , Denklem (3)’te yer alan varyans
eşitliğinden elde edilmiştir. , Denklem (1)’deki artıkların varyansını ifade etmektedir. Diğer
bir ifade ile
, piyasa getirisi volatilitesini göstermektedir. GARCH modelinden farklı olarak
’nin parametresi negatif olsa bile
pozitif olacak, yapay kısıtlamaya gerek
kalmayacaktır (Brooks, 2014). sabit terim olmak üzere,
önceki dönemlerdeki artıkların
varyansı (önceki dönemlerdeki volatilite) olarak tanımlanmaktadır. , koşullu varyans için
şokların kısa dönemdeki etkisini, , şokların sürekliliğini, √
⁄
ise standartlaşmış hata
terimini ifade etmektedir. Diğer bir ifade ile , ARCH parametresini, ise GARCH
parametresini ifade etmektedir. parametresi, istatistiki açıdan anlamlı ve sıfıra eşit değilse
( asimetrik volatilite var demektir. olması pozitif ve negatif şoklar volatilite üzerinde
aynı etkiye sahip olduğunu; -1< <0 durumu ise negatif şokların pozitif şoklardan daha fazla
etki ettiğini göstermektedir (Brooks, 2014). EGARCH(1,1) modeline EMV-ID dışsal şoku
eklendiğinde modelin denklemi (Risteski, Sadoghi ve Davcev, 2013): (
√
√
)
√
t=1,...,n (4) (4) şeklinde ifade edilebilir. parametresi, dışsal şok olarak modele eklenen EMV-ID’nin etki
derecesini göstermektedir. şeklinde ifade edilebilir. parametresi, dışsal şok olarak modele eklenen EMV-ID’nin etki
derecesini göstermektedir. 4. Ampirik Bulgular Çalışmada piyasa getirisi volatilitesini modellemek ve bu volatiliteye EMV-ID
endeksinin etkisini incelemek amacıyla tek değişkenli EGARCH modeli kullanılmıştır. Modelde yer alan değişkenlerin durağanlığının kontrol edilmesi amacıyla Dickey ve Fuller
(1981) tarafından geliştirilen Augmented Dickey Fuller (ADF), Phillips ve Perron'un (1988)
Phillips-Perron (PP) ve Enders ve Lee (2012) tarafından geliştirilen Fourier ADF birim kök
testleri yapılmıştır. EMV-ID değişkeninin verileri her ülke için farklı periyodlardan
oluştuğundan her bir ülke için durağanlığı kontrol edilmiştir. Tablo 2a ve 2b’de sonuçları
verilen birim kök testlerine göre tüm ülke piyasa getirileri ve EMV-ID endeksi düzeyde
durağandır. ablo 2a. Değişkenlere Ait ADF ve PP Birim Kök Testi Sonuçları
ADF I(0)
ADF I(1)
PP I(0)
PP Tablo 2a. Değişkenlere Ait ADF ve PP Birim Kök Testi Sonuçları
ADF I(0)
ADF I(1)
PP I(0) Tablo 2a. Değişkenlere Ait ADF ve PP Birim Kök Testi Sonuçları
ADF I(0)
ADF I(1)
PP I(0)
PP I(1)
r
EMV-ID
r
EMV-ID
r
EMV-ID
r
EMV-ID
ABD
-19.311* -2.900**
-9.306*
-11.570* -19.116*
-4.498* -167.893* -25.302*
Hindistan
-14.960*
-3.514*
-9.159*
-13.412* -14.857*
-4.665* -206.510* -28.377*
Brezilya
-16.844*
-7.180*
-9.050*
-9.561* -16.381*
-4.798* -128.551* -32.035*
Rusya
-13.471* -2.773***
-13.666* -14.575* -13.519*
-4.866*
-54.853* -25.423*
Kolombiya
-9.169*
-7.371*
-6.160*
-10.738* -9.224*
-7.823*
-82.145* -46.508*
Peru
-14.091*
-5.063*
-9.442*
-9.903* -13.875*
-4.849*
-83.549* -32.213*
Meksika
-12.735*
-7.194*
-12.777*
-9.486* -12.707*
-5.349*
-64.391* -34.213*
Arjantin
-11.700*
-7.120*
-11.575*
-9.992* -11.722*
-7.039*
-65.196* -54.307*
İspanya
-7.547*
-2.986**
-8.669*
-11.550* -14.164*
-4.553* -107.691* -27.054*
Güney Afrika -13.174*
-7.328*
-6.569*
-9.860* -13.129*
-7.788*
-52.854* -28.725*
İngiltere
-13.611* -2.983**
-10.038* -11.458* -13.597*
-4.429*
-65.863* -25.900*
İtalya
-7.801*
-2.925**
-10.751* -11.667* -14.399*
-4.621*
-98.391* -26.450*
Fransa
-13.388* -2.929**
-9.743*
-11.742* -13.476*
-4.516*
-64.292* -26.573*
*,**,*** t-istatistikleri sırasıyla %1,%5 ve %10 anlamlılık düzeylerine göre değişkenlerin durağan
olduğunu göstermektedir. Tablo 2b. Değişkenlere Ait Fourier ADF Birim Kök Testi Sonuçları
Fourier ADF
r
EMV-ID
ABD
-3.640*** (8)
-3.966* (3)
Hindistan
-3.989* (6)
-3.641*** (7)
Brezilya
-5.143* (6)
-4.862* (12)
Rusya
-13.696* (1)
-3.370*** (4)
Kolombiya
-8.357* (11)
-3.188** (10)
Peru
-3.546** (12)
-2.788*** (8)
Meksika
-12.790* (0)
-4.9982* (3)
Arjantin
-3.687** (0)
-4.089*** (7)
İspanya
-3.338* (9)
-3.974** (12)
Güney Afrika
-7.070* (12)
-3.485** (4)
İngiltere
-4.937* (7)
-3.895** (12)
İtalya
-5.182* (7)
-3.667*** (12)
Fransa
-4.744* (8)
-3.349*** (12)
*, **, *** sırasıyla %1, %5 ve %10 kritik değerlerde anlamlılığı ifade etmektedir. Parantez
içinde verilen değerler model tarafından belirlenen optimal gecikmeleri göstermektedir. Tablo 2b. Değişkenlere Ait Fourier ADF Birim Kök Testi Sonuçları 65 D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ GARCH modellerinin kullanıldığı çalışmalarda zaman serilerinde ARCH etkisi olduğu
varsayılmaktadır. Bu nedenle, EGARCH modeli uygulanmadan önce Denklem (1)’de ARCH
etkisinin olup olmadığı test edilmiştir. Tablo 3a ve 3b, ARCH-LM (Lagrange Multiplier Test
for ARCH) testinin sonuçlarını göstermektedir. Bu sonuçlara göre ARCH etkisinin olmadığını
ifade eden H0 hipotezi tüm ülkeler için reddedilmiş, serilerde ARCH etkisi olduğu sonucuna
ulaşılmıştır. GARCH modellerinin kullanıldığı çalışmalarda zaman serilerinde ARCH etkisi olduğu
varsayılmaktadır. Bu nedenle, EGARCH modeli uygulanmadan önce Denklem (1)’de ARCH
etkisinin olup olmadığı test edilmiştir. Tablo 3a ve 3b, ARCH-LM (Lagrange Multiplier Test
for ARCH) testinin sonuçlarını göstermektedir. Bu sonuçlara göre ARCH etkisinin olmadığını
ifade eden H0 hipotezi tüm ülkeler için reddedilmiş, serilerde ARCH etkisi olduğu sonucuna
ulaşılmıştır. Tablo 3a. ARCH LM Testi
ABD
Hindistan Brezilya Rusya Kolombiya
Peru
Meksika
ARCH(2) 29.272*
30.246*
23.417* 16.130*
19.404*
20*255*
10.291*
ARCH(4) 32.845*
39.078*
32.179* 26.284*
30.487*
52.985*
37.641*
ARCH(6) 36.454*
40.120*
35.520* 36.812*
53.559*
49.654*
52.956*
ARCH(8) 45.475*
50.486*
47.667* 40.622*
68.869*
17.805**
53.764*
ARCH-LM(n): n sayıdaki gecikmede ARCH - Lagrange Çarpanı testlerini ifade etmektedir. *,**,*** sırasıyla %1,%5 ve %10 anlamlılık düzeylerinde Ki-kare olasılık değerlerini ifade
etmektedir. Tablo 3b. ARCH LM Testi
Arjantin
İspanya
Güney Afrika
İngiltere
İtalya
Fransa
ARCH(2)
8.802**
8.904**
46.239*
20.401*
19.818*
6.784**
ARCH(4)
21.014*
26.812*
57.852*
20.823*
19.711*
32.285*
ARCH(6)
27.453*
26.912*
96.770*
20.647*
21.069*
31.941*
ARCH(8)
33.698*
31.015*
88.255*
37.758*
28.979*
38.639*
ARCH-LM(n): n sayıdaki gecikmede ARCH - Lagrange Çarpanı testlerini ifade etmektedir. *,**,*** sırasıyla %1,%5 ve %10 anlamlılık düzeylerinde Ki-kare olasılık değerlerini ifade
etmektedir. Serilerde ARCH etkisinin olduğu belirlendikten sonra EGARCH(1,1) modeli ile ortalama
denklemi (denklem 1) ve varyans denklemi (denklem 4) modellenmiş, sonuçları Tablo 4a ve
4b’de verilmiştir. Model üzerinde uygulanan ARCH-LM, Ljung-Box Q ve Jarque-Bera testleri
sonuçların tutarlı olduğunu göstermektedir. Tablo 4a ve 4b’ye göre ABD, Hindistan, Brezilya, Kolombiya, Peru, Meksika, Arjantin
ve İspanya piyasa getirileri için ARCH etkisi mevcutken Rusya, Güney Amerika, İngiltere,
İtalya ve Fransa için anlamlı bir etki bulunmamaktadır. GARCH parametresi ise tüm ülkelerin
piyasa getirileri için anlamlıdır. ARCH etkisi şokun geçici etkisini gösterirken, GARCH
parametresinin anlamlı olması şokun kalıcılığını ifade etmektedir. Diğer bir ifade ile tüm piyasa 66 Ekonomi, Politika & Finans Araştırmaları Dergisi, 2020, 5(Özel Sayı):59-75
Journal of Research in Economics, Politics & Finance, 2020, 5(Special Issue): 59-75 getirileri
volatilitesi
kendi
şoklarından
kısa
veya
uzun
vadede
etkilenmektedir
(ARCH/GARCH). g
ş
y
(ARCH/GARCH). Tablo 4a. ARCH-LM(n): n sayıdaki gecikmede ARCH - Lagrange Çarpanı testlerini ifade etmektedir. Parantez içinde
olasılık değerleri ile desteklenmektedir. ğ
B: Jarque-Bera normallik testini ifade etmektedir. Parantez içinde olasılık değerleri ile desteklenmektedir.
**,*** sırasıyla %1,%5 ve %10 anlamlılık düzeylerini göstermektedir. değerleri parantez içinde verilmiştir. ğ
JB: Jarque-Bera normallik testini ifade etmektedir. Parantez içinde olasılık değerleri ile desteklenmektedir.
*,**,*** sırasıyla %1,%5 ve %10 anlamlılık düzeylerini göstermektedir. Q(n): n sayıdaki gecikmeden Ljung-Box Q test istatistiklerini ifade etmektedir. Parantez içinde olasılık
değerleri ile desteklenmektedir. D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ EGARCH Modeli Sonuçları
ABD
Hindistan
Brezilya
Rusya
Kolombiya
Peru
Meksika
Ortalama Denklemi
C
0.0028*
(2.8929)
-0.0017**
(-2.3223)
0.0005*
(3.5453)
0.0031***
(-1.7403)
0.0029***
(1.7773)
0.0019**
(2.5096)
0.0399*
(117.11341)
rt-1
-0.2723*
(-3.2776)
-0.0703
(-0.8813)
-0.2026*
(-25.6057)
-0.0275
(-0.2419)
0.2433**
(2.0104)
-0.1129***
(-1.8975)
-0.0010*
(-144.8578)
Varyans Denklemi
C
-3.2434**
(-2.2960)
-0.9597*
(-158.4638)
-3.1831***
(-1.9185)
-5.4539*
(-4.5676)
-1.6349
(-1.3929)
-0.4466*
(-7.4201)
-0.6902
-0.8995)
ARCH(-1)
0.3992***
(1.7391)
-0.2876*
(-10.5029)
0.3178***
(1.8263)
-0.1121
(-0.6430)
0.2837***
(1.7029)
-0.2415*
(-3.1655)
0.3634**
(2.0007)
GARCH(-1)
0.7047*
(4.9214)
0.8819*
(5.8E+103)
0.6926*
(4.0292)
0.3710*
(2.6817)
0.8587*
(8.0334)
0.9400*
(4.1E+103)
0.9463*
(12.2432)
-0.2529**
(-2.0997)
-0.2078*
(-7.3000)
-0.1364
(-1.3548)
-0.1867***
(-1.7337)
0.0202
(0.1345)
0.0792***
(1.8091)
-0.1120
(-0.8441)
EMV-ID
0.02251***
(1.7046)
0.0105*
(17.1018)
0.02378
(1.5313)
0.0402
(3.4468)
0.01224
(1.2369)
0.0034*
(4.8139)
0.0007
(0.1102)
Log-likelihood
466.6477
461.4427
359.9678
383.7031
353.0567
424.3035
404.0330
ARCH-LM(2)
0.0857
(0.9541)
3.4448
(0.1786)
1.7460
(0.4177)
0.3232
(0.8508)
0.06953
(0.7064)
0.3570
(0.8365)
2.0964
(0.3506)
ARCH-LM(4)
1.1248
(0.8903)
3.4223
(0.4896)
2.6572
(0.6167)
1.6812
(0.7941)
1.8611
(0.7613)
6.1210
(0.1903)
5.6602
(0.2260)
ARCH-LM(6)
1.6607
(0.9481)
4.0303
(0.6726)
4.6774
(0.5858)
2.4273
(0.8765)
4.4695
(0.6134)
9.0142
(0.1728)
7.9566
(0.2413)
ARCH-LM(8)
2.4696
(0.9631)
4.4064
(0.8187)
9.5138
(0.3008)
2.6161
(0.9561)
5.0714
(0.7499)
11.8597
(0.1576)
8.0593
(0.4277)
Q(2)
4.0427
(0.132)
2.2188
(0.330)
0.3204
(0.571)
0.0281
(0.986)
8.1009
(0.151)
1.6446
(0.439)
0.0363
(0.982)
Q(4)
-0.039
(0.338)
2.8397
(0.585)
2.6048
(0.457)
1.2266
(0.874)
8.1945
(0.415)
5.8558
(0.210)
0.3185
(0.989)
Q(6)
-0.108
(0.337)
9.5544
(0.145)
5.6202
(0.445)
2.6395
(0.853)
11.554
(0.398)
8.8608
(0.439)
0.6234
(0.987)
Q(8)
0.015
(0.545)
10.010
(0.264)
11.501
(0.175)
3.4235
(0.905)
13.447
(0.337)
6.5758
(0.583)
0.8246
(0.999)
JB
29.9496
(0.000)
9.0044
(0.011)
1.2911
(0.524)
6.8987
(0.032)
4.5336
(0.106)
6.4744
(0.039)
0.9173
(0.664)
t-değerleri parantez içinde verilmiştir. Tablo 4a. EGARCH Modeli Sonuçları ğ
(n): n sayıdaki gecikmeden Ljung-Box Q test istatistiklerini ifade etmektedir. Parantez içinde olasılık
eğerleri ile desteklenmektedir. 67 D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ parametresi pozitif ve negatif şokların volatiliteye farklı etkisi olup olmadığını
göstermektedir. ABD, Hindistan, Rusya, İspanya, İtalya ve Fransa piyasa getirilerinde negatif
şokların volatiliteye pozitif şoklardan daha fazla etki ettiği belirlenmiştir. Diğer piyasalarda ise
pozitif ve negatif şokların volatiliteyi aynı derecede etkilediği söylenebilir. Tablo 4b. EGARCH Modeli Sonuçları
Arjantin
İspanya
Güney Afrika
İngiltere
İtalya
Fransa
Ortalama Denklemi
C
0.0027*
(9.2818)
0.0006*
(-11.1719)
0.0015**
(2.0886)
0.0011***
(-1.7162)
0.0010**
(-2.5159)
0.0011*
(-2.6469)
rt-1
0.0376*
(5.1964)
0.0253*
(10.0832)
-0.0938**
(-2.3052)
-0.1591*
(-4.6492)
-0.1102**
(-2.4884)
-0.0920**
(-2.0633)
Varyans Denklemi
-5.4858*
(-3.0467)
-1.9498
(-1.1321)
-0.6472*
(-4.9770)
-5.0676*
(-2.9141)
-6.4231***
(-1.8343)
-7.4977**
(-2.4179)
ARCH(-1)
0.2991***
(1.7058)
0.2161***
(1.6576)
-0.3274
(-1.4497)
-0.6098
(-1.3878)
-0.0454
(-0.0977)
-0.0403
(-0.6055)
GARCH(-1)
0.3525**
(1.9603)
0.7989*
(4.1841)
0.9167*
(74.2442)
0.4499**
(2.3871)
0.9672*
(12.9542)
0.8918*
(9.0960)
-0.1637
(-1.5621)
-0.1826**
(-2.4378)
-0.1885
(-1.0932)
0.1131
(0.4512)
-0.2697*
(-4.4312)
-0.2088*
(-6.8216)
EMV-ID
0.0378*
(2.6103)
0.0089
(0.7038)
0.0075*
(5.9659)
0.0347*
(2.8314)
0.01539**
(2.5259)
0.0575**
(2.2021)
Log-likelihood
272.1181
423.9958
363.2797
434.5006
425.9478
447.2951
ARCH-LM(2)
0.0189
(0.9906)
0.9552
(0.6203)
1.4801
(0.4771)
0.3350
(0.8458)
0.0307
(0.9848)
3.1086
(0.2113)
ARCH-LM(4)
0.5314
(0.9704)
3.5793
(0.4659)
4.1409
(0.3873)
4.7582
(0.3130)
3.1344
(0.5356)
7.1316
(0.1291)
ARCH-LM(6)
1.1230
(0.9805)
3.7613
(0.7089)
14.0712
(0.0289)
7.3508
(0.2896)
3.4320
(0.7530)
7.5592
(0.2722)
ARCH-LM(8)
2.6445
(0.9547)
3.7299
(0.8806)
15.3650
(0.0524)
10.6637
(0.2215)
4.8219
(0.7764)
7.5042
(0.4833)
Q(2)
0.3834
(0.826)
2.0720
(0.355)
0.5758
(0.750)
0.7664
(0.125)
9.0215
(0.108)
2.1560
(0.340)
Q(4)
3.3511
(0.501)
2.1164
(0.714)
0.6269
(0.960)
4.8602
(0.302)
9.6759
(0.208)
2.1961
(0.533)
Q(6)
4.3905
(0.624)
4.4715
(0.613)
4.9130
(0.427)
7.2369
(0.299)
12.754
(0.121)
2.5339
(0.639)
Q(8)
4.8394
(0.775)
9.4880
(0.303)
7.0124
(0.320)
9.4639
(0.305)
14.154
(0.121)
6.0779
(0.415)
JB
0.6769
(0.713)
9.9036
(0.007)
0.1470
(0.929)
29.9642
(0.000)
39.3941
(0.000)
15.4688
(0.004)
t-değerleri parantez içinde verilmiştir. ARCH LM( )
d ki
ik
d
ARCH
L
Ç
l i i if d
k di
P Tablo 4b. EGARCH Modeli Sonuçları Q(n): n sayıdaki gecikmeden Ljung-Box Q test istatistiklerini ifade etmektedir. Parantez içinde olasılık
değerleri ile desteklenmektedir. JB: Jarque-Bera normallik testini ifade etmektedir. Parantez içinde olasılık değerleri ile desteklenmektedir. *,**,*** sırasıyla %1,%5 ve %10 anlamlılık düzeylerini göstermektedir. JB: Jarque-Bera normallik testini ifade etmektedir. Parantez içinde olasılık değerleri ile desteklenmektedir. *,**,*** sırasıyla %1,%5 ve %10 anlamlılık düzeylerini göstermektedir. D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ 68 Ekonomi, Politika & Finans Araştırmaları Dergisi, 2020, 5(Özel Sayı):59-75
Journal of Research in Economics, Politics & Finance, 2020, 5(Special Issue): 59-75 Ekonomi, Politika & Finans Araştırmaları Dergisi, 2020, 5(Özel Sayı):59-75
Journal of Research in Economics, Politics & Finance, 2020, 5(Special Issue): 59-75 EMV-ID endeksinin ise ABD, Hindistan, Peru, Arjantin, Güney Afrika, İngiltere, İtalya
ve Fransa piyasa getirilerinin volatilitesini pozitif etkilediği; Brezilya, Rusya, Kolombiya,
Meksika ve İspanya’da ise anlamlı bir etkisinin bulunmadığı görülmektedir. Bu çalışma Covid-19 pandemisi sırasında piyasa getirilerinin volatilitesini ve Covid-19
haberlerinden derlenen EMV-ID endeksinin bu volatiliteyi nasıl etkilediğini test etmeyi
amaçlamaktadır. Analiz sonucunda Covid-19’un her piyasayı farklı şekilde etkilediği tespit
edilmiştir. Analiz sonuçları önceki çalışmaları desteklemektedir (Ali vd., 2020; Ashraf, 2020;
Bai vd., 2020; Baker vd., 2020; Barut ve Yerdelen Kaygın, 2020; Capelle-Blancard ve
Desroziers, 2020; Hacıevliyagil ve Gümüş, 2020; Haroon ve Rizvi, 2020; Li vd., 2020; Mishra
vd., 2020; Zeren ve Hızarcı, 2020). D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ Kaynakça Adıgüzel, M. (2020). Covid-19 pandemisinin Türkiye ekonomisine etkilerinin makroekonomik analizi. İstanbul Ticaret Üniversitesi Sosyal Bilimler Dergisi, 19(37), 191–221. Erişim adresi:
https://dergipark.org.tr/tr/pub/iticusbe Albulescu, C. (2020). Coronavirus and financial volatility: 40 days of fasting and fear. Capital Markets:
Asset Pricing & Valuation eJournal, 1-7. doi:10.2139/ssrn.3550630 Albuquerque, R. A., Koskinen, Y. J., Yang, S. and Zhang, C. (2020). Love in the time of COVID-19: The
resiliency of environmental and social stocks (CEPR Discussion Paper No. DP14661). doi:10.2139/ssrn.3583611 Alfaro, L., Chari, A., Greenland, A. and Schott, P. (2020). Aggregate and firm-level stock returns durin
pandemics, in real time (NBER Working Paper No. 26950). doi:10.3386/w26950 Ali, M., Alam, N. and Rizvi, S. A. R. (2020). Coronavirus (COVID-19) - An epidemic or pandemic for
financial
markets. Journal
of
Behavioral
and
Experimental
Finance, 27, 100341. doi:10.1016/j.jbef.2020.100341 Altig, D., Baker, S., Barrero, J. M., Bloom, N., Bunn, P., Chen, S., Davis, S., ... Thwaites, G. (2020). Economic uncertainty before and during the COVID-19 pandemic (NBER Working Paper No. 27418). doi:10.3386/w27418 Ashraf, B. N. (2020). Stock markets’ reaction to COVID-19: Cases or fatalities? Research in
International Business and Finance, 54, 101249. doi:10.1016/j.ribaf.2020.101249 Bai, L., Wei, Y., Wei, G., Li, X. and Zhang, S. (2020). Infectious disease pandemic and permanent
volatility of international stock markets: A long-term perspective. Finance Research Letters,
101709. doi:10.1016/j.frl.2020.101709 Baker, S., Bloom, N., Davis, S., Kost, K., Sammon, M. and Viratyosin, T. (2020). The Unprecedented
Stock Market Impact of COVID-19 (NBER Working Paper No. 26945). doi:10.3386/w26945 Barut, A. ve Yerdelen Kaygın, C. (2020). Covid-19 pandemisinin seçilmiş borsa endeksleri üzerine
etkisinin incelenmesi [Özel Sayı]. Gaziantep Üniversitesi Sosyal Bilimler Dergisi, 19(COVID-19
Özel Sayısı), 59–70. doi:10.21547/jss.773237 Bollerslev, T. (1986). Generalized autoregressive conditional heteroskedasticity. Journal of
Econometrics, 31(3), 307–327. doi:10.1016/0304-4076(86)90063-1 Breinlich, H., Leromain, E., Novy, D., Sampson, T. and Usman, A. (2018). The economic effects of
Brexit: Evidence from the stock market. Fiscal Studies, 39(4), 581–623. doi:10.1111/1475-
5890.12175 Brooks, C. (2014). Intoductry econometrics for finance (3rd Edition). Cambridge, MA: Cambridge
University Press. Burggraf, T., Fendel, R. and Huynh, T. L. D. (2020). Political news and stock prices: Evidence from
Trump’s
trade
war. Applied
Economics
Letters,
27(18),
1485–1488. doi:10.1080/13504851.2019.1690626 Campello, M., Kankanhalli, G. and Muthukrishnan, P. (2020). Corporate hiring under COVID-19: labor
market concentration, downskilling, and income inequality (NBER Working Paper No. 27208). doi:10.3386/w27208 Capelle-Blancard, G. and Desroziers, A. (2020). The stock market is not the economy? Insights from the
COVID-19 crisis. CEPR Covid Economics, 28, 29-69. https://dx.doi.org/10.2139/ssrn.3638208 Çetin, A. C. 5. Sonuç ve Değerlendirme Covid-19’un ortaya çıkmasından itibaren ülke ekonomileri ve piyasaları ciddi bir şekilde
etkilenmiştir. Bu etkiler akademik literatürde ve finans basınında önceki salgınlarla ve krizlerle
karşılaştırılmaktadır. Ancak salgının halen devam etmesi ve etkilerini tam olarak göstermemesi
küresel belirsizliği beraberinde getirmekte, riski artırmaktadır. Pay piyasaları ekonominin
gidişatına yönelik bir gösterge olarak kullanılmakta, Covid-19’un sosyal ve ekonomik sonuçları
da direkt olarak bu piyasalardaki volatiliteyi etkilemektedir. Bu çalışmada, Covid-19 pandemisinin kamuoyu üzerindeki etkilerini sayısal olarak ölçen,
Baker vd. (2020) tarafından geliştirilen EMV-ID endeksinin pay piyasalarındaki volatiliteyi
nasıl etkilediği araştırılmıştır. Bu etkinin daha güçlü bir şekilde incelenmesi için WHO
verilerine göre vaka ve ölüm sayısı en fazla olan ülkeler seçilmiştir. Çalışmada, pandemi
dönemi boyunca piyasa volatilitelerinin kendi şoklarından kalıcı bir şekilde etkilendiği
görülmüş, bazı piyasalarda negatif şokların volatiliteyi daha fazla artırdığı tespit edilmiştir. EMV-ID endeksinin ise çoğu piyasada volatiliteyi yükselttiği; Brezilya, Rusya, Kolombiya,
Meksika ve İspanya’da ise anlamlı bir etkisinin bulunmadığı görülmüştür. Özetle, analiz
sonuçları beklendiği gibi önceki çalışmaları destekler nitelikte olup (Bai vd., 2020; Barut ve
Yerdelen Kaygın, 2020; Hacıevliyagil ve Gümüş, 2020; Haroon ve Rizvi, 2020; Li vd., 2020;
Zeren ve Hızarcı, 2020; Zhang vd., 2020), Covid-19’un farklı piyasalarda farklı etkilere sahip
olduğunu göstermektedir. Bu sonucun en büyük nedeni, farklı devletlerin Covid-19 için farklı
politikalar izlemesi ve pay piyasalarının pandemiye karşı farklı tepkiler vermesidir. Ancak
Covid-19 tüm dünyayı sosyal, ekonomik ve finansal açıdan etkilemiştir. Özellikle epidemiden
pandemiye geçildiği dönemde küresel piyasaların öncüsü olan ABD ve Avrupa pay
piyasalarında yüksek dalgalanmalara neden olmuştur. Bu piyasaların da diğer gelişmiş ve
gelişmekte olan pay piyasalarını etkilediği bilinmektedir. Bu nedenle analiz sonucuna göre
EMV-ID endeksinin anlamsız çıktığı ülkelerde Covid-19’un bu piyasaları etkilemediği
çıkarımını yapmak doğru olmayacaktır. Covid-19 pandemisinin halen devam etmesi ve ne zaman sona ereceğine dair belirsizlik,
küresel olarak tüm etkilerinin henüz görülmesini engellemekte, etkisinin önümüzdeki yıllarda,
önceki salgın hastalıklardan ve krizlerden daha fazla olacağı tahmin edilmektedir. Bu nedenle,
ileriki yıllarda Covid-19 pandemisinin daha fazla tartışılacağı düşünülmektedir. Gelecek 69 D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ çalışmalar, Covid-19’un diğer piyasalara ve alt sektör piyasalarına olan etkisini inceleyebilir. Bunun yanında EMV-ID endeksinin etkisi daha uzun bir dönemde analiz edilebilir. alışmalar, Covid-19’un diğer piyasalara ve alt sektör piyasalarına olan etkisini inceleyebilir. Bunun yanında EMV-ID endeksinin etkisi daha uzun bir dönemde analiz edilebilir. 70 Ekonomi, Politika & Finans Araştırmaları Dergisi, 2020, 5(Özel Sayı):59-75
Journal of Research in Economics, Politics & Finance, 2020, 5(Special Issue): 59-75 Kaynakça (2020). Koronavirüs (Covid-19) salgınının türkiye’de genel ekonomik faaliyetlere ve hisse
senedi borsa endeksine etkisi. Mehmet Akif Ersoy Üniversitesi Uygulamalı Bilimler Dergisi, 4(2),
341–362. doi:10.31200/makuubd.766901 Corbet, S., Larkin, C. and Lucey, B. (2020). The contagion effects of the COVID-19 pandemic: Evidence
from
gold
and
cryptocurrencies. Finance
Research
Letters,
35,
101554. doi:10.1016/j.frl.2020.101554 71 D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ Demir, E., Bilgin, M. H., Karabulut, G. and Doker, A. C. (2020). The relationship between
cryptocurrencies and COVID-19 pandemic. Eurasian Economic Review, 10, 349-360. doi:10.2139/ssrn.3585147 Dickey, D. A. and Fuller, W. A. (1981). Likelihood ratio statistics for autoregressive time series with a
unit root. Econometrica, 49(4), 1057-1072. doi:10.2307/1912517 Duran, M. S. ve Acar, M. (2020). Bir virüsün dünyaya ettikleri: Covid-19 pandemisinin makroekonomik
etkileri. International Journal of Social and Economic Sciences, 10(1), 54–67. Retrieved from
http://www.ijses.org/ Enders, W. and Lee, J. (2012). A unit root test using a fourier series to approximate smooth breaks. Oxford
Bulletin
of
Economics
and
Statistics,
74(4),
574–599. doi:10.1111/j.1468-
0084.2011.00662.x Engle, R. F. (1982). Autoregressive conditional heteroscedasticity with estimates of the variance of
United Kingdom inflation. Econometrica, 50(4), 987-1007. doi:10.2307/1912773 Fernandes, N. (2020). Economic effects of Coronavirus outbreak (COVID-19) on the World economy
(IESE Business School Working Paper No. WP-1240-E). doi:10.2139/ssrn.3557504 Goodell, J. W. (2020). COVID-19 and finance: Agendas for future research. Finance Research Letters,
35, 101512. doi:10.1016/j.frl.2020.101512 Gopinath, G. (2020). The great lockdown: worst economic downturn since the Great Depression (IMF
Press Release No. 20/98). Retrieved from https://blogs.imf.org/2020/04/14/the-great-lockdown-
worst-economic-downturn-since-the-great-depression/ Gupta, R., Subramaniam, S., Bouri, E. and Ji, Q. (2021). Infectious disease-related uncertainty and the
safe-haven characteristic of US treasury securities. International Review of Economics & Finance,
71, 289–298. doi:10.1016/j.iref.2020.09.019 Hacıevliyagil, N. ve Gümüş, A. (2020). Covid-19’un en etkili olduğu ülkelerde salgın-borsa ilişkisi [Özel
Sayı]. Gaziantep Üniversitesi Sosyal Bilimler Dergisi, 19(COVID-19 Özel Sayı), 354-364. doi:10.21547/jss.742893 Haroon, O. and Rizvi, S. A. R. (2020). COVID-19: Media coverage and financial markets behavior—A
sectoral
inquiry. Journal
of
Behavioral
and
Experimental
Finance,
27,
100343. doi:10.1016/j.jbef.2020.100343 Harvey,
C. (2020). The
economic
and
financial
implications
of
COVID-19
[Video]. https://www.fma.org/virtual-seminar Ichev, R. and Marinč, M. (2018). Stock prices and geographic proximity of information: Evidence from
the
Ebola
outbreak. International
Review
of
Financial
Analysis,
56,
153–166. doi:10.1016/j.irfa.2017.12.004 Kargar, M., Lester, B., Lindsay, D., Liu, S., Weill, P. O. and Zúñiga, D. (2020). Corporate bond liquidity
during the COVID-19 crisis (NBER Working Paper No. 27355). doi:10.3386/w27355 Keleş, E. (2020). Covid-19 ve BİST-30 endeksi üzerine kısa dönemli etkileri. Kaynakça Marmara Üniversitesi
İktisadi ve İdari Bilimler Dergisi, 42(1), 91-105. doi:10.14780/muiibd.763962 Kenourgios, D., Dadinakis, E. and Tsakalos, I. (2020). Brexit referendum and European stock markets: A
sector analysis. Managerial Finance, 46(7), 913–933. doi:10.1108/MF-07-2019-0366 Kwan, S. H. and Mertens, T. M. (2020). Market assessment of COVID-19 ( FRBSF Economic Letter No:
2020–14). Retrieved from https://www.frbsf.org/economic-research/ Li, Y., Liang, C., Ma, F. and Wang, J. (2020). The role of the IDEMV in predicting European stock
market volatility during the COVID-19 pandemic. Finance Research Letters, 36, 101749. doi:10.1016/j.frl.2020.101749 Mishra, A. K., Rath, B. N. and Dash, A. K. (2020). Does the indian financial market nosedive because of
the COVID-19 outbreak, in comparison to after demonetisation and the GST? Emerging Markets
Finance and Trade, 56(10), 2162–2180. doi:10.1080/1540496X.2020.1785425 72 Ekonomi, Politika & Finans Araştırmaları Dergisi, 2020, 5(Özel Sayı):59-75
Journal of Research in Economics, Politics & Finance, 2020, 5(Special Issue): 59-75 Nelson, D. B. (1991). Conditional heteroskedasticity in asset returns: A new approach. Econometrica,
59(2), 347–370. doi:10.2307/2938260 Nippani, S. and Washer, K. M. (2004). SARS: A non-event for affected countries’ stock markets? Applied Financial Economics, 14(15), 1105–1110. doi:10.1080/0960310042000310579 Özden, Ü. H. (2008). İMKB bileşik 100 endeksi getiri volatilitesinin analizi. İstanbul Ticaret Üniversitesi
Sosyal Bilimler Dergisi, 7(13), 339–350. .Erişim adresi: https://www.ticaret.edu.tr/ Phillips, P. C. B. and Perron, P. (1988). Testing for a unit root in time series regression. Biometrika,
75(2), 335–346. doi:10.1093/biomet/75.2.335 Risteski, D., Sadoghi, A. and Davcev, D. (2013). Improving Predicting Power of EGARCH models for
financial time series volatility by using google trend. Paper presented at the Proceedings of 2013
International Conference on Frontiers of Energy,Environmental Materials and Civil Engineering. Shangai,
China. Retrieved
from
http://cstm.cnki.net/stmt/TitleBrowse/KnowledgeNet/XYSW201311001052?db=STMI8319 Sharif, A., Aloui, C. and Yarovaya, L. (2020). COVID-19 pandemic, oil prices, stock market, geopolitical
risk and policy uncertainty nexus in the US economy: Fresh evidence from the wavelet-based
approach. International Review of Financial Analysis, 70, 101496. doi:10.1016/j.irfa.2020.101496 Soylu, Ö. B. (2020). Türkiye ekonomisinde Covid-19’un sektörel etkileri. Avrasya Sosyal ve Ekonomi
Araştırmaları Dergisi, 7(6), 169–185. Erişim adresi: https://dergipark.org.tr/tr/pub/asead Tayar, T., Gümüştekin, E., Dayan, K. ve Mandi, E. (2020). Covid-19 krizinin Türkiye’deki sektörler
üzerinde etkileri: Borsa İstanbul sektör endeksleri araştırması [Özel Sayı]. Yüzüncü Yıl
Üniversitesi
Sosyal
Bilimler
Enstitüsü
Dergisi,
1(6),
293–320. Erişim
adresi:
https://dergipark.org.tr/en/pub/yyusbed Wagner, A., Zeckhauser, R. and Ziegler, A. (2017). Company stock reactions to the 2016 election shock:
Trump,
taxes
and
trade
(NBER
Working
Paper
No. 23152). Retrieved
from
https://www.nber.org/system/files/working_papers/w23152/w23152.pdf Zeren, F. ve Hızarcı, A. (2020). Kaynakça Covid-19 Coronavirüsün hisse senedi piyasalarına etkisi: seçilmiş
ülkelerden
kanıtlar. Muhasebe
ve
Finans
İncelemeleri
Dergisi,
3(1),
78–84. doi:10.32951/mufider.706159 Zhang, D., Hu, M. and Ji, Q. (2020). Financial markets under the global pandemic of COVID-19. Finance Research Letters, 36, 101528. doi:10.1016/j.frl.2020.101528 73 D. Sevinç, ―Covid-19’un Uluslararası Pay Piyasalarına Etkisi‖ Aim of The Study With the emergence of the Covid-19 virus in China in December 2019 and the World
Health Organization (WHO) declaring a pandemic on March 11, 2020, this epidemic has a great
impact on social, political, economic and finance globally. Since the emergence of Covid-19 and the measures taken, its impact on the economy and
financial system has been investigated in the literature. However, since the Covid-19 epidemic
is still new and continues, empirical studies examining its impact on financial markets are
limited in the literature. This study is to examine the relationship between Covid-19 and
volatilities of stock markets with EMV-ID (Infectious Infectious Disease Equity Market
Volatility Tracker) index which generated by Baker et al. (2020). Literature Review Baker et al. (2020) found that Covid-19 caused the largest market volatility to date in
their research which they developed the daily EMV-ID index. Bai, Wei, Wei, Li, and Zhang
(2020), Baker et al. (2020), by converting the EMV-ID index to a monthly series, examined the
impact of the epidemic pandemic on international markets. In the study, the period from January
4, 2005 to April 30, 2020, which includes the emergence and spread of bird flu, swine flu
(H1N1), Ebola, Polio, Zika virus and Covid-19, was analyzed. In their GARCH-MIDAS
analysis by Bai, Wei, Wei, Li, and Zhang (2020), it was concluded that epidemic pandemics
increased volatility in the US, UK and Japan markets, but the Chinese market was more stable
during these epidemics. Li, Liang, Ma and Wang (2020) analyzed the effect of EMV-ID on
stock market returns in France, England and Germany. EMV-ID has a strong influence in
predicting volatility in the UK and France. Capelle-Blancard and Desroziers (2020) analyzed
the effect of the EMV-ID index on the market returns of 74 countries, and in the panel model,
they concluded that the index had no effect on market returns, but stated that Covid-19 had a
different effect on each country. Empirical Results According to the model results, while there is an ARCH effect for the market returns of
USA, India, Brazil, Colombia, Peru, Mexico, Argentina and Spain, there is no significant effect
for Russia, South America, England, Italy and France. GARCH parameter is significant for
market returns of all countries. In other words, all market returns volatility is affected by their
own shocks in the short or long term (ARCH or GARCH). parameter indicates whether
positive and negative shocks have more effects on volatility. EMV-ID index positively affects
the volatility of market returns from the USA, India, Peru, Argentina, South Africa, England,
Italy and France when there is no significant effect in Brazil, Russia, Colombia, Mexico and
Spain. Methodology Nelson (1991) developed the EGARCH (Exponential GARCH) model, which eliminates
the necessity of the conditional variance parameters to be positive with logarithmic
transformation and shows the asymmetric effect (leverage effect) of shocks in order to eliminate
these deficiencies in the GARCH model. The EGARCH (1,1) model used in the study can be
expressed as follows: (
√
√
)
√
t=1,...,n (1) (1) Ekonomi, Politika & Finans Araştırmaları Dergisi, 2020, 5(Özel Sayı):59-75
Journal of Research in Economics, Politics & Finance, 2020, 5(Special Issue): 59-75 Ekonomi, Politika & Finans Araştırmaları Dergisi, 2020, 5(Özel Sayı):59-75
Journal of Research in Economics, Politics & Finance, 2020, 5(Special Issue): 59-75 , expresses the variance of residuals in equation (1). is defined as the variance of
residuals in previous periods (volatility in previous periods), where is a constant term. , is
the short-run effect of shocks for conditional variance, , is the continuity of shocks, and , expresses the variance of residuals in equation (1). is defined as the variance of
residuals in previous periods (volatility in previous periods), where is a constant term. , is
the short-run effect of shocks for conditional variance, , is the continuity of shocks, and √
⁄
is the standardized error term. If the parameter is statistically significant and not
equal to zero ( , there is asymmetric volatility. means that positive and negative
shocks have the same effect on volatility; -1< <0 ndicates that negative shocks affect more
than positive shocks (Brooks, 2014). Conclusion In this study, it is investigated how the EMV-ID index which is generated by Baker et al. (2020), affects the volatility in the stock markets. It was seen that market volatilities were
permanently affected by their own shocks during the pandemic period, and it was found that
negative shocks increased volatility more in some markets at the analysis. EMV-ID index
increased volatility in most markets; it has been observed that there is no significant effect in
some. The biggest reason for this result is that different states follow different policies for
Covid-19 and stock markets react differently to the pandemic. However, Covid-19 has affected
the whole world socially, economically and financially. Therefore, it would not be correct to
deduce that Covid-19 does not affect these markets in countries where the EMV-ID index is
found to be insignificant. 75
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https://openalex.org/W3191311031
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https://www.researchsquare.com/article/rs-752911/latest.pdf
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English
| null |
Clinical Significance and Genetic Characteristics in HER2 Low Expression Tumors of Hormone Receptor Positive Breast Cancer Patients
|
Research Square (Research Square)
| 2,021
|
cc-by
| 5,642
|
Clinical Significance and Genetic Characteristics in
HER2 Low Expression Tumors of Hormone
Receptor Positive Breast Cancer Patients Mengdi Chen
Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital
Deyue Liu
Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital
Weilin Chen
Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital
Weiguo Chen
Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital
Kunwei Shen
Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital
Jiayi Wu
Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital
Li Zhu
(
zhuli8@yeah.net
)
Ruijin Hospital,Shanghai Jiao Tong University School of Medicine
https://orcid.org/0000-0002-9294-
5727 Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital
Weiguo Chen
Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital
Kunwei Shen
Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital
Jiayi Wu
Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital
Li Zhu
(
zhuli8@yeah.net
)
Ruijin Hospital,Shanghai Jiao Tong University School of Medicine
https://orcid.org/0000-0002-9294-
5727 Conclusion Our study found that HER2 low expression couldn’t be a prognostic factor in HR + patients. HER2-low
patients had a higher proportion of ER high expressed tumors than HER2- ones did. The 21-gene assay
and its proliferation module might be less applicable to HER2-low patients compared with HER2- patients. Results The HER2 low tumors had a higher ratio of concurrent estrogen receptor (ER) high expression than HER2-
patients both at protein level (ER > 90%: 78.2% vs 58.6%, p < 0.01) and mRNA level (Spearman R = 0.5 vs
0.3). Analysis about DFS showed no significant difference between HER2 negative and low subgroups (5-
year DFS: 92.3% vs 93.3%, p = 0.83). However, RS range (cut-off: 18 and 30) didn’t maintain its predictive
value in HER2 low patients (p = 0.11) unlike that in HER2- group (p = 0.003). Further research for
respective gene suggested that proliferation related genes performed well in predicting DFS in HER2-
patients but lost its value in HER2 low group (p for interaction < 0.01). Contrarily, higher HER2 module was
associated with worse DFS only in HER2 low patients (p = 0.04). DOI: https://doi.org/10.21203/rs.3.rs-752911/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/19 Background Human epidermal growth factor receptor 2 (HER2) low expressed breast cancer was considered as a
distinct subtype different from HER2 negative tumors. We investigated the clinicopathological features
and recurrence score (RS) of HER2-low and HER2- patients and their prognostic value in hormone
receptor (HR) positive breast cancer. Methods A total of 2,099 HR-positive primary female breast cancer patients between Jan 2009 and Jan 2019 were
collected and tumors with immunohistochemistry 1 + or 2 + with negative in situ hybridization results was
defined as HER2 low. We retrospectively compared the clinical and genetical features of HER2-low (n =
1,732) and HER2- (n = 367) breast cancer and theirs impacts on disease-free survival (DFS). Introduction Human epidermal growth factor receptor 2 (HER2) was an important prognostic and predictive factor for
breast cancer. In general, HER2 positive breast cancer could benefit from anti-HER2 agents such as
trastuzuamb, pertuzumab, lapatinib, etc1–3, while HER2 negative tumors couldn’t. In addition to this
conventional binary classification, evolving evidence has indicated HER2 intratumor and intertumor
heterogeneity4–6. Cases with immunohistochemistry (IHC) 1 + or 2 + with negative in situ hybridization
(ISH) results, which had been categorized as HER2 negative before, were re-classified into a distinct new
subtype: HER2-low breast cancer7. Page 2/19 Page 2/19 About 45%-55% breast cancers were HER2 protein low expression8. So far, studies about these tumors
were insufficient for clinicians to differentiate them from other traditional subtypes. Previous evidence
also contradicted with each other when investigating the survival outcomes of these patients9–12. The
result of NASBP B-47 suggested that HER2-targeted therapy didn’t produce effect on HER2 low breast
cancer13 and this subset was treated as luminal-like or triple negative subtype according to the hormone
receptor (HR) status. However, the recent emergence of novel anti-HER2 agents brought striking benefit to
HER2 low metastatic breast cancer14,15 possibly due to the bystander killing effect16. Therefore, further
investigation might change the pattern of diagnosis and treatment of HER2-low breast cancer. Approximately 50–80% of traditional HER2-negative breast cancer were HR-positive simultaneously11,17. Importantly, previous analysis suggested that the difference between HER2 low expression and HER2
negativity had more clinical impact in HR + breast cancer than in triple negative breast cancer17. In the
past two decades, the 21-gene recurrence-score (RS)18 has provided prognostic information for
HR+/HER2- patients in clinical practice, including HER2-low tumors. Whether low expression of HER2
gene or protein would influence the predictive value of the clinicopathological factors and RS has been
never investigated before. Furthermore, two HER2 amplification related genes, growth factor receptor
bound protein 7 (GRB7) and HER2, were important components of the RS assay, and they might play a
different role in tumors with HER2 low status or complete HER2 negativity. Since HER2-low breast cancer
began to come into sight as a separate entity, it is necessary to re-evaluate the prognostic roles of
traditional features and the use of RS in HR+/HER2-low breast cancer patients. In our study, we attempted to make a comparison of basic clinicopathological factors between HER2-low
and HER2- breast cancer patients. Introduction We also analyzed the prognostic value of the RS for the survival
outcome of these two subgroups. Patients In total, clinical data of 2,099 HR-positive primary female breast cancer patients between Jan 2009 and
Jan 2019 was collected from Shanghai Jiao Tong University Breast Cancer Data Base (SJTU-BCDB). Inclusion criteria were listed as follows: (1) estrogen receptor (ER) positivity with ≥ 1% immunoreactive
tumor cell nuclei determined by IHC test19; (2) detailed IHC and ISH results of HER2; (3) record of a 21-
gene test. The median follow-up time was 50.19 (range 2.54–120.00) months. HER2 Status HER2 negativity was defined as score 0 of IHC. HER2-low expression was defined as IHC 1 + or 2 +
combined with non-amplification of HER2 gene on fluorescence in situ hybridization. According to
American Society of Clinical Oncology (ASCO) guidelines20, HER2 non-amplification was Page 3/19 Page 3/19 HER2/centromeric probe for chromosome 17 (CEP17) ratio < 2.0 with average HER2 gene copy number <
6.0 signals/cell, or average HER2 gene copy number < 4.0 signals/cell regardless of the ratio. HER2/centromeric probe for chromosome 17 (CEP17) ratio < 2.0 with average HER2 gene copy number <
6.0 signals/cell, or average HER2 gene copy number < 4.0 signals/cell regardless of the ratio. 21-gene Assay Testing 21-gene Assay Testing The 21-gene tests were performed using formalin-fixed, paraffin-embedded tissue. Hematoxylin and
eosin-stained slides were deparaffinized into two 10µm unstained sections using xylene followed by
ethanol21. RNA was extracted using the RNeasy FFPE kit (QIAGEN, Hilden, Germany). Total RNA content
was quantified after confirmed the absence of DNA contamination. Then, gene-specific reverse
transcription was conducted followed by standardized quantitative reverse transcriptase-polymerase
chain reactions (RT-PCR) in 96-well plates with Applied Biosystems (Foster City, CA) 7500 Real-Time PCR
system. The PCR cycling went as follows: 95◦C for 10 min for one cycle, 95◦C for 20 s, and 60◦C. We
used 18 and 30 as the cutoff of RS range18. Statistical Analysis We used disease-free survival (DFS) as our endpoint. DFS was defined as the length of time from the first
treatment for breast cancer to the time of first event as follows: local, regional or distant recurrence,
contralateral breast cancer, secondary malignancy or death as a result of any cause. The Kaplan-Meier
survival analysis, univariate and multivariate Cox regression analysis was performed in patients
according to different genetic risks and HER2 expression. All our tests were performed using R Studio
version 1.2.5019 based on R version 4.0.3. Baseline characteristics according to HER2 expression Abbreviation: IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; ER, estrogen receptor;
PR, progesterone receptor; RS, recurrence score; AI, aromatase inhibitor; SERM, selective estrogen
receptor modulator; OFS, ovarian function suppression. Baseline characteristics according to HER2 expression The majority of 2,099 HR positive cases were HER2 low expressed tumor (n = 1,732, 82.5%). No
significant difference was found when comparing the age, menopause status, histological grade,
progesterone receptor (PR status, American Joint Committee on Cancer (AJCC) stage, RS range and
treatment after surgery between HER2-low and HER2 negative subgroups (Table 1). The HER2 negative
subgroup had a higher proportion of specific pathologic types beyond invasive ductal and lobular cancer
(16.1% vs 10.5%, p = 0.01) and tumors with high histologic grade (p = 0.02). We also divided ER IHC
expression into two categories based on the median percentage of 90%. The HER2 low tumors showed a
higher ratio of concurrent ER high expression than HER2 negative patients (78.2% vs 58.6%, p < 0.01). Page 4/19 Table 1
Basic Characteristics of 2,099 HR + breast cancer patients from SJTU-BCDB
Characteristics
HER2 negative
n = 367
HER2 low
n = 1,732
p-value
Age
≤50
>50
110(30.0)
257(70.0)
562(32.4)
1,170(67.6)
0.39
Menopause status
Premenopausal
Postmenopausal
123(33.5)
244(66.5)
607(35.0)
1,125(65.0)
0.61
Pathology
IDC
ILC
others
294(80.1)
14(3.8)
59(16.1)
1,478(85.3)
72(4.2)
182(10.5)
0.01
Grade
I
II
III
Unknown
36(9.8)
173(47.1)
77(21.0)
81(22.1)
147(8.5)
1,027(59.3)
312(18.0)
246(14.2)
0.02
ER positivity(%)
<90
≥90
152(41.4)
215(58.6)
377(21.8)
1355(78.2)
< 0.01
PR
Positive
Negative
313(85.3)
54(14.7)
1,540(88.9)
192(11.1)
0.06
Ki67(%)
<20
≥20
213(58.0)
154(42.0)
951(54.9)
781(45.1)
0.30 Abbreviation: IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; ER, estrogen receptor;
PR, progesterone receptor; RS, recurrence score; AI, aromatase inhibitor; SERM, selective estrogen
receptor modulator; OFS, ovarian function suppression. Page 5/19 Page 5/19 Characteristics
HER2 negative
n = 367
HER2 low
n = 1,732
p-value
pT
1
2–4
262(71.4)
105(28.6)
1,215(70.2)
517(29.8)
0.68
pN
0
1–2
313(85.3)
54(14.7)
1,448(83.6)
284(16.4)
0.47
RS
<18
18–30
>30
83(22.6)
185(50.4)
99(27.0)
361(20.8)
914(52.8)
457(26.4)
0.67
Endocrine therapy
AI(± OFS)
SERM(± OFS)
others
246(67.0)
120(32.7)
1(0.3)
1,114(64.3)
616(35.6)
2(0.1)
0.29
Chemotherapy
No
Yes
196(53.4)
170(46.6)
859(49.6)
872(50.4)
0.19
Radiotherapy
No
Yes
197(53.7)
169(46.3)
903(52.2)
828(47.8)
0.60
Abbreviation: IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; ER, estrogen receptor;
PR, progesterone receptor; RS, recurrence score; AI, aromatase inhibitor; SERM, selective estrogen
receptor modulator; OFS, ovarian function suppression. The survival outcomes of patients according to different
HER2 status Abbreviation: IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; ER, estrogen receptor;
PR, progesterone receptor; RS, recurrence score; AI, aromatase inhibitor; SERM, selective estrogen
receptor modulator; OFS, ovarian function suppression. The survival outcomes of patients according to different
HER2 status A total of 146 DFS events happened during the follow-up period (33 and 123 in HER2 negative and low
subgroups respectively). The Kaplan-Meier curves (Fig. 1) revealed no significant difference between
HER2 negative and low subgroups in HR + patients. We further performed survival analyses according to Page 6/19 Page 6/19 Page 6/19 different RS, and the subpopulation treatment effect pattern plot (STEPP) analysis22 (Fig. 3) showed that
in patients with RS > 30, HER2 low subgroup showed a better DFS than HER2 negative subgroup. We
compared the predictive value of RS in HR + patients with different HER2 status (Fig. 2). Surprisingly, in
HER2 negative patients, the Kaplan-Meier curves showed significant different survival outcomes between
patients with high risk and those with low/intermediate risk (p = 0.003). However, the recurrence score
was not significantly correlated with survival outcomes in HER2 low patients (p = 0.11). Univariate And Multivariate Analysis Of Clinicopathological
Predictors For Dfs We further investigated the impact of clinicopathological factors on DFS in subgroups divided by HER2
status respectively (Table 2). In HER2 negative population, higher pN grade (hazard ratio = 2.59, 95% CI
1.08–6.26, p = 0.03) and RS > 30 (hazard ratio = 2.39, 95% CI 1.14–5.04, p = 0.02) was associated with
worse survival outcomes after adjustment. However, in patients with HER2 low tumors, higher RS and pN
stage lost their prognostic value both in univariate and multivariate analysis. Page 7/19 Table 2
The impact of clinicopathological factors on DFS according to HER2 status
Factors
Univariate Analysis
p-value
Multivariate Analysis
p-value
HER2 negative
Age(y)
>50 vs ≤ 50
1.08(0.51–2.66)
0.85
Menopause
Post vs Pre
1.37(0.65–2.88)
0.42
Grade
III vs I-II
1.67(0.76–3.65)
0.20
ER positivity(%)
≥90 vs < 90
0.46(0.21–1.02)
0.06
0.53(0.23–1.20)
0.13
PR status
Pos vs Neg
0.39(0.18–0.81)
0.01
0.62(0.28–1.39)
0.24
Ki67 positivity(%)
≥20 vs < 20
1.68(0.82–3.43)
0.16
pT
2–4 vs 1
1.99(0.98–4.01)
< 0.01
1.51(0.70–3.24)
0.29
pN
≥ 1 vs 0
2.84(1.24–6.49)
0.01
2.59(1.08–6.26)
0.03
RS
>30 vs ≤ 30
3.10(1.56–6.17)
< 0.01
2.39(1.14–5.04)
0.02
Chemotherapy
No vs Yes
1.44(0.72–2.86)
0.30
HER2 low
Age(y)
>50 vs ≤ 50
0.90(0.62–1.31)
0.60
Menopause
Post vs Pre
0.91(0.63–1.30)
0.60 Abbreviation: ER, estrogen receptor; PR, progesterone receptor; RS, recurrence score; HER2, human
epidermal growth factor receptor 2. Page 8/19 Page 8/19 Factors
Univariate Analysis
p-value
Multivariate Analysis
p-value
Grade
III vs I-II
1.73(1.15–0.62)
0.01
1.30(0.82–2.05)
0.27
ER positivity(%)
≥90 vs < 90
0.66(0.45–0.98)
0.04
0.52(0.23–1.19)
0.12
PR status
Pos vs Neg
0.76(0.47–1.24)
0.27
Ki67 positivity(%)
≥20 vs < 20
1.71(1.20–2.45)
< 0.01
1.47(0.93–2.34)
0.10
pT
2–4 vs 1
2.19(1.54–3.13)
< 0.01
1.39(0.91–2.11)
0.13
pN
≥ 1 vs 0
1.15(0.70–1.91)
0.58
1.20 (0.68–2.10)
0.53
RS
> 30 vs ≤ 30
1.47(1.02–2.12)
0.04
0.86(0.49–1.52)
0.60
Chemotherapy
No vs Yes
1.44(1.00-2.06)
0.05
1.13(0.70–1.81)
0.62
Abbreviation: ER, estrogen receptor; PR, progesterone receptor; RS, recurrence score; HER2, human
epidermal growth factor receptor 2. The distribution of genetic risks and gene expression between HER2 negative and HER2-low subgroups Abbreviation: ER, estrogen receptor; PR, progesterone receptor; RS, recurrence score; HER2, human
epidermal growth factor receptor 2. The distribution of genetic risks and gene expression between HER2 negative and HER2 low subgroups We compared the expression of individual RS genes between HER2 low and negative groups. The results
showed no significant difference of gene expression except for HER2 gene. Univariate And Multivariate Analysis Of Clinicopathological
Predictors For Dfs The level of HER2 gene mRNA
was slightly higher in HER2 low subgroup than in HER2 negative one (Fig. 2). The impact of RS modules on DFS according to HER2 status We further evaluated the predictive roles of separate RS modules and genes in patients according to
HER2 status and observed different patterns (Table 4). Page 9/19 Table 4 The impact of RS modules and genes on DFS according to HER2 status
Factors
HER2-negative
p-value
HER2-low
p-value
pinteraction
ER Module
ER
PgR
Bcl2
SCUBE2
ER Module
1.20(0.88-1.65)
0.84(0.67-1.06)
0.88(0.58-1.32)
0.89(0.70-1.13)
0.84(0.58-1.21)
0.25
0.15
0.53
0.34
0.34
1.01(0.85-1.19)
0.97(0.87-1.08)
0.87(0.71-1.06)
0.97(0.86-1.09)
0.92(0.76-1.11)
0.95
0.55
0.16
0.56
0.38
0.36
0.30
0.67
0.39
0.52
Proliferation Module
Ki67
STK15
Survivin
CCNB1
MYBL2
Prol Module
2.20(1.46-3.30)
1.85(1.26-2.72)
1.72(1.25-2.37)
1.48(0.90-2.44)
1.52(1.15-2.02)
7.22(2.49-20.93)
<0.01
<0.01
<0.01
0.12
<0.01
<0.01
1.02(0.88-1.19)
1.02(0.89-1.17)
1.06(0.89-1.26)
1.19(0.95-1.50)
1.08(0.90-1.28)
0.81(0.40-1.66)
0.80
0.78
0.53
0.14
0.41
0.57
<0.01
<0.01
0.03
0.64
0.12
<0.01
HER2 Module
GRB7
HER2
HER2 Module
1.39(1.04-1.87)
1.02(0.70-1.49)
1.32(0.71-2.48)
0.03
0.91
0.38
1.01(0.82-1.24)
0.99(0.81-1.23)
1.89(1.02-3.48)
0.92
0.98
0.04
0.27
0.76
0.32
Invasion Module
MMP11
CTSL2
Inv Module
1.11(0.83-1.48)
1.39(1.06-1.83)
1.58(1.04-2.38)
0.49
0.02
0.03
1.20(1.04-1.39)
1.08(0.90-1.29)
1.30(1.05-1.61)
0.02
0.42
0.02
0.53
0.30
0.68
GSTM1
1.15(0.82-1.62)
0.43
0.85(0.70-1.02)
0.08
0.32
CD68
1.57(1.02-2.41)
0.04
1.02(0.80-1.30)
0.87
0.20
BAG1
1.13(0.73-1.77)
0.58
1.01(0.82-1.24)
0.96
0.99 The impact of RS modules and genes on DFS according to HER2 status In HER2 negative subgroup, most of the proliferation related genes played important parts in predicting
DFS, as well as the whole module. Although GRB7 in HER2 module acted as a possible predictor of Page 10/19 poorer survival (hazard ratio = 1.39, 95%CI 1.04–1.87, p = 0.03), HER2 mRNA expression and HER2
module didn’t had significant prognostic value. The increased invasion module score was also
associated with worse survival outcome. No significance was association was found between ER module
and DFS. In HER2 low subgroup, the proliferation related genes lost prognostic value. In addition, unlike in the HER2
negative subgroup, the higher score of HER2 module might predict increased risk of DFS events (hazard
ratio = 1.89, 95%CI 1.02–3.48, p = 0.04). The invasion module still served as a negative prognostic factor,
while the correlation between ER related genes and DFS was not obvious in HER2 low as in HER2
negative patients The interaction test for HER2 status and proliferation module showed that there were significant
interactive effects of most proliferation related genes and HER2 status on DFS. Discussion This subtype was found to make up for a dominated proportion
of luminal A/B breast cancer in current data. In addition, our study found its strong correlation with high
ER expression, which corresponded to the hypothesis above. The 21-gene test was one of the most widely accepted assay for making treatment strategies in HR +
breast cancer patients and the HER2-related module was an important part of 21 gene assay. As
expected, the expression of HER2 was higher in HER2 low patients than in HER2 negative patients. Higher
HER2 module also had an association with poor DFS in HER2 low patients, while we failed to find any
predictive value of HER2 gene expression in HER2- group. Of note, almost all of the proliferation related
genes were strong unfavorable prognostic factors only in HER2 negative patients. However, these factors
had no significant predictive value in HER2 low patients. Further analysis for interaction confirmed that
most of proliferation related genes, as well as the whole module, had different prognostic value with
HER2 status changed. An implication of this difference was that the recurrence score might somewhat
amplify the impact of proliferation related genes in HER2 low breast cancer. We conducted survival analysis using RS cutoff points of 18 and 30 in HER2 negative and low patients
respectively to test whether the RS range maintained its prognostic value in HR + patients regardless of
HER2 status. The Kaplan-Meier curve of HER2 negative patients was in concordance with previous
results, which suggested that RS had a good performance as a DFS predictor in this cohort. Meanwhile,
when focusing on HER2 low patients, we noticed that no significant difference was found between these
three subgroups. Further STEPP analysis showed an obvious survival disparity between HER2 low and
negative patients with RS > 30. It should be pointed out that the corresponding coefficient of proliferation
module was the highest among the four modules in the RS algorithm18. We hypothesized that the 21-
gene assay was more applicable to HR+/HER2- patients than to HR+/HER2-low patients since the
proliferation module contributed predominantly to the recurrence score while its predictive value actually
varied according to different HER2 status. A stricter range of RS might compensate for the absence of
proliferation related predictability in HER2 low breast cancer. Our study had several limitations. First, we conducted our study based on retrospective data which meant
that potential bias was unavoidable. Discussion Recently, the outstanding therapeutic effects of novel antibody-drug conjugates (ADCs) 14,15to HER2 low
expressed breast cancer had arouse the interest in this potential new subtype. Our study compared the
clinicopathological characteristics and 21-gene modules of HER2 low and HER2 negative subgroups of
HR + early breast cancer. We found that the expression of HER2 had a positive correlation with ER. Although the distribution of genes and modules was almost similar between these two subgroups, the
proliferation had a better performance when predicting DFS in HER2- patients than in HER2 low patients. Our results provided important suggestion for use of clinicopathological factors and 21-gene assay in
HER2-low patients. The distributions of most of basic clinical and pathological features were similar in HER2 low and
negative patients. However, HER2 low breast cancer constituted a higher proportion of ER high expressed
tumors compared with HER2 negative subgroup. The expression data derived from tests of 21-gene
assay also showed a direct relation between HER2 and ER at the mRNA levels (supple Fig. 1). This was in
consistency with the previous evidence based on HER2 negative patients23. Furthermore, this correlation
was stronger in HER2-low cases than in pure HER2-negative ones (Spearman R = 0.5 vs 0.3). In the analysis of survival outcomes, we noticed the possible protective effect of high ER expression24,25,
which was observed in plenty of studies. The Kaplan-Meier Curve showed no significant difference was
found between HER2 low and negative patients. As a whole, despite that some of the previous findings
demonstrated that moderate HER2 expression might be an unfavorable prognostic factor for survival
outcome9,26, our study showed that HER2 status (low/negative) couldn’t be routinely used as an
independent prognostic factor in HR + breast cancer patients. Two recent studies also discovered that
HER2-low/HR + patients had similar survival outcomes compared with HER2-/HR + patients. Furthermore,
a recent research based on PAM50 subtype demonstrated that instead of low HER2 expression, it was
hormone receptor and its related genes expression that might be the actually dominated oncological Page 11/19 Page 11/19 driver for HER2-low/HR + breast cancer10. This subtype was found to make up for a dominated proportion
of luminal A/B breast cancer in current data. In addition, our study found its strong correlation with high
ER expression, which corresponded to the hypothesis above. driver for HER2-low/HR + breast cancer10. Discussion Secondly, the follow-up time of our patients might be not long
enough for HR + breast cancer. We also limited our analysis to basic molecular subtypes and the 21-gene
assay. It might be better to incorporate the PAM50 subtypes. In addition, in subpopulation analysis, we
used 18 and 30 as cutoff points of RS because the majority of our patients was diagnosed and treated
before the 11/25 cutoff point from the TAILORx study27. Author Contributions: Author Contributions: LZ, JY and MC made study design. DL, WC and KS participated in data acquisition. JW and MC
conducted statistical analysis and manuscript preparation. KS and LZ helped to review the manuscript. All authors read and approved the final manuscript. Competing Interests statement: The authors disclose that no financial support has been received from
any person or organization for this study. Competing Interests statement: The authors disclose that no financial support has been received from
any person or organization for this study. Availability of supporting data: The data used to support the findings of this study are available from the
corresponding author upon request. Conclusion In conclusion, our study found that HER2 low expression couldn’t be used an individual prognostic factor
in HR + patients. HER2-low patients had a higher proportion of ER high expressed tumors than HER2
negative ones did. The 21-gene assay and its proliferation module might be less applicable to HER2-low
patients compared with HER2 negative patients, which should be noticed in clinical practice. Page 12/19 Page 12/19 Declarations Ethics approval and consent to participate: NA
Funding: NA Ethics approval and consent to participate: NA Consent for publication: Written informed consent for publication was obtained from all participants. Acknowledgements: We appreciate all the patients for their participation and study coordinators, nurses,
and physicians for their assistance. Acknowledgements: We appreciate all the patients for their participation and study coordinators, nurses,
and physicians for their assistance. References 1. Cameron D, Piccart-Gebhart MJ, Gelber RD, et al. 11 years' follow-up of trastuzumab after adjuvant
chemotherapy in HER2-positive early breast cancer: final analysis of the HERceptin Adjuvant (HERA)
trial. Lancet. 2017;389(10075):1195-1205.doi:10.1016/S0140-6736(16)32616-2 1. Cameron D, Piccart-Gebhart MJ, Gelber RD, et al. 11 years' follow-up of trastuzumab after adjuvant
chemotherapy in HER2-positive early breast cancer: final analysis of the HERceptin Adjuvant (HERA)
trial. Lancet. 2017;389(10075):1195-1205.doi:10.1016/S0140-6736(16)32616-2 2. von Minckwitz G, Procter M, de Azambuja E, et al. Adjuvant Pertuzumab and Trastuzumab in Early
HER2-Positive Breast Cancer. N Engl J Med. 2017;377(2):122-131.doi:10.1056/NEJMoa1703643 2. von Minckwitz G, Procter M, de Azambuja E, et al. Adjuvant Pertuzumab and Trastuzumab in Early
HER2-Positive Breast Cancer. N Engl J Med. 2017;377(2):122-131.doi:10.1056/NEJMoa1703643 3. Johnston SRD, Hegg R, Im SA, et al. Phase III, Randomized Study of Dual Human Epidermal Growth
Factor Receptor 2 (HER2) Blockade With Lapatinib Plus Trastuzumab in Combination With an
Aromatase Inhibitor in Postmenopausal Women With HER2-Positive, Hormone Receptor-Positive
Metastatic Breast Cancer: ALTERNATIVE. J Clin Oncol. 2018;36(8):741-
748.doi:10.1200/JCO.2017.74.7824 4. Lee HJ, Kim JY, Park SY, et al. Clinicopathologic Significance of the Intratumoral Heterogeneity of
HER2 Gene Amplification in HER2-Positive Breast Cancer Patients Treated With Adjuvant
Trastuzumab. Am J Clin Pathol. 2015;144(4):570-578.doi:10.1309/AJCP51HCGPOPWSCY 4. Lee HJ, Kim JY, Park SY, et al. Clinicopathologic Significance of the Intratumoral Heterogeneity of
HER2 Gene Amplification in HER2-Positive Breast Cancer Patients Treated With Adjuvant
Trastuzumab. Am J Clin Pathol. 2015;144(4):570-578.doi:10.1309/AJCP51HCGPOPWSCY 5. Kurozumi S, Padilla M, Kurosumi M, et al. HER2 intratumoral heterogeneity analyses by concurrent
HER2 gene and protein assessment for the prognosis of HER2 negative invasive breast cancer
patients. Breast Cancer Res Treat. 2016;158(1):99-111.doi:10.1007/s10549-016-3856-2 5. Kurozumi S, Padilla M, Kurosumi M, et al. HER2 intratumoral heterogeneity analyses by concurrent
HER2 gene and protein assessment for the prognosis of HER2 negative invasive breast cancer
patients. Breast Cancer Res Treat. 2016;158(1):99-111.doi:10.1007/s10549-016-3856-2 5. Kurozumi S, Padilla M, Kurosumi M, et al. HER2 intratumoral heterogeneity analyses by concurrent
HER2 gene and protein assessment for the prognosis of HER2 negative invasive breast cancer
patients. Breast Cancer Res Treat. 2016;158(1):99-111.doi:10.1007/s10549-016-3856-2 Page 13/19 Page 13/19 6. Hou Y, Nitta H, Wei L, et al. HER2 intratumoral heterogeneity is independently associated with
incomplete response to anti-HER2 neoadjuvant chemotherapy in HER2-positive breast carcinoma. Breast Cancer Res Treat. 2017;166(2):447-457.doi:10.1007/s10549-017-4453-8 7. Tarantino P, Hamilton E, Tolaney SM, et al. HER2-Low Breast Cancer: Pathological and Clinical
Landscape. J Clin Oncol. 2020;38(17):1951-1962.doi:10.1200/JCO.19.02488 7. Tarantino P, Hamilton E, Tolaney SM, et al. References HER2-Low Breast Cancer: Pathological and Clinical
Landscape. J Clin Oncol. 2020;38(17):1951-1962.doi:10.1200/JCO.19.02488 8. Schalper KA, Kumar S, Hui P, Rimm DL, Gershkovich P. A retrospective population-based comparison
of HER2 immunohistochemistry and fluorescence in situ hybridization in breast carcinomas: impact
of 2007 American Society of Clinical Oncology/College of American Pathologists criteria. Arch
Pathol Lab Med. 2014;138(2):213-219.doi:10.5858/arpa.2012-0617-OA 8. Schalper KA, Kumar S, Hui P, Rimm DL, Gershkovich P. A retrospective population-based comparison
of HER2 immunohistochemistry and fluorescence in situ hybridization in breast carcinomas: impact
of 2007 American Society of Clinical Oncology/College of American Pathologists criteria. Arch
Pathol Lab Med. 2014;138(2):213-219.doi:10.5858/arpa.2012-0617-OA 9. Eggemann H, Ignatov T, Burger E, et al. Moderate HER2 expression as a prognostic factor in hormone
receptor positive breast cancer. Endocr Relat Cancer. 2015;22(5):725-733.doi:10.1530/ERC-15-0335 9. Eggemann H, Ignatov T, Burger E, et al. Moderate HER2 expression as a prognostic factor in hormone
receptor positive breast cancer. Endocr Relat Cancer. 2015;22(5):725-733.doi:10.1530/ERC-15-0335 10. Agostinetto E, Rediti M, Fimereli D, et al. HER2-Low Breast Cancer: Molecular Characteristics and
Prognosis. Cancers (Basel). 2021;13(11).doi:10.3390/cancers13112824 10. Agostinetto E, Rediti M, Fimereli D, et al. HER2-Low Breast Cancer: Molecular Characteristics and
Prognosis. Cancers (Basel). 2021;13(11).doi:10.3390/cancers13112824 11. Schettini F, Chic N, Braso-Maristany F, et al. Clinical, pathological, and PAM50 gene expression
features of HER2-low breast cancer. NPJ Breast Cancer. 2021;7(1):1.doi:10.1038/s41523-020-00208-
2 11. Schettini F, Chic N, Braso-Maristany F, et al. Clinical, pathological, and PAM50 gene expression
features of HER2-low breast cancer. NPJ Breast Cancer. 2021;7(1):1.doi:10.1038/s41523-020-00208-
2 12. Kim MH, Kim GM, Kim JH, et al. Intermediate HER2 expression is associated with poor prognosis in
estrogen receptor-positive breast cancer patients aged 55 years and older. Breast Cancer Res Treat. 2020;179(3):687-697.doi:10.1007/s10549-019-05505-4 12. Kim MH, Kim GM, Kim JH, et al. Intermediate HER2 expression is associated with poor prognosis in
estrogen receptor-positive breast cancer patients aged 55 years and older. Breast Cancer Res Treat. 2020;179(3):687-697.doi:10.1007/s10549-019-05505-4 13. Fehrenbacher L, Cecchini RS, Geyer CE, Jr., et al. NSABP B-47/NRG Oncology Phase III Randomized
Trial Comparing Adjuvant Chemotherapy With or Without Trastuzumab in High-Risk Invasive Breast
Cancer Negative for HER2 by FISH and With IHC 1+ or 2. J Clin Oncol. 2020;38(5):444-
453.doi:10.1200/JCO.19.01455 13. Fehrenbacher L, Cecchini RS, Geyer CE, Jr., et al. NSABP B-47/NRG Oncology Phase III Randomized
Trial Comparing Adjuvant Chemotherapy With or Without Trastuzumab in High-Risk Invasive Breast
Cancer Negative for HER2 by FISH and With IHC 1+ or 2. J Clin Oncol. 2020;38(5):444-
453.doi:10.1200/JCO.19.01455 14. References Modi S, Park H, Murthy RK, et al. Antitumor Activity and Safety of Trastuzumab Deruxtecan in
Patients With HER2-Low-Expressing Advanced Breast Cancer: Results From a Phase Ib Study. J Clin
Oncol. 2020;38(17):1887-1896.doi:10.1200/JCO.19.02318 14. Modi S, Park H, Murthy RK, et al. Antitumor Activity and Safety of Trastuzumab Deruxtecan in
Patients With HER2-Low-Expressing Advanced Breast Cancer: Results From a Phase Ib Study. J Clin
Oncol. 2020;38(17):1887-1896.doi:10.1200/JCO.19.02318 15. Banerji U, van Herpen CML, Saura C, et al. Trastuzumab duocarmazine in locally advanced and
metastatic solid tumours and HER2-expressing breast cancer: a phase 1 dose-escalation and dose-
expansion study. Lancet Oncol. 2019;20(8):1124-1135.doi:10.1016/S1470-2045(19)30328-6 15. Banerji U, van Herpen CML, Saura C, et al. Trastuzumab duocarmazine in locally advanced and
metastatic solid tumours and HER2-expressing breast cancer: a phase 1 dose-escalation and dose-
expansion study. Lancet Oncol. 2019;20(8):1124-1135.doi:10.1016/S1470-2045(19)30328-6 16. van der Lee MM, Groothuis PG, Ubink R, et al. The Preclinical Profile of the Duocarmycin-Based
HER2-Targeting ADC SYD985 Predicts for Clinical Benefit in Low HER2-Expressing Breast Cancers. Mol Cancer Ther. 2015;14(3):692-703.doi:10.1158/1535-7163.MCT-14-0881-T 16. van der Lee MM, Groothuis PG, Ubink R, et al. The Preclinical Profile of the Duocarmycin-Based
HER2-Targeting ADC SYD985 Predicts for Clinical Benefit in Low HER2-Expressing Breast Cancers. Mol Cancer Ther. 2015;14(3):692-703.doi:10.1158/1535-7163.MCT-14-0881-T 17. Denkert C, Seither F, Schneeweiss A, et al. Clinical and molecular characteristics of HER2-low-positive
breast cancer: pooled analysis of individual patient data from four prospective, neoadjuvant clinical
trials. Lancet Oncol. 2021.doi:10.1016/S1470-2045(21)00301-6 17. Denkert C, Seither F, Schneeweiss A, et al. Clinical and molecular characteristics of HER2-low-positive
breast cancer: pooled analysis of individual patient data from four prospective, neoadjuvant clinical
trials. Lancet Oncol. 2021.doi:10.1016/S1470-2045(21)00301-6 18. Paik S, Shak S, Tang G, et al. A multigene assay to predict recurrence of tamoxifen-treated, node-
negative breast cancer. N Engl J Med. 2004;351(27):2817-2826.doi:10.1056/NEJMoa041588 18. Paik S, Shak S, Tang G, et al. A multigene assay to predict recurrence of tamoxifen-treated, node-
negative breast cancer. N Engl J Med. 2004;351(27):2817-2826.doi:10.1056/NEJMoa041588 18. Paik S, Shak S, Tang G, et al. A multigene assay to predict recurrence of tamoxifen-treated, node-
negative breast cancer. N Engl J Med. 2004;351(27):2817-2826.doi:10.1056/NEJMoa041588 Page 14/19 19. Hammond ME, Hayes DF, Dowsett M, et al. American Society of Clinical Oncology/College Of
American Pathologists guideline recommendations for immunohistochemical testing of estrogen
and progesterone receptors in breast cancer. J Clin Oncol. 2010;28(16):2784-
2795.doi:10.1200/JCO.2009.25.6529 20. Wolff AC, Hammond MEH, Allison KH, et al. References Adjuvant Chemotherapy Guided by a 21-Gene Expression
Assay in Breast Cancer. N Engl J Med. 2018;379(2):111-121.doi:10.1056/NEJMoa1804710 27. Sparano JA, Gray RJ, Makower DF, et al. Adjuvant Chemotherapy Guided by a 21-Gene Expression
Assay in Breast Cancer. N Engl J Med. 2018;379(2):111-121.doi:10.1056/NEJMoa1804710 Figures References Human Epidermal Growth Factor Receptor 2 Testing in
Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical
Practice Guideline Focused Update. J Clin Oncol. 2018;36(20):2105-
2122.doi:10.1200/JCO.2018.77.8738 21. Lin C, Wu J, Lin L, et al. A Novel Prognostic Scoring System Integrating Gene Expressions and
Clinicopathological Characteristics to Predict Very Early Relapse in Node-Negative Estrogen Receptor-
Positive/HER2-Negative Breast Cancer. Front Oncol. 2020;10:1335.doi:10.3389/fonc.2020.01335 21. Lin C, Wu J, Lin L, et al. A Novel Prognostic Scoring System Integrating Gene Expressions and
Clinicopathological Characteristics to Predict Very Early Relapse in Node-Negative Estrogen Receptor-
Positive/HER2-Negative Breast Cancer. Front Oncol. 2020;10:1335.doi:10.3389/fonc.2020.01335 22. Lazar AA, Cole BF, Bonetti M, Gelber RD. Evaluation of treatment-effect heterogeneity using
biomarkers measured on a continuous scale: subpopulation treatment effect pattern plot. J Clin
Oncol. 2010;28(29):4539-4544.doi:10.1200/JCO.2009.27.9182 22. Lazar AA, Cole BF, Bonetti M, Gelber RD. Evaluation of treatment-effect heterogeneity using
biomarkers measured on a continuous scale: subpopulation treatment effect pattern plot. J Clin
Oncol. 2010;28(29):4539-4544.doi:10.1200/JCO.2009.27.9182 23. Pinhel I, Hills M, Drury S, et al. ER and HER2 expression are positively correlated in HER2 non-
overexpressing breast cancer. Breast Cancer Res. 2012;14(2):R46.doi:10.1186/bcr3145 23. Pinhel I, Hills M, Drury S, et al. ER and HER2 expression are positively correlated in HER2 non-
overexpressing breast cancer. Breast Cancer Res. 2012;14(2):R46.doi:10.1186/bcr3145 24. Ma H, Lu Y, Marchbanks PA, et al. Quantitative measures of estrogen receptor expression in relation
to breast cancer-specific mortality risk among white women and black women. Breast Cancer Res. 2013;15(5):R90.doi:10.1186/bcr3486 24. Ma H, Lu Y, Marchbanks PA, et al. Quantitative measures of estrogen receptor expression in relation
to breast cancer-specific mortality risk among white women and black women. Breast Cancer Res. 2013;15(5):R90.doi:10.1186/bcr3486 25. Bartlett JM, Brookes CL, Robson T, et al. Estrogen receptor and progesterone receptor as predictive
biomarkers of response to endocrine therapy: a prospectively powered pathology study in the
Tamoxifen and Exemestane Adjuvant Multinational trial. J Clin Oncol. 2011;29(12):1531-
1538.doi:10.1200/JCO.2010.30.3677 25. Bartlett JM, Brookes CL, Robson T, et al. Estrogen receptor and progesterone receptor as predictive
biomarkers of response to endocrine therapy: a prospectively powered pathology study in the
Tamoxifen and Exemestane Adjuvant Multinational trial. J Clin Oncol. 2011;29(12):1531-
1538.doi:10.1200/JCO.2010.30.3677 26. Rossi V, Sarotto I, Maggiorotto F, et al. Moderate immunohistochemical expression of HER-2 (2+)
without HER-2 gene amplification is a negative prognostic factor in early breast cancer. Oncologist. 2012;17(11):1418-1425.doi:10.1634/theoncologist.2012-0194 27. Sparano JA, Gray RJ, Makower DF, et al. Figures Page 15/19 Page 15/19 Figure 1
Association between HER2 status and DFS in HR+ breast cancer patients. Figure 1
Association between HER2 status and DFS in HR+ breast cancer patients. Figure 1 Association between HER2 status and DFS in HR+ breast cancer patients. Association between HER2 status and DFS in HR+ breast cancer patients. Page 16/19 Figure 2
DFS of patients with different genetic risks in HER2 negative (A) and low (B) subgroups. Figure 2 DFS of patients with different genetic risks in HER2 negative (A) and low (B) subgroups. DFS of patients with different genetic risks in HER2 negative (A) and low (B) subgroups. Figure 3
5-year DFS of subpopulation with different HER2 status by median RS Figure 3 Figure 4 The expression of RS genes in HER2 low and negative subgroup respectively. The expression of RS genes in HER2 low and negative subgroup respectively. Figure 3 5-year DFS of subpopulation with different HER2 status by median RS Page 17/19 Page 17/19 Figure 4
The expression of RS genes in HER2 low and negative subgroup respectively. Supplementary Files Page 18/19
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 d Page 18/19
This is a list of supplementary files associated with this preprint. Click to download. supfig1.tif supfig1.tif Page 19/19
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Data from an International Multi-Centre Study of Statistics and Mathematics Anxieties and Related Variables in University Students (the SMARVUS Dataset)
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RSHQSV\FKRORJ\GDWD
-RXUQDORI RSHQSV\FKRORJ\GDWD
-RXUQDORI Data from an International
Multi-Centre Study of
Statistics and Mathematics
Anxieties and Related
Variables in University
Students (the SMARVUS
Dataset) JENNY TERRY
ROBERT M. ROSS
TAMÁS NAGY
MAURICIO SALGADO
PATRICIA GARRIDO-VÁSQUEZ
JACOB O. SARFO
SUSAN COOPER
ANKE C. BUTTNER
TIAGO J. S. LIMA
İBRAHİM ÖZTÜRK
NAZLI AKAY
FLAVIA H. SANTOS
CHRISTINA ARTEMENKO
LEE T. COPPING
MAHMOUD M. ELSHERIF
ILIJA MILOVANOVIĆ
ROBERT A. CRIBBIE
MARINA G. DRUSHLYAK
KATHERINE SWAINSTON
YIYUN SHOU
JUAN DAVID LEONGÓMEZ
NICOLA PALENA
FITRI A. ABIDIN
MARIA F. REYES-RODRÍGUEZ
YUNFENG HE
JUNEMAN ABRAHAM
ARGIRO VATAKIS
KRISTIN JANKOWSKY
STEPHANIE N. L. SCHMIDT
ELISE GRIMM
DESIRÉE GONZÁLEZ
PHILIPP SCHMID
ROBERTO A. FERREIRA
DMITRI ROZGONJUK
NESLIHAN ÖZHAN
PATRICK A. O’CONNOR
ANDRAS N. ZSIDO
GREGOR STIGLIC
DARREN RHODES
CRISTINA RODRÍGUEZ
IVAN ROPOVIK
VIOLETA ENEA
RATRI NURWANTI
ALEJANDRO J. ESTUDILLO
NATALY BERIBISKY
KAREL K. HIMAWAN
LINDA M. GEVEN
ANNE H. VAN HOOGMOED
AMÉLIE BRET
JODIE E. CHAPMAN
UDI ALTER
ZOE M. FLACK
DONNCHA HANNA
MOJTABA SOLTANLOU
GABRIEL BANIK
MATÚŠ ADAMKOVIČ
SANNE H. G. VAN DER VEN
JOCHEN A. MOSBACHER
HILAL H. ŞEN
JOEL R. ANDERSON
MICHAEL BATASHVILI
KRISTEL DE GROOT
MATTHEW O. PARKER
MAI HELMY
MARIIA M. OSTROHA
KATIE A. GILLIGAN-LEE CORRESPONDING AUTHOR:
Jenny Terry
School of Psychology,
University of Sussex, UK
jenny.terry@sussex.ac.uk KEYWORDS: Statistics; mathematics;
anxiety; education; jangle
fallacy Statistics; mathematics;
anxiety; education; jangle
fallacy TO CITE THIS ARTICLE: Terry, J., Ross, R. M., Nagy, T.,
Salgado, M., Garrido-Vásquez,
P., Sarfo, J. O., Cooper, S.,
Buttner, A. C., Lima, T. J. S.,
Öztürk, I˙., Akay, N., Santos, F. H., Artemenko, C., Copping, L. T., Elsherif, M. M., Milovanović,
I., Cribbie, R. A., Drushlyak,
M. G., Swainston, K., Shou, Y.,
Leongómez, J. D., Palena, N.,
Abidin, F. A., Reyes-Rodríguez,
M. F., He, Y., Abraham, J.,
Vatakis, A., Jankowsky, K.,
Schmidt, K., S. N. L., Grimm,
E., González, D., Schmid, P.,
Ferreira, R. A., Rozgonjuk,
D., Özhan, N., O’Connor, P. A., Zsido, A. N., Stiglic, G.,
Rhodes, D., Rodríguez, C.,
Ropovik, I., Enea, V., Nurwanti,
R., Estudillo, A. J., Beribisky,
N., Himawan, K. K., Geven,
L. M., van Hoogmoed, A. H., Bret, A., Chapman, J. E.,
Alter, U., Flack, Z. M., Hanna,
D., Soltanlou, M., Banik, G., D., Özhan, N., O’Connor, P. Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 2 Adamkovič, M., van der
Ven, S. H. G., Mosbacher,
J. A., Şen, H. H., Anderson,
J. R., Batashvili, M., de
Groot, K., Parker, M. O.,
Helmy, M., Ostroha, M. M.,
Gilligan-Lee, K. A., Egara,
F. O., Barwood, M. J.,
Thomas, K., McMahon, G.,
Griffin, S. M., Nuerk, H.-C.,
Counsell, A., Lindemann,
O., Van Rooy, D., Wege, T. E., Lewis, J. E., Aczel, B.,
Monaghan, C., Al-Hoorie,
A. H., Huber, J. F., Yapan,
S., Garrido Vásquez, M. E., Callea, A., Ergiyen, T.,
Clay, J. M., Mertens, G.,
Topçu, F., Tutlu, M. G.,
Täht, K., Mikkor, K., Caso,
L., Karner, A., Storm, M. M. C., Daroczy, G., Zein, R. A., Greco, A., Buchanan,
E. M., Schmid, K., Hunt, T. E., De keersmaecker, J.,
Branney, P. E., Randell, J.,
Clark, O. J., Steltenpohl,
C. N., Malu, B., Tekeş, B.,
Ramis, T., Agrigoroaei,
S., Badcock, N. A.,
McAloney-Kocaman, K.,
Semenikhina, O. V., Graf,
E. W., Lea, C., Ogba, K. T. U., Guppy, F. M., Warhurst,
A. C., Lindsay, S., Al
Khateeb, A., Scharnowski,
F., de Kwaadsteniet, L.,
Francis, K. B., Lecompte,
M., Webster, L. A. D.,
Morsanyi, K., Forwood, S. E., Walters, E. R., Tip, L. K., Wagge, J. R., Lai, H. Y.,
Crossland, D. S., Darda, K. M., Flack, T. R., Leviston,
Z., Brolly, M., Hills, S. P., Collins, E., Roberts,
A. J., Cheung, W.-Y.,
Leonard, S., Verschuere,
B., Stanley, S. K., Xenidou-
Dervou, I., Ghasemi,
O., Liew, T., Ansari, D.,
Guilaran, J., Penny, S. G.,
Bahnmueller, J., Hand,
C. TO CITE THIS ARTICLE: J., Rahajeng, U. W.,
Peterburg, D., Takacs, Z. K., Platow, M. J., & Field,
A. P. (2023). Data from
an International Multi-
Centre Study of Statistics
and Mathematics
Anxieties and Related
Variables in University
Students (the SMARVUS
Dataset). Journal of Open
Psychology Data, 11: 8,
pp. 1–25. DOI: https://doi. org/10.5334/jopd.80 FELIX O. EGARA
MARTIN J. BARWOOD
KARUNA THOMAS
GRACE MCMAHON
SIOBHÁN M. GRIFFIN
HANS-CHRISTOPH NUERK
ALYSSA COUNSELL
OLIVER LINDEMANN
DIRK VAN ROOY
THERESA E. WEGE
JOANNA E. LEWIS
BALAZS ACZEL
CONAL MONAGHAN
ALI H. AL-HOORIE
JULIA F. HUBER
SAADET YAPAN
MAURICIO E. GARRIDO VÁSQUEZ
ANTONINO CALLEA
TOLGA ERGIYEN
JAMES M. CLAY
GAETAN MERTENS
FEYZA TOPÇU
MERVE G. TUTLU
KARIN TÄHT
KRISTEL MIKKOR
LETIZIA CASO
ALEXANDER KARNER
MAXINE M. C. STORM
GABRIELLA DAROCZY
RIZQY A. ZEIN
ANDREA GRECO
ERIN M. BUCHANAN
KATHARINA SCHMID
THOMAS E. HUNT
JONAS DE KEERSMAECKER
PETER E. BRANNEY
JORDAN RANDELL
OLIVER J. CLARK
CRYSTAL N. STELTENPOHL
BHASKER MALU
BURCU TEKEŞ
TAMILSELVAN RAMIS
STEFAN AGRIGOROAEI
OLENA V. SEMENIKHINA
ERICH W. GRAF
CHARLIE LEA
KALU T. U. OGBA
FERGUS M. GUPPY
AMY C. WARHURST
SHANE LINDSAY
AHMED AL KHATEEB
FRANK SCHARNOWSKI
LEONTIEN DE KWAADSTENIET
KATHRYN B. FRANCIS
MARIAH LECOMPTE
LISA A. D. WEBSTER
KINGA MORSANYI
SUZANNA E. FORWOOD
ELIZABETH R. WALTERS
LINDA K. TIP
JORDAN R. WAGGE
HO YAN LAI
DEBORAH S. CROSSLAND
KOHINOOR M. DARDA
TESSA R. FLACK
ZOE LEVISTON
MATTHEW BROLLY
SAMUEL P. HILLS
ELIZABETH COLLINS
ANDREW J. ROBERTS
WING-YEE CHEUNG
SOPHIE LEONARD
BRUNO VERSCHUERE
SAMANTHA K. STANLEY
IRO XENIDOU-DERVOU
OMID GHASEMI
TIMOTHY LIEW
DANIEL ANSARI
JOHNREV GUILARAN
SAMUEL G. PENNY
JULIA BAHNMUELLER
CHRISTOPHER J. HAND
UNITA W. RAHAJENG
DAR PETERBURG
ZSOFIA K. TAKACS
MICHAEL J. PLATOW
A
V
J. J. G
H
G
F. T
G
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A
S. E
C
To
Tä
L. M
A
E
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R
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Se
E
U
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Z
P
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B
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a
A
V
St FELIX O. EGARA
MARTIN J. BARWOOD
KARUNA THOMAS
GRACE MCMAHON
SIOBHÁN M. GRIFFIN
HANS-CHRISTOPH NUERK
ALYSSA COUNSELL
OLIVER LINDEMANN
DIRK VAN ROOY
THERESA E. WEGE
JOANNA E. LEWIS
BALAZS ACZEL
CONAL MONAGHAN
ALI H. AL-HOORIE
JULIA F. HUBER
SAADET YAPAN
MAURICIO E. GARRIDO VÁSQUEZ
ANTONINO CALLEA
TOLGA ERGIYEN
JAMES M. CLAY
GAETAN MERTENS
FEYZA TOPÇU
MERVE G. (1) BACKGROUND statistics anxiety is related to mathematics anxiety, but
some variance remains unaccounted for, suggesting a
unique component (r = 0.41 to r = 0.67; Baloğlu, 2002;
Birenbaum & Eylath, 1994; Paechter et al., 2017; Zeidner,
1991). What this unique component is remains unclear. It is possible the unexplained variance does not reflect
differences in statistics and mathematics anxieties,
but differences in the scales’ dimensions. For example,
because the Statistics Anxiety Rating Scale (STARS; Cruise
et al., 1985) includes a “fear of asking for help” subscale
and the Revised Maths Anxiety Rating Scale (R-MARS;
Baloğlu & Zelhart, 2007) does not, the unique variance
may have been driven by the fear of asking for help only
captured by the STARS. Many
university
students
on
non-mathematics-
based degrees report feeling anxious about learning
mathematics and statistics (e.g., Field, 2014). Statistics
anxiety was initially assumed to be the same as
mathematics anxiety, but many now consider it distinct
(Chew & Dillon, 2014). Statistics anxiety has been
defined as “a negative state of emotional arousal
experienced by individuals as a result of encountering
statistics in any form and at any level [...] and is related
to but distinct from mathematics anxiety” (Chew &
Dillon, 2014, p. 199). Mathematics anxiety is similarly
defined as involving “feelings of tension and anxiety
that interfere with the manipulation of numbers and
the solving of mathematical problems in [...] ordinary
life and academic situations” (Richardson & Suinn, 1972,
p. 551). Neither definition is clear about how these two
constructs differ, and students may perceive them to
be the same because both mathematics and statistics
involve the manipulation and interpretation of numerical
information. This conflation could be a shared root of
students’ anxiety, rather than their anxiety being specific
to mathematics or statistics. It is important to use a range of methods to study the
constructs’ independence, such as various confirmatory
factor analysis techniques (Lawson & Robins, 2021),
extrinsic convergent validity analysis (Gonzalez et al.,
2020), and multi-trait-multi-method designs (Campbell
& Fiske, 1959). However, previous studies that compared
measures of mathematics and statistics anxiety (e.g.,
Baloğlu, 2002; Paechter et al., 2017) have based their
conclusions on correlations, which are only one of the
10 criteria that can determine the extent that two scales
overlap (Lawson & Robins, 2021). These definitions have informed the scales that
measure these constructs (Baloğlu & Zelhart, 2007; Cruise
et al., 1985). ABSTRACT This large, international dataset contains survey responses from N = 12,570
students from 100 universities in 35 countries, collected in 21 languages. We
measured anxieties (statistics, mathematics, test, trait, social interaction,
performance, creativity, intolerance of uncertainty, and fear of negative
evaluation), self-efficacy, persistence, and the cognitive reflection test, and
collected demographics, previous mathematics grades, self-reported and official
statistics grades, and statistics module details. Data reuse potential is broad,
including testing links between anxieties and statistics/mathematics education
factors, and examining instruments’ psychometric properties across different
languages and contexts. Data and metadata are stored on the Open Science
Framework website [https://osf.io/mhg94/]. TO CITE THIS ARTICLE: TUTLU
KARIN TÄHT
KRISTEL MIKKOR
LETIZIA CASO
ALEXANDER KARNER
MAXINE M. C. STORM
GABRIELLA DAROCZY
RIZQY A. ZEIN
ANDREA GRECO
ERIN M. BUCHANAN
KATHARINA SCHMID
THOMAS E. HUNT
JONAS DE KEERSMAECKER
PETER E. BRANNEY
JORDAN RANDELL
OLIVER J. CLARK
CRYSTAL N. STELTENPOHL
BHASKER MALU
BURCU TEKEŞ
TAMILSELVAN RAMIS
STEFAN AGRIGOROAEI
OLENA V. SEMENIKHINA
ERICH W. GRAF
CHARLIE LEA
KALU T. U. OGBA
FERGUS M. GUPPY
AMY C. WARHURST
SHANE LINDSAY
AHMED AL KHATEEB
FRANK SCHARNOWSKI
LEONTIEN DE KWAADSTENIET
KATHRYN B. FRANCIS
MARIAH LECOMPTE
LISA A. D. WEBSTER
KINGA MORSANYI
SUZANNA E. FORWOOD
ELIZABETH R. WALTERS
LINDA K. TIP
JORDAN R. WAGGE
HO YAN LAI
DEBORAH S. CROSSLAND
KOHINOOR M. DARDA
TESSA R. FLACK
ZOE LEVISTON
MATTHEW BROLLY
SAMUEL P. HILLS
ELIZABETH COLLINS
ANDREW J. ROBERTS
WING-YEE CHEUNG
SOPHIE LEONARD
BRUNO VERSCHUERE
SAMANTHA K. STANLEY
IRO XENIDOU-DERVOU
OMID GHASEMI
TIMOTHY LIEW
DANIEL ANSARI
JOHNREV GUILARAN
SAMUEL G. PENNY
JULIA BAHNMUELLER
CHRISTOPHER J. HAND
UNITA W. RAHAJENG
DAR PETERBURG
ZSOFIA K. TAKACS
MICHAEL J. PLATOW Thomas, K., McMahon, G.,
Griffin, S. M., Nuerk, H.-C., Counsell, A., Lindemann, O., Van Rooy, D., Wege, T. E., Lewis, J. E., Aczel, B., Monaghan, C., Al-Hoorie, A. H., Huber, J. F., Yapan, M. C., Daroczy, G., Zein, R. A., Greco, A., Buchanan, E. M., Schmid, K., Hunt, T. E., De keersmaecker, J., Branney, P. E., Randell, J., Clark, O. J., Steltenpohl, C. N., Malu, B., Tekeş, B., Ramis, T., Agrigoroaei, McAloney-Kocaman, K., Khateeb, A., Scharnowski, F., de Kwaadsteniet, L., Francis, K. B., Lecompte, M., Webster, L. A. D., Morsanyi, K., Forwood, S. E., Walters, E. R., Tip, L. K., Wagge, J. R., Lai, H. Y., Crossland, D. S., Darda, K. M., Flack, T. R., Leviston, Z., Brolly, M., Hills, S. P., Collins, E., Roberts, 3 Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 3 2.1 STUDY DESIGN The data were collected via a cross-sectional, online,
self-report questionnaire-based, multi-centre study. The final dataset was generated from the following
three sources (see Section 2.5 for full details of each
variable): However, construct validation work should be
conducted for all populations that use a given measure
(Flake, 2021) and with statistics being a required module1
for undergraduate students of most social and physical
sciences in universities throughout the world (Schwab-
McCoy, 2019), the extent to which these findings are
generalisable should be examined. 1) The student survey, containing survey responses
from university students on measures of statistics
and mathematics anxieties (including the modified
versions from Terry et al., 2023), test anxiety,
trait anxiety, fear of negative evaluation, social
interaction anxiety and performance anxiety,
intolerance of uncertainty, creativity anxiety, self-
efficacy, persistence, analytical thinking, and belief
in God/s. Students also provided demographic
information (age, gender/sex, ethnicity, and any
specific learning difficulties), information about
their pre-university mathematics qualifications
(highest level, grades, and how long ago they
were taken), self-reported grades for completed
statistics modules, and their degree course details
(university, major, year of study, and whether they
are studying any non-statistics mathematics-
based modules). Student survey data also include
selected information auto-recorded by Qualtrics
(Qualtrics, Provo, UT; the start and end dates,
duration, and completion percentage for each
response) and key identifiers added by the lead
author (participant ID, survey ID, country, and
language). 1) The student survey, containing survey responses
from university students on measures of statistics
and mathematics anxieties (including the modified
versions from Terry et al., 2023), test anxiety,
trait anxiety, fear of negative evaluation, social
interaction anxiety and performance anxiety,
intolerance of uncertainty, creativity anxiety, self-
efficacy, persistence, analytical thinking, and belief
in God/s. Students also provided demographic
information (age, gender/sex, ethnicity, and any
specific learning difficulties), information about
their pre-university mathematics qualifications
(highest level, grades, and how long ago they
were taken), self-reported grades for completed
statistics modules, and their degree course details
(university, major, year of study, and whether they
are studying any non-statistics mathematics-
based modules). Student survey data also include
selected information auto-recorded by Qualtrics
(Qualtrics, Provo, UT; the start and end dates,
duration, and completion percentage for each
response) and key identifiers added by the lead
author (participant ID, survey ID, country, and
language). (2) METHODS results suggested a jangle fallacy. Specifically, the
factor analyses and latent profile analyses of the four
measures, as well as their experimental studies, found
converging evidence that the scales were measuring
the same construct. (1) BACKGROUND For these scales to be valid, we need clarity
about whether they measure facets of anxiety specific
to statistics/mathematics or reflect a common numeric
anxiety. In short, we must rule out a jangle fallacy, where
two scales are incorrectly assumed to measure different
constructs (Kelley, 1927). Jangle fallacies can lead to
independently evolving theoretical literatures for each
construct that should instead be mutually informative
(Block, 1995). To address these concerns, Terry et al. (2023) explored
these constructs’ distinctiveness in two samples of
UK-based undergraduate psychology students (N =
465 and N = 245). They measured statistics anxiety
with the STARS (Cruise et al., 1985) and mathematics
anxiety with the R-MARS (Baloğlu & Zelhart, 2007), and
developed versions of each scale modified to reflect
the other construct (i.e., a mathematics version of the
STARS and a statistics version of the R-MARS). By doing
so, Terry et al. (2023) created equivalent, comparable
subscales (e.g., there was now a mathematics version
of the “Fear of asking for help” subscale). Their Few studies have tested the distinctiveness of statistics
and mathematics anxiety scales. Most concluded Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 4 (2) METHODS 2.1 STUDY DESIGN Therefore, the first aim of the present study was to
assess generalisability by repeating Terry et al.’s (2023)
study in a large, international sample of university
students from different academic disciplines for whom
statistics was part of their degrees. Our second aim was to explore whether specific facets
of the STARS and R-MARS are driven by a superordinate
parent construct (Lawson & Robins, 2021). For example,
scores on the scales’ test anxiety items might be driven
by general test anxiety, and not specific to mathematics
or statistics tests. Therefore, we added further measures
of fear of negative evaluation (Carleton et al., 2011),
intolerance of uncertainty (Carleton et al., 2007), social
interaction and performance anxiety (Baker et al., 2002;
Liebowitz, 1987), creativity anxiety (Daker et al., 2020),
test anxiety (Benson & El‐Zahhar, 1994), and trait anxiety
(Ree et al., 2008) to assess whether they underpin STARS
and R-MARS items. Our third aim was to examine the constructs’ extrinsic
convergent validity (ECV; the extent two measures
correlate with other constructs in the same ways;
Gonzalez et al., 2020). The more similar the correlations
are, the more probable it is that the measures are
tapping the same construct (Gonzalez et al., 2020). To
examine ECV, we included five additional variables
shown to correlate with statistics and/or mathematics
anxieties: Self-efficacy (e.g., Z = |0.52|; Trassi et al.,
2022), persistence (e.g., r = –.75; González et al., 2016),
analytic thinking (using a revised version of the Cognitive
Reflection Test; CRT; Shenhav et al., 2012),2 pre-university
mathematics qualifications (e.g., r = –.27; Beurze et al.,
2013), and university statistics module grades (although,
this relationship varies from r = –.56 to r = .10; Terry &
Field, 2023). 2) An instructor survey, containing information about
the statistics modules students were taking at
the time of completing the survey. The instructor
survey recorded dates of the student participants’
statistics module, mode of teaching delivery (e.g.,
lectures/workshops, online/face-to-face), module
content, and types and dates of assessments. Instructors also indicated how assessments were
graded, necessary to standardise grades across
institutions. 3) Students’ grade data from university records (where
permitted to obtain and share). 2.2 TIME OF DATA COLLECTION Besides our core aims, we maximised the reuse
potential of this dataset – particularly its capacity to
address important questions in the statistics education
literature (see Section 4 – Reuse Potential) – by
collecting data from the student participants’ statistics
instructors about their module format, content, and
assessment. Data
were
collected
between
January
2021
and September 2021.3 Due to the differences in
term/semester dates cross-nationally, different research
teams had different start and end dates. The date
participants began and finished the survey is included in
the dataset. Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 5 5 2.3 LOCATION OF DATA COLLECTION 2.3 LOCATION OF DATA COLLECTION 2.3 LOCATION OF DATA COLLECTION
COUNTRY (ISO CODE; N)
LANGUAGE
UNIVERSITY
N
Australia (AU; N = 315)
English
Macquarie University
237
English
Australian National University
53
English
University of Western Australia
25
Austria (AT; N = 230)
German
University of Vienna
120
German
University of Graz
108
German
Medical University of Graz
1
German
Technical University of Graz
1
Belgium (BE; N = 184)
French
Catholic University of Louvain (UCLouvain)
184
Brazil (BR; N = 68)
Portuguese
University of Brasília
58
Portuguese
UNESP – São Paulo State University
10
Canada (CA; N = 986)
English
Toronto Metropolitan University (formerly Ryerson University)
520
English
York University
228
English
Memorial University of Newfoundland
127
English
Western University
111
Chile (CL; N = 191)
Spanish
Andrés Bello National University
98
Spanish
University of Concepción
93
China (CN; N = 323)
Chinese
Tianjin Normal University
196
Chinese
Qufu Normal University
127
Colombia (CO; N = 114)
Spanish
El Bosque University
113
Spanish
Other (unspecified)
1
Egypt (EG; N = 1,390)
Arabic
Menoufia University
1,390
Estonia (EE; N = 98)
Estonian
University of Tartu
91
Estonian
Tallinn University
7
France (FR; N = 248)
French
University of Nantes
248
Germany (DE; N = 506)
German
University of Erfurt
231
German
University of Konstanz
114
German
University of Tübingen
110
German
University of Kassel
50
German
International University of Applied Sciences
1
(C
d ) Terry et al. 2.3 LOCATION OF DATA COLLECTION Journal of Open Psychology Data DOI: 10.5334/jopd.80 6 y
f
p
y
gy
j p
COUNTRY (ISO CODE; N)
LANGUAGE
UNIVERSITY
N
Ghana (GH; N = 41)
English
University of Education, Winneba
19
English
University of Cape Coast
9
English
University of Ghana
7
English
All Nations University
2
English
Kwame Nkrumah University of Science and Technology
1
English
Other (unspecified)
1
English
University of Health and Allied Sciences
1
English
University of Professional Studies, Accra
1
Greece (GR; N = 99)
Greek
Panteion University
94
Greek
Aristotle University of Thessaloniki
2
Greek
National and Kapodistrian University of Athens
2
Greek
University of Crete
1
Hungary (HU; N = 206)
Hungarian
ELTE Eötvös Loránd University
184
Hungarian
University of Pécs
22
India (IN; N = 41)
English
CHRIST (deemed to be) University
41
Indonesia (ID; N = 697)
Bahasa Indonesia
Bina Nusantara University
223
Bahasa Indonesia
Brawijaya University
171
Bahasa Indonesia
Airlangga University
131
Bahasa Indonesia
Pelita Harapan University
96
Bahasa Indonesia
Padjadjaran University
62
Bahasa Indonesia
Atma Jaya Catholic University of Indonesia
14
Ireland (IE; N = 82)
English
University of Limerick
60
English
University College Dublin
22
Israel (IL; N = 285)
Hebrew
Reichman University (née Interdisciplinary Center Herzliya)
285
Italy (IT; N = 248)
Italian
University of Bergamo
176
Italian
LUMSA University
72
Malaysia (MY; N = 369)
English
HELP University
369
Netherlands (NL; N = 508)
Dutch
Radboud University
165
Dutch
Tilburg University
133
English
University of Amsterdam
114
Dutch
Erasmus University Rotterdam
96
Nigeria (NG; N = 255)
English
University of Nigeria
255
Philippines (PH; N = 47)
English
University of the Philippines Visayas
47 Terry et al. 2.3 LOCATION OF DATA COLLECTION Journal of Open Psychology Data DOI: 10.5334/jopd.80 7 COUNTRY (ISO CODE; N)
LANGUAGE
UNIVERSITY
N
Poland (PO; N = 69)
Polish
University of Silesia
58
Polish
WSB University, Poznan
11
Romania (RO; N = 317)
Romanian
Alexandru Ioan Cuza University
317
Saudi Arabia (SA; N = 100)
Arabic
King Faisal University
100
Serbia (RS; N = 117)
Serbian
University of Novi Sad
117
Slovakia (SL; N = 88)
Slovakian
University of Prešov
88
Slovenia (SL; N = 94)
Slovenian
University of Maribor
94
Spain (ES; N = 346)
Spanish
University of La Laguna
218
English
ESADE Business School, Universitat Ramon Llull
128
Turkey (TR; N = 834)
Turkish
Hasan Kalyoncu University
339
Turkish
MEF University
160
Turkish
Baskent University
158
Turkish
Izmir University of Economics
100
Turkish
Middle East Technical University
77
UK (GB; N = 2,962)
English
University of Sussex
413
English
University of Birmingham
363
English
Bournemouth University
214
English
Nottingham Trent University
202
English
University of Southampton
163
English
Kingston University
157
English
Queen’s University Belfast
137
English
Loughborough University
134
English
University of Stirling
125
English
University of Lincoln
124
English
University of Hull
123
English
University of Portsmouth
116
English
University of Winchester
107
English
University of Brighton
99
English
University of Surrey
99
English
Teesside University
90
English
University of Derby
90
English
Glasgow Caledonian University
60
English
University of Bradford
56
English
Anglia Ruskin University
36
English
Manchester Metropolitan University
32
English
Leeds Trinity University
22
(C
d ) Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 8 COUNTRY (ISO CODE; N)
LANGUAGE
UNIVERSITY
N
Ukraine (UA; N = 25)
Ukrainian
Sumy Makarenko State Pedagogical University
25
USA (US; N = 87)
English
University of Southern Indiana
51
English
University of Northern Colorado
33
English
Avila University
3
Total
12,570
Table 1 A table detailing the universities data were collected from, the country they were in,4 associated survey language, and the
number of responses at the country and university level (after exclusions). English
University of Southern Indiana
51
English
University of Northern Colorado
33
English
Avila University
3
Total
12,570
Table 1 A table detailing the universities data were collected from, the country they were in,4 associated survey language, and the
number of responses at the country and university level (after exclusions). Student Survey Convenience sampling was used to recruit student
participants. Most students (at least 80.4%)5 were invited
to take part by collaborating researchers (or those with
access to the sample on researchers’ behalf, such as
statistics module instructors) via email, virtual learning
environments, university-specific student social media
platforms, and university participant pools6 and took
part in their own time. Some students (at least 2.9%)
were invited to complete the survey as part of an in-
class exercise. Participation was always voluntary, and
students were able to withdraw during the study and
up to four weeks after taking part. The study was hosted
via Qualtrics online survey software (Qualtrics, Provo, UT)
and students completed it using a suitable electronic
device (e.g., laptop, mobile phone, or tablet)7 with
internet access. Participants’ ages ranged from 18 to 67 years
(M = 21.01, SD = 4.12); with 3,119 participants choosing
not to respond to this question and 14 values (≥99 years)
recoded as implausible. The majority of participants
identified as8 a woman/female (n = 8,298, 66.0%), with
a further 2,002 identifying as a man/male (15.9%), 74
as non-binary (0.6%), 13 preferred to describe their
gender in another way (0.1%),9 and 2,183 (17.4%)
did not respond to this question. Most participants
(n = 9,026, 71.8%) reported they did not have a diagnosis
of any of the following Specific Learning Differences
(SpLDs): ADHD/ADD, Dyslexia, Dyscalculia, Dyspraxia,
or Dysgraphia/Dysorthography. However, 738 (5.9%)
participants reported having one or more SpLD, whilst
a further 111 (0.8%) responded “other” (including self-
diagnosis), 3 were unsure (<0.1%), and 2,692 (21.4%) did
not respond. A total of N = 18,841 student survey responses
were recorded. For the present version of the data,
we have excluded any cases where the participant
began the survey but withdrew before the first block of
measurement scale items; n = 6,199) and any duplicates
(n = 72), which were identified using a combination
of participant-generated ID code and demographic
responses. In line with our pre-registration (https://osf. io/xs5wf), the case with the most complete data was
retained, or, if both cases contained the same amount
of data, the case with the earliest start date (i.e., the
participant’s first attempt) was retained. In line with
our goal to provide the data in as raw a form as possible,
we have not excluded any other data. 2.3 LOCATION OF DATA COLLECTION Figure 1 The top panel is a map showing the countries from which data were collected and their respective sample sizes. The bottom
panel is a treemap of sample sizes for each country, organised by continents (see Table 1 for ISO country codes). Total Table 1 A table detailing the universities data were collected from, the country they were in,4 associated survey language, and the
number of responses at the country and university level (after exclusions). Figure 1 The top panel is a map showing the countries from which data were collected and their respective sample sizes. The bottom
panel is a treemap of sample sizes for each country, organised by continents (see Table 1 for ISO country codes). Figure 1 The top panel is a map showing the countries from which data were collected and their respective sample sizes. The bottom
panel is a treemap of sample sizes for each country, organised by continents (see Table 1 for ISO country codes). Figure 1 The top panel is a map showing the countries from which data were collected and their respective sample sizes. The bottom
panel is a treemap of sample sizes for each country, organised by continents (see Table 1 for ISO country codes). 9 Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 2.4 SAMPLING, SAMPLE AND DATA
COLLECTION have included these responses in the present dataset
to afford other researchers the opportunity to set their
own exclusion criteria. Similarly, the pre-registration for
the primary empirical study (https://osf.io/xs5wf) states
that we would only retain responses that passed the
attention checks, but we have not removed them in the
present data (n = 8,597 responded to and passed all
seven; see Quality Control for more details). Collaborating research teams were recruited via
Twitter and word-of-mouth, with efforts made to invite
researchers from geographically and culturally diverse
countries with varying education systems to produce
more generalisable results. In the end, data were
collected from 100 universities in 35 countries and in 21
languages. After exclusions, the final sample presented here
contains n = 12,570 responses (68.2% of initial
responses). Table 1 contains a breakdown of the number
of responses from each university and Figure 1 offers a
visual summary of how sample sizes varied worldwide. Student Survey Note that for
our primary research study, we planned to recruit
undergraduate students that had taken or were taking
statistics as part of their research methods training
on any degree course that is not typically associated
with mathematics. For example, we would exclude
courses such as physics, engineering, and data science,
whilst courses such as social sciences, business, and
geography were eligible. Despite this stipulation, some
responses were received from postgraduate students
(n = 3), and from those on mathematics and statistics
degrees (n = 2), mathematics-adjacent degrees (e.g.,
physics, engineering, computer sciences; n = 151), and
degrees that are unlikely to have included a statistics
module (e.g., arts & humanities subjects; n = 232). We Most participants indicated they were in the first
year of their degree course (n = 4,505, 35.8%), with a
further 3,126 in second year (24.9%), 1,859 in third
year (14.8%), 689 in fourth year (5.5%), and 40 in
fifth year (0.3%). An additional 61 participants (0.5%)
indicated their degree year as ‘other’, three participants
(0.02%) were postgraduates, and 2,287 (18.2%) did not
respond. Psychology was the most common degree
major amongst participants (n = 8,759, 69.7%), followed
by Business and Finance (n = 768, 6.1%), Education
(n = 397, 3.2%), Health and Medical Sciences (n = 273,
2.2%), and Computer Sciences (n = 128, 1.0%).10 A further
1,526 (12.1%) of students did not indicate their degree
major. Each university provided their own participation
incentives based on local norms and availability. Half
of participants were offered ungraded course credits
(50.0%) and around a third were offered no incentive
(33.0%), with the remaining being offered either a prize
draw (up to a maximum of £50 or local equivalent per
100 participants; 10.0%), payment (maximum £5 or local
equivalent; 3.1%), a choice of a prize draw or course
credits (3.3%), or both payment and course credits
(0.5%). Incentive information is unavailable for 0.1% of
participants. Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 10 education locally), and the demographic questions
(e.g., adapting the ethnicity options to reflect local
populations). Researchers could also adapt it to award
participant incentives (e.g., linking to local course credit
systems). The measurement scales were not altered,
with minor exceptions (detailed in the Measures section
below). Student Survey Where permitted by the student participants and by
their universities, we also collected grades (and grading
scales) for the statistics module students were taking
at the time of completing the survey from university
records.11 A total of N = 20 universities provided this data,
corresponding to n = 1,804 student participants in n = 41
modules in n = 9 countries. Statistics Anxiety. Statistics anxiety was measured by
the Statistics Anxiety Rating Scale (STARS; Cruise et al.,
1985). The three anxiety subscales (Hanna et al., 2008;
Papousek et al., 2012) of the STARS were used (23 items
in total): test and class anxiety (8 items), interpretation
anxiety (11 items), and fear of asking for help (4 items). Each item describes a situation involving statistics such
as “Doing an examination in a statistics course” (test and
class anxiety), “Interpreting the meaning of a table in a
journal article” (interpretation anxiety), or “Going to ask
my statistics teacher for individual help with material I
am having difficulty understanding” (fear of asking for
help). Participants were asked to indicate how much
anxiety they feel in those situations on a Likert scale
ranging from 1 = “no anxiety” to 5 = “a great deal of
anxiety”.12 Student Survey Very rarely, and where it did not impact our core
research aims, questions were removed altogether to
meet the requirements of the local ethics boards and/
or to be appropriate in the local context (e.g., some
ethics boards requested we did not ask about ethnicity). Data that are missing from the student survey because
a question was omitted is recorded in the data as ‘Not
Administered’. Instructor Survey The student participants’ statistics module instructors
were invited to take part by email (either by the lead
researcher, where the collaborating researchers were also
module instructors, or by the collaborating researchers
where there were not). In some cases, someone other
than the primary instructor completed the survey
(e.g., graduate teaching assistants). Participation was
voluntary and not a condition of involvement with
the project. The instructor survey was also hosted via
Qualtrics online survey software (Qualtrics, Provo, UT). A total of N = 176 instructor survey responses were
recorded. We have excluded responses given in error (e.g.,
for a postgraduate course or for more than one module
per response; n = 21) and any cases where no data was
entered (n = 36). The instructor survey was not adapted, and all
respondents took the same English-language version of
the survey. After exclusions, the final sample contained n = 119
responses (67.6% of initial responses), representing
n = 96 modules in n = 57 universities in n = 27
countries, corresponding to n = 4,867 student survey
responses. All
materials,
including
copies
of
all
adapted/modified
surveys
are
available
on
the
project’s OSF page (https://osf.io/3bmqz/). 2.5 MATERIALS/SURVEY INSTRUMENTS
Survey Adaptations The student survey was prepared in stages. First, a
generic master version of the survey was created in
English by the lead researcher (available here: https://osf. io/enc29). This version was then adapted from English
into the local language by collaborating research teams
as required, resulting in a generic master version for each
language. A translation guide was provided (available
here: https://osf.io/v3qxf), which advised translators to
adopt a team-based approach (Behr & Shishido, 2016). This approach involved a minimum of two people
translating the scales individually and resolving any
differences as a team. It was chosen over the more
widespread back-translation technique, because it is
more effective in producing equivalent scales across
languages (Behr, 2017). Several items use outdated language and were
modified to reflect modern equivalents (e.g., “Asking
one of my teachers for help in understanding a printout”
was changed to “Asking one of my teachers for help in
understanding statistical output”). These modifications
are the same as those made in Terry et al. (2023). An attention check was also included in this scale,
which asked participants to “Please select ‘1 – no anxiety’
for this question”. The generic master version for each language was
then copied for each research team for modification
to the local context, following guidelines provided to
encourage consistency (available here: https://osf.io/
t2pc5). Modifications were kept minimal and primarily
pertained to course/module details (e.g., the names of
the statistics modules), the mathematics education
questions (e.g., to reflect the structure of pre-university Mathematics Anxiety. Mathematics anxiety was
measured with the Revised Mathematics Anxiety Rating
Scale (R-MARS; Baloğlu & Zelhart, 2007). There are three
subscales in the R-MARS which measure mathematics
test anxiety (15 items), numerical task anxiety (5 original
items plus 4 modified items – see the Modified STARS
and R-MARS section below for modification details),
and mathematics course anxiety (5 items). Each item Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 11 onto separate factors due to differences in perceived
complexity, we re-modified the numerical task anxiety
items and added these to the present version (as
well as the original modifications, for comparison). 2.5 MATERIALS/SURVEY INSTRUMENTS
Survey Adaptations The trait subscale is further broken down into cognitive
(10 items) and somatic symptoms (11 items). Cognitive
symptoms are measured with statements such as
“I cannot concentrate without irrelevant thoughts
intruding” and somatic symptoms are measured with
statements such as “My heart beats fast”. Participants
are asked to indicate the extent to which each item is
true of them on a Likert scale ranging from 1 = “not at all”
to 4 = “very much so”. Three items in the original STARS were not easily
distinguishable as being about either mathematics or
statistics so equivalent items were not created (“Arranging
to have a body of data put into the computer”, “Reading
an advertisement for a car which includes figures on
miles per gallon, depreciation, etc.”, and “Trying to
understand the odds in a lottery”). Additionally, one item
on the original R-MARS was deemed untranslatable to a
statistics context so, again, an equivalent was not created
(“Reading a cash register receipt after your purchase”). These items are, therefore, identical to the original scales. When creating composites of the STARS-M and the
R-MARS-S, the original items should be included instead
so that both modified scales have the same number of
items as their originals (23 for the STARS-M and 20 for
the R-MARS-S). Test Anxiety. Test anxiety was measured with the
Revised Test Anxiety Scale (R TAS; (Benson & El‐Zahhar,
1994). The scale contains four subscales: 7 worry items
(e.g., “During tests I find myself thinking about the
consequences of failing”), 6 tension items (e.g., “During
tests I feel very tense”), 5 test-irrelevant thinking items
(e.g., “During tests I find I am distracted by thoughts of
upcoming events”), and 7 bodily symptoms items (e.g., “I
get a headache during an important test”). We included
5 items later removed by Benson and El-Zahhar (1994) to
form a 20-item scale, which secondary researchers may
also wish to remove (see Benson and El-Zahhar, 1994, for
details). Participants were asked to respond to each item
in terms of how they feel when taking tests in general
on a scale of 1 = “almost never” to 4 = “almost always”. Test Anxiety. Test anxiety was measured with the
Revised Test Anxiety Scale (R TAS; (Benson & El‐Zahhar,
1994). 2.5 MATERIALS/SURVEY INSTRUMENTS
Survey Adaptations Four items (R-MARS-S-NUM) were modified from
mathematics items to statistics items whilst keeping
the language more consistent (e.g., “Being given a set
of numerical problems involving addition to solve on
paper” was modified to “Being given a set of statistical
problems involving addition to solve on paper”) and four
items (R-MARS-NUM) were changed from our original
modifications back to mathematics but matching the
more complex language used (e.g., “Calculating the sum
of squared deviances by adding the squared deviances
together” has been modified to “Finding the codomain
of the function h(x, y) = x + y when x = {3,4,5,6} and
y = {5,7,9,13}”). describes a situation involving mathematics such as
“Taking an exam in a math course” (mathematics test
anxiety), “Being given a set of division problems to solve”
(numerical task anxiety), or “Listening to another student
explain a math formula” (mathematics course anxiety). Participants were asked to indicate how much anxiety
they feel in those situations on a Likert-type scale ranging
from 1 = “no anxiety” to 5 = “a great deal of anxiety”. Where the local context required it, items were
modified to reflect local equivalents of US terms (e.g., in
the UK, “Taking the math section of a college entrance
exam” was changed to “Taking the maths section of a
university entrance exam”). Modified STARS and R-MARS. The modified versions
of the STARS (STARS-M) and R-MARS (R-MARS-S) used
in Terry et al. (2023) were also included. In these
versions, the original STARS items were revised to
reflect mathematics-related situations (e.g., “Doing
the coursework for a statistics course” was changed
to “Doing the coursework for a mathematics course”)
and the original R-MARS statements were revised to
reflect statistics-related situations (e.g., “Walking into
a mathematics class” was changed to “Walking into
a statistics class”). The response scales were kept the
same as the originals. An attention check was also included in the STARS-M,
which asked participants to “Please select ‘5 – a great
deal of anxiety’ for this question”. Trait Anxiety. Trait anxiety was measured using the
trait subscale of the State Trait Inventory for Cognitive
and Somatic Anxiety (STICSA; Ree et al., 2008). The STICSA
has been developed and evidenced to differentiate
anxiety from depression more effectively than other
popular anxiety measures (e.g., the State-Trait Anxiety
Inventory (STAI); Spielberger, 1983; Tindall et al., 2021). 2.5 MATERIALS/SURVEY INSTRUMENTS
Survey Adaptations The scale contains four subscales: 7 worry items
(e.g., “During tests I find myself thinking about the
consequences of failing”), 6 tension items (e.g., “During
tests I feel very tense”), 5 test-irrelevant thinking items
(e.g., “During tests I find I am distracted by thoughts of
upcoming events”), and 7 bodily symptoms items (e.g., “I
get a headache during an important test”). We included
5 items later removed by Benson and El-Zahhar (1994) to
form a 20-item scale, which secondary researchers may
also wish to remove (see Benson and El-Zahhar, 1994, for
details). Participants were asked to respond to each item
in terms of how they feel when taking tests in general
on a scale of 1 = “almost never” to 4 = “almost always”. The exploratory factor analysis in Terry et al. (2023)
indicated that the R-MARS numerical task anxiety
subscale was the only subscale where the revised items
did not load onto the same factor as the corresponding
original items. We believe the inconsistency in factor
loadings in the original study could be because the
modifications were not equivalent. For example, “Being
given a set of numerical problems involving addition to
solve on paper” was modified for the statistical context
to “Calculating the sum of squared deviances by adding
the squared deviances together” and, although the
two both involved addition, the latter would be less
familiar to participants and, thus, could be perceived
as more a complex mathematical task. To rule out the
possibility that the original and modified items loaded Fear
of
Negative
Evaluation. Following
recommendations by Carleton et al. (2011), fear of Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 12 anxious on a Likert scale ranging from 0 = “not at all” to
4 = “very much”. negative evaluation was measured using the Brief Fear
of Negative Evaluation Scale – Straightforward (BNFE-S;
Leary, 1983; Rodebaugh et al., 2004). The scale contains
8 items, including statements such as, “I am afraid that
people will find fault with me” and “I often worry that I
will say or do the wrong things”. The BNFE-S omits the
reverse-scored items in the original BNFE scale (items 2, 4,
7, and 10), which were found to be measuring a different
construct (Carleton et al., 2011). 2.5 MATERIALS/SURVEY INSTRUMENTS
Survey Adaptations Specifically, “Drinking with
others” was reworded to “Drinking coffee with others”,
“Urinating in a public bathroom” was changed to “Using
a public bathroom”, and “Trying to make someone’s
acquaintance for the purpose of a romantic/sexual
relationship” was changed to “Making someone’s
acquaintance for the purpose of making a marriage
proposal”. Self-Efficacy. Self-efficacy was measured with the
8-item New General Self Efficacy Scale (NGSE; Chen et
al., 2001), which contains items such as “When facing
difficult tasks, I am certain that I will accomplish them”. Participants were asked to indicate the extent to which
they agree with each statement on a Likert scale of 1 =
“strongly disagree” to 5 = “strongly agree”. An attention check was also included in this scale
which asked participants to “Please select ‘4 – agree’ for
this question”. Self-Efficacy. Self-efficacy was measured with the
8-item New General Self Efficacy Scale (NGSE; Chen et
al., 2001), which contains items such as “When facing
difficult tasks, I am certain that I will accomplish them”. Participants were asked to indicate the extent to which
they agree with each statement on a Likert scale of 1 =
“strongly disagree” to 5 = “strongly agree”. An attention check was also included in this scale
which asked participants to “Please select ‘4 – agree’ for
this question”. Self-Efficacy. Self-efficacy was measured with the
8-item New General Self Efficacy Scale (NGSE; Chen et
al., 2001), which contains items such as “When facing
difficult tasks, I am certain that I will accomplish them”. Participants were asked to indicate the extent to which
they agree with each statement on a Likert scale of 1 =
“strongly disagree” to 5 = “strongly agree”. Intolerance of Uncertainty. Intolerance of uncertainty
was measured using the Intolerance of Uncertainty Scale y
y
was measured using the Intolerance of Uncertainty Scale
– Short Form (IUS-SF; Carleton et al., 2007). The scale
contains 2 subscales, Prospective Anxiety and Inhibitory
Anxiety, each with 6 items. The Prospective Anxiety
subscale includes statements such as, “The smallest
doubt can stop me from acting”. The Inhibitory Anxiety
subscale includes statements such as, “It frustrates me
not having all the information I need”. Participants were
asked to indicate how characteristic each item is of them
on a Likert scale ranging from 1 = “not at all characteristic
of me” to 5 = “entirely characteristic of me”. – Short Form (IUS-SF; Carleton et al., 2007). 2.5 MATERIALS/SURVEY INSTRUMENTS
Survey Adaptations Participants were asked
to indicate how characteristic each item is of them on a
Likert scale ranging from 1 = “not at all characteristic of
me” to 5 = “extremely characteristic of me”. An attention check was also included in this scale,
which asked participants to “Please select ‘2 – a little’ for
this question”. Analytic Thinking. Analytic thinking was measured
using a revised version of the Cognitive Reflection Test
(CRT; Frederick, 2005), developed by Shenhav et al. (2012). We selected a revised version because participants were
less likely to be familiar with it than the original. Like the
original, the revised CRT contains three word-problems,
each of which requires a numerical response. Questions
are open-ended, but respondents typically give either
the correct response (indicating greatest analytic
thinking), a single incorrect and intuitively compelling
response, or varying incorrect and unintuitive responses. The data set contains the raw numerical responses
given by participants so that researchers can code them
according to their chosen criteria. An attention check was also included in this scale,
which asked participants to “Please select ‘3 – moderately
characteristic of me’ for this question”. Social Interaction Anxiety and Performance Anxiety. Social interaction anxiety and performance anxiety were
measured using the experienced fear/anxiety dimension
of the Liebowitz Social Anxiety Scale – Self Report (LSAS-
SR; Baker et al., 2002; Liebowitz, 1987). The scale is
broken down into social interaction anxiety (12 items,
e.g., “Talking with people you don’t know very well”)
and performance anxiety (12 items, e.g., “Participating
in small groups”). Participants were asked to indicate
how anxious they would feel in each situation on a Likert
scale ranging from 0 = “not at all” to 3 = “very much so”. Belief in God/s. Participants’ belief in God/s was
recorded using a single item. Participants were asked,
“How strongly do you believe in God (or gods) from
0–100? If you are certain that God (or gods) does not
exist, then enter “0” and if you are certain that God (or
gods) does exist then enter “100”.” Possible responses
ranged between 0 and 100. Some LSAS-SR items were adapted to respect local
laws/norms in Saudi Arabia. Instructor Survey
i
i Instructor Survey
Statistics Module Details. The instructor survey asked
for the following information about each module: Name
and/or code, start and end dates, the statistical software
taught, the approximate content of the modules (via
a checklist of different statistics topics), the primary
academic discipline of the instructors, the mode(s) of
teaching and number of hours per format (e.g., 1-hour
online lecture, 2-hour in-person workshop), the types,
format, and date of assessments, how assessments were
graded, opportunities for formative feedback, average
grade from previous cohorts, and any other information
that would be useful to contextualise the assessment
information. Statistics
Grades
(Self-Reported). We
asked
participants whether they had previously taken any
university-level statistics modules and, for those that
had, to self-report their grades for these modules. Grades are in their raw form, but we also provide grading
scale/system information for each university to enable
standardisation (see Grade Data, below). Degree Course Details. Participants were asked
to indicate their university, (intended) major subject
of study (i.e., the subject of the degree that they are
pursuing), their current year of study, and whether
they were studying any other (i.e., non-statistics)
mathematics-based modules on their degree. Where the
local researchers already knew these details (e.g., they
were only sharing the survey with their own students)
these questions were omitted to reduce the length of the
survey and the information was instead added into the
data during data processing. 2.5 MATERIALS/SURVEY INSTRUMENTS
Survey Adaptations Journal of Open Psychology Data DOI: 10.5334/jopd.80 4, “If I have trouble solving a homework problem in the
book, I copy down the answer in the back of the book
if it is available”, was removed because the required
modifications would have changed the meaning too far
from the original. All items except 1 and 7 are reverse
scored. Participants were asked to indicate the extent to
which they agree with each statement on a Likert scale
of 1 = “strongly disagree” to 5 = “strongly agree”. An attention check was also included in this scale,
which asked participants to “Please select ‘4 – agree’ for
this question”. 4, “If I have trouble solving a homework problem in the
book, I copy down the answer in the back of the book
if it is available”, was removed because the required
modifications would have changed the meaning too far
from the original. All items except 1 and 7 are reverse
scored. Participants were asked to indicate the extent to
which they agree with each statement on a Likert scale
of 1 = “strongly disagree” to 5 = “strongly agree”. Survey Metadata. The dataset also contains selected
metadata that was automatically collected by Qualtrics
(Qualtrics, Provo, UT), which researchers may find useful. Specifically, we include the percentage of the survey
completed, the time it took to complete the survey, and
the dates participants began and finished the survey. Identifiers. We have also added relevant identifiers. Specifically, the country in which the survey was taken,
the language in which the survey was taken, the survey
ID (because some surveys were made available to
students in more than one university), and a randomly
generated participant ID, which replaced the participant-
generated ID code for anonymisation purposes. The type
of incentive offered to students and the context (inside or
outside of class) in which the survey was completed has
also been recorded. An attention check was also included in this scale,
which asked participants to “Please select ‘4 – agree’ for
this question”. An attention check was also included in this scale,
which asked participants to “Please select ‘4 – agree’ for
this question”. Mathematics Education. Participants were asked
for their highest level of pre-university mathematics
education (GCSE or A Level or international equivalents),
the grade they received at each level, and how long ago
(in months) they took each qualification. 2.5 MATERIALS/SURVEY INSTRUMENTS
Survey Adaptations These questions
were modified for the local context of each partner
university and, consequently, some include additional
questions (see codebook for full details). Note that grades
are in their raw form and will need to be standardised
before they can be compared. Mathematics Education. Participants were asked
for their highest level of pre-university mathematics
education (GCSE or A Level or international equivalents),
the grade they received at each level, and how long ago
(in months) they took each qualification. These questions
were modified for the local context of each partner
university and, consequently, some include additional
questions (see codebook for full details). Note that grades
are in their raw form and will need to be standardised
before they can be compared. Procedure
Student Surve Attention Check. In addition to the attention
checks embedded within the measurement scales,
participants were presented with the following at the
end of the survey: “Please indicate whether you feel you
have answered the previous questions carefully and
truthfully. Answering ‘yes’ will ensure that your data is
included in our analyses. Answering ‘no’ will mean that
your data is excluded from our analyses but will have no
impact upon you (i.e., you will still earn your incentive
for taking part)”. Participants could respond “Yes, I
have answered all questions carefully and truthfully” or
“No, I have not answered the questions carefully and
truthfully”. Grade Data
i
i Statistics Grades (Official). At universities where it was
approved by the local ethics committees, we asked
student participants to provide their names and/or
student ID codes, so that the grades for the statistics
modules they were taking at the time they completed
the survey could be obtained from their university
records. Note that grades are in their raw form, but we
also provide grading scale/system information to enable
standardisation. Demographics. Participants were also invited to
provide their age (in years), gender identity, ethnicity,
and whether they have been diagnosed with a specific
learning difference (SpLD), such as dyslexia or dyscalculia. 2.5 MATERIALS/SURVEY INSTRUMENTS
Survey Adaptations The scale
contains 2 subscales, Prospective Anxiety and Inhibitory
Anxiety, each with 6 items. The Prospective Anxiety
subscale includes statements such as, “The smallest
doubt can stop me from acting”. The Inhibitory Anxiety
subscale includes statements such as, “It frustrates me
not having all the information I need”. Participants were
asked to indicate how characteristic each item is of them
on a Likert scale ranging from 1 = “not at all characteristic
of me” to 5 = “entirely characteristic of me”. Persistence. Persistence was measured with the
persistence subscale of the Attitude Towards Mathematics
Survey (ATMS; Miller et al., 1996), which contains 8 items
such as “If I have trouble understanding a problem, I go
over it again until I understand it”. Although the ATMS
as a whole focusses on mathematics, the persistence
subscale items refer to academic persistence more
generally. Some items were modified to make them more
appropriate for the higher education context. Specifically,
in item 3 the words “in the book” were removed, in item
6 the words “hope that the teacher explains it” were
changed to “hope that it is explained”, and the word
“homework” was removed from items 2, 7, and 8. Item Persistence. Persistence was measured with the
persistence subscale of the Attitude Towards Mathematics
Survey (ATMS; Miller et al., 1996), which contains 8 items
such as “If I have trouble understanding a problem, I go
over it again until I understand it”. Although the ATMS
as a whole focusses on mathematics, the persistence
subscale items refer to academic persistence more
generally. Some items were modified to make them more
appropriate for the higher education context. Specifically,
in item 3 the words “in the book” were removed, in item
6 the words “hope that the teacher explains it” were
changed to “hope that it is explained”, and the word
“homework” was removed from items 2, 7, and 8. Item Creativity/Non-Creativity Anxiety. Creativity/Non-
Creativity Anxiety was measured using the Creativity
Anxiety Scale (Daker et al., 2020). The scale contains 16
items: 8 creativity anxiety items (e.g., “Having to solve a
problem for which the solution is open-ended”) paired
with 8 non-creativity items (e.g., “Working in a situation
where there is an established correct and incorrect way
of doing things”). Participants were asked to indicate
how much each situation would make them feel 13 Terry et al. CRT Check To help ensure the integrity of the revised CRT, participants
were asked “You have just answered three reasoning
problems. Did you look any of the answers up online?”, to
which they could respond “Yes” or “No”. There were 382
(3.0%) participants that answered “Yes’ to this question. Instructor Survey Upon receiving the invitation to take part, statistics
module instructors were directed to the online survey
where they read the information sheet and provided
consent before continuing. Participants were first
reminded that they should complete the instructor
survey once for every statistics module that the student
participants were taking at the time of completing the
student survey and provided with a unique code they
could use if they later wished to remove their data. The
survey then requested (in order) the university name, the
statistics module name and/or code, and the start and
end dates of the module. Participants could then select
the software(s) taught on the module, whether the
module was frequentist, Bayesian, both, or other, and
select the topics taught from a checklist (e.g., ANOVA,
Bayes factors, Data visualisation). We then asked whether
the module was taught by the mathematics/statistics
department or from the students’ main discipline (e.g.,
psychology faculty that teach statistics). The survey
then requested the percentage of in-person teaching
and whether there was less than usual due to COVID-19. We then asked for details about the mode of teaching
(e.g., lectures, practicals), including how many hours per
week were spent on each, whether they were online or
in-person, and synchronous or asynchronous. Additionally, six attention checks were embedded
within the measurement scales which asked participants
to select a specific response (e.g., “please select ‘1 –
strongly disagree’”). There were two in the first block
which contained the statistics and mathematics anxiety
measures and four in the second block which contained
all other scales. In the first block, 2,052 (16.3%) of
participants responded incorrectly to the first check and
2,194 (17.5%) responded incorrectly to the second. In
the second block, the number of students responding
incorrectly to each check were 2,779 (22.1%) to the third,
2,812 (22.4%) to the fourth, 2,872 (22.9%) to the fifth,
and 2,752 (21.9%) to the sixth. Student Survey Upon receiving the invitation to take part, students
were directed to the online survey where they read
the information sheet and provided consent before
continuing. Participants were then asked to complete
an eligibility check (if they had not been pre-screened),
and to provide their name and/or student ID code (to
obtain grade data from student records, where relevant),
a unique participant ID code (to withdraw their data, if
desired), and their primary degree subject and statistics
module names (if researchers were unsure of these
details in advance). All participants then completed the Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 14 first block of measurement scales containing all four
measures of statistics anxiety and mathematics anxiety,
randomised at the measure and item level. This block
was presented first because it contained the measures
most critical to the primary study and if participants did
not proceed to the next block, their data would still be
useful. The second block of measurement scales – also
randomised at the question and item level – contained
measures of trait anxiety, test anxiety, fear of negative
evaluation,
social
interaction/performance
anxiety,
creativity anxiety, intolerance of uncertainty, self-efficacy,
persistence, and the revised cognitive reflection test (CRT). The question asking about participants’ belief in God/s
was randomly presented before or after the revised CRT. The two follow-up questions about the revised CRT were
then asked. Participants were next asked about their pre-
university mathematics education, their statistics grades
from previous modules at university (if applicable), the
year of their degree course, and demographics. Finally,
participants answered the final attention check question,
were debriefed, and if required, redirected to collect their
survey incentives. The median completion time for the
survey was 30 minutes. coursework, and the scale used for grading (e.g., numeric
continuous, letter grades). Where respondents reported
using regular testing, we also asked for the frequency
and format (e.g., quizzes, tasks) of testing, whether they
were timed, and whether all grades counted towards the
final, overall grade. Next, instructors could indicate the
types of any formative assessment (e.g., verbal/written,
peer/instructor), what the average final overall grade
for the module usually is, and, finally, instructors were
invited to record any additional information about their
assessments that could be useful for contextualising
their data. Attention Checks At the end of the student survey, participants were asked
whether they had answered all questions truthfully and
carefully, to which 10,281 (81.8%) responded “yes”, 172
(1.4%) responded “no”, and 2,117 (16.8%) responses are
missing (where participants did not reach that stage of
the survey). Grade Data Where permitted by collaborating universities’ ethics
committees and legal teams, grade data was obtained
by the collaborating researchers and shared with the
lead researcher using password-protected files. 3.8 PUBLICATION DATE
1st of October, 2024 1st of October, 2024 3.9 FAIR DATA/CODEBOOK Data are stored in .csv format on the OSF, along with a
detailed codebook and all materials, using a CC BY 4.0
licence. 3.3 DATA TYPE Partially processed primary data.13 3.6 LICENSE The data and supplementary materials are licenced
under a CC BY 4.0 licence. Ethics The next section was about module assessments. We asked for the type of assessment (e.g., exams,
coursework), the percentage of the final grade each type
contributed to, the length of any timed assessments,
whether assessments were online/in-person (where
appropriate),
the
date
of
exams/deadlines
for This study was approved (ER/JLT26/7) by the Sciences
& Technology Cross-Schools Research Ethics Committee
(C-REC) at the University of Sussex in adherence to the
British Psychological Society’s Code of Human Research
Ethics (2018). Partner universities were covered by the Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 15 • SMARVUS_measures.csv – id, measurement scales • SMARVUS_measures.csv – id, measurement scales overarching University of Sussex ethics approval, but
were asked to check with their own ethics boards whether
further approval was required at the local level and, if
necessary, to obtain it before beginning data collection. Ethics approval documentation is available here: https://
osf.io/2aumd/. • SMARVUS_maths_edu.csv – id, prior mathematics
education data • SMARVUS_stats_edu.csv – id, statistics education
data (from official records and self-reported) • SMARVUS_codebook.csv – the codebook • SMARVUS_codebook.csv – the codebook For those universities that shared students’ grade
data with us, a data protection agreement was in place
to allow the legal transfer of the non-anonymised data
(i.e., student names and/or ID codes) required to obtain,
share, and link grades to participants’ survey data. Anonymisation y
Raw data have and will only ever be available to the
research leads at the University of Sussex. To anonymise
the data for sharing, the student names and ID codes
have been replaced with a randomly generated unique
ID code, and the demographic variables and some
course/module details from the student surveys have
been edited as required to ensure that participants
are not identifiable via a combination of these data. Specifically, students’ age, degree major, and any
specified non-statistics mathematics modules have
been categorised, gender identities and SpLDs have
been partially re-categorised, and ethnicity data have
been removed completely. Full details on how the data
have been processed for anonymisation is available in
the codebook and data processing notes (available here:
https://osf.io/374vn/). 3.5 LANGUAGE All data are stored in English (UK), except proper
nouns (e.g., names of pre-university mathematics
qualifications). Free-text responses were mostly short
and straightforward to translate (e.g., degree major or
gender identity) so were translated back into English
by the lead author using Google Translate. Where a
translation was ambiguous, it was clarified with native
speakers. 3.4 FORMAT NAMES AND VERSIONS All versions of the dataset are available as .csv files, which
can be opened using most spreadsheet and statistics
software. 2.8 EXISTING USE OF DATA Data will be under embargo until 1st of October, 2024
to allow the authors sufficient time to publish from
it first. During this time, data will be made available
upon request, provided the intended research does not
overlap with projects being undertaken by the present
authors. At the time of writing, there are no published articles
or other outputs originating from this data. However,
following the embargo period, researchers will be able
to register their planned secondary analyses on an
open document, which we encourage use of to prevent
duplication of efforts. (4) REUSE POTENTIAL The dataset is available in its complete form (i.e., the
combined and matched student survey, instructor survey,
and grades) and – due to its size – also in its component
parts, which can be rematched using the `unique_id`
variable. Accompanying the data is a detailed codebook. The files are named as follows: The SMARVUS dataset has the potential to address many
important questions, particularly regarding statistics and
mathematics education, anxiety, psychometrics, and
survey methodology. It uniquely facilitates cross-lingual
and cross-cultural comparisons and the larger-than-
usual sample size is more likely to produce reliable and
robust estimates. Below, we highlight just some of the
ways this could benefit specific fields. • SMARVUS_complete.csv – all data • SMARVUS_demo_meta.csv – id, demographics, • SMARVUS_demo_meta.csv – id, demographics,
Qualtrics meta-data, and key identifiers Qualtrics meta-data, and key identifiers Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 16 Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 STATISTICS EDUCATION Our data could also be used for measurement invariance
testing. There is a dearth of invariance testing for most
psychological scales (D’Urso et al., 2022), so there are many
gaps to be filled. For example, we know that mathematics
anxiety scores vary between cultures (Hunt et al., 2021),
which could be indicative of cultural non-invariance. If that
is the case, the generalisability of predominantly Western
research findings should not be assumed. Such findings
might be misleading for other cultures, with consequences
for education. This dataset could address this problem via
the cross-cultural investigation of mathematics anxiety
scale properties. These data enable the exploration of relationships
between many constructs. For example, a much-
debated question is whether statistics anxiety affects
achievement (e.g., statistics module grades). A recent
meta-analysis of this relationship produced a non-
significant effect size of just Z = |0.07| (Trassi et al., 2022). However, the authors noted considerable variability in
their systematic review, explaining it may be attributed
to moderators, such as pre-university mathematics
grades and self-efficacy. Another review identified
mode of assessment as a potential moderator (Terry &
Field, 2023). These moderators could be tested with the
SMARVUS dataset. COGNITIVE REFLECTION TEST (CRT) The data includes responses to a revised version of the
Cognitive Reflection Test (CRT; Shenhav et al., 2012), a
hugely popular measure of reflective thinking tendencies,
the original (Frederick, 2005), having been cited over
6000 times, according to Google Scholar. Projects
are underway to test the psychometric properties of
Shenhav et al.’s (2012) version and, assuming the scale
shares key properties of the original (e.g., excellent
validity, reasonable reliability, and incorrect responses
converging on the same typical response), the SMARVUS
opens up opportunities for research into cross-cultural
and gender comparisons of cognitive reflection and its
relationship to various types of anxiety. Variability
could
also
be
due
to
the
multi-
dimensionality of the STARS (Cruise et al., 1985). There
are three subscales that measure statistics attitudes, not
statistics anxiety (Hanna et al., 2008; Papousek et al.,
2012), thus should not be conflated. The data required
for Trassi et al. (2022) to separate these subscales were
unavailable, forcing them to use composite scores. A
large-scale analysis of the relationship between statistics
anxiety and achievement using the anxiety subscales
alone is possible with the SMARVUS dataset. Trassi et al. (2022) further note that studies in their
meta-analysis mainly tested psychology students within
Europe and North America and many had low sample
sizes, which the SMARVUS dataset addresses. Such
limitations are pervasive in psychology (Ioannidis, 2005;
Rad et al., 2018), so these data could benefit many
other research questions in the same ways. Furthermore,
the sample is sufficiently large to enable multi-level
modelling
to
estimate
variation
across
different
languages, geographic regions, or educational systems. SURVEY METHODOLOGY For survey-based research to be robust, it is essential
that care and attention are employed by respondents. One study found 10–12% of responses to long surveys
by undergraduates completing it for course credit are
given without such care (Meade & Craig, 2012). Some
researchers have proposed using attention checks to
help identify and eliminate such responses (Huang et
al., 2012). The present study included attention checks
within the survey measures, asking participants to
choose a particular response option, and an ‘amnesty’
at the end, asking if they had answered carefully and
truthfully throughout. SMARVUS data could be used to
compare the effectiveness of these checks with other
measures of careless responding, such as response time
and ‘long-string analysis’ (providing the same response
to all items on a scale; Curran, 2016). CONSTRUCT VALIDITY To understand how generalisable research is, the
scales we use to measure constructs must be validated
in different populations (Flake, 2021). This includes
ensuring adaptations (e.g., translations) are valid and
reliable, and that different groups respond to measures
in the same ways, such that the factor structure,
loadings, and item intercepts are equivalent (i.e., are
measurement invariant). Our student survey included eight scales adapted
to 21 languages. We also modified some scales to be
appropriate for the local context. In most cases, this
was minimal (e.g., changing “college” to “university”). However, we made more substantial modifications to our
measure of social interaction and performance anxiety –
the LSAS-SR (Baker et al., 2002) – for use in Saudi Arabia
(e.g., modifying inappropriate references to alcohol and
dating). Validating adapted scales would ensure these
versions are appropriate for use in different countries
and cultural contexts, opening up fresh opportunities for
cross-cultural research. FUNDING INFORMATION 2 We also added a single item measure of participants’ belief in
God/s to test a CRT-related research question, outside of our
core aims. 2 We also added a single item measure of participants’ belief in
God/s to test a CRT-related research question, outside of our
core aims. Kristel de Groot was supported by the Dutch Research
Council (NWO); James M. Clay was supported by the
Economic and Social Research Council (ES/P000673/1);
Elizabeth Collins was supported by the Economic and
Social Research Council; Philipp Schmid was supported
by a European Commission Horizon 2020 Grant (964728
JITSUVAX); Robert M. Ross was supported the Australian
Research Council (grant number: DP180102384), a
Macquarie University Research Fellowship (MQRF), and
the John Templeton Foundation (grant ID: 62631);
Andras N. Zsido was supported by the New National
Excellence Program of the Ministry for Innovation
and Technology from the source of the National
Research, Development and Innovation Fund (OTKA
PD-137588; ÚNKP-21-4); Jenny Terry was supported
by a School of Psychology PhD Studentship, University
of Sussex; Gabriel Banik was supported by a Slovak
Research and Development Agency (APVV-17-0418);
Matúš Adamkovič was supported by a Slovak Research
and
Development
Agency
(APVV-20-0319);
Ivan
Ropovik was supported by the Systemic Risk Institute
(PRIMUS/20/HUM/009; LX22NPO5101); Mahmoud M. Elsherif was supported by the Baily Thomas Charitable
Fund; Mauricio Salgado was supported by the National
Agency for Research and Development (ANID), Ministry
of Science, Technology, Knowledge and Innovation,
through the Centre for Research in Inclusive Education
(PIA-ANID CIE160009; 2017); Sophie Leonard was
supported by the University College Dublin Ad
Astra Scholarship; Flavia H. Santos was supported
by the University College Dublin Ad Astra Start Up
Scholarship. 3 The period of data collection coincided with the COVID-19
pandemic, which affected teaching delivery (e.g., the move to
online learning, some details of which were captured by the
instructor survey) and general anxiety levels may have been
higher than usual. 4 Here, the UK includes the devolved nations of England, Scotland,
and Northern Ireland (we did not collect data in Wales). Note,
however, that the devolved nations have different education
systems both pre-university (e.g., different mathematics exams)
and during university (e.g., different degree durations). 5 16.7% of collaborating researchers did not provide this data
(typically, because they were unable to). NOTES 1 We use the term ‘module’ to describe the smaller units that make
up a degree course (often lasting one semester) to distinguish
them from full degree programmes, which we refer to as ‘courses’. 1 We use the term ‘module’ to describe the smaller units that make
up a degree course (often lasting one semester) to distinguish
them from full degree programmes, which we refer to as ‘courses’. PEDAGOGY Finally, we suggest the SMARVUS dataset has unique
pedagogical reuse potential. First, students might find a
dataset related to mathematics and statistics anxieties
to be relatable, something qualitative evidence suggests
can aid learning (e.g., Blackburn, 2015) and reduce anxiety
(e.g., Trakulphadetkrai, 2017) in statistics education. Second, using these data in a statistics class would give
instructors an opportunity to make students conscious
of any anxieties, show them they are far from alone, and Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 17 ACKNOWLEDGEMENTS encourage students to notice and, subsequently, challenge
the influence anxiety may be having on their attitudes
and behaviours regarding learning statistics. Third, there
are general benefits of using authentic, secondary data in
statistics education that could further enhance the specific
benefits. For example, students can learn data processing
strategies that are usually unavailable with pre-prepared
datasets – such as dealing with missing data – alongside
statistical procedures and tests. Additionally, students who
use this data for research projects (e.g., undergraduate
dissertations) could do so without needing to apply for
ethics approval or worrying about recruiting a large enough
sample, and could present their work at conferences and
in publications, as previously done by Long & Chalk (2020)
with Grahe et al.’s (2018) Emerging Adulthood Measured at
Multiple Institutions 2 (EAMMi2) data. The authors would like to thank everyone that contributed
to administration and data collection for this project,
including: Alexandria Goveia (Toronto Metropolitan
University); Alissa Beath (Macquarie University); Andrés
Castellanos-Chacón (Universidad El Bosque); Christiany
Suwartono (Universitas Katolik Indonesia Atma Jaya);
Darcy Hallett and Brittany Pitt (Memorial University of
Newfoundland); Edoardus Gilang Wardana, Muhammad
Raihan, and Isyraq Qurratul’Aini (Brawijaya University);
Leona Cilar Budler (University of Maribor); Maria Flakus
(Polish Academy of Sciences in Warsaw); Martina Daiser,
Alexander Soell, and Tang Kuan (University of Tübingen);
Megan Davies (University of Surrey); Roberto Capelli,
Lucrezia Cavagnis, Jacopo Stringo, and Mariantonella
Todaro (University of Bergamo); Bethany Lewington,
Robert Spell, and Iga Ewa Zlotucha (University of Sussex). We also thank Peter Lugtig (Utrect University) for his
advice on translation. 1 We use the term ‘module’ to describe the smaller units that make
up a degree course (often lasting one semester) to distinguish
them from full degree programmes, which we refer to as ‘courses’. FUNDING INFORMATION 6 A limitation of these modes of recruitment is that it is usually
not possible to determine how many students were exposed
to the invitation to take part. Consequently, we do not provide
participation rates as the data would be incomplete and/or
inaccurate. 7 We did not collect data on which device was used, but note that
the device used could be a source of heterogeneity. 8 Although we recognise that “female” and “male” refer to
biological sex and that “woman” and “man” refer to gender
identity, the adapted response options to the question asking
participants’ gender identity were inconsistent with some listing
“woman” and “man”, others listing “female” and “male”, and
others listing “woman/female” and “man/male”, so we have
merged the responses. 9 An anonymised list of the ways participants described their
gender is available in the OSF supplementary materials. 10 See the supplementary materials for the frequency of degree
major categories below 1%. 11 Some universities also provided students’ grades from their
previous modules. This data will be provided as a supplementary
file on the OSF at a later date. 12 We do not provide reliability coefficients for this or any of the
other measures because such coefficients should be calculated
for the specific subsample chosen for any secondary research
studies. 13 Our aim was to provide data as close to its raw form as possible,
after translations and anonymisation. Whether and how each
variable has been processed is detailed in the ‘Data Processing
Notes’ column of the code book and is summarised here:
https://osf.io/6n9ye. 18 Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 AUTHOR CONTRIBUTIONS van der
Ven, Jochen A. Mosbacher, Hilal H. Şen, Joel R. Anderson,
Michael Batashvili, Kristel de Groot, Katie A. Gilligan-
Lee, Felix O. Egara, Martin J. Barwood, Karuna Thomas,
Grace McMahon, Siobhán M. Griffin, Hans-Christoph
Nuerk, Alyssa Counsell, Oliver Lindemann, Dirk Van Rooy,
Theresa E. Wege, Joanna E. Lewis, Mauricio, E. Garrido
Vásquez, Thomas E. Hunt, Peter E. Branney, TamilSelvan
Ramis, Nicholas A. Badcock, Kareena McAloney-Kocaman,
Olena V. Semenikhina, Frank Scharnowski, Mariah
Lecompte, Kinga Morsanyi, Suzanna E. Forwood, Ho Yan
Lai, Deborah S. Crossland, Tessa R. Flack, Samuel P. Hills,
Timothy Liew, Daniel Ansari, and Julia Bahnmueller. Data curation: Jenny Terry and Tamás Nagy. Data curation: Jenny Terry and Tamás Nagy. Funding acquisition: Mauricio Salgado and Patricia
Garrido-Vásquez. Funding acquisition: Mauricio Salgado and Patricia
Garrido-Vásquez. Investigation: Jenny Terry, Robert M. Ross, Tamás
Nagy, Mauricio Salgado, Patricia Garrido-Vásquez, Jacob
O. Sarfo, Susan Cooper, Anke C. Buttner, Tiago J. S. Lima,
İbrahim Öztürk, Nazlı Akay, Flavia H. Santos, Christina
Artemenko, Lee T. Copping, Mahmoud M. Elsherif, Ilija
Milovanović, Robert A. Cribbie, Marina G. Drushlyak,
Katherine Swainston, Yiyun Shou, Juan David Leongómez,
Nicola Palena, Fitri A. Abidin, Maria F. Reyes-Rodríguez,
Yunfeng He, Juneman Abraham, Argiro Vatakis, Kristin
Jankowsky, Stephanie N. L. Schmidt, Elise Grimm, Desirée
González, Philipp Schmid, Roberto A. Ferreira, Dmitri
Rozgonjuk, Neslihan Özhan, Patrick A. O’Connor, Andras
N. Zsido, Gregor Stiglic, Darren Rhodes, Cristina Rodríguez,
Ivan Ropovik, Violeta Enea, Ratri Nurwanti, Alejandro
J. Estudillo, Nataly Beribisky, Karel K. Himawan, Linda
M. Geven, Anne H. van Hoogmoed, Amélie Bret, Jodie
E. Chapman, Udi Alter, Zoe M. Flack, Donncha Hanna,
Mojtaba Soltanlou, Gabriel Banik, Matúš Adamkovič,
Sanne H. G. van der Ven, Jochen A. Mosbacher, Hilal H. Şen, Joel R. Anderson, Michael Batashvili, Kristel de Groot,
Matthew O. Parker, Mai Helmy, Mariia M. Ostroha, Katie
A. Gilligan-Lee, Felix O. Egara, Martin J. Barwood, Karuna
Thomas, Grace McMahon, Siobhán M. Griffin, Hans-
Christoph Nuerk, Alyssa Counsell, Oliver Lindemann,
Dirk Van Rooy, Theresa E. Wege, Joanna E. Lewis, Balazs
Aczel, Conal Monaghan, Ali H. Al-Hoorie, Julia F. Huber,
Saadet Yapan, Mauricio, E. Garrido Vásquez, Antonino
Callea, Tolga Ergiyen, James M. Clay, Gaetan Mertens,
Feyza Topçu, Merve G. Tutlu, Karin Täht, Kristel Mikkor,
Letizia Caso, Alexander Karner, Maxine M. C. Storm,
Gabriella Daroczy, Rizqy A. Zein, Andrea Greco, Erin M. Buchanan, Katharina Schmid, Thomas E. Hunt, Jonas
De keersmaecker, Peter E. Branney, Jordan Randell,
Oliver J. Clark, Crystal N. AUTHOR CONTRIBUTIONS Methodology: Jenny Terry, Robert M. Ross, and Andy
P. Field. Project administration: Jenny Terry, Robert M. Ross,
Tamás Nagy, Mauricio Salgado, Jacob O. Sarfo, Susan
Cooper, Anke C. Buttner, Tiago J. S. Lima, İbrahim Öztürk,
Nazlı Akay, Flavia H. Santos, Christina Artemenko, Lee T. Copping, Mahmoud M. Elsherif, Ilija Milovanović, Robert
A. Cribbie, Marina G. Drushlyak, Katherine Swainston,
Yiyun Shou, Juan David Leongómez, Nicola Palena, Fitri A. Abidin, Maria F. Reyes-Rodríguez, Yunfeng He, Juneman
Abraham, Argiro Vatakis, Kristin Jankowsky, Stephanie
N. L. Schmidt, Elise Grimm, Desirée González, Philipp
Schmid, Roberto A. Ferreira, Dmitri Rozgonjuk, Neslihan
Özhan, Patrick A. O’Connor, Andras N. Zsido, Gregor
Stiglic, Darren Rhodes, Cristina Rodríguez, Ivan Ropovik,
Violeta Enea, Ratri Nurwanti, Alejandro J. Estudillo,
Nataly Beribisky, Karel K. Himawan, Linda M. Geven, Anne
H. van Hoogmoed, Amélie Bret, Jodie E. Chapman, Udi
Alter, Zoe M. Flack, Donncha Hanna, Mojtaba Soltanlou,
Gabriel Banik, Matúš Adamkovič, Sanne H. G. van der
Ven, Jochen A. Mosbacher, Hilal H. Şen, Joel R. Anderson,
Michael Batashvili, Kristel de Groot, Katie A. Gilligan-
Lee, Felix O. Egara, Martin J. Barwood, Karuna Thomas,
Grace McMahon, Siobhán M. Griffin, Hans-Christoph
Nuerk, Alyssa Counsell, Oliver Lindemann, Dirk Van Rooy,
Theresa E. Wege, Joanna E. Lewis, Mauricio, E. Garrido
Vásquez, Thomas E. Hunt, Peter E. Branney, TamilSelvan
Ramis, Nicholas A. Badcock, Kareena McAloney-Kocaman,
Olena V. Semenikhina, Frank Scharnowski, Mariah
Lecompte, Kinga Morsanyi, Suzanna E. Forwood, Ho Yan
Lai, Deborah S. Crossland, Tessa R. Flack, Samuel P. Hills,
Timothy Liew, Daniel Ansari, and Julia Bahnmueller. Conceptualisation: Jenny Terry, Robert M. Ross, and
Andy P. Field. Project administration: Jenny Terry, Robert M. Ross,
Tamás Nagy, Mauricio Salgado, Jacob O. Sarfo, Susan
Cooper, Anke C. Buttner, Tiago J. S. Lima, İbrahim Öztürk,
Nazlı Akay, Flavia H. Santos, Christina Artemenko, Lee T. Copping, Mahmoud M. Elsherif, Ilija Milovanović, Robert
A. Cribbie, Marina G. Drushlyak, Katherine Swainston,
Yiyun Shou, Juan David Leongómez, Nicola Palena, Fitri A. Abidin, Maria F. Reyes-Rodríguez, Yunfeng He, Juneman
Abraham, Argiro Vatakis, Kristin Jankowsky, Stephanie
N. L. Schmidt, Elise Grimm, Desirée González, Philipp
Schmid, Roberto A. Ferreira, Dmitri Rozgonjuk, Neslihan
Özhan, Patrick A. O’Connor, Andras N. Zsido, Gregor
Stiglic, Darren Rhodes, Cristina Rodríguez, Ivan Ropovik,
Violeta Enea, Ratri Nurwanti, Alejandro J. Estudillo,
Nataly Beribisky, Karel K. Himawan, Linda M. Geven, Anne
H. van Hoogmoed, Amélie Bret, Jodie E. Chapman, Udi
Alter, Zoe M. Flack, Donncha Hanna, Mojtaba Soltanlou,
Gabriel Banik, Matúš Adamkovič, Sanne H. G. COMPETING INTERESTS Elizabeth Collins, Andrew J. Roberts, Wing-Yee Cheung,
Sophie Leonard, Bruno Verschuere, Samantha K. Stanley,
Iro Xenidou-Dervou, Omid Ghasemi, Timothy Liew,
Daniel Ansari, Johnrev Guilaran, Samuel G. Penny, Julia
Bahnmueller, and Christopher J. Hand. Elizabeth Collins, Andrew J. Roberts, Wing-Yee Cheung,
Sophie Leonard, Bruno Verschuere, Samantha K. Stanley,
Iro Xenidou-Dervou, Omid Ghasemi, Timothy Liew,
Daniel Ansari, Johnrev Guilaran, Samuel G. Penny, Julia
Bahnmueller, and Christopher J. Hand. The authors have no competing interests to declare. The authors have no competing interests to declare. AUTHOR CONTRIBUTIONS Rahajeng,
Dar Peterburg, Zsofia K. Takacs, Michael J. Platow, and
Andy P. Field. Kristel Mikkor, Letizia Caso, Alexander Karner, Maxine
M. C. Storm, Gabriella Daroczy, Rizqy A. Zein, Andrea
Greco, Erin M. Buchanan, Katharina Schmid, Thomas E. Hunt, Jonas De keersmaecker, Peter E. Branney, Jordan
Randell, Oliver J. Clark, Crystal N. Steltenpohl, Bhasker
Malu, Burcu Tekeş, TamilSelvan Ramis, Stefan Agrigoroaei,
Nicholas A. Badcock, Kareena McAloney-Kocaman,
Olena V. Semenikhina, Erich W. Graf, Charlie Lea, Kalu
T. U. Ogba, Fergus M. Guppy, Amy C. Warhurst, Shane
Lindsay, Ahmed Al Khateeb, Frank Scharnowski, Leontien
de Kwaadsteniet, Kathryn B. Francis, Mariah Lecompte,
Lisa A. D. Webster, Kinga Morsanyi, Suzanna E. Forwood,
Elizabeth R. Walters, Linda K. Tip, Jordan R. Wagge, Ho
Yan Lai, Deborah S. Crossland, Kohinoor M. Darda, Tessa
R. Flack, Zoe Leviston, Matthew Brolly, Samuel P. Hills,
Elizabeth Collins, Andrew J. Roberts, Wing-Yee Cheung,
Sophie Leonard, Bruno Verschuere, Samantha K. Stanley,
Iro Xenidou-Dervou, Omid Ghasemi, Timothy Liew,
Daniel Ansari, Johnrev Guilaran, Samuel G. Penny, Julia
Bahnmueller, Christopher J. Hand, Unita W. Rahajeng,
Dar Peterburg, Zsofia K. Takacs, Michael J. Platow, and
Andy P. Field. Anderson, Michael Batashvili, Kristel de Groot, Matthew
O. Parker, Mai Helmy, Mariia M. Ostroha, Balazs Aczel,
Conal Monaghan, Ali H. Al-Hoorie, Julia F. Huber, Saadet
Yapan, Mauricio, E. Garrido Vásquez, Antonino Callea,
Tolga Ergiyen, James M. Clay, Gaetan Mertens, Feyza
Topçu, Merve G. Tutlu, Karin Täht, Kristel Mikkor, Letizia
Caso, Alexander Karner, Maxine M. C. Storm, Gabriella
Daroczy, Rizqy A. Zein, Andrea Greco, Tessa R. Flack, Unita
W. Rahajeng, Dar Peterburg, and Zsofia K. Takacs. Anderson, Michael Batashvili, Kristel de Groot, Matthew
O. Parker, Mai Helmy, Mariia M. Ostroha, Balazs Aczel,
Conal Monaghan, Ali H. Al-Hoorie, Julia F. Huber, Saadet
Yapan, Mauricio, E. Garrido Vásquez, Antonino Callea,
Tolga Ergiyen, James M. Clay, Gaetan Mertens, Feyza
Topçu, Merve G. Tutlu, Karin Täht, Kristel Mikkor, Letizia
Caso, Alexander Karner, Maxine M. C. Storm, Gabriella
Daroczy, Rizqy A. Zein, Andrea Greco, Tessa R. Flack, Unita
W. Rahajeng, Dar Peterburg, and Zsofia K. Takacs. Supervision: Jenny Terry, Robert M. Ross, Tamás Nagy,
Mauricio Salgado, Jacob O. Sarfo, Susan Cooper, Anke
C. Buttner, Tiago J. S. Lima, İbrahim Öztürk, Nazlı Akay,
Flavia H. Santos, Christina Artemenko, Lee T. Copping,
Mahmoud M. Elsherif, Ilija Milovanović, Robert A. Cribbie,
Marina G. Drushlyak, Katherine Swainston, Yiyun Shou,
Juan David Leongómez, Nicola Palena, Fitri A. Abidin,
Maria F. Reyes-Rodríguez, Yunfeng He, Argiro Vatakis,
Matthew O. AUTHOR CONTRIBUTIONS Parker, Mai Helmy, Mariia M. Ostroha, Katie
A. Gilligan-Lee, Felix O. Egara, Martin J. Barwood, Karuna
Thomas, Grace McMahon, Siobhán M. Griffin, Hans-
Christoph Nuerk, Alyssa Counsell, Oliver Lindemann, Dirk
Van Rooy, Theresa E. Wege, Joanna E. Lewis, Mauricio, E. Garrido Vásquez, Stefan Agrigoroaei, Erich W. Graf, Lisa A. D. Webster, and Andy P. Field. AUTHOR CONTRIBUTIONS Steltenpohl, Bhasker Malu,
Burcu Tekeş, TamilSelvan Ramis, Stefan Agrigoroaei,
Nicholas A. Badcock, Kareena McAloney-Kocaman,
Olena V. Semenikhina, Erich W. Graf, Charlie Lea, Kalu
T. U. Ogba, Fergus M. Guppy, Amy C. Warhurst, Shane
Lindsay, Ahmed Al Khateeb, Frank Scharnowski, Leontien
de Kwaadsteniet, Kathryn B. Francis, Mariah Lecompte,
Lisa A. D. Webster, Kinga Morsanyi, Suzanna E. Forwood,
Elizabeth R. Walters, Linda K. Tip, Jordan R. Wagge, Ho
Yan Lai, Deborah S. Crossland, Kohinoor M. Darda, Tessa
R. Flack, Zoe Leviston, Matthew Brolly, Samuel P. Hills, Resources: Jenny Terry, Robert M. Ross, Tamás Nagy,
Mauricio Salgado, Patricia Garrido-Vásquez, Jacob O. Sarfo, Susan Cooper, Anke C. Buttner, Tiago J. S. Lima,
İbrahim Öztürk, Nazlı Akay, Flavia H. Santos, Christina
Artemenko, Lee T. Copping, Mahmoud M. Elsherif, Ilija
Milovanović, Robert A. Cribbie, Marina G. Drushlyak,
Katherine Swainston, Yiyun Shou, Juan David Leongómez,
Nicola Palena, Fitri A. Abidin, Maria F. Reyes-Rodríguez,
Yunfeng He, Juneman Abraham, Argiro Vatakis, Kristin
Jankowsky, Stephanie N. L. Schmidt, Elise Grimm,
Desirée González, Philipp Schmid, Roberto A. Ferreira,
Dmitri Rozgonjuk, Neslihan Özhan, Patrick A. O’Connor,
Andras N. Zsido, Gregor Stiglic, Darren Rhodes, Cristina
Rodríguez, Ivan Ropovik, Violeta Enea, Ratri Nurwanti,
Alejandro J. Estudillo, Nataly Beribisky, Karel K. Himawan,
Linda M. Geven, Anne H. van Hoogmoed, Amélie Bret,
Jodie E. Chapman, Udi Alter, Donncha Hanna, Mojtaba
Soltanlou, Gabriel Banik, Matúš Adamkovič, Sanne H. G. van der Ven, Jochen A. Mosbacher, Hilal H. Şen, Joel R. Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 19 Kristel Mikkor, Letizia Caso, Alexander Karner, Maxine
M. C. Storm, Gabriella Daroczy, Rizqy A. Zein, Andrea
Greco, Erin M. Buchanan, Katharina Schmid, Thomas E. Hunt, Jonas De keersmaecker, Peter E. Branney, Jordan
Randell, Oliver J. Clark, Crystal N. Steltenpohl, Bhasker
Malu, Burcu Tekeş, TamilSelvan Ramis, Stefan Agrigoroaei,
Nicholas A. Badcock, Kareena McAloney-Kocaman,
Olena V. Semenikhina, Erich W. Graf, Charlie Lea, Kalu
T. U. Ogba, Fergus M. Guppy, Amy C. Warhurst, Shane
Lindsay, Ahmed Al Khateeb, Frank Scharnowski, Leontien
de Kwaadsteniet, Kathryn B. Francis, Mariah Lecompte,
Lisa A. D. Webster, Kinga Morsanyi, Suzanna E. Forwood,
Elizabeth R. Walters, Linda K. Tip, Jordan R. Wagge, Ho
Yan Lai, Deborah S. Crossland, Kohinoor M. Darda, Tessa
R. Flack, Zoe Leviston, Matthew Brolly, Samuel P. Hills,
Elizabeth Collins, Andrew J. Roberts, Wing-Yee Cheung,
Sophie Leonard, Bruno Verschuere, Samantha K. Stanley,
Iro Xenidou-Dervou, Omid Ghasemi, Timothy Liew,
Daniel Ansari, Johnrev Guilaran, Samuel G. Penny, Julia
Bahnmueller, Christopher J. Hand, Unita W. AUTHOR AFFILIATIONS Visualisation: Jenny Terry, Tamás Nagy, Erin M. Buchanan, and Andy P. Field. Writing – original draft: Jenny Terry. Jenny Terry
orcid.org/0000-0002-6843-7116
School of Psychology, University of Sussex, UK Jenny Terry
orcid.org/0000-0002-6843-7116
School of Psychology, University of Sussex, UK Writing – review & editing: Jenny Terry, Robert M. Ross, Tamás Nagy, Mauricio Salgado, Patricia Garrido-
Vásquez, Jacob O. Sarfo, Susan Cooper, Anke C. Buttner,
Tiago J. S. Lima, İbrahim Öztürk, Nazlı Akay, Flavia H. Santos, Christina Artemenko, Lee T. Copping, Mahmoud
M. Elsherif, Ilija Milovanović, Robert A. Cribbie, Marina
G. Drushlyak, Katherine Swainston, Yiyun Shou, Juan
David Leongómez, Nicola Palena, Fitri A. Abidin, Maria F. Reyes-Rodríguez, Yunfeng He, Juneman Abraham, Argiro
Vatakis, Kristin Jankowsky, Stephanie N. L. Schmidt,
Elise Grimm, Desirée González, Philipp Schmid, Roberto
A. Ferreira, Dmitri Rozgonjuk, Neslihan Özhan, Patrick A. O’Connor, Andras N. Zsido, Gregor Stiglic, Darren Rhodes,
Cristina Rodríguez, Ivan Ropovik, Violeta Enea, Ratri
Nurwanti, Alejandro J. Estudillo, Nataly Beribisky, Karel
K. Himawan, Linda M. Geven, Anne H. van Hoogmoed,
Amélie Bret, Jodie E. Chapman, Udi Alter, Zoe M. Flack,
Donncha Hanna, Mojtaba Soltanlou, Gabriel Banik,
Matúš Adamkovič, Sanne H. G. van der Ven, Jochen
A. Mosbacher, Hilal H. Şen, Joel R. Anderson, Michael
Batashvili, Kristel de Groot, Matthew O. Parker, Mai Helmy,
Mariia M. Ostroha, Katie A. Gilligan-Lee, Felix O. Egara,
Martin J. Barwood, Karuna Thomas, Grace McMahon,
Siobhán M. Griffin, Hans-Christoph Nuerk, Alyssa Counsell,
Oliver Lindemann, Dirk Van Rooy, Theresa E. Wege,
Joanna E. Lewis, Balazs Aczel, Conal Monaghan, Ali H. Al-
Hoorie, Julia F. Huber, Saadet Yapan, Mauricio, E. Garrido
Vásquez, Antonino Callea, Tolga Ergiyen, James M. Clay,
Gaetan Mertens, Feyza Topçu, Merve G. Tutlu, Karin Täht, Writing – review & editing: Jenny Terry, Robert M. Ross, Tamás Nagy, Mauricio Salgado, Patricia Garrido-
Vásquez, Jacob O. Sarfo, Susan Cooper, Anke C. Buttner,
Tiago J. S. Lima, İbrahim Öztürk, Nazlı Akay, Flavia H. Santos, Christina Artemenko, Lee T. Copping, Mahmoud
M. Elsherif, Ilija Milovanović, Robert A. Cribbie, Marina
G. Drushlyak, Katherine Swainston, Yiyun Shou, Juan
David Leongómez, Nicola Palena, Fitri A. Abidin, Maria F. Reyes-Rodríguez, Yunfeng He, Juneman Abraham, Argiro
Vatakis, Kristin Jankowsky, Stephanie N. L. Schmidt,
Elise Grimm, Desirée González, Philipp Schmid, Roberto
A. Ferreira, Dmitri Rozgonjuk, Neslihan Özhan, Patrick A. O’Connor, Andras N. Zsido, Gregor Stiglic, Darren Rhodes,
Cristina Rodríguez, Ivan Ropovik, Violeta Enea, Ratri
Nurwanti, Alejandro J. Estudillo, Nataly Beribisky, Karel
K. Himawan, Linda M. Geven, Anne H. AUTHOR AFFILIATIONS Zsido
orcid.org/0000-0003-0506-6861
Institute of Psychology, University of Pécs, Hungary Mahmoud M. Elsherif
orcid.org/0000-0002-0540-3998
School of Psychology, University of Birmingham, UK Ilija Milovanović
orcid.org/0000-0001-5560-8695
Department of Psychology, Faculty of Philosophy, University of
Novi Sad, Serbia Gregor Stiglic
orcid.org/0000-0002-0183-8679
Faculty of Health Sciences, University of Maribor, Slovenia;
Usher Institute, University of Edinburgh, UK Robert A. Cribbie
orcid.org/0000-0002-9247-497X
Department of Psychology, York University, Canada Darren Rhodes
orcid.org/0000-0001-5859-4567
NTU Psychology, Nottingham Trent University, UK Marina G. Drushlyak
orcid.org/0000-0002-9648-2248
Mathematics Department, Makarenko Sumy State Pedagogical
University, Ukraine Cristina Rodríguez
orcid.org/0000-0001-9929-9767
Facultad de Ciencias de la Educación, Universidad Católica del
Maule, Chile; Millennium Nucleus for the Science of Learning
(MiNSoL), Chile Katherine Swainston
orcid.org/0000-0002-6548-7441
School of Psychology, Newcastle University, UK Ivan Ropovik
orcid.org/0000-0001-5222-1233
Charles University, Faculty of Education, Institute for Research
and Development of Education, Czech Republic; Faculty of
Education, University of Presov, Slovakia Yiyun Shou
orcid.org/0000-0002-7386-0031
Research School of Psychology, Australian National University,
Australia; Saw Swee Hock School of Public Health, National
University of Singapore, Singapore Violeta Enea
orcid.org/0000-0003-3789-2017
Department of Psychology, Alexandru Ioan Cuza University,
Romania Juan David Leongómez
orcid.org/0000-0002-0092-6298
Faculty of Psychology, Universidad El Bosque, Colombia Ratri Nurwanti
orcid.org/0000-0003-2793-9870
Department of Psychology, Brawijaya University, Indonesia Nicola Palena
orcid.org/0000-0002-2746-1208
Department of Human and Social Sciences, University of
Bergamo, Italy Alejandro J. Estudillo
orcid.org/0000-0002-7760-318X
Department of Psychology, Bournemouth University, UK;
School of Psychology, University of Nottingham, Malaysia Fitri A. Abidin
orcid.org/0000-0002-2755-9651
Faculty of Psychology, Universitas Padjadjaran, Indonesia Nataly Beribisky
orcid.org/0000-0002-1081-0125
Department of Psychology, York University, Canada Maria F. Reyes-Rodríguez
orcid.org/0000-0002-2645-5092
Department of Psychology, Universidad de los Andes, Colombia Karel K. Himawan
orcid.org/0000-0003-4778-7325
Faculty of Psychology, Universitas Pelita Harapan, Indonesia;
RELASI Research Lab, Indonesia Yunfeng He
Liaoning Key Laboratory of Psychological Testing and Behavior
Analysis, Liaoning Univeristy, China; Department of Psychology,
University of Tuebingen, Germany Linda M. Geven
orcid.org/0000-0001-5075-5223
Institute of Criminal Law and Criminology, Leiden University,
the Netherlands; Department of Clinical Psychology, University
of Amsterdam, the Netherlands Juneman Abraham
orcid.org/0000-0003-0232-2735
Department of Psychology, Faculty of Humanities, Bina
Nusantara University, Indonesia Argiro Vatakis
orcid.org/0000-0002-6545-376X
Department of Psychology, Panteion University of Social and
Political Sciences, Greece Anne H. van Hoogmoed
orcid.org/0000-0001-9038-9993
Behavioural Science Institute, Radboud University, the Netherlands Amélie Bret
orcid.org/0000-0002-9129-0415
LPPL, Nantes University, France Kristin Jankowsky
orcid.org/0000-0002-4847-0760
Psychological Assessment, University of Kassel, Germany Jodie E. Chapman
orcid.org/0000-0002-1094-6196
School of Psychology, Australian Catholic University, Australia Stephanie N. L. AUTHOR AFFILIATIONS van Hoogmoed,
Amélie Bret, Jodie E. Chapman, Udi Alter, Zoe M. Flack,
Donncha Hanna, Mojtaba Soltanlou, Gabriel Banik,
Matúš Adamkovič, Sanne H. G. van der Ven, Jochen
A. Mosbacher, Hilal H. Şen, Joel R. Anderson, Michael
Batashvili, Kristel de Groot, Matthew O. Parker, Mai Helmy,
Mariia M. Ostroha, Katie A. Gilligan-Lee, Felix O. Egara,
Martin J. Barwood, Karuna Thomas, Grace McMahon,
Siobhán M. Griffin, Hans-Christoph Nuerk, Alyssa Counsell,
Oliver Lindemann, Dirk Van Rooy, Theresa E. Wege,
Joanna E. Lewis, Balazs Aczel, Conal Monaghan, Ali H. Al-
Hoorie, Julia F. Huber, Saadet Yapan, Mauricio, E. Garrido
Vásquez, Antonino Callea, Tolga Ergiyen, James M. Clay,
Gaetan Mertens, Feyza Topçu, Merve G. Tutlu, Karin Täht, Robert M. Ross
orcid.org/0000-0001-8711-1675
Department of Philosophy, Macquarie University, Australia Tamás Nagy
orcid.org/0000-0001-5244-0356
Institute of Psychology, ELTE Eötvös Loránd University, Hungary Mauricio Salgado
orcid.org/0000-0002-8108-6154
School of Social Sciences, Universidad Andres Bello, Chile;
Centre for Research in Inclusive Education, Universidad Andres
Bello, Chile Mauricio Salgado
orcid.org/0000-0002-8108-6154
School of Social Sciences, Universidad Andres Bello, Chile;
Centre for Research in Inclusive Education, Universidad Andres
Bello, Chile Patricia Garrido-Vásquez
orcid.org/0000-0002-9561-8983
Department of Psychology, University of Concepción, Chile Patricia Garrido-Vásquez
orcid.org/0000-0002-9561-8983
Department of Psychology, University of Concepción, Chile Jacob O. Sarfo
orcid.org/0000-0003-2859-7278
Department of Health, Physical Education, and Recreation,
University of Cape Coast, Ghana Jacob O. Sarfo
orcid.org/0000-0003-2859-7278
Department of Health, Physical Education, and Recreation,
University of Cape Coast, Ghana Susan Cooper
orcid.org/0000-0003-1211-6214
Department of Psychology, Kingston University, UK Susan Cooper
orcid.org/0000-0003-1211-6214
Department of Psychology, Kingston University, UK Anke C. Buttner
orcid.org/0000-0001-7449-4777
School of Psychology, University of Birmingham, UK Anke C. Buttner
orcid.org/0000-0001-7449-4777
School of Psychology, University of Birmingham, UK Tiago J. S. Lima
orcid.org/0000-0001-8840-4285
Department of Social and Work Psychology, University of
Brasília, Brazil Tiago J. S. Lima
orcid.org/0000-0001-8840-4285
Department of Social and Work Psychology, University of
Brasília, Brazil İbrahim Öztürk
orcid.org/0000-0002-5113-1225
Department of Psychology, Middle East Technical University,
Turkey Nazlı Akay
orcid.org/0000-0002-4470-219X
Department of Psychology, Middle East Technical University,
Turkey Flavia H. Santos
orcid.org/0000-0003-2592-9038
School of Psychology, University College Dublin, Ireland Flavia H. Santos
orcid.org/0000-0003-2592-9038
School of Psychology, University College Dublin, Ireland Christina Artemenko
orcid.org/0000-0001-5947-7960
Department of Psychology, University of Tuebingen, Germany Christina Artemenko
orcid.org/0000-0001-5947-7960
Department of Psychology, University of Tuebingen, Germany 20 Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 Lee T. Copping
orcid.org/0000-0003-3252-0353
Department of Psychology, Teesside University, UK Patrick A. O’Connor
orcid.org/0000-0003-0936-0478
School of Psychology, Queen’s University Belfast, UK Andras N. AUTHOR AFFILIATIONS Schmidt
orcid.org/0000-0003-1579-5881
Department of Psychology, University of Konstanz, Germany Udi Alter
orcid.org/0000-0003-3133-839X
Department of Psychology, Toronto Metropolitan University,
Canada; Department of Psychology, York University, Canada Elise Grimm
orcid.org/0000-0002-2508-1228
Psychological Sciences Research Institute, UCLouvain, Belgium Desirée González
orcid.org/0000-0003-3462-1254
Departamento de Didáctica e Investigación Educativa,
Universidad de la Laguna, Spain Zoe M. Flack
orcid.org/0000-0001-8123-5589
School of Humanities and Social Science, University of Brighton,
UK Philipp Schmid
orcid.org/0000-0003-2966-0806
Media and Communication Science, University of Erfurt,
Germany; Implementation Research, Bernhard-Nocht-Insitute
for Tropical Medicine, Germany Donncha Hanna
orcid.org/0000-0003-3382-4842
School of Psychology, Queen’s University Belfast, UK Mojtaba Soltanlou
orcid.org/0000-0003-1959-1384
School of Psychology, University of Surrey, UK; Brain and Mind
Institute & Department of Psychology, Western University, Canada Roberto A. Ferreira
orcid.org/0000-0002-2097-5759
Facultad de Ciencias de la Educación, Universidad Católica del
Maule, Chile; Millennium Nucleus for the Science of Learning
(MiNSoL), Chile Gabriel Banik
orcid.org/0000-0002-6601-3619
University of Presov, Slovakia Matúš Adamkovič
orcid.org/0000-0002-9648-9108
Institute of Social Sciences, CSPS, Slovak Academy of Sciences,
Slovakia; Faculty of Humanities and Social Sciences, University
of Jyväskylä, Finland Dmitri Rozgonjuk
orcid.org/0000-0002-1612-2040
Institute of Mathematics and Statistics, University of Tartu,
Estonia; Department of Molecular Psychology, Ulm University,
Germany Sanne H. G. van der Ven
orcid.org/0000-0002-4761-0741
Behavioural Science Institute, Radboud University, the Netherlands Neslihan Özhan
orcid.org/0000-0001-8648-2458
School of Psychology, University of Southampton, UK Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 21 Jochen A. Mosbacher
orcid.org/0000-0001-8507-9966
Institute of Psychology, University of Graz, Austria Hilal H. Şen
orcid.org/0000-0002-9877-5108
Department of Psychology, MEF University, Turkey; Faculty of
Psychology, University of Akureyri, Iceland Julia F. Huber
orcid.org/0009-0006-0505-240X
Department of Psychology, University of Tuebingen, Germany Joel R. Anderson
orcid.org/0000-0003-3649-2003
School of Psychology, Australian Catholic University, Australia;
Australian Research Centre in Sex, Health and Society, Australia Saadet Yapan
orcid.org/0000-0002-1772-7324
Department of Psychology, Hasan Kalyoncu University, Turkey Michael Batashvili
orcid.org/0000-0003-4328-6312
Baruch Ivcher School of Psychology, Reichman University, Israel Mauricio E. Garrido Vásquez
orcid.org/0000-0001-6451-4308
Department of Psychology, University of Concepción, Chile Kristel de Groot
orcid.org/0000-0003-1123-3056
Department of Psychology, Erasmus School of Social and
Behavioural Sciences, Erasmus University Rotterdam, the
Netherlands; Department of Applied Economics, Erasmus
School of Economics, Erasmus University Rotterdam, the
Netherlands Kristel de Groot
orcid.org/0000-0003-1123-3056
Department of Psychology, Erasmus School of Social and
Behavioural Sciences, Erasmus University Rotterdam, the Antonino Callea
orcid.org/0000-0002-9984-6123
Department of Human Sciences, Libera Università Maria SS. Assunta University, Italy School of Economics, Erasmus University Rotterdam, the
Netherlands Tolga Ergiyen
orcid.org/0000-0002-5892-0198
Izmir University of Economics, Turkey Matthew O. AUTHOR AFFILIATIONS Parker
orcid.org/0000-0002-7172-5231
School of Pharmacy and Biomedical Science, University of
Portsmouth, UK James M. Clay
orcid.org/0000-0002-3392-5099
Department of Psychology, University of Portsmouth, UK Gaetan Mertens
orcid.org/0000-0003-1923-6657
Department of Medical and Clinical Psychology, Tilburg
University, the Netherlands Mai Helmy
orcid.org/0000-0002-7649-1358
Department of Psychology, College of Education, Sultan
Qaboos University, Oman; Department of Psychology, Faculty
of Arts, Menoufia University, Egypt Feyza Topçu
orcid.org/0000-0002-5853-2670
Department of Psychology, Hasan Kalyoncu University, Turkey Mariia M. Ostroha
orcid.org/0000-0003-0044-8801
Computer Science Department, Makarenko Sumy State
Pedagogical University, Ukraine Merve G. Tutlu
orcid.org/0000-0003-4225-7982
Department of Psychology, Hasan Kalyoncu University, Turkey Karin Täht
orcid.org/0000-0001-8719-0924
Institute of Mathematics and Statistics, University of Tartu,
Estonia; Institute of Psychology, University of Tartu, Estonia Katie A. Gilligan-Lee
orcid.org/0000-0002-5406-2149
School of Psychology, University of Surrey, UK; School of
Psychology, University College Dublin, Ireland Kristel Mikkor
Institute of Mathematics and Statistics, University of Tartu, Estonia Felix O. Egara
orcid.org/0000-0002-6249-9615
Department of Science Education, University of Nigeria, Nsukka Letizia Caso
orcid.org/0000-0001-9703-903X
Department of Human Sciences, Libera Università Maria SS. Assunta University, Italy Martin J. Barwood
orcid.org/0000-0002-1409-2191
School of Health, Sport, and Life Sciences, Leeds Trinity
University, UK Alexander Karner
orcid.org/0000-0001-5000-3195
Department of Cognition, Emotion and Methods in Psychology,
University of Vienna, Austria Karuna Thomas
orcid.org/0000-0002-2082-5523
Department of Psychology, HELP University, Malaysia Grace McMahon
orcid.org/0000-0002-3563-5888
Department of Psychology, University of Limerick, Ireland Maxine M. C. Storm
orcid.org/0000-0002-8847-8310
Department of Psychology, Erasmus School of Social and
Behavioural Sciences, Erasmus University Rotterdam, the
Netherlands Siobhán M. Griffin
orcid.org/0000-0002-3613-2844
Department of Psychology, University of Limerick, Ireland Hans-Christoph Nuerk
orcid.org/0000-0002-0331-7498
Department of Psychology, University of Tuebingen, Germany Gabriella Daroczy
orcid.org/0000-0002-7154-4940
Department of Psychology, University of Tuebingen, Germany Alyssa Counsell
orcid.org/0000-0001-9449-6630
Department of Psychology, Toronto Metropolitan University,
Canada Rizqy A. Zein
orcid.org/0000-0001-7840-0299
Department of Psychology, Universitas Airlangga, Indonesia Andrea Greco
orcid.org/0000-0002-8086-2801
Department of Human and Social Sciences, University of
Bergamo, Italy Oliver Lindemann
orcid.org/0000-0003-3789-5373
Department of Psychology, Erasmus School of Social and
Behavioural Sciences, Erasmus University Rotterdam, the
Netherlands Erin M. Buchanan
orcid.org/0000-0002-9689-4189
Harrisburg University of Science and Technology, USA Dirk Van Rooy
orcid.org/0000-0003-2525-5408
Research School of Psychology, Australian National University,
Australia; Institute for Climate, Energy, and Disaster Solutions,
Australia Katharina Schmid
orcid.org/0000-0001-6018-9245
Universitat Ramon Llull, Esade Business School, Spain Thomas E. Hunt
orcid.org/0000-0001-5769-1154
School of Psychology, University of Derby, UK Theresa E. Wege
orcid.org/0000-0002-6201-4104
Centre for Mathematical Cognition, Loughborough University,
UK Jonas De keersmaecker
orcid.org/0000-0002-8062-7422
Ramon Llull University, Esade Business School, Spain Peter E. AUTHOR AFFILIATIONS Branney
orcid.org/0000-0002-2084-461X
Department of Psychology, University of Bradford, UK Peter E. Branney
orcid.org/0000-0002-2084-461X
Department of Psychology, University of Bradford, UK Joanna E. Lewis
orcid.org/0000-0003-0974-2230
University of Northern Colorado, USA Jordan Randell
orcid.org/0000-0002-5451-1186
Department of Psychology, University of Winchester, UK Balazs Aczel
orcid.org/0000-0001-9364-4988
Institute of Psychology, ELTE Eötvös Loránd University, Hungary 22 Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 Oliver J. Clark
orcid.org/0000-0002-1294-7360
Department of Psychology, Manchester Metropolitan University,
UK Suzanna E. Forwood
orcid.org/0000-0002-8406-5121
School of Psychology & Sport Science, Anglia Ruskin University,
UK Crystal N. Steltenpohl
orcid.org/0000-0001-5049-9354
University of Southern Indiana, USA; Dartmouth Center for
Program Design and Evaluation, USA Elizabeth R. Walters
orcid.org/0000-0003-4009-3996
Department of Psychology, University of Bradford, UK Linda K. Tip
orcid.org/0000-0002-2973-4046
School of Humanities and Social Science, University of Brighton,
UK Bhasker Malu
orcid.org/0000-0002-2592-6972
O P Jindal Global University, India Burcu Tekeş
orcid.org/0000-0002-6601-1023
Department of Psychology, Başkent University, Turkey Jordan R. Wagge
orcid.org/0000-0002-5105-2084
School of Psychology and Cognitive Science, Avila University,
USA TamilSelvan Ramis
orcid.org/0000-0003-3646-984X
Centre for American Education, Sunway University, Malaysia Ho Yan Lai
Department of Psychology, HELP University, Malaysia Stefan Agrigoroaei
orcid.org/0000-0002-4935-688X
Psychological Sciences Research Institute, UCLouvain,
Belgium Deborah S. Crossland
orcid.org/0000-0002-8968-2073
Department of Psychology, University of Winchester, UK Nicholas A. Badcock
orcid.org/0000-0001-6862-4694
School of Psychological Science, University of Western Australia,
Australia; School of Psychological Sciences, Macquarie
University, Australia Kohinoor M. Darda
orcid.org/0000-0002-2692-7360
University of Pennsylvania, USA Tessa R. Flack
orcid.org/0000-0002-4115-4466
School of Psychology, University of Lincoln, UK Kareena McAloney-Kocaman
orcid.org/0000-0003-4561-9619
Department of Psychology, Glasgow Caledonian University, UK Zoe Leviston
orcid.org/0000-0002-4969-7916
Research School of Psychology, Australian National University,
Australia; School of Arts & Humanities, Edith Cowan University,
Perth, WA, Australia Olena V. Semenikhina
orcid.org/0000-0002-3896-8151
Computer Science Depatment, Makarenko Sumy State
Pedagogical University, Ukraine Matthew Brolly
orcid.org/0000-0002-3576-9675
School of Applied Sciences, University of Brighton, UK Erich W. Graf
orcid.org/0000-0002-3162-4233
School of Psychology, University of Southampton, UK Samuel P. Hills
orcid.org/0000-0002-8622-4333
Faculty of Health and Social Sciences, Bournemouth University,
UK Charlie Lea
orcid.org/0000-0003-3271-2769
School of Humanities and Social Science, University of Brighton,
UK Elizabeth Collins
orcid.org/0000-0002-2707-4646
Division of Psychology, Faculty of Natural Sciences, University of
Stirling, UK Kalu T. U. Ogba
orcid.org/0000-0003-0018-9029
Psychology Department, University of Nigeria, Nigeria Fergus M. Guppy
orcid.org/0000-0002-8526-9169
School of Applied Sciences, University of Brighton, UK; School
of Energy, Geoscience, Infrastructure and Society, Heriot-Watt
University, UK Andrew J. Roberts
orcid.org/0000-0002-8058-299X
Department of Philosophy, Macquarie University, Australia Wing-Yee Cheung
orcid.org/0000-0003-2395-1872
Department of Psychology, University of Winchester, UK Amy C. AUTHOR AFFILIATIONS Warhurst
orcid.org/0000-0002-9828-2127
School of Psychology, University of Winchester, UK Sophie Leonard
orcid.org/0000-0002-2846-5366
School of Psychology, University College Dublin, Ireland Shane Lindsay
orcid.org/0000-0002-0068-3468
Department of Psychology and Social Work, University of Hull,
UK Bruno Verschuere
orcid.org/0000-0002-6161-4415
Department of Clinical Psychology, University of Amsterdam,
the Netherlands Ahmed Al Khateeb
orcid.org/0000-0003-4196-5338
Independent Researcher, Saudi Arabia Samantha K. Stanley
orcid.org/0000-0002-1272-5768
Research School of Psychology, Australian National University,
Australia Frank Scharnowski
orcid.org/0000-0002-9973-2521
Department of Cognition, Emotion and Methods in Psychology,
University of Vienna, Austria; Psychiatric Hospital, University of
Zürich, Switzerland Iro Xenidou-Dervou
orcid.org/0000-0002-1511-6191
Centre for Mathematical Cognition, Loughborough University,
UK Leontien de Kwaadsteniet
orcid.org/0000-0002-0988-8523
Behavioural Science Institute, Radboud University, the
Netherlands Omid Ghasemi
orcid.org/0000-0001-7511-5580
School of Psychological Sciences, Macquarie University,
Australia; School of Psychology, University of New South Wales,
Australia Kathryn B. Francis
orcid.org/0000-0002-3875-8904
School of Psychology, Keele University, UK Timothy Liew
Department of Psychology, HELP University, Malaysia Mariah Lecompte
orcid.org/0000-0001-5231-4210
Department of Psychology, Toronto Metropolitan University,
Canada Daniel Ansari
orcid.org/0000-0002-7625-618X
Department of Psychology, Western University, Canada Lisa A. D. Webster
orcid.org/0000-0002-2269-4942
School of Psychology and Therapeutic Studies, Leeds Trinity
University, UK Johnrev Guilaran
orcid.org/0000-0001-6607-8001
Division of Social Sciences, College of Arts and Sciences,
University of the Philippines Visayas, Philippines Kinga Morsanyi
orcid.org/0000-0002-7550-1454
Centre for Mathematical Cognition, Loughborough University,
UK Samuel G. Penny
orcid.org/0000-0002-7485-7897
School of Applied Sciences, University of Brighton, UK; Bristol
Zoological Society, Bristol, UK Samuel G. Penny
orcid.org/0000-0002-7485-7897
School of Applied Sciences, University of Brighton, UK; Bristol
Zoological Society, Bristol, UK 23 Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 Julia Bahnmueller
orcid.org/0000-0001-9207-5074
Centre for Mathematical Cognition, Loughborough University, UK
Christopher J. Hand
orcid.org/0000-0002-1536-4005
School of Education, University of Glasgow, UK
Unita W. Rahajeng
orcid.org/0000-0002-6911-4861
Department of Psychology, Brawijaya University, Indonesia
Dar Peterburg
Baruch Ivcher School of Psychology, Reichman University, Israel
Zsofia K. Takacs
orcid.org/0000-0003-0190-0628
School of Health in Social Science, University of Edinburgh, UK
Michael J. Platow
orcid.org/0000-0002-1721-0827
Research School of Psychology, Australian National University,
Australia Blackburn, G. (2015). Effectiveness of eLearning in
statistics: Pictures and stories. E-Learning and
Digital Media, 12(5–6), 459–480. DOI: https://doi. org/10.1177/2042753016653704 Block, J. (1995). A contrarian view of the five-factor approach to
personality description. Psychological Bulletin, 117(2), 187–
215. DOI: https://doi.org/10.1037/0033-2909.117.2.187 Campbell, D. T., & Fiske, D. W. (1959). Convergent and
discriminant validation by the multitrait-multimethod
matrix. Psychological Bulletin, 56(2), 81–105. DOI: https://
doi.org/10.1037/h0046016 Michael J. Platow
orcid.org/0000-0002-1721-0827
Research School of Psychology, Australian National University,
Australia Carleton, R. N., Collimore, K. C., McCabe, R. REFERENCES Carleton, R. N., Norton, P., & Asmundson, G. (2007). Fearing
the unknown: A short version of the Intolerance of
Uncertainty Scale. Journal of Anxiety Disorders, 21, 105–
117. DOI: https://doi.org/10.1016/j.janxdis.2006.03.014 Baker, S. L., Heinrichs, N., Kim, H.-J., & Hofmann, S. G. (2002). The Liebowitz social anxiety scale as a self-
report instrument: A preliminary psychometric analysis. Behaviour Research and Therapy, 40(6), 701–715. DOI:
https://doi.org/10.1016/S0005-7967(01)00060-2 Chen, G., Gully, S. M., & Eden, D. (2001). Validation of
a New General Self-Efficacy Scale. Organizational
Research Methods, 4(1), 62–83. DOI: https://doi. Baloğlu, M. (2002). Psychometric Properties of the
Statistics Anxiety Rating Scale. Psychological Reports,
90(1), 315–325. DOI: https://doi.org/10.2466/
pr0.2002.90.1.315 Chew, P. K. H., & Dillon, D. B. (2014). Statistics Anxiety
Update: Refining the Construct and Recommendations
for a New Research Agenda. Perspectives on
Psychological Science, 9(2), 196–208. DOI: https://doi. org/10.1177/1745691613518077 Baloğlu, M., & Zelhart, P. F. (2007). Psychometric Properties
of the Revised Mathematics Anxiety Rating Scale. The
Psychological Record, 57(4), 593–611. DOI: https://doi. org/10.1007/BF03395597 Cruise, R., J., Cash, R. W., & Bolton, D., L. (1985). Development
and Validation of an Instrument to Measure Statistical
Anxiety. American Statistical Association Proceedings of the
Section on Statistical Education, 4, 92–97. Behr, D. (2017). Assessing the use of back translation: The
shortcomings of back translation as a quality testing
method. International Journal of Social Research
Methodology, 20(6), 573–584. DOI: https://doi.org/10.1080
/13645579.2016.1252188 Curran, P. G. (2016). Methods for the detection of carelessly
invalid responses in survey data. Journal of Experimental
Social Psychology, 66, 4–19. DOI: https://doi.org/10.1016/j. jesp 2015 07 006 Behr, D., & Shishido, K. (2016). The Translation of
Measurement Instruments for Cross-Cultural Surveys. In C. Wolf, D. Joye, T. Smith, & Y. Fu (Eds.), The SAGE Handbook
of Survey Methodology (pp. 269–287). SAGE Publications
Ltd. DOI: https://doi.org/10.4135/9781473957893.n19 D’Urso, E. D., Maassen, E., van Assen, M. A. L. M., Nuijten, M. B.,
De Roover, K., & Wicherts, J. M. (2022). The Dire Disregard
of Measurement Invariance Testing in Psychological Science
[Preprint]. PsyArXiv. DOI: https://doi.org/10.31234/osf.io/
n3f5u Benson, J., & El‐Zahhar, N. (1994). Further refinement
and validation of the revised test anxiety scale. Structural Equation Modeling: A Multidisciplinary
Journal, 1(3), 203–221. DOI: https://doi. org/10.1080/10705519409539975 Daker, R. J., Cortes, R. A., Lyons, I. M., & Green, A. E. (2020). Creativity anxiety: Evidence for anxiety that is specific
to creative thinking, from STEM to the arts. Journal of
Experimental Psychology: General, 149(1), 42–57. DOI:
https://doi.org/10.1037/xge0000630 Beurze, S. M., Donders, A. R. AUTHOR AFFILIATIONS E., & Antony, M. M. (2011). Addressing revisions to the Brief Fear of Negative
Evaluation scale: Measuring fear of negative evaluation
across anxiety and mood disorders. Journal of Anxiety
Disorders, 25(6), 822–828. DOI: https://doi.org/10.1016/j. janxdis.2011.04.002 Andy P. Field
orcid.org/0000-0003-3306-4695
School of Psychology, University of Sussex, UK REFERENCES T., Zielhuis, G. A., de Vegt, F., &
Verbeek, A. L. M. (2013). Statistics Anxiety: A Barrier for
Education in Research Methodology for Medical Students? Medical Science Educator, 23(3), 377–384. DOI: https://doi. org/10.1007/BF03341649 Field, A. P. (2014). Skills in Mathematics and Statistics in
Psychology and Tackling Transition (Higher Education
Academy STEM Series). Birenbaum, M., & Eylath, S. (1994). Who is afraid of statistics? Correlates of statistics anxiety among students of
educational sciences. Educational Research, 36(1), 93–98. DOI: https://doi.org/10.1080/0013188940360110 Flake, J. K. (2021). Strengthening the foundation of educational
psychology by integrating construct validation into open
science reform. Educational Psychologist. 56, 132–141. DOI:
https://doi.org/10.1080/00461520.2021.1898962 Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 24 Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 Frederick, S. (2005). Cognitive Reflection and Decision Making. Journal of Economic Perspectives, 19(4), 25–42. DOI: Meade, A. W., & Craig, S. B. (2012). Identifying careless
responses in survey data. Psychological Methods, 17(3),
437–455. DOI: https://doi.org/10.1037/a0028085 González, A., Rodríguez, Y., Faílde, J. M., & Carrera, M. V. (2016). Anxiety in the statistics class: Structural relations
with self-concept, intrinsic value, and engagement in
two samples of undergraduates. Learning and Individual
Differences, 45, 214–221. DOI: https://doi.org/10.1016/j. lindif.2015.12.019 Miller, R. B., Greene, B. A., Montalvo, G. P., Ravindran, B., &
Nichols, J. D. (1996). Engagement in Academic Work: The
Role of Learning Goals, Future Consequences, Pleasing
Others, and Perceived Ability. Contemporary Educational
Psychology, 21(4), 388–422. DOI: https://doi.org/10.1006/
ceps.1996.0028 Paechter, M., Macher, D., Martskvishvili, K., Wimmer, S., &
Papousek, I. (2017). Mathematics Anxiety and Statistics
Anxiety. Shared but Also Unshared Components and
Antagonistic Contributions to Performance in Statistics. Frontiers in Psychology, 8. DOI: https://doi.org/10.3389/
fpsyg.2017.01196 Gonzalez, O., MacKinnon, D. P., & Muniz, F. B. (2020). Extrinsic
Convergent Validity Evidence to Prevent Jingle and Jangle
Fallacies. Multivariate Behavioral Research, 56(1), 3–19. DOI: https://doi.org/10.1080/00273171.2019.1707061 Grahe, J. E., Chalk, H. M., Alvarez, L. D. C., Faas, C. S., Hermann,
A. D., & McFall, J. P. (2018). Emerging Adulthood
Measured at Multiple Institutions 2: The Data. Journal
of Open Psychology Data, 6(1), 4. DOI: https://doi. org/10.5334/jopd.38 Papousek, I., Ruggeri, K., Macher, D., Paechter, M., Heene,
M., Weiss, ElisabethM., Schulter, G., & Freudenthaler,
H. H. (2012). Psychometric Evaluation and Experimental
Validation of the Statistics Anxiety Rating Scale. Journal of
Personality Assessment, 94(1), 82–91. DOI: https://doi.org/
10.1080/00223891.2011.627959 Hanna, D., Shevlin, M., & Dempster, M. (2008). REFERENCES The structure
of the statistics anxiety rating scale: A confirmatory
factor analysis using UK psychology students. Personality
and Individual Differences, 45(1), 68–74. DOI: https://doi. org/10.1016/j.paid.2008.02.021 Rad, M. S., Martingano, A. J., & Ginges, J. (2018). Toward
a psychology of Homo sapiens: Making psychological
science more representative of the human population. Proceedings of the National Academy of Sciences of the
United States of America, 115(45), 11401–11405. DOI:
https://doi.org/10.1073/pnas.1721165115 Huang, J. L., Curran, P. G., Keeney, J., Poposki, E. M., & DeShon,
R. P. (2012). Detecting and Deterring Insufficient Effort
Responding to Surveys. Journal of Business and Psychology,
27(1), 99–114. DOI: https://doi.org/10.1007/s10869-011-
9231-8 Ree, M. J., French, D., MacLeod, C., & Locke, V. (2008). Distinguishing Cognitive and Somatic Dimensions of State
and Trait Anxiety: Development and Validation of the
State-Trait Inventory for Cognitive and Somatic Anxiety
(STICSA). Behavioural and Cognitive Psychotherapy, 36(03). DOI: https://doi.org/10.1017/S1352465808004232 Hunt, T. E., Simms, V., Cahoon, A., & Muwonge, C. M. (2021). Socio-Cognitive-Affective Barriers to Mathematics
Education in Developing Nations. In W. Leal Filho, A. M. Azul, L. Brandli, P. G. Özuyar & T. Wall (Eds.), Quality
Education (pp. 1–11). Springer International Publishing. DOI: https://doi.org/10.1007/978-3-319-69902-8_128-1 Richardson, F. C., & Suinn, R. M. (1972). The Mathematics
Anxiety Rating Scale: Psychometric data. Journal of
Counseling Psychology, 19(6), 551–554. DOI: https://doi. org/10.1037/h0033456 Ioannidis, J. P. A. (2005). Why Most Published Research
Findings Are False. PLOS Medicine, 2(8), e124. DOI: https:// Ioannidis, J. P. A. (2005). Why Most Published Research
Findings Are False. PLOS Medicine, 2(8), e124. DOI: https://
doi.org/10.1371/journal.pmed.0020124 Kelley, T. L. (1927). Interpretation of educational
measurements. New York, NY: World Book. Rodebaugh, T. L., Woods, C. M., Thissen, D. M., Heimberg, R. G.,
Chambless, D. L., & Rapee, R. M. (2004). More Information
From Fewer Questions: The Factor Structure and Item
Properties of the Original and Brief Fear of Negative
Evaluation Scale. Psychological Assessment, 16(2), 169–
181. DOI: https://doi.org/10.1037/1040-3590.16.2.169 Lawson, K. M., & Robins, R. W. (2021). Sibling Constructs:
What Are They, Why Do They Matter, and How
Should You Handle Them? Personality and Social
Psychology Review, 25(4), 344–366. DOI: https://doi. org/10 1177/10888683211047101 Schwab-McCoy, A. (2019). The State of Statistics Education
Research in Client Disciplines: Themes and Trends Across
the University. Journal of Statistics Education, 27(3),
253–264. DOI: https://doi.org/10.1080/10691898.2019.1
687369 • PR File 1. Peer Review History. DOI: https://doi.
org/10.5334/jopd.80.pr1 Terry, J., Ross, R. M., Nagy, T., Salgado, M., Garrido-Vásquez, P., Sarfo, J. O., Cooper, S., Buttner, A. C., Lima, T. J. S., Öztürk, I˙., Akay,
N., Santos, F. H., Artemenko, C., Copping, L. T., Elsherif, M. M., Milovanović, I., Cribbie, R. A., Drushlyak, M. G., Swainston, K., Shou, Y.,
Leongómez, J. D., Palena, N., Abidin, F. A., Reyes-Rodríguez, M. F., He, Y., Abraham, J., Vatakis, A., Jankowsky, K., Schmidt, K., S. N.
L., Grimm, E., González, D., Schmid, P., Ferreira, R. A., Rozgonjuk, D., Özhan, N., O’Connor, P. A., Zsido, A. N., Stiglic, G., Rhodes, D.,
Rodríguez, C., Ropovik, I., Enea, V., Nurwanti, R., Estudillo, A. J., Beribisky, N., Himawan, K. K., Geven, L. M., van Hoogmoed, A. H., Bret,
A., Chapman, J. E., Alter, U., Flack, Z. M., Hanna, D., Soltanlou, M., Banik, G., Adamkovič, M., van der Ven, S. H. G., Mosbacher, J. A., Şen,
H. H., Anderson, J. R., Batashvili, M., de Groot, K., Parker, M. O., Helmy, M., Ostroha, M. M., Gilligan-Lee, K. A., Egara, F. O., Barwood,
M. J., Thomas, K., McMahon, G., Griffin, S. M., Nuerk, H.-C., Counsell, A., Lindemann, O., Van Rooy, D., Wege, T. E., Lewis, J. E., Aczel,
B., Monaghan, C., Al-Hoorie, A. H., Huber, J. F., Yapan, S., Garrido Vásquez, M. E., Callea, A., Ergiyen, T., Clay, J. M., Mertens, G., Topçu,
F., Tutlu, M. G., Täht, K., Mikkor, K., Caso, L., Karner, A., Storm, M. M. C., Daroczy, G., Zein, R. A., Greco, A., Buchanan, E. M., Schmid, K.,
Hunt, T. E., De keersmaecker, J., Branney, P. E., Randell, J., Clark, O. J., Steltenpohl, C. N., Malu, B., Tekeş, B., Ramis, T., Agrigoroaei, S.,
Badcock, N. A., McAloney-Kocaman, K., Semenikhina, O. V., Graf, E. W., Lea, C., Ogba, K. T. U., Guppy, F. M., Warhurst, A. C., Lindsay, S.,
Al Khateeb, A., Scharnowski, F., de Kwaadsteniet, L., Francis, K. B., Lecompte, M., Webster, L. A. D., Morsanyi, K., Forwood, S. E., Walters,
E. R., Tip, L. K., Wagge, J. R., Lai, H. Y., Crossland, D. S., Darda, K. M., Flack, T. R., Leviston, Z., Brolly, M., Hills, S. P., Collins, E., Roberts, A. J.,
Cheung, W.-Y., Leonard, S., Verschuere, B., Stanley, S. K., Xenidou-Dervou, I., Ghasemi, O., Liew, T., Ansari, D., Guilaran, J., Penny, S. G.,
Bahnmueller, J., Hand, C. J., Rahajeng, U. W., Peterburg, D., Takacs, Z. K., Platow, M. J., & Field, A. P. (2023). Data from an International
Multi-Centre Study of Statistics and Mathematics Anxieties and Related Variables in University Students (the SMARVUS Dataset).
Journal of Open Psychology Data, 11: 8, pp. 1–25. DOI: https://doi.org/10.5334/jopd.80 org/10.1177/10888683211047101 Leary, M. R. (1983). Brief Fear of Negative Evaluation Scale
[Data set]. American Psychological Association. DOI:
https://doi.org/10.1037/t00048-000 Liebowitz, M. R. (1987). Social Phobia. Modern Problems
in Pharmacopsychiatry, 22, 141–173. DOI: https://doi. org/10.1159/000414022 Shenhav, A., Rand, D. G., & Greene, J. D. (2012). Divine
intuition: Cognitive style influences belief in God. Journal of
Experimental Psychology: General, 141(3), 423–428. DOI:
https://doi.org/10.1037/a0025391 Long, O., & Chalk, H. M. (2020). Belonging and Marital
Perception Variances in Emerging Adults With
Differing Disability Identities. Psi Chi Journal of
Psychological Research, 25(1), 22–29. DOI: https://doi. org/10.24839/2325-7342.JN25.1.22 Spielberger, C. D. (1983). State-Trait Anxiety Inventory for
Adults (STAI-AD) [Database record]. APA PsycTests. DOI:
https://doi.org/10.1037/t06496-000 Terry et al. Journal of Open Psychology Data DOI: 10.5334/jopd.80 25 Terry, J., & Field, A. P. (2023). A comparison of theoretical
explanations for the relationship between maths and
statistics anxieties on academic performance. Manuscript
in Preparation. Trassi, A. P., Leonard, S. J., Rodrigues, L. D., Rodas, J. A., &
Santos, F. H. (2022). Mediating factors of statistics
anxiety in university students: A systematic review and
meta-analysis. Annals of the New York Academy of Sciences,
1512(1), 76–97. DOI: https://doi.org/10.1111/nyas.14746 Terry, J., Lea, C., & Field, A. P. (2023). Statistics Anxiety
and Maths Anxiety: Evidence of another Jangle Fallacy. Manuscript in Preparation. Zeidner, M. (1991). Statistics and Mathematics Anxiety in
Social Science Students: Some Interesting Parallels. British
Journal of Educational Psychology, 61(3), 319–328. DOI:
https://doi.org/10.1111/j.2044-8279.1991.tb00989.x Tindall, I. K., Curtis, G. J., & Locke, V. (2021). Dimensionality
and Measurement Invariance of the State-Trait
Inventory for Cognitive and Somatic Anxiety
(STICSA) and Validity Comparison With Measures
of Negative Emotionality. Frontiers in Psychology,
12. https://www.frontiersin.org/articles/10.3389/
fpsyg.2021.644889. DOI: https://doi.org/10.3389/
fpsyg.2021.644889 TO CITE THIS ARTICLE: Terry, J., Ross, R. M., Nagy, T., Salgado, M., Garrido-Vásquez, P., Sarfo, J. O., Cooper, S., Buttner, A. C., Lima, T. J. S., Öztürk, I˙., Akay,
N., Santos, F. H., Artemenko, C., Copping, L. T., Elsherif, M. M., Milovanović, I., Cribbie, R. A., Drushlyak, M. G., Swainston, K., Shou, Y.,
Leongómez, J. D., Palena, N., Abidin, F. A., Reyes-Rodríguez, M. F., He, Y., Abraham, J., Vatakis, A., Jankowsky, K., Schmidt, K., S. N. L., Grimm, E., González, D., Schmid, P., Ferreira, R. A., Rozgonjuk, D., Özhan, N., O’Connor, P. A., Zsido, A. N., Stiglic, G., Rhodes, D.,
Rodríguez, C., Ropovik, I., Enea, V., Nurwanti, R., Estudillo, A. J., Beribisky, N., Himawan, K. K., Geven, L. M., van Hoogmoed, A. H., Bret,
A., Chapman, J. E., Alter, U., Flack, Z. M., Hanna, D., Soltanlou, M., Banik, G., Adamkovič, M., van der Ven, S. H. G., Mosbacher, J. A., Şen,
H. H., Anderson, J. R., Batashvili, M., de Groot, K., Parker, M. O., Helmy, M., Ostroha, M. M., Gilligan-Lee, K. A., Egara, F. O., Barwood,
M. J., Thomas, K., McMahon, G., Griffin, S. M., Nuerk, H.-C., Counsell, A., Lindemann, O., Van Rooy, D., Wege, T. E., Lewis, J. E., Aczel,
B., Monaghan, C., Al-Hoorie, A. H., Huber, J. F., Yapan, S., Garrido Vásquez, M. E., Callea, A., Ergiyen, T., Clay, J. M., Mertens, G., Topçu,
F., Tutlu, M. G., Täht, K., Mikkor, K., Caso, L., Karner, A., Storm, M. M. C., Daroczy, G., Zein, R. A., Greco, A., Buchanan, E. M., Schmid, K.,
Hunt, T. E., De keersmaecker, J., Branney, P. E., Randell, J., Clark, O. J., Steltenpohl, C. N., Malu, B., Tekeş, B., Ramis, T., Agrigoroaei, S.,
Badcock, N. A., McAloney-Kocaman, K., Semenikhina, O. V., Graf, E. W., Lea, C., Ogba, K. T. U., Guppy, F. M., Warhurst, A. C., Lindsay, S.,
Al Khateeb, A., Scharnowski, F., de Kwaadsteniet, L., Francis, K. B., Lecompte, M., Webster, L. A. D., Morsanyi, K., Forwood, S. E., Walters,
E. R., Tip, L. K., Wagge, J. R., Lai, H. Y., Crossland, D. S., Darda, K. M., Flack, T. R., Leviston, Z., Brolly, M., Hills, S. P., Collins, E., Roberts, A. J.,
Cheung, W.-Y., Leonard, S., Verschuere, B., Stanley, S. K., Xenidou-Dervou, I., Ghasemi, O., Liew, T., Ansari, D., Guilaran, J., Penny, S. G.,
Bahnmueller, J., Hand, C. J., Rahajeng, U. W., Peterburg, D., Takacs, Z. K., Platow, M. J., & Field, A. P. (2023). TO CITE THIS ARTICLE: Data from an International
Multi-Centre Study of Statistics and Mathematics Anxieties and Related Variables in University Students (the SMARVUS Dataset). Journal of Open Psychology Data, 11: 8, pp. 1–25. DOI: https://doi.org/10.5334/jopd.80 Published: 29 May 2023 Published: 29 May 2023 © 2023 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0
International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
author and source are credited. See http://creativecommons.org/licenses/by/4.0/.
Journal of Open Psychology Data is a peer reviewed open access journal published by Ubiquity Press PEER REVIEW COMMENTS Journal of Open Psychology Data has blind peer review,
which is unblinded upon article acceptance. The editorial
history of this article can be downloaded here: Trakulphadetkrai, N. V. (2017, February). Undergraduate
students’ perceptions of learning introductory statistics
through producing a statistical picture book. CERME 10. https://hal.archives-ouvertes.fr/hal-01927710 COPYRIGHT: © 2023 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0
International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
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author and source are credited. See http://creativecommons.org/licenses/by/4.0/. Journal of Open Psychology Data is a peer-reviewed open access journal published by Ubiquity Press. Journal of Open Psychology Data is a peer-reviewed open access journal published by Ubiquity Press.
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Challenges and Opportunities of Very Light High-Performance Electric Drives for Aviation
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Challenges and Opportunities of Very Light
High-Performance Electric Drives for Aviation Markus Henke 1,*, Gerrit Narjes 2 ID , Jan Hoffmann 1, Constantin Wohlers 2, Stefan Urbanek 2 ID ,
Christian Heister 1 ID , Jörn Steinbrink 2, Wolf-Rüdiger Canders 1 and Bernd Ponick 2 Markus Henke 1,*, Gerrit Narjes 2 ID , Jan Hoffmann 1, Constantin Wohlers 2, Stefan Urbane
Christian Heister 1 ID , Jörn Steinbrink 2, Wolf-Rüdiger Canders 1 and Bernd Ponick 2 Christian Heister 1 ID , Jörn Steinbrink 2, Wolf-Rüdiger Canders 1 and Bernd Ponick 2
1
Institut für Elektrische Maschinen, Antriebe und Bahnen, Technische Universität Braunschweig,
38106 Braunschweig, Germany; j.hoffmann@tu-bs.de (J.H.); c.heister@tu-braunschweig.de (C.H.);
w.canders@tu-braunschweig.de (W.-R.C.) 1
Institut für Elektrische Maschinen, Antriebe und Bahnen, Technische Universität Braunschweig,
38106 Braunschweig, Germany; j.hoffmann@tu-bs.de (J.H.); c.heister@tu-braunschweig.de (C.H.);
w.canders@tu-braunschweig.de (W.-R.C.) 1
Institut für Elektrische Maschinen, Antriebe und Bahnen, Technische Universität Braunschweig,
38106 Braunschweig, Germany; j.hoffmann@tu-bs.de (J.H.); c.heister@tu-braunschweig.de (C.H.);
w.canders@tu-braunschweig.de (W.-R.C.) g
2
Institut für Antriebssysteme und Leistungselektronik, Leibniz Universität Hannover, 30167 Hannover,
Germany; gerrit.narjes@ial.uni-hannover.de (G.N.); constantin.wohlers@ial.uni-hannover.de (C.W.);
stefan.urbanek@ial.uni-hannover.de (S.U.); steinbrink@ial.uni-hannover.de (J.S.);
ponick@ial.uni-hannover.de (B.P.) 2
Institut für Antriebssysteme und Leistungselektronik, Leibniz Universität Hannover, 30167 Hannover,
Germany; gerrit.narjes@ial.uni-hannover.de (G.N.); constantin.wohlers@ial.uni-hannover.de (C.W.);
stefan.urbanek@ial.uni-hannover.de (S.U.); steinbrink@ial.uni-hannover.de (J.S.);
ponick@ial.uni-hannover.de (B.P.) *
Correspondence: markus.henke@tu-braunschweig.de; Tel.: +49-531-391-3914 Received: 3 January 2018; Accepted: 26 January 2018; Published: 2 February 2018 Received: 3 January 2018; Accepted: 26 January 2018; Published: 2 February 2018 Abstract: The demand for alternative fueling methods to reduce the need for fossil fuels is not
limited to the electrification of ground vehicles. More-electric and all-electric aircraft pose challenges,
with extensive requirements in terms of power density, efficiency, safety, and environmental
sustainability. This paper focuses on electrical machines and their components, especially for
high-power applications like the main propulsion. The electrical machine is evaluated from different
aspects, followed by a closer look at the components and materials to determine the suitability of the
current standard materials and advanced technologies. Furthermore, the mechanical and thermal
aspects are reviewed, including new and innovative concepts for the cooling of windings and for
the use of additive manufacturing. Aircraft have special demands regarding weight and installation
space. Following recent developments and looking ahead to the future, the need and the possibilities
for light and efficient electrical machines are addressed. All of the approaches and developments
presented lead to a better understanding of the challenges to be expected and highlight the upcoming
opportunities in electrical machine design for the use of electric motors and generators in future
aircraft. Challenges and Opportunities of Very Light
High-Performance Electric Drives for Aviation Several prototypes of electrical machines for smaller aircraft already exist, such as the electric
drive of the Siemens powered Extra 330LE. The focus of this paper is to provide an overview of
current technical possibilities and technical interrelations of high performance electric drives for
aviation. A 1 MW drive is exemplified to present the possibilities for future drives for airplanes
carrying a larger number of passengers. All presented techniques can also be applied to other drive
power classes. Keywords: electrical machines; high-performance electric drive; all-electric aircraft; more-electric
aircraft; electric propulsion; additive manufacturing; superconductors; mechanical modelling;
thermal modelling; direct liquid cooling www.mdpi.com/journal/energies energies energies energies www.mdpi.com/journal/energies 1. Challenges and Requirements Concerning Electric Drives in Aviation Studies of the future trends in aviation concluded that in the next few years, more-electric aircraft
using hybrid propulsion systems and all-electric aircraft incorporating pure electric propulsion will
be technically feasible, offering benefits for the environment in terms of pollution, noise, and for
business [1,2]. The potential of future technologies covered in this study are displayed in Figure 1. The advances in technology that have had several impacts on the flight-range of electric aircraft are
presented using a Dornier 328 aircraft as an example. At the top of the graph, a conventionally powered Energies 2018, 11, 344; doi:10.3390/en11020344 www.mdpi.com/journal/energies 2 of 25
drives
ent of Energies 2018, 11, 344
y
which instantly de aircraft is shown. The bars below indicate several flight ranges when incorporating electric drive
components, improving aircraft design, and using batteries with different energy to weight ratios. another benefit: they work independent of the surrounding air pressure. Additionally, electric
systems are increasingly incorporated into on-board systems. Figure 1. Example and comparison between conventional and all-electric aircraft according to Roland
Berger LTD, London, UK [1]. Figure 1. Example and comparison between conventional and all-electric aircraft according to Roland
Berger LTD, London, UK [1]. Figure 1. Example and comparison between conventional and all-electric aircraft according to Roland
Berger LTD, London, UK [1]. Figure 1. Example and comparison between conventional and all-electric aircraft according to Roland
Berger LTD, London, UK [1]. Studies concerning the possibilities of applications to aircraft to replace hydraulics have already
been completed by several companies, such as the United Technologies Research Center. Lightweight
energy storage systems are crucial for energy efficient operation and flight distance. Lightweight
energy storage systems are also important for electric drives, which lower the take-off mass. Thus, a
future task is to further optimize electric drives and gas turbine generators in terms of the power to
weight ratio. Changing the machine design from conventional principles to more advanced
technologies will likely also be required, such as superconducting machines with high-temperature
superconductors. A current university project of Technische Universität Braunschweig (TU Braunschweig), the
Collaborative Research Centre 880 (CRC 880) funded by Deutsche Forschungsgemeinschaft (DFG)
unites a number of university institutes in aircraft research. The scope is future aircraft design. With the implementation of different future technologies, the flight range of an electric aircraft
could reach or outperform a conventional aircraft burning fossil fuel. 1. Challenges and Requirements Concerning Electric Drives in Aviation Turbofan plane of DFG-funded CRC 880 [2]. Fi u e 2 Tu bofa
la e of DFG fu ded CRC 880 [2] Today, the most efficient aircraft engines are turbofan engines with a high bypass ratio. This
ype of engine can be found in nearly all commercial aircraft. As an example, consider the 1.1 MW
cruising power) turbofan engine (PW 300 Series, Pratt & Whitney, East Hartford, CT, USA) for a
business aircraft. The overall weight is 470 kg, with a fan diameter of 800 mm, and a fan speed of
10,000–11,000 rpm (circumferential speed 466 m/s) [3,4] (Figure 3). At cruising operation, the
rotational speed is reduced to 80%, reducing the power output to approximately 30% of the
maximum power. The maximum speed of the turbine’s high-pressure part is 27,500 rpm, driving the
compressor, whereas the low-pressure part of the expansion turbine is directly coupled to the fan. To
adapt to the fan speed, a gearbox can be used (geared turbofan). This one-stage, low-weight planetary
gear is calculated as 0.005 kg/kW. By exchanging the turbine for an electrical drive, we obtain an
“electrofan” engine as a competing system that allows carbon dioxide-free operation of airplanes,
which is one of the big challenges in the future of air transport. Today, the most efficient aircraft engines are turbofan engines with a high bypass ratio. This type
of engine can be found in nearly all commercial aircraft. As an example, consider the 1.1 MW (cruising
power) turbofan engine (PW 300 Series, Pratt & Whitney, East Hartford, CT, USA) for a business aircraft. The overall weight is 470 kg, with a fan diameter of 800 mm, and a fan speed of 10,000–11,000 rpm
(circumferential speed 466 m/s) [3,4] (Figure 3). At cruising operation, the rotational speed is reduced
to 80%, reducing the power output to approximately 30% of the maximum power. The maximum speed
of the turbine’s high-pressure part is 27,500 rpm, driving the compressor, whereas the low-pressure
part of the expansion turbine is directly coupled to the fan. To adapt to the fan speed, a gearbox can
be used (geared turbofan). This one-stage, low-weight planetary gear is calculated as 0.005 kg/kW. By exchanging the turbine for an electrical drive, we obtain an “electrofan” engine as a competing
system that allows carbon dioxide-free operation of airplanes, which is one of the big challenges in the
future of air transport. 1. Challenges and Requirements Concerning Electric Drives in Aviation Today, the most efficient aircraft engines are turbofan engines with a high bypass ratio. This
type of engine can be found in nearly all commercial aircraft. As an example, consider the 1.1 MW
(cruising power) turbofan engine (PW 300 Series, Pratt & Whitney, East Hartford, CT, USA) for a
business aircraft. The overall weight is 470 kg, with a fan diameter of 800 mm, and a fan speed of
10,000–11,000 rpm (circumferential speed 466 m/s) [3,4] (Figure 3). At cruising operation, the
rotational speed is reduced to 80%, reducing the power output to approximately 30% of the
maximum power. The maximum speed of the turbine’s high-pressure part is 27,500 rpm, driving the
compressor, whereas the low-pressure part of the expansion turbine is directly coupled to the fan. To
adapt to the fan speed, a gearbox can be used (geared turbofan). This one-stage, low-weight planetary
gear is calculated as 0.005 kg/kW. By exchanging the turbine for an electrical drive, we obtain an
“electrofan” engine as a competing system that allows carbon dioxide-free operation of airplanes,
which is one of the big challenges in the future of air transport. Figure 3. PW 305A turbofan engine, 470 kg weight on the left (source Pratt & Whitney [5]) and a
Figure 3. PW 305A turbofan engine, 470 kg weight on the left (source Pratt & Whitney [5]) and a
possible substitution of the gas turbine with a high efficient electric drive on the right
Figure 3. PW 305A turbofan engine, 470 kg weight on the left (source Pratt & Whitney [5]) and
a possible substitution of the gas turbine with a high-efficient electric drive on the right. Figure 3. PW 305A turbofan engine, 470 kg weight on the left (source Pratt & Whitney [5]) and a
Figure 3. PW 305A turbofan engine, 470 kg weight on the left (source Pratt & Whitney [5]) and a
ibl
b tit ti
f th
t
bi
ith
hi h
ffi i
t l
t i d i
th
i ht
Figure 3. PW 305A turbofan engine, 470 kg weight on the left (source Pratt & Whitney [5]) and
a possible substitution of the gas turbine with a high-efficient electric drive on the right. p
g
g
g
Assuming the electrofans of the aircraft are operated on a DC bus, each electric drive will include
an electrical machine and an inverter. 1. Challenges and Requirements Concerning Electric Drives in Aviation In this study, the increase in
gravimetric battery density roadmap shows that the required level for aviation of 500 Wh/kg will be
reached by 2025. At present, densities of 250 to 320 Wh/kg have been produced, which is close to
the requirement. The motivation in researching this subject area is based on significantly reducing
energy usage, noise, and emissions [2,3]. Electrifying the propulsion of aircraft is possible with hybrid
systems or electric propulsion. Future all-electric aircraft will have highly dynamic electric drives
which instantly develop torque over a wide range of speeds. The instantaneous development of torque
can increase security, such as when performing a go-around procedure. Electric drives have another
benefit: they work independent of the surrounding air pressure. Additionally, electric systems are
increasingly incorporated into on-board systems. Detailed data, including turbofan engines for a 100 pax plane, have already been gathered. This data
can be used for dimensioning purposes of future electric aircraft and electric propulsion systems
(Figure 2). Studies concerning the possibilities of applications to aircraft to replace hydraulics have
already been completed by several companies, such as the United Technologies Research Center. Lightweight energy storage systems are crucial for energy efficient operation and flight distance. Lightweight energy storage systems are also important for electric drives, which lower the take-off
mass. Thus, a future task is to further optimize electric drives and gas turbine generators in
terms of the power to weight ratio. Changing the machine design from conventional principles
to more advanced technologies will likely also be required, such as superconducting machines with
high-temperature superconductors. A current university project of Technische Universität Braunschweig (TU Braunschweig),
the Collaborative Research Centre 880 (CRC 880) funded by Deutsche Forschungsgemeinschaft (DFG)
unites a number of university institutes in aircraft research. The scope is future aircraft design. Detailed
data, including turbofan engines for a 100 pax plane, have already been gathered. This data can be
used for dimensioning purposes of future electric aircraft and electric propulsion systems (Figure 2). 3 of 25
3 of 25
3 of 25 Energies 2018, 11, 344
Energies 2017, 10, x FOR P
Energies 2017, 10, x FO Figure 2 Turbofan plane of DFG-funded CRC 880 [2]
Figure 2. Turbofan plane of DFG-funded CRC 880 [2]. Figure 2 Turbofan plane of DFG-funded CRC 880 [2] Figure 2 Tu bofa
la e of DFG fu ded CRC 880 [2]
Figure 2. 2. Design Space for Aviation Drives
2. Design Space for Aviation Drives The electrical machine for aviation must be thermally robust and highly efficient to limit the
energy consumption and cooling [7]. A close dependency exists between energy consumption and
machine weight. The machine’s weight originates from the mass of the soft-magnetic components
that can be reduced by the higher saturation of the material (i.e., iron or cobalt-iron), and the weight
of the winding that can be reduced by higher current densities or the use of aluminium instead of
copper. Higher losses in the machine must be covered by increased power consumption, thus raising
either battery mass or tank volume, when using fuel cells. The electrical machine for aviation must be thermally robust and highly efficient to limit the
energy consumption and cooling [7]. A close dependency exists between energy consumption and
machine weight. The machine’s weight originates from the mass of the soft-magnetic components that
can be reduced by the higher saturation of the material (i.e., iron or cobalt-iron), and the weight of the
winding that can be reduced by higher current densities or the use of aluminium instead of copper. Higher losses in the machine must be covered by increased power consumption, thus raising either
battery mass or tank volume, when using fuel cells. Different electrical machine concepts could be used to design an all-electric aircraft. Especially
for the main engine, a motor is needed that has either high torque density or high-power density. Krishnan and Bharadwaj [7] list three possible machine concepts that could achieve these goals: (1)
permanent magnet synchronous machine (PMSM), (2) switched reluctance machine (SRM), and (3)
cage induction machine (IM). In this paper, we compare these three concepts and the electrically
excited synchronous machine (ESM) as well. Table 1 shows the different key characteristics of the
aforementioned electrical machine concepts. Given this comparison the permanent magnet synchronous machine is the most feasible
Different electrical machine concepts could be used to design an all-electric aircraft. Especially
for the main engine, a motor is needed that has either high torque density or high-power density. Krishnan and Bharadwaj [7] list three possible machine concepts that could achieve these goals:
(1) permanent magnet synchronous machine (PMSM), (2) switched reluctance machine (SRM), and (3)
cage induction machine (IM). In this paper, we compare these three concepts and the electrically
excited synchronous machine (ESM) as well. Table 1 shows the different key characteristics of the
aforementioned electrical machine concepts. 1. Challenges and Requirements Concerning Electric Drives in Aviation They should not increase the weight of the engine at a
comparable power rating, having an extremely lightweight, often used electrical machine is essential. Additionally, the difference between apparent power consumption and real power output should be
Assuming the electrofans of the aircraft are operated on a DC bus, each electric drive will include
an electrical machine and an inverter. They should not increase the weight of the engine at a
comparable power rating, having an extremely lightweight, often used electrical machine is essential. Additionally, the difference between apparent power consumption and real power output should be
as small as possible to minimize the size and weight of the inverter. Assuming the electrofans of the aircraft are operated on a DC bus, each electric drive will include
an electrical machine and an inverter. They should not increase the weight of the engine at a comparable
power rating, having an extremely lightweight, often used electrical machine is essential. Additionally,
the difference between apparent power consumption and real power output should be as small as
possible to minimize the size and weight of the inverter. as small as possible to minimize the size and weight of the inverter. As design approach, we see two main areas of focus: a direct drive without a gearbox for the fan
as the simplest approach, and a high-speed drive of approximately the same speed as an actual
t
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i
d
ith
b
d
d t
b f
T
h i
l h ll
ill i
l d th
p
g
As design approach, we see two main areas of focus: a direct drive without a gearbox for the fan
as the simplest approach, and a high-speed drive of approximately the same speed as an actual
turbine, equipped with a gearbox as used on geared turbofans. Technical challenges will include the
As design approach, we see two main areas of focus: a direct drive without a gearbox for the
fan as the simplest approach, and a high-speed drive of approximately the same speed as an actual
turbine, equipped with a gearbox as used on geared turbofans. Technical challenges will include the 4 of 25 Energies 2018, 11, 344 overload capacity of the electric drive during take-off and the material fatigue over the lifetime of
the aircraft. 1. Challenges and Requirements Concerning Electric Drives in Aviation Operating a fluid flow engine, we have a quadratic function between torque and speed,
so exploiting the advantages of field weakening (reduced power rating) will not be possible. overload capacity of the electric drive during take-off and the material fatigue over the lifetime of the
aircraft. Operating a fluid flow engine, we have a quadratic function between torque and speed, so
exploiting the advantages of field weakening (reduced power rating) will not be possible In Figure 4, a schematic overview of a modern turbofan engine is shown. Before changing to
all-electric aircraft, more-electric aircraft are likely to be developed. As independently rotating spools
are implemented to develop torque or to generate power, electrifying a part of the spools is a possible
method to create a highly integrated hybrid turbofan engine. exploiting the advantages of field weakening (reduced power rating) will not be possible. In Figure 4, a schematic overview of a modern turbofan engine is shown. Before changing to all-
electric aircraft, more-electric aircraft are likely to be developed. As independently rotating spools
are implemented to develop torque or to generate power, electrifying a part of the spools is a possible
method to create a highly integrated hybrid turbofan engine. Figure 4. Typical modern three-spool turbofan jet engine: compressor on the left, turbine on the right
[6]. Figure 4. Typical modern three-spool turbofan jet engine: compressor on the left, turbine on the
right [6]. Figure 4. Typical modern three-spool turbofan jet engine: compressor on the left, turbine on the right
[6]. Figure 4. Typical modern three-spool turbofan jet engine: compressor on the left, turbine on the
right [6]. 2. Design Space for Aviation Drives
2. Design Space for Aviation Drives Given this comparison, the permanent magnet synchronous machine is the most feasible
solution for implementation into an aircraft. Ganev [8], however, worked under the premise that the
machines will be used as auxiliary units to the main engine, e.g., by replacing hydraulic pumps. For
higher power demands in the megawatt range, this comparison may not be fully applicable. Also,
characteristics like functional safety, and a comparison of the machine concepts regarding the
constant power range, are missing. Arkkio et al. [9], Binder et al. [10], and Gerada et al. [11] compared
many designs and concepts over a wide power range. Figure 5 shows many of the highlighted
machine designs as output power over their rotational speed. Given this comparison, the permanent magnet synchronous machine is the most feasible solution
for implementation into an aircraft. Ganev [8], however, worked under the premise that the machines
will be used as auxiliary units to the main engine, e.g., by replacing hydraulic pumps. For higher power
demands in the megawatt range, this comparison may not be fully applicable. Also, characteristics like
functional safety, and a comparison of the machine concepts regarding the constant power range,
are missing. Arkkio et al. [9], Binder et al. [10], and Gerada et al. [11] compared many designs and
concepts over a wide power range. Figure 5 shows many of the highlighted machine designs as output
power over their rotational speed. 5 of 25
5 of 25 Energies 2018, 11, 344
Energies 2017, 10, x FOR Table 1. Comparison of key characteristics for different electrical machine concepts [8]. (– unfavourable;
- disadvantageous; o neutral; + beneficial; ++ very beneficial). Table 1. Comparison of key characteristics for different electrical machine concepts [8]. (
unfavourable; - disadvantageous; o neutral; + beneficial; ++ very beneficial). 2. Design Space for Aviation Drives
2. Design Space for Aviation Drives Key Characteristic
ESM
IM
SRM
PMSM Key Characteristic
ESM
IM
SRM
PMSM
Rotor losses
–
o
o
++
Stator losses
++
o
o
o
Windage Losses
-
o
–
++
Rotor thermal limitations
o
+
++
o
Cooling options
–
o
o
++
Rotor mechanical limitations
–
o
+
++
Torque-to-inertia ratio
o
o
o
++
Compatibility with bearings
-
o
o
++
High-speed capability
–
o
+
++
Short-circuit behaviour
–
++
++
-
Machine complexity
o
+
++
+
Current density
–
+
+
+
Power density
–
+
+
+
Key Characteristic
ESM
IM
SRM
PMSM
Rotor losses
--
o
o
++
Stator losses
++
o
o
o
Windage Losses
-
o
--
++
Rotor thermal limitations
o
+
++
o
Cooling options
--
o
o
++
Rotor mechanical limitations
--
o
+
++
Torque-to-inertia ratio
o
o
o
++
Compatibility with bearings
-
o
o
++
High-speed capability
--
o
+
++
Short-circuit behaviour
--
++
++
-
Machine complexity
o
+
++
+
Current density
--
+
+
+
Power density
--
+
+
+
Figure 5 Overview of electrical machine designs from the literature
Figure 5. Overview of electrical machine designs from the literature. Figure 5 Overview of electrical machine designs from the literature
Figure 5. Overview of electrical machine designs from the literature. Figure 5. Overview of electrical machine designs from the literature. Figure 5. Overview of electrical machine designs from the literature. The general suitability of a machine design can be described in different ways. Often, the power
to weight ratio is used. As this number compares the power to the overall weight, high-speed designs
have an advantage, since they need a much lower volume for the same power output. The reason
why this value was not used to quantify the following machine designs is that, apart from the one
mentioned before, this value is greatly dependent on the used materials, and the used weight includes
the housing and bearings. As these differ considerably depending on the application, the power to
weight ratio does not allow a fair comparison; an alternative ratio would be the power to volume
ratio. This value is just slightly more suitable, as it is not material-dependent but does not consider
cut-outs in the rotor body, which are used in machines with high bore diameters to reduce the weight
and the moment of inertia. 2. Design Space for Aviation Drives
2. Design Space for Aviation Drives A more fitting choice is Esson’s number [12], as it incorporates the
rotational speed into the power to volume ratio, thus providing a torque-to-volume ratio:
The general suitability of a machine design can be described in different ways. Often, the power
to weight ratio is used. As this number compares the power to the overall weight, high-speed designs
have an advantage, since they need a much lower volume for the same power output. The reason
why this value was not used to quantify the following machine designs is that, apart from the one
mentioned before, this value is greatly dependent on the used materials, and the used weight includes
the housing and bearings. As these differ considerably depending on the application, the power to
weight ratio does not allow a fair comparison; an alternative ratio would be the power to volume ratio. This value is just slightly more suitable, as it is not material-dependent but does not consider cut-outs
in the rotor body, which are used in machines with high bore diameters to reduce the weight and the
moment of inertia. A more fitting choice is Esson’s number [12], as it incorporates the rotational speed
into the power to volume ratio, thus providing a torque-to-volume ratio: ߳=
ܲே
ܦ
ଶ⋅݈⋅݊ே
ܽ݊݀ሾ߳ሿ= kW ⋅min
mଷ
(1)
ϵ =
PN
D2
i ·l f e·nN
and [ϵ] = kW·min
m3
(1) (1) (1) where PN is the rated power, Di is the bore diameter, lfe is the length of the active parts, and nN is the
rated rotational speed. This number describes the torque density of the machine and allows a more
distinctive view of a wide range of rotational speeds. where PN is the rated power, Di is the bore diameter, lfe is the length of the active parts, and nN is the
rated rotational speed. This number describes the torque density of the machine and allows a more
distinctive view of a wide range of rotational speeds. where PN is the rated power, Di is the bore diameter, lfe is the length of the active parts, and nN is the
rated rotational speed. This number describes the torque density of the machine and allows a more
distinctive view of a wide range of rotational speeds. 2.1.1. Electrically Excited Synchronous Machines Synchronous machines equipped with field windings in the rotor are used for a variety of
applications in the multi-megawatt range. These machines are mostly used at relatively low rotational
speeds, up to 15,000 rpm, as generators at power plants or as motors, such as for turbo compressors. With respect to the mechanical stability of the rotor system, electrically excited synchronous machines
achieve higher circumferential speeds than induction machines, since they can be manufactured with
solid rotors that can also be used as a start-up cage in many cases. However, for aircraft applications,
synchronous machines with field winding are not used because the excitation is mostly realized
through brushes and slip rings that need to be maintained regularly. This is even more true if such a
machine is designed as a high-speed concept, as higher rotational speed increases the abrasion of the
brushes. Additionally, the rated speed approaches the first bending frequency, which serves as another
limit [8,13]. 2.1.2. Reluctance Machines Reluctance machines, especially synchronous reluctance machines, have received increased
attention because of the developments in inverter technology and the absence of permanent magnets
or a rotor winding. The latter is the reason for low rotor losses compared to induction motors,
and better fault management due to the lack of permanent magnets that could demagnetize or
induce voltages above the DC-link voltage when the machine operates in the field weakening
range. Additionally, reluctance machines are cheap to produce because of the lack of a rotor cage,
field winding, and permanent magnets. Nonetheless, the dimensions of both synchronous reluctance
machines and switched reluctance machines are about 50% larger than those of PM synchronous
machines due to the reactive power needed [14]. 2. Design Space for Aviation Drives
2. Design Space for Aviation Drives where PN is the rated power, Di is the bore diameter, lfe is the length of the active parts, and nN is the
rated rotational speed. This number describes the torque density of the machine and allows a more
distinctive view of a wide range of rotational speeds. Energies 2018, 11, 344 6 of 25 2.1. Machine Concepts 2.1. Machine Concepts In the following, different electrical machine concepts are compared in terms of their usability for
a multi-megawatt motor design. 2.1.4. Permanent Magnet Synchronous Machine
2.1.4. Permanent Magnet Synchronous Machine 2.1.4. Permanent Magnet Synchronous Machine
2.1.4. Permanent Magnet Synchronous Machine Permanent magnet synchronous machines (PMSM) are widely used and are being applied in an
increasing number of fields, including electromobility, railway traction, ship propulsion, generators etc.,
since the missing field winding allows for higher efficiency, higher power density, lower heat
production in the rotor, and sensorless control, although the magnets are comparatively expensive. Nonetheless, the absence of an exciter machine with rotating rectifier or slip rings to supply the field
winding means less maintenance is required. The different rotor designs for permanent magnet excited
machines include: v-shaped buried magnets, beam-like buried magnets, surface-mounted magnets,
or multi-layer buried magnets (Figure 6). Permanent magnet synchronous machines (PMSM) are widely used and are being applied in an
increasing number of fields, including electromobility, railway traction, ship propulsion, generators
etc., since the missing field winding allows for higher efficiency, higher power density, lower heat
production in the rotor, and sensorless control, although the magnets are comparatively expensive. Nonetheless, the absence of an exciter machine with rotating rectifier or slip rings to supply the field
winding means less maintenance is required. The different rotor designs for permanent magnet
excited machines include: v-shaped buried magnets, beam-like buried magnets, surface-mounted
magnets, or multi-layer buried magnets (Figure 6). Figure 6. Different rotor concepts of permanent magnet synchronous machines [18]: (a) V-shaped
buried; (b) Beam-like buried; (c) Surface-mounted; (d) Multi-layer. (a)
(c)
(d)
(b)
Figure 6. Different rotor concepts of permanent magnet synchronous machines [18]: (a) V-shaped
buried; (b) Beam-like buried; (c) Surface-mounted; (d) Multi-layer. (c) (b) (d) (a) (b) (a) (d) (c) Figure 6. Different rotor concepts of permanent magnet synchronous machines [18]: (a) V-shaped
buried; (b) Beam-like buried; (c) Surface-mounted; (d) Multi-layer. Figure 6. Different rotor concepts of permanent magnet synchronous machines [18]: (a) V-shaped
buried; (b) Beam-like buried; (c) Surface-mounted; (d) Multi-layer. Each has its benefits concerning flux density, flux distribution, mechanical stability, or weight. Bacaro et al. [18] compared these different rotor concepts for an internal permanent magnet
synchronous machine that was used as railway traction drive. The power output was 630 kW and it
operated at 1200 rpm, categorizing it as a low-speed machine with a low circumferential speed of 22
m/s. As is the case for most mobility applications, the installation space was limited, resulting in an
active length of 500 mm. 2.1.4. Permanent Magnet Synchronous Machine
2.1.4. Permanent Magnet Synchronous Machine Esson’s number for this PMSM was 8.6 kW·min/m3, which, in comparison,
is considerably higher than the highlighted induction machines. In the end, the chosen rotor design
was the buried multi-layer design with three layers, where the amount of magnet material was
optimized to determine the best ratio of copper and iron losses. Each has its benefits concerning flux density, flux distribution, mechanical stability, or weight. Bacaro et al. [18] compared these different rotor concepts for an internal permanent magnet
synchronous machine that was used as railway traction drive. The power output was 630 kW and
it operated at 1200 rpm, categorizing it as a low-speed machine with a low circumferential speed of
22 m/s. As is the case for most mobility applications, the installation space was limited, resulting in an
active length of 500 mm. Esson’s number for this PMSM was 8.6 kW·min/m3, which, in comparison,
is considerably higher than the highlighted induction machines. In the end, the chosen rotor design was
the buried multi-layer design with three layers, where the amount of magnet material was optimized
to determine the best ratio of copper and iron losses. Higher rotational speeds combined with high power requirements lead to electrical machine
designs with high rotor diameters and thus high circumferential speeds. Zhang et al. [19] investigated
different surface magnet concepts and filler materials for the pole gap to ensure safe operation by
developing a hybrid rotor armor design. The machine rotates at 18,000 rpm and delivers 1.12 MW. With an active length of 400 mm, its Esson’s number is 6.4 kW·min/m3. Higher rotational speeds combined with high power requirements lead to electrical machine
designs with high rotor diameters and thus high circumferential speeds. Zhang et al. [19] investigated
different surface magnet concepts and filler materials for the pole gap to ensure safe operation by
developing a hybrid rotor armor design. The machine rotates at 18,000 rpm and delivers 1.12 MW. With an active length of 400 mm, its Esson’s number is 6.4 kW·min/m3. 2.1.3. Induction Machines Many applications use induction machines (IM) because of their overall robustness and low
production cost. The absence of permanent magnets benefits the IM in the field weakening range. In comparison to reluctance machines, IMs have a higher power factor making the machine suitable
for high-speed operation. The critical aspects of the machine design include the mechanical stress of
the cage, especially for its end-rings, and the thermal evaluation as a result of the conducting rotor
parts [15]. Different rotor designs have been developed and compared, but for high power applications
with restricted installation space and the low weight requirement, laminated and caged rotors are most
feasible [16]. Caprio et al. [15] presented a 2 MW high-speed flywheel machine that operates at 15,000 rpm
with an Esson’s number of 2.6 kW·min/m3. The challenge of the high rotational speed lies in the
mechanical design of the rotor. At the high circumferential speed of 290 m/s, the end-ring design and
the mechanical stress at the rotor ends and its joints to the cage are crucial. Mechanical and thermal
aspects must be considered to choose the best design and material. During the 1990s, the first high-speed high-power electrical machines were developed for industry
applications, such as compressor drives in refineries. Therefore, Wood et al. [17] designed an induction
motor with a solid rotor and aluminium bars that operates at 11,000 rpm at a power output of
2.6 MW. The Esson’s number achieved with this design was 1.9 kW·min/m3. In their design, the large
bearing span of 2.4 m required special attention to the rotor dynamics. A simulation was completed to
investigate the critical bending frequencies and the torsional resonance frequencies. Energies 2018, 11, 344
E
i
2017 10
FOR 7 of 25
7 of 25 7 of 25
7 of 25 3. Winding Technology The armature winding is one of the key components in the electromechanical energy conversion
process. The number of turns and the number of pole pairs significantly impact the shape of the
speed-torque operation map. To achieve a high overall system efficiency, these parameters have to be
chosen to meet the system requirements of the aircraft as well as the needs of the power electronics. Furthermore, the winding production method directly influences the winding properties like the slot
filling factor and the end-winding length. The former affects the electrical and thermal resistance and
therefore the losses, whereas the latter determines the available space for energy conversion, which in
turn determines the power density of the overall system. In addition to conventional round wires, by using rectangular wire windings, the slot fill factor can
be significantly increased, resulting in smaller motors with excellent performance. The most popular
approach is rectangular wire, which is well-suited to and widely used for mass production, bent into a
hairpin shape on one axial side of the stator. The ends of the wire on the other side are welded to form
connected coils in a wave shape [20]. Large copper cross-sections may lead to higher losses if the slots
are exposed to higher frequencies. Loss effects on the system must be examined and balanced in a
holistic design. However, in addition to a proper pole pair number, winding turns, and a suitable production
technology, additional degrees of freedom exist: by increasing the number of phases, the fundamental
wave in a machine can be further strengthened compared to a three-phase system, which results in
higher torque and efficiency. Furthermore, certain higher order harmonics can be significantly reduced
or even completely eliminated using this approach. These higher harmonic orders are parasitic in
nature, since they do not contribute to the constant torque, but their occurrence leads to an increase in
iron losses, eddy current losses in magnets, local saturation, pulsating torque, noise, and vibrations. Moreover, using more than three phases improves fault tolerance by adding redundancy to the system. 4. Materials The selection of materials used for an electrical machine is an important aspect of each design
process. The following sections will highlight the different components of an electrical machine in
terms of the different concepts to show the state-of-the-art material choices as well as some other
materials as references for comparison. Energies 2018, 11, 344 Energies 2018, 11, 344 Energies 2018, 11, 344 8 of 25 The direct-driven solution should incorporate a permanent magnet synchronous machine, since
the optimal efficiency of a PMSM lies within the constant flux range that would require a high DC-link
voltage to be used given the demand of the high rotational speeds. Furthermore, in the field weakening
range, the induced voltage is higher than the maximum allowable DC-link voltage, resulting in
additional safety measures being required. High centrifugal forces caused by a high bore diameter can
be covered through rotor armoring, which will be described below. 2.2. Concepts and Design Choice
2.2. Concepts and Design Choice For travel ranges of several thousand kilometres, current aircraft engines are mostly high bypass
ratio or ultra-high bypass ratio concepts. Given the specifications of the PW305A, manufactured by
Pratt and Whitney and used in the Lear Jet 60 by Bombardier [4], a direct-driven engine would need
a motor at the same speed as the fan. A rotational speed of 10,608 rpm is not a typical low-speed
design. According to Figure 5, both permanent magnet synchronous machines and induction
machines would be feasible solutions. The use of an additional gear box would broaden the
For travel ranges of several thousand kilometres, current aircraft engines are mostly high bypass
ratio or ultra-high bypass ratio concepts. Given the specifications of the PW305A, manufactured by
Pratt and Whitney and used in the Lear Jet 60 by Bombardier [4], a direct-driven engine would need a
motor at the same speed as the fan. A rotational speed of 10,608 rpm is not a typical low-speed design. According to Figure 5, both permanent magnet synchronous machines and induction machines would
be feasible solutions. The use of an additional gear box would broaden the possibilities. possibilities. For a high-speed machine concept, the induction motor with laminated or solid rotor core and a
squirrel-cage, depending on the rated power, is most suitable according to Section 2.1.3, due to its
strength in the field weakening range and the low maintenance requirements. The optimal operating
point with maximum efficiency occurs at a higher rotational speed for IM than the PMSM, which
For a high-speed machine concept, the induction motor with laminated or solid rotor core and
a squirrel-cage, depending on the rated power, is most suitable according to Section 2.1.3, due to its
strength in the field weakening range and the low maintenance requirements. The optimal operating
point with maximum efficiency occurs at a higher rotational speed for IM than the PMSM, which makes
it the better choice for high-speed operation by exploiting the field weakening range. 4.1. Magnetically Hard Materials Magnetically hard materials supply the rotor field in permanent magnet synchronous machines. Figure 7 shows the different magnet materials that are currently used in electrical machines. A wide
variety of materials have been developed since the 1990s, allowing permanent magnet machines to be
viable for a variety of applications [21]. Rare earth magnets, with their high energy densities and their
high remanence field strengths, are especially used in high-efficiency electrical machines with high
power factors, such as in wind turbine generators or traction drives for vehicles. Synchronous machines
with high rotational speeds are prone to demagnetization leading to failure, which is dependent on
the coercive field strength. Figure 8 shows different permanent magnets and their coercive field
strengths plotted against their remanence flux densities [22–26]. The plot demonstrates why hard
ferrites are not used in high-efficiency applications. AlNiCo magnets are not suitable either, since they
are highly susceptible to demagnetize during failure. Therefore, the rare earth materials NdFeB and 9 of 25 Energies 2018, 11, 344 SmCo, in different specifications, are the best choices from a technical standpoint, even though they
are rather expensive. NdFeB and SmCo, in different specifications, are the best choices from a technical standpoint, even
though they are rather expensive
NdFeB and SmCo, in different specifications, are the best choices from a technical standpoint, even
though they are rather expensive. g
y
p
Figure 7. Permanent magnet materials. Figure 7. Permanent magnet materials. Figure 7. Permanent magnet materials. Figure 7. Permanent magnet materials. Figure 7. Permanent magnet materials. Figure 7. Permanent magnet materials. Figure 8. Overview of different magnetically hard materials. Figure 8. Overview of different magnetically hard materials. Figure 8. Overview of different magnetically hard materials. Figure 8. Overview of different magnetically hard materials. Figure 8. Overview of different magnetically hard materials. Figure 8. Overview of different magnetically hard materials. Depending on the application, high mechanical strength, high energy density, or a wide
temperature range are required in the magnetically hard materials. Figure 9 provides an overview of
magnetically hard materials and their maximum operating temperatures. Depending on the application, high mechanical strength, high energy density, or a wide
temperature range are required in the magnetically hard materials. Figure 9 provides an overview of
magnetically hard materials and their maximum operating temperatures. 4.2. Magnetically Soft Materials
4.2. Magnetically Soft Materials
4.2. Magnetically Soft Materials
ll
f
l g
y
f
Magnetically soft materials, and electrical sheets in particular, are another high-tech component
of electrical machines. Given the increasing demand for efficiency and the growing interest in high-
speed motors, efforts have been made to develop new and better alloys. These consist mostly of iron
and a varying percentage of either silicon or cobalt and other metals. Depending on the requirements,
high saturation magnetization, high yield strength, and/or low specific iron losses is desired. For
high-speed machines, a material with good values for all three properties would be optimal but this
is impossible. Cobalt-iron (CoFe) sheets are the best choice for high saturation magnetization. Unfortunately, it has poor mechanical characteristics. Figure 10 shows several electrical sheet
materials and their yield strength against the specific iron loss [27] with VACOFLUX and VACODUR
as specialized CoFe-materials produced by VACUUMSCHMELZE GmbH & Co. KG, Hanau,
Germany [28]. Magnetically soft materials, and electrical sheets in particular, are another high-tech component
of electrical machines. Given the increasing demand for efficiency and the growing interest in
high-speed motors, efforts have been made to develop new and better alloys. These consist mostly
of iron and a varying percentage of either silicon or cobalt and other metals. Depending on the
requirements, high saturation magnetization, high yield strength, and/or low specific iron losses
is desired. For high-speed machines, a material with good values for all three properties would
be optimal but this is impossible. Cobalt-iron (CoFe) sheets are the best choice for high saturation
magnetization. Unfortunately, it has poor mechanical characteristics. Figure 10 shows several electrical
sheet materials and their yield strength against the specific iron loss [27] with VACOFLUX and
VACODUR as specialized CoFe-materials produced by VACUUMSCHMELZE GmbH & Co. KG,
Hanau, Germany [28]. Magnetically soft materials, and electrical sheets in particular, are another high-tech component
of electrical machines. Given the increasing demand for efficiency and the growing interest in high-
speed motors, efforts have been made to develop new and better alloys. These consist mostly of iron
and a varying percentage of either silicon or cobalt and other metals. Depending on the requirements,
high saturation magnetization, high yield strength, and/or low specific iron losses is desired. For
high-speed machines, a material with good values for all three properties would be optimal but this
is impossible. Cobalt-iron (CoFe) sheets are the best choice for high saturation magnetization. Unfortunately, it has poor mechanical characteristics. 4.1. Magnetically Hard Materials C
i
NdF B
d S
C
h
diff
h i
h
i
l
f
il
Depending on the application, high mechanical strength, high energy density, or a wide
temperature range are required in the magnetically hard materials. Figure 9 provides an overview of
magnetically hard materials and their maximum operating temperatures. Depending on the application, high mechanical strength, high energy density, or a wide
temperature range are required in the magnetically hard materials. Figure 9 provides an overview of
magnetically hard materials and their maximum operating temperatures. Depending on the application, high mechanical strength, high energy density, or a wide
temperature range are required in the magnetically hard materials. Figure 9 provides an overview of
magnetically hard materials and their maximum operating temperatures. C
i
NdF B
d S
C
h
diff
h i
h
i
l
f
il
Depending on the application, high mechanical strength, high energy density, or a wide
temperature range are required in the magnetically hard materials. Figure 9 provides an overview of
magnetically hard materials and their maximum operating temperatures. g
y
p
g
p
Comparing NdFeB and SmCo, the differences are their mechanical parameters for tensile
strength and compressive strength. The SmCo variant is much more viable for high-speed motors,
particularly when the rotor armor exerts pressure on the underlying surface-mounted permanent
magnets. Comparing NdFeB and SmCo, the differences are their mechanical parameters for tensile
strength and compressive strength. The SmCo variant is much more viable for high-speed motors,
particularly when the rotor armor exerts pressure on the underlying surface-mounted permanent
magnets. Comparing NdFeB and SmCo, the differences are their mechanical parameters for tensile
strength and compressive strength. The SmCo variant is much more viable for high-speed
motors, particularly when the rotor armor exerts pressure on the underlying surface-mounted
permanent magnets. 10 of 25
10 of 25 Energies 2018, 11, 344
E e gie
,
, Figure 9. Maximum operating temperature of magnetically hard materials. Figure 9. Maximum operating temperature of magnetically hard materials. Figure 9. Maximum operating temperature of magnetically hard materials. Figure 9. Maximum operating temperature of magnetically hard materials. Figure 9. Maximum operating temperature of magnetically hard materials. Figure 9. Maximum operating temperature of magnetically hard materials. 4.2. Magnetically Soft Materials
4.2. Magnetically Soft Materials
4.2. Magnetically Soft Materials
ll
f
l Figure 10 shows several electrical sheet
materials and their yield strength against the specific iron loss [27] with VACOFLUX and VACODUR
as specialized CoFe-materials produced by VACUUMSCHMELZE GmbH & Co. KG, Hanau,
Germany [28]. Figure 10. Different electrical sheets: yield strength against specific iron loss. Figure 10. Different electrical sheets: yield strength against specific iron loss. Figure 10 Different electrical sheets: yield strength against specific iron loss
Figure 10. Different electrical sheets: yield strength against specific iron loss. Figure 10. Different electrical sheets: yield strength against specific iron loss. The thickest sheets (1 mm) exhibit the highest losses. The sheet thickness is decreased to reduce
the eddy current losses. In conjunction with the high saturation magnetization, the cobalt-iron
The thickest sheets (1 mm) exhibit the highest losses. The sheet thickness is decreased to reduce
the eddy current losses. In conjunction with the high saturation magnetization, the cobalt-iron
The thickest sheets (1 mm) exhibit the highest losses. The sheet thickness is decreased to reduce the
eddy current losses. In conjunction with the high saturation magnetization, the cobalt-iron materials
exist in different variations. For example, VACOFLUX 48 has very low specific losses and is available 11 of 25
sses and Energies 2018, 11, 344
materials exist in in different thicknesses, from less than 0.1 mm to 0.5 mm. VACODUR, on the other hand, is the choice
for high mechanical requirements due to its high yield strength. The iron losses in the rotor are much
lower than the loss in the stator, thus a lamination material with higher specific iron loss but higher
yield strength can be chosen. For high-power applications such as aircraft, the use of different materials
for the stator and rotor could be advantageous. is the choice for high mechanical requirements due to its high yield strength. The iron losses in the
rotor are much lower than the loss in the stator, thus a lamination material with higher specific iron
loss but higher yield strength can be chosen. For high-power applications such as aircraft, the use of
different materials for the stator and rotor could be advantageous. During the design phase of an electrical machine, the iron losses and, even more importantly, During the design phase of an electrical machine, the iron losses and, even more importantly,
the mechanical stability of the rotor system, have to be considered, but these are not the only restrictions. 4.2. Magnetically Soft Materials
4.2. Magnetically Soft Materials
4.2. Magnetically Soft Materials
ll
f
l Especially for aircraft applications, the weight must be considered. Most silicon-iron sheets have
a density of approximately 7800 kg/m3. The cobalt in the CoFe sheets is about 50%, resulting in
densities around 8120 kg/m3, thus making the electrical machine heavier. Usually, this disadvantage
is countered by the fact that these sheets have a high saturation magnetization. Hence, less material is
needed to create the same amount of magnetic flux (Figure 11). For future electrical machine designs,
which are in need of a high power to weight ratio or a high torque to weight ratio, these cobalt-iron
alloys must provide the same magnetic flux but with lower material in the yokes and teeth. g
g
p
p
y
the mechanical stability of the rotor system, have to be considered, but these are not the only
restrictions. Especially for aircraft applications, the weight must be considered. Most silicon-iron
sheets have a density of approximately 7800 kg/m³. The cobalt in the CoFe sheets is about 50%,
resulting in densities around 8120 kg/m³, thus making the electrical machine heavier. Usually, this
disadvantage is countered by the fact that these sheets have a high saturation magnetization. Hence,
less material is needed to create the same amount of magnetic flux (Figure 11). For future electrical
machine designs, which are in need of a high power to weight ratio or a high torque to weight ratio,
these cobalt-iron alloys must provide the same magnetic flux but with lower material in the yokes
and teeth. Figure 11. Comparison of SiFe and CoFe sheets. Figure 11. Comparison of SiFe and CoFe sheets. g
p
Another material option with low eddy current losses is soft magnetic composites (SMC) that
consist of small iron particles that are isolated by a thin insulation layer from each other. As the name
suggests, this composite is moulded. Therefore, its use in mechanically challenging applications is
not currently advisable
Another material option with low eddy current losses is soft magnetic composites (SMC) that
consist of small iron particles that are isolated by a thin insulation layer from each other. As the name
suggests, this composite is moulded. Therefore, its use in mechanically challenging applications is not
currently advisable. 4 4 S
d
t
4.4. Superconductors 4.4. Superconductors
Alternatives to classic conductor materials like copper are superconductors. Superconductors
have nearly no electrical resistance if operated below a certain threshold temperature. Depending on
the threshold, the superconductors are further separated into superconductors and high-temperature
superconductors (HTS). The name HTS was created for the first superconductors with a threshold
temperature above approximately 20 K. The reason these materials are superconducting is not yet
fully understood [30,31]. The most common high-temperature superconductors are composed of rare
earth, barium, and copper oxides (YBCO) that have a temperature threshold of 93 K and are usually
operated at 70 K. The material is available either as bulk or as tape conductor several kilometers in
Alternatives to classic conductor materials like copper are superconductors. Superconductors have
nearly no electrical resistance if operated below a certain threshold temperature. Depending on the
threshold, the superconductors are further separated into superconductors and high-temperature
superconductors (HTS). The name HTS was created for the first superconductors with a threshold
temperature above approximately 20 K. The reason these materials are superconducting is not yet
fully understood [30,31]. The most common high-temperature superconductors are composed of rare
earth, barium, and copper oxides (YBCO) that have a temperature threshold of 93 K and are usually
operated at 70 K. The material is available either as bulk or as tape conductor several kilometers in
length. Some of the currently used HTS and their threshold temperatures are shown in Table 2. of the currently used HTS and their threshold temperatures are shown in T
Table 2 Threshold temperatures of high temperature superconductors (HTS) [32]
Table 2. Threshold temperatures of high-temperature superconductors (HTS) [32]. of the currently used HTS and their threshold temperatures are shown in Ta
Table 2. Threshold temperatures of high-temperature superconductors (HTS) [32]. of the currently used HTS and their threshold temperatures are shown in T
Table 2. Threshold temperatures of high-temperature superconductors (HTS) [32]. Material
Threshold Temperature
YBaCuO
93 K
BiSrCaCuO
110 K
TlBaCaCuO
125 K
Table 2. Threshold temperatures of high-temperature superconductors (HTS) [32]
Material
Threshold Temperature
YBaCuO
93 K
BiSrCaCuO
110 K
TlBaCaCuO
125 K old temperatures of high temperature superconducto
Material
Threshold Temperature
YBaCuO
93 K
BiSrCaCuO
110 K
TlBaCaCuO
125 K
Material
Threshold Temperature
YBaCuO
93 K
BiSrCaCuO
110 K
TlBaCaCuO
125 K Even at high temperatures of about −148 °C (125 K), cooling is a challenge. y
4 3 C
d
4.3. Conductors Induction motors 12 of 25
12 of 25 Energies 2018, 11, 344
Energies 2017, 10, x FOR need as small an air-gap as possible. Therefore, using rotor armor should be avoided. With these
materials and alloys combined with fitting electrical sheets, a high-speed rotor with high power density
can be designed. beryllium, or possibly aluminium alloys that have relatively high conductivity and high tensile
strength. Induction motors need as small an air-gap as possible. Therefore, using rotor armor should
be avoided. With these materials and alloys combined with fitting electrical sheets, a high-speed rotor
with high power density can be designed. Fi
12 C
i
f
d
t
t
i l
Figure 12. Comparison of conductor materials. Figure 12. Comparison of conductor materials. 4 4 S
d
t
4.4. Superconductors Some of the proposals
include liquid hydrogen at 20 K or liquid neon at 27 K [33]. Incorporating this system into an aircraft
would be difficult but the weight and size of the machine would be reduced by 70% [34]. Hence,
several superconducting machines have been designed to date. Applications range from ship
propulsion [35] to all-electric aircraft proposals [36], achieving power to weight ratios of up to 8
kW/kg and torque densities of up to 2 Nm/kg in the power range of single megawatts or single MVA,
respectively. The machines are either electrically excited by HTS-wound rotors or fitted with HTS
magnets. Even at high temperatures of about −148 ◦C (125 K), cooling is a challenge. Some of the proposals
include liquid hydrogen at 20 K or liquid neon at 27 K [33]. Incorporating this system into an
aircraft would be difficult but the weight and size of the machine would be reduced by 70% [34]. Hence, several superconducting machines have been designed to date. Applications range from
ship propulsion [35] to all-electric aircraft proposals [36], achieving power to weight ratios of up to
8 kW/kg and torque densities of up to 2 Nm/kg in the power range of single megawatts or single
MVA, respectively. The machines are either electrically excited by HTS-wound rotors or fitted with
HTS magnets. y
4 3 C
d
4.3. Conductors 4.3. Conductors
There are two types of conductors in an electrical machine: the stator winding that mostly
consists of copper and either a rotor winding, which is copper in most cases, or the cage of an
induction motor. For most industrial applications, the cage bars and end-rings of motors are
manufactured from aluminium, since it has a relatively low resistivity, and is relatively cheap and
easy to die cast. The advantages over copper and copper alloys are the cost and the low density,
which reduces the moment of inertia, whereas its conductivity is relatively high compared to many
other materials. For the stator winding, the number of possible alternatives to copper, like silver and
gold, are very limited, being generally too expensive for too little a technical advantage. As we have
excluded the electrically excited synchronous machines above, the main field of research on feasible
conductor materials focuses on the cage of induction machines. Figure 12 shows the different
materials with their resistivity against their tensile strength [29]. A copper cage seems to be most
suitable for high-speed operations, but other materials may be suitable, like phosphorus-copper,
There are two types of conductors in an electrical machine: the stator winding that mostly consists
of copper and either a rotor winding, which is copper in most cases, or the cage of an induction
motor. For most industrial applications, the cage bars and end-rings of motors are manufactured
from aluminium, since it has a relatively low resistivity, and is relatively cheap and easy to die cast. The advantages over copper and copper alloys are the cost and the low density, which reduces the
moment of inertia, whereas its conductivity is relatively high compared to many other materials. For the stator winding, the number of possible alternatives to copper, like silver and gold, are very
limited, being generally too expensive for too little a technical advantage. As we have excluded the
electrically excited synchronous machines above, the main field of research on feasible conductor
materials focuses on the cage of induction machines. Figure 12 shows the different materials with their
resistivity against their tensile strength [29]. A copper cage seems to be most suitable for high-speed
operations, but other materials may be suitable, like phosphorus-copper, beryllium, or possibly
aluminium alloys that have relatively high conductivity and high tensile strength. 5.1. Winding Design
5.1. Winding Design
A
l
d A general and deterministic method to determine multi-phase and multi-layer winding topologies
has been introduced [37–39]. In this approach, an N-slot machine and N-phase winding (or N/2-phase
if N is an even integer) arrangement, with an ideal current sheet space harmonics spectrum,
was considered as a starting point. However, from a practical point of view, the proper choice
of a winding usually is obtained from the choice of a phase number smaller than N Figure 13. A general and deterministic method to determine multi-phase and multi-layer winding
topologies has been introduced [37–39]. In this approach, an N-slot machine and N-phase winding
(or N/2-phase if N is an even integer) arrangement, with an ideal current sheet space harmonics
spectrum, was considered as a starting point. However, from a practical point of view, the proper
choice of a winding usually is obtained from the choice of a phase number smaller than N Figure 13. Figure 13. Spectra of winding factors for different topologies with N = 24 slots and p = 5. Figure 13. Spectra of winding factors for different topologies with N = 24 slots and p = 5. Figure 13 Spectra of winding factors for different topologies with N = 24 slots and p = 5
Figure 13. Spectra of winding factors for different topologies with N = 24 slots and p = 5. For a given number of phases, pole pairs, and slots, Cai et al. [37] demonstrated that an optimal
MMF spectrum exists as the best approximation of the ideal spectrum that can be determined by
solving a linear equation system. In this case, the best approximation means the unavoidable smallest
loss in fundamental winding factor and the smallest distortion of MMF space harmonics spectrum
due to the choice of a ms smaller than N. The resulting windings are usually the multi-layer type and
probably contain coils with different numbers of turns. This method is further improved by
considering and exploiting symmetry as one of the most important properties of symmetrical multi-
phase windings [38,39]. For a given number of phases, pole pairs, and slots, Cai et al. [37] demonstrated that an optimal
MMF spectrum exists as the best approximation of the ideal spectrum that can be determined
by solving a linear equation system. 5. Special Issues in Design In this section, crucial special issues and demands concerning high performance and lightweight
machines are addressed and exemplified. 13 of 25
htweight Energies 2018, 11, 344
In this section
machines are addr 13 of 25
g 5.1. Winding Design
5.1. Winding Design
A
l
d In this case, the best approximation means the unavoidable
smallest loss in fundamental winding factor and the smallest distortion of MMF space harmonics
spectrum due to the choice of a ms smaller than N. The resulting windings are usually the multi-layer
type and probably contain coils with different numbers of turns. This method is further improved
by considering and exploiting symmetry as one of the most important properties of symmetrical
multi-phase windings [38,39]. p
g [
,
]
In Domann and Henke [40], a machine featuring a six-phase winding configuration was built. The winding was considered superior to several three-phase windings with respect to copper and
iron losses. The spectrum of this winding was further improved by implementing multi-layer
topology with coils of different numbers of turns and coil pitches [41]. Compared to the winding
spectra [40], the seventh harmonic winding factor is not shown, further increasing the winding factor
of the working harmonic. In Domann and Henke [40], a machine featuring a six-phase winding configuration was built. The winding was considered superior to several three-phase windings with respect to copper and iron
losses. The spectrum of this winding was further improved by implementing multi-layer topology
with coils of different numbers of turns and coil pitches [41]. Compared to the winding spectra [40],
the seventh harmonic winding factor is not shown, further increasing the winding factor of the
working harmonic. 5.2. Rotor Armoring
5.2. Rotor Armoring Synchronous machines with very high rotational speeds, and thus high circumferential speeds,
often use additional rotor armoring to secure the permanent magnets against centrifugal force. In
Table 3, several rotor armor materials are shown with their respective mechanical properties [42]. Synchronous machines with very high rotational speeds, and thus high circumferential speeds,
often use additional rotor armoring to secure the permanent magnets against centrifugal force. In Table 3, several rotor armor materials are shown with their respective mechanical properties [42]. Energies 2018, 11, 344
Energies 2017, 10, x FOR 14 of 25
14 of 25 Table 3. Rotor armor materials. Young’s Modulus
(GPa)
Density (kg Material
Tensile
Strength
Young’s Modulus
(GPa)
Density (kg/m3)
Thermal Expansion
Coefficient (K−1)
Ti6Al4
895 MPa
110
4430
8.6 × 10–6
Glass-fibre
3 GPa
75
2500
5 × 10–6
Carbon-fibre
5.7 GPa
275
1750
−0.1 × 10–6
Kevlar
4.5 GPa
125
1440
–4 × 10–6
g
Ti6Al4
895 MPa
110
4430
8.6 × 10–6
Glass-fibre
3 GPa
75
2500
5 × 10–6
Carbon-fibre
5.7 GPa
275
1750
−0.1 × 10–6
Kevlar
4.5 GPa
125
1440
–4 × 10–6
The values of the tensile strength and the Young’s modulus for fibre materials are orthotropi
values in the direction of the fibres. In general, a material with high tensile strength would be chose The values of the tensile strength and the Young’s modulus for fibre materials are orthotropic
values in the direction of the fibres. In general, a material with high tensile strength would be chosen to
reduce the thickness of the armor. However, the differences in the Young’s modulus must be considered,
especially if the machine has a very tight air-gap. The lower the Young’s modulus, the higher the
elongation when under stress. Apart from countering the centrifugal force, the rotor armor should
help to hold the components of the rotor in place. If the radial elongation is too high, the rotor could
potentially close the distance of the air-gap and rub against the stator, which would damage the armor. to reduce the thickness of the armor. However, the differences in the Young’s modulus must be
considered, especially if the machine has a very tight air-gap. The lower the Young’s modulus, the
higher the elongation when under stress. Apart from countering the centrifugal force, the rotor armor
should help to hold the components of the rotor in place. 5.2. Rotor Armoring
5.2. Rotor Armoring If the radial elongation is too high, the rotor
could potentially close the distance of the air-gap and rub against the stator, which would damage
the armor. Other important parameters for the design of a rotor armor include the thermal expansion
coefficient resistivity and magnetic permeability As shown in Table 3 the thermal expansion Other important parameters for the design of a rotor armor include the thermal expansion
coefficient, resistivity, and magnetic permeability. As shown in Table 3, the thermal expansion
coefficients of the chosen armor materials differ not only in value but in sign, meaning that carbon
fibre and Kevlar shrink with increasing temperature. The shrinking would increase the stress on the
armor but would counteract the elongation due to the centrifugal force. coefficient, resistivity, and magnetic permeability. As shown in Table 3, the thermal expansion
coefficients of the chosen armor materials differ not only in value but in sign, meaning that carbon
fibre and Kevlar shrink with increasing temperature. The shrinking would increase the stress on the
armor but would counteract the elongation due to the centrifugal force. The resistivity and the magnetic permeability are needed to estimate the potential eddy current
losses in the armor due to the current harmonics in the stator winding and field harmonics in the air The resistivity and the magnetic permeability are needed to estimate the potential eddy current
losses in the armor due to the current harmonics in the stator winding and field harmonics in the
air-gap. Compound materials have an additional advantage, since the fibres are small and act like a
laminated core, which reduces eddy current losses, thus resulting in lower heat generation in the armor. losses in the armor due to the current harmonics in the stator winding and field harmonics in the air-
gap. Compound materials have an additional advantage, since the fibres are small and act like a
laminated core, which reduces eddy current losses, thus resulting in lower heat generation in the
armor. Energies 2018, 11, 344 Energies 2018, 11, 344 15 of 25 15 of 25 As the size of an electrical machine is mainly determined by the torque, a high-speed machine
is of comparable small size if the speed increases. However, for a certain rated power, the specific
losses increase due to higher additional losses in the iron and eddy current losses in the winding. So,
an increasing loss density must be expected, which requires more intensive cooling than a conventional
machine. If the circumferential speed exceeds approximately 180 m/s, the gas friction losses cannot
be neglected and additional measures are necessary. As permanent magnet synchronous machines
have very small rotor losses, they are preferable for aviation applications. Proven approaches to
handle the additional losses include: (1) sinusoidal flux distribution (surface magnets or protected
magnets with reluctance component), (2) distributed winding with low harmonic content, (3) inverter
with high switching frequency, thus feeding currents with low harmonic content, and (4) advanced
cooling concepts. g
p
In Section 5.2, an overview of different available banding materials and their mechanical
parameters was provided. In the following section, the use of rotor armoring will be further explained. The mechanical stabilization of a surface-mounted permanent magnet high-speed synchronous
machine requires the construction of a banding made from carbon fibre located in the air-gap. As the
magnetic flux density decreases with increasing air-gap, the carbon fibre banding should be as thin as
possible. The carbon fibre usually forms a unidirectionally wound cylinder that is resin-impregnated
(CFRP) and maintains the rotor structure under considerably high pressure, so that even at testing
speed, no “lift-off” of the permanent magnets can occur. By applying this measure, uncontrollable
unbalance of the rotor is avoided. For kinematic equations, a superposition is required of the elastic displacements due to the
gap pressures and the centrifugal forces. As a result, the displacements become speed-dependent. The displacements can be calculated from the elastic material properties, considering that the magnet
layer and carbon-fibre banding exhibit anisotropic properties. The design process incorporates Hooke’s
law for a polar orthotropic material like a unidirectional wound carbon fibre. Even the elastic
deformation of a polar orthotropic material can be calculated analytically, thus allowing a quick design
process and parameter variation. Knowing all elastic displacements, the necessary degree of shrinkage
can be calculated, yielding the gap pressures and the stresses in the components of the interference
fit (Figure 15). 5.3. Mechanical and Thermal Aspects
5.3. Mechanical and Thermal Aspects The design of high-speed machines is dominated by a close interaction between electromagnetic,
mechanical, and thermal parameters (Figure 14). The design of high-speed machines is dominated by a close interaction between electromagnetic,
mechanical, and thermal parameters (Figure 14). The design of high-speed machines is dominated by a close interaction between electromagnetic,
mechanical, and thermal parameters (Figure 14). The design of high-speed machines is dominated by a close interaction between electromagnetic,
mechanical, and thermal parameters (Figure 14). The design of high-speed machines is dominated by a close interaction between electromagnetic,
echanical, and thermal parameters (Figure 14). e
esig o
ig
spee
ac i es is
o
i a e
by a c ose i
e ac io be
ee e ec o
ag e ic,
mechanical, and thermal parameters (Figure 14). Figure 14. Task list for the design of high speed, high power density machines. Figure 14. Task list for the design of high speed, high power density machines. Figure 14. Task list for the design of high speed, high power density machines. Figure 14. Task list for the design of high speed, high power density machines. Energies 2018, 11, 344 Figure 16 demonstrates the dependency of the radial and stress components in the
rotor parts to speed. The compressive stresses in the magnets and the shaft decrease with increasing
speed, whereas the radial stress in the CFRP banding is nearly constant. At low speeds, the CFRP
banding delivers a compressive stress or a negative pressure value as the banding is prestressed as
mentioned above. The dimensioning figure is the gap pressure, which becomes positive beyond the
testing speed of 1.2 nominal speed. So, the glue joint between magnets and shaft is guaranteed to stay
in the pressure region at all operational speeds. Energies 2017, 10, x FOR PEER REVIEW
16 of 25 Figure 15. Rotor interference fit. Figure 15. Rotor interference fit. Figure 15. Rotor interference fit. Figure 15. Rotor interference fit. Energies 2018, 11, 344 16 of 25 (b)
16
Rotor interference fit. (a)
(b)
Figure 16. (a) the location of the occurrence of the pressures; and (b) Radial pressure of rotor gaps vs. rotational speed. Neg. press. is compressive stress, and pos. press. is tensile stress. Figure 16. (a) the location of the occurrence of the pressures; and (b) Radial pressure of rotor gaps vs. rotational speed. Neg. press. is compressive stress, and pos. press. is tensile stress. rgies 2017, 10, x FOR PEER REVIEW
16 of
Figure 15. Rotor interference fit. (a)
gies 2017, 10, x FOR PEER REVIEW
Figure 15. (b) (a) (b) Figure 16. (a) the location of the occurrence of the pressures; and (b) Radial pressure of rotor gaps vs. rotational speed. Neg. press. is compressive stress, and pos. press. is tensile stress. Figure 16. (a) the location of the occurrence of the pressures; and (b) Radial pressure of rotor gaps vs. rotational speed. Neg. press. is compressive stress, and pos. press. is tensile stress. Due to the high speed of the rotor, the influence of unbalance, and the natural frequencies of
the rotor become important. Additionally, the gyroscopic effects influence the natural frequencies of
the rotor that become speed-dependent. As unbalance forces increase with the square of the speed,
a high-quality balancing of the rotor according to ISO 1940 quality grade G 1 or G 0.4 is necessary. To show the influence of the rotor-dynamics, a rotor model with maximum speed of 40,000 rpm
was used. As the inverter is a co
5.4. Rotor Loss Mechanisms modulation, the current is not ideally sinusoidal. The inverter contains the fundamental frequency
and a spectrum of higher harmonics. Each harmonic oscillation of the current is transformed by the
machine winding into a spectrum of waves containing the fundamental wave and its higher
harmonics (Figures 19 and 20). The winding of a real electrical machine is placed into the slots of the
stator, usually manufactured equidistant to each other. As the purpose of the electrical winding is to
generate an electric loading to produce a magnetic field to generate torque in a rotating electric
machine, by distributing discrete conductors in the stator, only discrete parts of a magnetic field can
be provided by the stator when moving along the circumference. In an ideal state, the machine would
possess a sinusoidal magnetic field, but a real stator consists of discrete distributed conductors that
cause ramped magnetic field distributions, and thus the magnetic airgap field offers a spectrum of
harmonic waves. Superimposed, these waves result in a magnetic air-gap field, which is quite
different from a sinusoidal field. A Fourier-transformation of the magnetic field or the electric loading
shows that part of the harmonics travel in the rotational direction of the rotor, and part travel in
opposite direction. Furthermore, discrete current feeding by inverters causes an additional spectrum
as shown below. Around the bore of a rotating electrical machine, the current loading shows a
spectrum of different frequencies and spatial distributions. These phenomena cause additional losses
in electrical machines, such as in the magnets, inside the conductors and in the electric sheets. As the inverter is a component that controls the current by a switching pattern like a space vector
modulation, the current is not ideally sinusoidal. The inverter contains the fundamental frequency
and a spectrum of higher harmonics. Each harmonic oscillation of the current is transformed by
the machine winding into a spectrum of waves containing the fundamental wave and its higher
harmonics (Figures 19 and 20). The winding of a real electrical machine is placed into the slots of the
stator, usually manufactured equidistant to each other. As the purpose of the electrical winding is
to generate an electric loading to produce a magnetic field to generate torque in a rotating electric
machine, by distributing discrete conductors in the stator, only discrete parts of a magnetic field can be
provided by the stator when moving along the circumference. Energies 2018, 11, 344 The model shown in Figure 17 includes all the components that influence the rotor stiffness,
and a simple bearing model with radial stiffness of the bearings, and a viscous damping is added. The calculation of the natural frequencies using rotor dynamics software [41] provides an overview
of the vibrational behaviour of the rotor-bearing system. This is depicted in the Campbell diagram
(Figure 18), which shows the influence of speed on the natural frequencies. The straight red line in Figure 17 with a slope of one is the speed line that crosses the
frequency of the first bending mode of 288 Hz at 17,300 rpm. From there up to the maximum speed,
no further natural frequency has to be passed. The second bending mode, at 940 Hz, is well above
maximum speed. (a)
(b)
Figure 16. (a) the location of the occurrence of the pressures; and (b) Radial pressure of rotor gaps vs. rotational speed. Neg. press. is compressive stress, and pos. press. is tensile stress. Figure 17. Model for calculation of rotor stiffness and natural frequencies. Figure 17. Model for calculation of rotor stiffness and natural frequencies. Figure 17. Model for calculation of rotor stiffness and natural frequencies. Figure 17. Model for calculation of rotor stiffness and natural frequencies. Figure 17. Model for calculation of rotor stiffness and natural frequencies. 17 of 25
17 of 25 Energies 2018, 11, 344
Energies 2017, 10, x FO Figure 18. Campbell diagram of the rotor dynamics calculation. Figure 18. Campbell diagram of the rotor dynamics calculation. Figure 18. Campbell diagram of the rotor dynamics calculation. Figure 18. Campbell diagram of the rotor dynamics calculation. This mode also shows the stronger influence of the gyroscopic effects because the natural
frequency at zero speed is split by the increasing speed into a forward and backward mode. Whereas
the forward mode always stiffens the system, the backward mode reduces the natural frequency with
increasing speed. If the speed of the machine is increasing and the rotational speed reaches 17,300
min–1, the rotor passes its critical speed and rotates at the resonance frequency of the bending mode. The effect of this state can be further examined to determine the oscillation velocity and displacement. To reduce the amplitudes, several measures can be taken such as reducing the bearing stiffness. 5.4. Energies 2018, 11, 344 Rotor Loss Mechanisms
This mode also shows the stronger influence of the gyroscopic effects because the natural
frequency at zero speed is split by the increasing speed into a forward and backward mode. Whereas the forward mode always stiffens the system, the backward mode reduces the natural
frequency with increasing speed. If the speed of the machine is increasing and the rotational speed
reaches 17,300 min–1, the rotor passes its critical speed and rotates at the resonance frequency of the
bending mode. The effect of this state can be further examined to determine the oscillation velocity
and displacement. To reduce the amplitudes, several measures can be taken such as reducing the
bearing stiffness. As the inverter is a co
5.4. Rotor Loss Mechanisms In an ideal state, the machine would
possess a sinusoidal magnetic field, but a real stator consists of discrete distributed conductors that
cause ramped magnetic field distributions, and thus the magnetic airgap field offers a spectrum of
harmonic waves. Superimposed, these waves result in a magnetic air-gap field, which is quite different
from a sinusoidal field. A Fourier-transformation of the magnetic field or the electric loading shows
that part of the harmonics travel in the rotational direction of the rotor, and part travel in opposite
direction. Furthermore, discrete current feeding by inverters causes an additional spectrum as shown
below. Around the bore of a rotating electrical machine, the current loading shows a spectrum of
different frequencies and spatial distributions. These phenomena cause additional losses in electrical
machines, such as in the magnets, inside the conductors and in the electric sheets. 18 of 25
18 of 25 18 of 25
18 of 25 Energies 2018, 11, 344
Energies 2017, 10, x F Figure 19. Spatial harmonic spectrum of the machine resulting from sinusoidal feeding. Figure 19. Spatial harmonic spectrum of the machine resulting from sinusoidal feeding. Figure 19. Spatial harmonic spectrum of the machine resulting from sinusoidal feeding. Due to the higher frequency of the harmonic oscillations, the velocity of the correspondi
fundamental waves is much higher than that of the working fundamental wave. That is why th
harmonics produce significant losses in the permanent magnets, which in turn can increase
magnet temperature to levels where the magnets are demagnetized by the armature reaction. chine resulting from sinusoidal feeding. scillations the velocity of the correspond Figure 19 Spatial harmonic spectrum of the machine resulting from sinusoidal feeding
Figure 19. Spatial harmonic spectrum of the machine resulting from sinusoidal feeding. p
g
p
g
et temperature to levels where the magnets are demagnetized by the armature reaction. Due to the higher frequency of the harmonic oscillations, the velocity of the correspondi
undamental waves is much higher than that of the working fundamental wave. That is why the
armonics produce significant losses in the permanent magnets, which in turn can increase t
agnet temperature to levels where the magnets are demagnetized by the armature reaction. Figure 20. Spatial harmonics resulting from inverter feeding. Figure 20. Spatial harmonics resulting from inverter feeding. Figure 20. Spatial harmonics resulting from inverter feeding. Figure 20. Spatial harmonics resulting from inverter feeding. Figure 20. As the inverter is a co
5.4. Rotor Loss Mechanisms Spatial harmonics resulting from inverter feeding. For the current harmonics to stay small, the inverter uses the inductance of the machine. However, a high-speed machine with carbon fibre banding has a specifically small inductance, so
that a high switching frequency, of preferably more than 20 kHz, is necessary. A protected magnet
concept with a small iron layer above the magnets helps to prevent the harmonic waves of the small
pole pitch from intruding into the magnets and facilitates the production of the rotor A protected
Due to the higher frequency of the harmonic oscillations, the velocity of the corresponding
fundamental waves is much higher than that of the working fundamental wave. That is why these
harmonics produce significant losses in the permanent magnets, which in turn can increase the magnet
temperature to levels where the magnets are demagnetized by the armature reaction. Figure 20. Spatial harmonics resulting from inverter feeding. For the current harmonics to stay small, the inverter uses the inductance of the machine. However, a high-speed machine with carbon fibre banding has a specifically small inductance, so
that a high switching frequency, of preferably more than 20 kHz, is necessary. A protected magnet
concept with a small iron layer above the magnets helps to prevent the harmonic waves of the small
pole pitch from intruding into the magnets and facilitates the production of the rotor A protected
Due to the higher frequency of the harmonic oscillations, the velocity of the corresponding
fundamental waves is much higher than that of the working fundamental wave. That is why these
harmonics produce significant losses in the permanent magnets, which in turn can increase the magnet
temperature to levels where the magnets are demagnetized by the armature reaction. g
p
g
g
For the current harmonics to stay small, the inverter uses the inductance of the machine. However, a high-speed machine with carbon fibre banding has a specifically small inductance, so
that a high switching frequency, of preferably more than 20 kHz, is necessary. A protected magnet
concept with a small iron layer above the magnets helps to prevent the harmonic waves of the small
pole pitch from intruding into the magnets and facilitates the production of the rotor. A protected
magnet concept does not help with the fundamental waves of the harmonic oscillations. As the inverter is a co
5.4. Rotor Loss Mechanisms To reduce
th
l
i
th
t
bdi i i
f th
t
i t
d
d
pole pitch from intruding into the magnets and facilitates the production of the rotor. A protected
magnet concept does not help with the fundamental waves of the harmonic oscillations. To reduce
these losses in the magnets, subdivisions of the magnets were introduced. Particularly in electrical high-speed drives, the fundamental frequencies of the revolving
magnetic field are much higher than in conventional drives. This can also occur with lower-speed
For the current harmonics to stay small, the inverter uses the inductance of the machine. However,
a high-speed machine with carbon fibre banding has a specifically small inductance, so that a high
switching frequency, of preferably more than 20 kHz, is necessary. A protected magnet concept with a
small iron layer above the magnets helps to prevent the harmonic waves of the small pole pitch from
intruding into the magnets and facilitates the production of the rotor. A protected magnet concept
does not help with the fundamental waves of the harmonic oscillations. To reduce these losses in the
magnets, subdivisions of the magnets were introduced. these losses in the magnets, subdivisions of the magnets were introduced. Particularly in electrical high-speed drives, the fundamental frequencies of the revolving
magnetic field are much higher than in conventional drives. This can also occur with lower-speed
Particularly in electrical high-speed drives, the fundamental frequencies of the revolving magnetic
field are much higher than in conventional drives. This can also occur with lower-speed high-torque
drives when the machine topology exhibits a higher number of pole pairs. As stated above, the ideal
sinusoidal fundamental field distributions develop a number of revolving field waves in inverter-fed
machines, incorporating real winding distributions. These additionally-occurring waves move at lower 19 of 25
f 25
t d Energies 2018, 11, 344
Energies 2017, 10, x and higher speeds in relation to the fundamental working wave in the stator and rotor, causing iron and
copper losses. The aim of designing a compact electrical machine is influenced by the size of the stator
slots and amount of copper used. In this regard, a high copper filling factor inside the slot is desired. Highest filling factors can be realized by using solid undivided wires with larger cross-sectional
areas. 5.5. Direct Liquid-Cooled Windin
5.5. Direct Liquid-Cooled Winding For synchronous machines, the maximum allowable winding temperature, and hence the
temperature rise in the machine, is the main limiting factor when increasing torque density. Several
different approaches are available to enhance the heat dissipation capacity and achieve higher current
densities. A higher current density leads to a higher torque density. A higher heat dissipation capacity
leads to a lower operating temperature, and thus to reduced winding losses. Typically, medium-sized
electrical machines are cooled by either an air cooling jacket or a water jacket. Normally, electrical machines with water jacket cooling reach current densities of 13–18 A/mm2
For synchronous machines, the maximum allowable winding temperature, and hence the
temperature rise in the machine, is the main limiting factor when increasing torque density. Several different approaches are available to enhance the heat dissipation capacity and achieve higher
current densities. A higher current density leads to a higher torque density. A higher heat dissipation
capacity leads to a lower operating temperature, and thus to reduced winding losses. Typically,
medium-sized electrical machines are cooled by either an air cooling jacket or a water jacket. during steady-state operation [43], strongly depending on the size of the machine. Several thermal
resistances, like the resistance from winding to the stator core or from the stator core to the housing
[44], separate the cooling medium of a water jacket from the heat source. The aim of the novel
approach presented by Wohlers et al. [45] was to eliminate all but one thermal resistance between the
cooling medium and the heat source, i.e., the conductors, to allow a large increase in the possible
current density without exceeding the critical winding temperature. Due to innovative generative
manufacturing methods, including cooling channels into the coils of electrical machines is now
possible. Then, only the thermal resistance of the heat transfer between the single conductors of the
y
g j
j
Normally, electrical machines with water jacket cooling reach current densities of 13–18 A/mm2
during steady-state operation [43], strongly depending on the size of the machine. Several thermal
resistances, like the resistance from winding to the stator core or from the stator core to the housing [44],
separate the cooling medium of a water jacket from the heat source. The aim of the novel approach
presented by Wohlers et al. As the inverter is a co
5.4. Rotor Loss Mechanisms Conversely, skin and proximity effects that arise in copper wires of this type, due to higher
frequencies and under influence of oscillating magnetic fields across the slots, strongly contribute to
the amount of copper loss, especially under high load conditions. For this reason, the filling factor
must be compromised. Dividing the copper wire in the slot considerably reduces eddy currents in this
area, and thus contributes to increased efficiency. Figure 21 provides a graphical impression of the
current density in the cross-sectional area of copper conductors, for wires exposed to an alternating
magnetic field in the slot of an electrical machine. The highest values of current density occur at the
vertical borders of the conductors. To reduce the resulting copper losses, a further division of the
conductors is an appropriate measure. above, the ideal sinusoidal fundamental field distributions develop a number of revolving field
waves in inverter-fed machines, incorporating real winding distributions. These additionally-
occurring waves move at lower and higher speeds in relation to the fundamental working wave in
the stator and rotor, causing iron and copper losses. The aim of designing a compact electrical
machine is influenced by the size of the stator slots and amount of copper used. In this regard, a high
copper filling factor inside the slot is desired. Highest filling factors can be realized by using solid
undivided wires with larger cross-sectional areas. Conversely, skin and proximity effects that arise
in copper wires of this type, due to higher frequencies and under influence of oscillating magnetic
fields across the slots, strongly contribute to the amount of copper loss, especially under high load
conditions. For this reason, the filling factor must be compromised. Dividing the copper wire in the
slot considerably reduces eddy currents in this area, and thus contributes to increased efficiency. Figure 21 provides a graphical impression of the current density in the cross-sectional area of copper
conductors, for wires exposed to an alternating magnetic field in the slot of an electrical machine. The
highest values of current density occur at the vertical borders of the conductors. To reduce the
resulting copper losses, a further division of the conductors is an appropriate measure. Figure 21. Current density plotted over copper conductor height, slot middle. Figure 21. Current density plotted over copper conductor height, slot middle. Figure 21. Current density plotted over copper conductor height, slot middle. Figure 21. As the inverter is a co
5.4. Rotor Loss Mechanisms Current density plotted over copper conductor height, slot middle. 5.5. Direct Liquid-Cooled Windin
5.5. Direct Liquid-Cooled Winding [45] was to eliminate all but one thermal resistance between the cooling
medium and the heat source, i.e., the conductors, to allow a large increase in the possible current density
without exceeding the critical winding temperature. Due to innovative generative manufacturing
methods, including cooling channels into the coils of electrical machines is now possible. Then, only the
thermal resistance of the heat transfer between the single conductors of the coils and the cooling
medium remains. The cooled surface area, and hence the heat dissipation capacity, are appreciably
increased. Moreover, the coil geometry can be adapted to reduce losses due to current displacement
and to further increase the heat dissipation. Lastly, the slot filling factor can be increased using 20 of 25
tor can
oils A Energies 2018, 11, 344
to current displace
b
i
d
i hollow conductors due to the advantages of casted or 3-D-printed coils. A prototype of the coil
displayed in Figure 22 was casted with the help of a rapid-prototyped negative. The cast coil was
supplied with DC current densities up to 100 A/mm2. The used cooling medium was Galden HT135,
a perfluoropolyether with a boiling point of 135 ◦C. The steady-state temperatures of the coil with a
flow rate of 1.9 L/min and an inlet coolant temperature of 30 ◦C are shown in Figure 23. The average
coil temperature was measured as Tavg 135 ◦C. coils and the cooling medium remains. The cooled surface area, and hence the heat dissipation
capacity, are appreciably increased. Moreover, the coil geometry can be adapted to reduce losses due
to current displacement and to further increase the heat dissipation. Lastly, the slot filling factor can
be increased using hollow conductors due to the advantages of casted or 3-D-printed coils. A
prototype of the coil displayed in Figure 22 was casted with the help of a rapid-prototyped negative. The cast coil was supplied with DC current densities up to 100 A/mm2. The used cooling medium
was Galden HT135, a perfluoropolyether with a boiling point of 135 °C. The steady-state
prototype of the coil displayed in Figure 22 was casted with the help of a rapid prototyped negative. The cast coil was supplied with DC current densities up to 100 A/mm2. The used cooling medium
was Galden HT135, a perfluoropolyether with a boiling point of 135 °C. 5.5. Direct Liquid-Cooled Windin
5.5. Direct Liquid-Cooled Winding The steady-state
temperatures of the coil with a flow rate of 1.9 L/min and an inlet coolant temperature of 30 °C are
shown in Figure 23. The average coil temperature was measured as Tavg 135 °C. Figure 24 shows a schematic of a cast coil and the RMS current density distribution of two slots
at 500 Hz Due to the number of slots per phase and pole being 2/5 the current density distribution g
Figure 24 shows a schematic of a cast coil and the RMS current density distribution of two slots
at 500 Hz. Due to the number of slots per phase and pole being 2/5, the current density distribution
repeats itself every two slots. The average current density was 50.4 A/mm2. The current displacement
leads to a 15.4% higher loss. For comparison, the current displacement without cooling channels leads
to a 22.6% increase in losses [45]. temperatures of the coil with a flow rate of 1.9 L/min and an inlet coolant temperature of 30 °C are
shown in Figure 23. The average coil temperature was measured as Tavg 135 °C. Figure 24 shows a schematic of a cast coil and the RMS current density distribution of two slots
at 500 Hz. Due to the number of slots per phase and pole being 2/5, the current density distribution
repeats itself every two slots. The average current density was 50.4 A/mm². The current displacement
leads to a 15.4% higher loss. For comparison, the current displacement without cooling channels leads
to a 22.6% increase in losses [45]. at 500 Hz. Due to the number of slots per phase and pole being 2/5, the current density distribution
repeats itself every two slots. The average current density was 50.4 A/mm². The current displacement
leads to a 15.4% higher loss. For comparison, the current displacement without cooling channels leads
to a 22.6% increase in losses [45]. ats itself every two slots. The average current density was 50.4 A/mm2. The current displacem
s to a 15.4% higher loss. For comparison, the current displacement without cooling channels le
22.6% increase in losses [45]. at 500 Hz. Due to the number of slots per phase and pole being 2/5, the current density distribution
repeats itself every two slots. The average current density was 50.4 A/mm². The current displacement
leads to a 15.4% higher loss. 5.6. Metal Additive Manufacturing
ermanent magnet synchronous ma
otor and shaft which was introduc Through the use of metal additive manufacturing technologies, building electrical machine parts
layer per layer is possible. More specifically, fine metallic powder is fused by a high-power laser beam
to create complex 3-D metallic structures (Figure 25b), which has led to new design possibilities for
electrical machine parts [46]. otor and shaft, which was introduced from Lammers et al. [48], and was made of a soft magnetic
rro-silicon alloy, assembled and implemented into a conventionally manufactured stator, and
uccessfully tested under usual operating conditions. Finally, handling of common three-
imensional machine features like skewing or machine end-effects becomes possible during the
roduction process using metal additive manufacturing techniques [49]. (a)
(b)
Figure 25. (a) Additive manufacturing process: rotor active part and shaft; (b) rotor active part and
shaft fabricated by IAL and DMRC. Figure 25. (a) Additive manufacturing process: rotor active part and shaft; (b) rotor active part and
shaft fabricated by IAL and DMRC. (b) (a) (a) (b) gure 25. (a) Additive manufacturing process: rotor active part and shaft; (b) rotor active part and
haft fabricated by IAL and DMRC. Figure 25. (a) Additive manufacturing process: rotor active part and shaft; (b) rotor active part and
shaft fabricated by IAL and DMRC. Considering boundary conditions such as available space, layer thickness, or building time,
every imaginable structure is producible [47]. Currently, it is possible to process many different
materials like titanium, aluminium, stainless steel and, particularly interesting for the field of electrical
machines, soft magnetic materials, such as ferro-silicon or ferro-cobalt alloys [46]. Because of this,
the two-dimensional design limitation, due to conventionally laminated stator and rotor active
parts, is negated. More specifically, this could be used to improve the cooling concept by placing
cooling channels close to the origin of loss. Furthermore, implementing lightweight lattice structures
beyond the flux paths to increase the machine’s power density and dynamics is possible. The latter
improvement was successfully implemented in an additively manufactured rotor of a permanent
magnet synchronous machine [48]. Figure 25a, shows another additively manufactured rotor and shaft,
which was introduced from Lammers et al. [48], and was made of a soft magnetic ferro-silicon alloy,
assembled and implemented into a conventionally manufactured stator, and successfully tested under
usual operating conditions. Finally, handling of common three-dimensional machine features like
skewing or machine end-effects becomes possible during the production process using metal additive
manufacturing techniques [49]. 5.5. Direct Liquid-Cooled Windin
5.5. Direct Liquid-Cooled Winding For comparison, the current displacement without cooling channels leads
to a 22.6% increase in losses [45]. Figure 22. 3D-casted coil [45]. Figure 23. Temperature distribution in 3D-printed coil [45]. Figure 22. 3D-casted coil [45]. 22.6% increase in losses [45]. Figure 22. 3D-casted coil [45]. Figure 23. Temperature distribution in 3D-printed coil [45]. Figure 23. Temperature distribution in 3D-printed coil [45]. Energies 2017, 10, x FOR PEER REVIEW
21 of 25
(a)
(b)
Figure 24. (a) Schematic model of the cast coil [45]; (b) RMS values of the current density distribution
in the three conductors closest to the air gap at 500 Hz [45]. 5.6. Metal Additive Manufacturing
Figure 24. (a) Schematic model of the cast coil [45]; (b) RMS values of the current density distribution
in the three conductors closest to the air gap at 500 Hz [45]. Figure 22. 3D-casted coil [45]. Figure 23. Temperature distribution in 3D-printed coil [45]. Figure 22. 3D-casted coil [45]. Figure 22. 3D-casted coil [45]. Figure 23. Temperature distribution in 3D-printed coil [45]. Figure 23. Temperature distribution in 3D-printed coil [45]. Energies 2017, 10, x FOR PEER REVIEW Figure 22. 3D-casted coil [45]. Figure 22. 3D-casted coil [45]. Figure 22. 3D-casted coil [45]. Figure 22. 3D-casted coil [45]. Figure 22. 3D-casted coil [45]. Figure 22. 3D-casted coil [45]. Figure 23. Temperature distribution in 3D-printed coil [45]. Figure 23. Temperature distribution in 3D-printed coil [45]. Figure 23. Temperature distribution in 3D-printed coil [45]. R PEER REVIEW (b) (a)
(b)
Figure 24. (a) Schematic model of the cast coil [45]; (b) RMS values of the current density distribution
in the three conductors closest to the air gap at 500 Hz [45]. 5.6. Metal Additive Manufacturing
Figure 24. (a) Schematic model of the cast coil [45]; (b) RMS values of the current density distribution
in the three conductors closest to the air gap at 500 Hz [45]. (a) (a) (b) Figure 24. (a) Schematic model of the cast coil [45]; (b) RMS values of the current density distribution
in the three conductors closest to the air gap at 500 Hz [45]. 5 6 Metal Additive Manufacturing
Figure 24. (a) Schematic model of the cast coil [45]; (b) RMS values of the current density distribution
in the three conductors closest to the air gap at 500 Hz [45]. Energies 2018, 11, 344
lacing cooling chan
ructures beyond th 21 of 25
attice
sible. 21 of 25
attice
sible. 5.7. Insulation Apart from the active components of an electrical machine, passive components, like the insulation,
have to be considered for possible fatigue resulting from the environmental conditions during flight. The life span can be estimated from the amount of stress due to partial discharges. These stresses arise
because of air inclusions in the insulation and are increasingly important for higher DC voltages under
pulsed excitation. The voltage needed for a discharge can be calculated from Paschen’s law [50]: UD =
π·r2
I·λ·U
ln
π·r2
I ·pd
kB·T
−ln|ln|1 −β−1||
p
kB·T
(2) (2) where rI is the radius of an ion, λ is the mean free path, U is the voltage, p is the gas pressure, d is the
distance, kB is the Boltzmann constant, T is the temperature, and β is the second Townsend coefficient. Energies 2018, 11, 344 Energies 2018, 11, 344 Energies 2018, 11, 344 22 of 25 So, the breakdown voltage is a function of gas pressure and temperature. Both values change
during the flight time of an aircraft. Pressure and temperature decrease when gaining altitude. When we formulate the dependence as: eUD = A·
p·15 ◦C
T·101.325 kPa
(3) (3) by using an expression normed to standard atmospheric values and by assuming that the change of
term A is negligible, the temperature near the winding would be identical to the winding temperature. At cruising altitude, the air pressure is 19.1 kPa. For this case, a winding temperature of 120 ◦C was
assumed. This would lower the breakdown voltage to approximately 2.3% of that at sea level at
ambient temperature. The demand for low weight and high efficiency has led to the use of higher DC-link voltages and
increasing frequencies for electrical machines. With new developments in semiconductor materials,
the switching frequency has been significantly increased, which steepens the switching edges to
values above 50 kV/ns. Therefore, even at ground level, the common insulations may be insufficient. There is an immediate need for the development of better materials to operate electrical machines
in low pressure and high temperature environments at high frequencies with high DC voltages
under pulsed excitation. Changing environmental conditions during the flight, as well as increasing
switching frequencies in combination with the demand for low weight, encourages the use of Teflon or
Nomex-coated Kapton insulations instead of classical resin-based insulation materials. Conflicts of Interest: The authors declare no conflict of interest. Author Contributions: All authors contributed equally to this paper. References . Aircraft Electrical Propulsion—The Next Chapter of Aviation? Roland Berger Ltd.: London, UK, 2017. 2. Radespiel, R.; Heinze, W.; Bertsch, L. High-lift research for future transport aircraft. In Proceedings of the
Der 66, Deutscher Luft- und Raumfahrtkongress, Munich, Germany, 5–7 September 2017. 3. Heidmann, J. NASA’s AATT project and distributed propulsion perspectives. In Proceedings of the New
Engine Integration Concepts Symposium, Braunschweig, Germany, 23 August 2017. 4. Type-Certificate Data Sheet No. IM.E.051 for PW305&PW306 Series Engines; European Aviation Safety Agency:
Cologne, Germany, 2015; Issue 04. Picture from Pratt & Whitney. Available online: http://www.pw.utc.com/Content/Photos/Feed/Stories 5. Picture from Pratt & Whitney. Available online: http://www.pw.utc.com/Content/Photos/Feed/Stories/
PW300-01/pw305a_1_high.jpg (accessed on 5 December 2017). 6. Bräunling, W.J.G. Flugzeugtriebwerke, Grundlagen, Aero-Thermodynamik, ideale und reale Kreisprozesse,
Thermische Turbomaschinen, Komponenten, Emissionen und Systeme, 4th ed.; Springer: Berlin/Heidelberg,
Germany, 2015. 7. Krishnan, R.; Bharadwaj, A.S. A comparative study of various Motor Drive Systems for Aircraft
Applications. In Proceedings of the IEEE Industry Applications Society Annual Meeting, Dearborn, MI,
USA, 28 September–4 October 1991. 8. Ganev, E.D. High-performance electric drives for aerospace more electric architectures Part I—Electric
machines. In Proceedings of the IEEE Power Engineering Society General Meeting, Tampa, FL, USA,
24–28 June 2007. 9. Arkkio, A.; Jokinen, T.; Lantto, E. Induction and permanent-magnet synchronous machines for high-speed
applications. In Proceedings of the International Conference on Electrical Machines and Systems,
Nanjing, China, 27–29 September 2005. 10. Binder, A.; Schneider, T. High-speed inverter-fed AC drives. In Proceedings of the International Aegean
Conference on Electrical Machines and Power Electronics, Bodrum, Turkey, 10–12 September 2007. 11. Gerada, D.; Mebarki, A.; Brown, N.L.; Gerada, C.; Cavagnino, A.; Boglietti, A. High-speed electrical machines:
Technologies, trends and developments. IEEE Trans. Ind. Electron. 2014; 61, 2946–2959. 12. Binder, A. Elektrische Maschinen und Antriebe—Grundlagen, Betriebsverhalten; Springer: Berlin/Heidelberg,
Germany, 2012. 13. Kleiner, F.; de Wit, B.; Ponick, B. Choosing electric turbocompressor drivers. IEEE Ind. Appl. 2001, 7, 45–52. [CrossRef] 14. Cao, W.; Mecrow, B.C.; Atkinson, G.J.; Bennet, J.W.; Atkinson, D.J. Overview of electric motor technologies
used for more electric aircraft (MEA). IEEE Trans. Ind. Electron. 2012, 59, 3523–3531. 15. Caprio, M.; Lelos, V.; Herbst, J.; Upshaw, J. Advanced induction motor endring design features for high
speed applications. In Proceedings of the IEEE International Conference on Electric Machines and Drives,
San Antonio, TX, USA, 15 May 2005. 16. Lateb, R.; Enon, J.; Durantay, L. High-speed, high power electrical induction motor technologies for integrated
compressors. 6. Summary and Conclusions A short overview of the requirements and demands of electrical machines for more-electric
and all-electric aircraft, which will be significant in the coming years and decades, was presented. As an example, a currently available turbofan engine with a medium power demand was highlighted. Presently, different electric machine concepts need to be addressed and could prove to be feasible. Each was reviewed and evaluated in terms of their effectiveness and applicability. Eventually,
the permanent magnet synchronous machine and the induction machine with squirrel-cage rotor were
identified as the most promising electrical machine types for low-speed and high-speed operation,
respectively. However, the properties and future usability of the machine concept as well as the
components and materials need to be examined. This paper shows the possibilities of multi-phase,
multi-layer windings in addition to common three-phase windings. Different conductor materials
and superconducting materials were evaluated, as well as the flux exciting permanent magnets for
synchronous machines, and the potential for up-to-date materials as the magnetic core. In addition
to electromagnetic aspects, the mechanical evaluation, especially in the range of high circumferential
speeds at high rotational speeds or with large bore diameters, was considered with different rotor armor
materials, followed by a closer look at the challenges of the design of this banding. Thermal aspects
cannot be disregarded. Hence, the sources of heat, with a closer look at the additional rotor losses,
were shown, followed by the concept of an innovative cooling method for cast coils and arising
opportunities from the currently researched additive manufacturing of electrical steel rotors with new
design potentials. In considering all of the technical aspects and solutions, the focus can be shifted to
the objective of building lightweight electrical machines. The challenges for electrical machines emerging from the desire for more-electric or all-electric
aircraft are many and demanding. This paper shows state-of-the-art concepts and materials. Furthermore, an assessment of the direction of future research and designs was discussed regarding
current developments. Author Contributions: All authors contributed equally to this paper. Conflicts of Interest: The authors declare no conflict of interest. 23 of 25 Energies 2018, 11, 344 References In Proceedings of the IEEE International Conference on Electrical Machines and Systems,
Tokyo, Japan, 15–18 November 2009. 17. Wood, B.M.; Olsen, C.L.; Hartzo, G.D.; Rama, J.C.; Szenasi, F.R. Development of an 11,000-r/min 3500-HP
induction motor and adjustable-speed drive for refinery service. IEEE Trans. Ind. Appl. 1997, 33, 815–825. [CrossRef] 18. Barcaro, M.; Fornasiero, E.; Bianchi, N.; Bolognani, S. Design procedure of IPM motor drive for railway
traction. In Proceedings of the IEEE International Electrical Machines & Drives Conference, Niagara Falls,
ON, Canada, 15–18 May 2011. 19. Zhang, F.; Du, G.; Wang, T.; Liu, G.; Cao, W. Rotor retaining sleeve design for a 1.12-MW high-speed PM
machine. IEEE Trans. Ind. Appl. 2015, 51, 3675–3685. [CrossRef] 0. Ishigami, T.; Tanaka, Y.; Homma, H. Development of Motor Stator with Rectangular-Wire Lap Winding
an Automatic Process for Its Production. Electr. Eng. Jpn. 2014, 187, 51–59. [CrossRef] 21. Walmer, M.H.; Liu, J.F.; Dent, P.C. Current Status of Permanent Magnet Industriy in the United States. In Proceedings of the 20th International Workshop on Rare Earth Permanent Magnets and their Applications,
Crete, Greece, 8–10 September 2008. 24 of 25 24 of 25 Energies 2018, 11, 344 22. Arnold Magnetic Technologies Neodymium-Iron-Boron Magnet Grades—Summary Product List & Reference Guide;
REV Group Inc.: Milwaukee, WI, USA, 2015; p. 150410a. 23. Arnold Magnetic Technologies RECOMA—The Complete Range of SmCo5 and Sm2Co17 Alloys; REV Group Inc.:
Milwaukee, WI, USA, 2014; p. 131025. 24. Arnold Magnetic Technologies Cast AlNiCo Permanent Magnets; REV Group Inc.: Milwaukee, WI, USA,
2003; p. 6.1. 25. MANNEL MAGNETTECHNIK GmbH & Co. KG. DAUERMAGNETE—LANGLEBIG, HALTBAR UND
FLEXIBEL. Available online: http://www.mannel-magnet.com/images/pdf/Datenblaeter_Dauermagnete. pdf (accessed on 11 August 2017). p
g
26. VACUUMSCHMELZE GmbH & Co. KG. Selten-Erd-Dauermagnete—VACODYM, VACOMAX; PD002;
VACUUMSCHMELZE GmbH & Co. KG: Hanau, Germany, 2014. 27. C. D. Wälzholz KG Elektroband. Available online: https://www.waelzholz.com/fileadmin/Website-DE/
Documents/Broschueren/waelzholz elektroband 201310 de pdf (accessed on 13 November 2017) 27. C. D. Wälzholz KG Elektroband. Available online: https://www.waelzholz.com/fileadmin/Website-DE/ 27. C. D. Wälzholz KG Elektroband. Available online: https://www.waelzholz.com/fileadmin/Website-DE/
Documents/Broschueren/waelzholz-elektroband-201310-de.pdf (accessed on 13 November 2017). 28. VACUUMSCHMELZE GmbH & Co. KG. Soft Magnetic Cobalt-Iron Alloys—VACOFLUX and VACODUR;
PD002; VACUUMSCHMELZE GmbH & Co. KG: Hanau, Germany, 2014. 29. Copper Development Association. The Copper Advantage—A Guide to Working with Copper and Copper Alloys;
Antimicrobial Copper: Dusseldorf, Germany, 2010; p. A1360. 30. Ginzburg, V.L. Once again about high-temperature superconductivity. Contemp. Phys. 1992, 33, 15–23. [CrossRef] 31. Krabbes, G.; Fuchs, G.; Canders, W.-R.; May, H.; Palka, R. References High Temperature Superconductor Bulk Materials;
Wiley-VCH: Weinheim, Germany, 2006; ISBN 3-527-40383-3. 32. Schwaigerer, F.; Sailer, B.; Glaser, J.; Meyer, H.-J. Supraleitfähigkeit—Strom eiskalt serviert. Chem. Unserer Zeit
2002, 36, 108–124. [CrossRef] 33. Oberly, C.E. Lightweight superconducting generators for mobile military platforms. In Proceedings of the
IEEE Power Engineering Society General Meeting, Montreal, QC, Canada, 18–22 June 2006. 34. Neumüller, H.-W.;
Siemens, C.T. Synchronous HTS machines—Benefits and future applications. Paper Presented at the 6th European Conference of Applied Superconductivity (EUCAS 03), Sorrento,
Italy, 14–18 September 2003; IOP Punblishing Ltd.: Bristow, UK, 2004. 35. Klaus, G.; Nick, W.; Neumüller, H.-W.; Nerowski, G.; McCown, W. Advances in the development of
synchronous machines with high-temperature superconducting field winding at siemens AG. In Proceedings
of the IEEE Power Engineering Society General Meeting, Montreal, QC, Canada, 18–22 June 2006. 36. Masson, P.J.; Luongo, C.A. HTS machines for applications in all-electric aircraft. In Proceedings of the IEEE
Power Engineering Society General Meeting, Tampa, FL, USA, 24–28 June 2007. 37. Cai, M.; Henke, M.; Canders, W.R. A deterministic method for finding multi phase winding topologies with
quasi-ideal current sheet space harmonics spectrum. In Proceedings of the 9th International Symposium on
Linear Drives for Industry Application (LDIA), Hangzhou, China, 7–10 July 2013. 38. Cai, M.; Henke, M.; Canders, W.R. An improved method for design of symmetrical multiphase winding
with optimal space harmonics spectrum. In Proceedings of the 17th International Conference on Electrical
Machines and Systems (ICEMS), Hangzhou, China, 22–25 October 2014. y
(
),
g
,
,
39. Cai, M. Contribution to the Winding Theory—Introduction of a Unified Method for the Treatment of Winding
Topology; Cuvillier Verlag: Göttingen, Germany, 2017. 40. Domann, N.; Henke, M. Design and build-up of a high performance six-phase machine for an automotive
application. In Proceedings of the International Conference on Electrical Machines (ICEM), Berlin, Germany,
2–5 September 2014. 41. Madyn 2000; Technoparkstrasse 1, 8005; DELTA JS AG: Zurich, Switzerland, 2005. 42. Roos, E.; Maile, K. Werkstoffkunde für Ingenieure—Grundlagen, Anwendung, Prüfung, 5th ed.; Springer:
Berlin, Germany, 2015. 43. Müller, G.; Vogt, K.; Ponick, B. Berechnung Elektrischer Maschinen, 6th ed.; Maschinen, E., Ed.; Wiley-VCH:
Weinheim, Germany, 2011. 44. Huang, Z. Thermal Design of Electrical Machines-Investigation and Evaluation of Cooling Performances. Ph.D. Thesis, Lund University, Lund, Sweden, 2013. 45. Wohlers, C.; Juris, P.; Kabelac, S.; Ponick, B. Design and Direct Liquid Cooling of Tooth-Coil Windings. Electr. Eng. in press. 25 of 25 Energies 2018, 11, 344 46. References Bremer, S.; Meiner, W.; Diatlov, A. Selective Laser Melting—A manufacturing technology for future? Laser Tech. J. 2012, 9, 33–38. [CrossRef] 47. Adam, G.A.O.; Zimmer, D. Design for Additive Manufacturing—Element transitions and aggregated
structures. CIRP J. Manuf. Sci. Technol. 2014, 7, 20–28. [CrossRef] 48. Lammers, S.;
Adam, G.;
Schmid, H.J.;
Morzek, R.;
Hoffmann, M.J.;
Quattrone, F.;
Ponick, B. Additive manufacturing of a lightweight rotor for a permanent magnet synchronous machine. In Proceedings
of the International Electric Drives Production Conference (EDPC) 2016, Nuremberg, Germany,
30 November–1 December 2016. [CrossRef] 49. Ponick, B. Der Einfluss der Nutschrägung auf die axiale Verteilung des Luftspaltfelds elektrischer Maschinen. Elektrie 2000, 54, 248–252. 0. Lehr, J.; Ron, P. Foundations of Pulsed Power Technology; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 201 © 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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Regulation of Reentrainment Function Is Dependent on a Certain Minimal Number of Intact Functional ipRGCs in rd Mice
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Journal of ophthalmology
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Research Article
Regulation of Reentrainment Function Is Dependent on a Certain
Minimal Number of Intact Functional ipRGCs in rd Mice Jingxue Zhang,1 Huaizhou Wang,2 Shen Wu,1 Qian Liu,1 and Ningli Wang1,2
1Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Ophthalmology & Visual Sciences Key Laboratory,
Beijing Tongren Hospital, Capital Medical University, Beijing, China
2Beijing Tongren Eye Center, Beijing Ophthalmology & Visual Sciences Key Laboratory, Beijing Tongren Hospital,
Capital Medical University, Beijing, China Correspondence should be addressed to Ningli Wang; wningli@vip.163.com Received 8 June 2017; Accepted 10 October 2017; Published 22 November 2017 Academic Editor: Ji-jing Pang Copyright © 2017 Jingxue Zhang et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited. Purpose. To investigate the effect of partial ablation of melanopsin-containing retinal ganglion cells(mcRGCs) on nonimage-forming
(NIF) visual functions in rd mice lacking rods. Methods. The rd mice were intravitreally injected with different doses (100 ng/μl,
200 ng/μl, and 400 ng/μl) of immunotoxin melanopsin-SAP. And then, the density of ipRGCs was examined. After establishing
the animal models with different degrees of ipRGC damage, a wheel-running system was used to evaluate their reentrainment
response. Results. Intravitreal injection of melanopsin-SAP led to partial ablation of ipRGCs in a dose-dependent manner. The
survival rates of ipRGCs in the 100 ng/μl, 200 ng/μl, and 400 ng/μl groups were 74.14% ± 4.15%, 39.25% ± 2.29%, and
38.38% ± 3.74%, respectively. The wheel-running experiments showed that more severe ipRGC loss was associated with a longer
time needed for reentrainment. When the light/dark cycle was delayed by 8 h, the rd mice in the PBS control group took
4.67 ± 0.79 days to complete the synchronization with the shifted cycle, while those in the 100 ng/μl and 200 ng/μl groups
required 7.90 ± 0.55 days and 11.00 ± 0.79 days to complete the synchronization with the new light/dark cycle, respectively. Conclusion. Our study indicates that the regulation of some NIF visual functions is dependent on a certain minimal number of
intact functional ipRGCs. Hindawi
Journal of Ophthalmology
Volume 2017, Article ID 6804853, 8 pages
https://doi.org/10.1155/2017/6804853 Hindawi
Journal of Ophthalmology
Volume 2017, Article ID 6804853, 8 pages
https://doi.org/10.1155/2017/6804853 Hindawi
Journal of Ophthalmology
Volume 2017, Article ID 6804853, 8 pages
https://doi.org/10.1155/2017/6804853 1. Introduction antibodies against melanopsin and compared the number
of ipRGCs on flat-mounted retina among 1-, 3-, and 6-
month-old rd mice (n = 6 in each age group) (the detailed
methodology is described below). 2.3. Eye Injections. Three-month-old C3H/HeJ mice were
divided into 3 dose groups (n = 6 in each group): 100 ng/μl,
200 ng/μl, and 400 ng/μl. All animals were anesthetized
with 5% chloraldurat (8μl/g), and the eyes were topically
anesthetized with one drop of 0.5% proparacaine (Alcon
Laboratories Inc., Fort Worth, TX, USA). The left eye of
each animal was intravitreally injected with different doses
of immunotoxin melanopsin-SAP (2 μl/eye) (Advanced
Targeting Systems, San Diego, CA, USA), consisting of
saporin conjugated to a melanopsin polyclonal antibody. A Hamilton syringe with a 30 gauge needle (BD Medical
Systems, Franklin Lakes, NJ, USA) was used for the intra-
vitreal injections, and the needle was left in place for about
3 minutes after the injections. The right eye of each animal
was injected with 2μl phosphate-buffered saline (PBS) vehi-
cle as control. All animals were sacrificed by CO2 asphyxi-
ation 4 weeks after injection. The sampling time of each
mouse was fixed at 14:00. The density of ipRGCs was
examined on flat-mounted retina. In the above-mentioned studies, however, as the ablation
of ipRGCs occurred in the presence of rods and cones, there
was a possibility that the signals generated by the rods and
cones might be carried to the brain to mediate NIF functions
through other pathways. So the respective roles of the three
types of photoreceptors, that is, ipRGCs, rods, and cones, in
multiple NIF functions cannot be clearly established. p
y
Moreover, in prior studies, only partial ablation of
ipRGCs already led to impairment of NIF functions, suggest-
ing that a minimal number of ipRGCs may be required for
NIF visual responses. Thus, our study aimed to clarify and
quantify the specific contributions of ipRGCs to NIF func-
tions. In order to exclude the possible auxiliary functions of
rods and cones, we selected rd mice (C3H/Hej) lacking rods
and cones. Animal models with different degrees of ipRGC
damage were established via intravitreal injection of different
doses of immunotoxin melanopsin-SAP. And then, a wheel-
running system was used to evaluate the reentrainment func-
tion (one of the most important NIF functions) of these
animals. 1. Introduction The discovery of ipRGCs has allowed for rapid progress in
the past decade toward understanding the NIF visual system,
especially the three types of photoreceptors [4–13]. Mammalian eyes mediate both image-forming (IF) and
nonimage-forming (NIF) visual functions. NIF vision pro-
vides a measure of the ambient light for the purposes of
synchronization of circadian clocks to light/dark cycles and
regulation of pupil size, pineal melatonin production, and
other functions. People traditionally believe that the classical
photoreceptors (rods and cones) regulate both IF and NIF
visual pathways. But recently, a small subset of retinal
ganglion cells (RGCs) called intrinsically photosensitive
RGCs (ipRGCs) or melanopsin-containing RGCs (mcRGCs),
projecting to the suprachiasmatic nucleus and other pretectal
areas, has been identified to be a third type of mammalian
photoreceptors and determined to be photosensitive [1–3]. Previous studies have attempted to elucidate the respec-
tive roles of these photoreceptor types in NIF visual func-
tions, most of which showed that NIF vision was almost
not affected after the degeneration of rods and cones
[14–17]. Melanopsin-knockout mice (opn4−/−) retained
generally normal NIF functions expect for an attenuated
phase-shifting response to light and a diminished pupillary
constriction at high irradiance levels. In addition, the effect
of light on circadian rhythm disappeared after the loss of
both melanopsin and rods and cones [18–21]. It therefore
seems that NIF visual functions can be supported by
photoreception in either rods and cones or ipRGCs. 2 Journal of Ophthalmology It should be noted that most of the available data comes
from studies using animals carrying genetic lesions. These
studies only abolished the genetic expression of melanopsin,
while the functions of ipRGCs as ganglion cells remained
unchanged, especially the projection to brain areas and the
transduction of photic information. In order to fully clarify
the roles of ipRGCs, it is imperative to ablate the whole cell
rather than the expression of melanopsin alone. Recent stud-
ies reported that after targeted destruction of ipRGCs, all of
the light-regulated NIF functions were dramatically impaired
or even disappeared [22–24]. This indicates that in addition
to their intrinsic photosensitivity, ipRGCs also function as a
conduit through which rods and cones can access brain
circuits mediating NIF functions. Thus, ipRGCs seem to play
a unique role in NIF visual responses. 1. Introduction Based on the changes in the reentrainment
response, we attempted to elucidate the roles of ipRGCs in
NIF functions and determine whether the regulation of such
functions is dependent on a certain number of ipRGCs. 2.4. Retinal Processing and Staining. The procedures of
retinal processing and staining were performed as described
in our previous study [25]. Briefly, for flat mount, a slit was
cut in the sclera close to the cornea. The eyes were then sub-
merged in 0.01 mol/l PBS. The front part (cornea, lens, and
vitreous) of the eye was cut away, and the retina was carefully
isolated from the pigment epithelium. The retinas were fixed
in fresh 4% paraformaldehyde in PBS for 30 minutes and
then washed three times in PBS for 5 minutes each. The
free-floating retinas were incubated in a blocking solution
(0.3% Triton X-100 and 5% bovine serum albumin in PBS)
for 1 hour at room temperature and were then incubated
with a primary melanopsin antibody (polyclonal rabbit
anti-melanopsin; Affinity BioReagents, Golden, CO, USA)
at 1:500 dilution in PBS/0.3% Triton X-100/5% bovine serum
albumin for 72 hours at 4°C. After three washes in PBS of 15
minutes each, the fluorescence-conjugated secondary anti-
body (Alexa Fluor 488 goat antibody to rabbit immunoglob-
ulin G; Molecular Probes, Eugene, OR, USA) was applied to
the sample as previously described, except that incubation
was for 2 hours at room temperature. The free-floating ret-
inas were washed again as described above and flat mounted
onto glass slides, and coverslips were applied using Vect
Mount Permanent Mounting Medium (Vector Laboratories,
Burlingame, CA, USA). 2. Materials and Methods Wheel revolutions were moni-
tored by computer, stored as revolutions/min, then summed
across each 5 min interval, and plotted in raster format using
ActiView (Mini Mitter Co. Inc., Sunriver, OR, USA) software. 3.3. INL Thickness. In order to determine whether the injec-
tion of immunotoxin melanopsin-SAP had any other effect
on the retina, we compared the thickness of the inner
nuclear layer (INL) of the retina between the highest dose
(400 ng/μl) group and the control group. Based on the
H&E stained slides, the morphology of the retina was found
to be similar between the two groups (Figure 3(a)); nearly
all photoreceptors in the outer nuclear layer (ONL) disap-
peared, while the structure from the INL to the ganglion
cell layer (GCL) remained intact. The results of the image
pro plus (IPP) software analysis showed that the INL
thickness of the control group was 133.40 ± 11.61 pixels
and that of the highest dose group was 122.00 ± 9.70 pixels;
the difference was not statistically significant (P > 0 05)
(Figure 3(b)). When entrainment stabilized, in order to assess the
adjustment function of the circadian clock to light conditions
(reentrainment), an important NIF charged by ipRGCs, mice
were subjected to a jet lag test with 8 h delay in the time of
lights on and lights offto evaluate whether they could resyn-
chronize with the shifted cycle. The light condition was still
12 h light: 12 h dark. After the 8 h delay of the LD cycle, the
number of days to reentrain was defined for each animal
as the number of days required to shift activity midpoint by
8 ±0.25 h. The reentrainment was analyzed using the Acti-
View software. 2.7. Statistical Analysis. All data were expressed as mean ±SD
(standard deviation). An independent sample t-test was used
to compare the differences in the mean INL thickness. One-
way ANOVA with Tukey’s multiple comparison test was
used to compare the differences in the mean number of
ipRGCs and the survival rate of ipRGCs among different
groups. For behavioral analysis, we used two-way ANOVA,
followed by Fisher’s LSD post hoc tests. P < 0 05 was consid-
ered statistically significant. 3.4. Wheel-Running Experiments. 2. Materials and Methods 2.1. Animals. C3H/HeJ male mice (Jackson Laboratories,
Bar Harbor, ME, USA) were used in this study. These
mice were homozygous for the retinal degeneration 1
mutation (Pde6b rd1), which caused blindness by weaning
age. All experimental and animal care procedures were
strictly in accordance with the institutional guidelines and
the ARVO Statement for the Use of Animals in Ophthalmic
and Vision Research. The study protocol was approved by
the Animal Care and Use Committee at the Capital Medical
University (permit number 2010-X-30). All surgery was per-
formed under sodium pentobarbital anesthesia, and all
efforts were made to minimize suffering. For Hematoxylin and Eosin (H&E) staining, the eyes
were removed as above and the eyecups were fixed in 4%
paraformaldehyde for 2 hours, washed three times with
PBS, and embedded in paraffin. Eight-micron-thick paraffin
sections were used for H&E staining. Stained slides were
visualized under a Leica microscope. The thickness of the
inner nuclear layer (INL) was measured using image pro
plus (IPP) software. 2.2. Comparison of the Number of ipRGCs in rd Mice at
Different Ages. In order to determine whether all damages
to ipRGCs were caused by the injection of immunotoxin,
or whether theses damages were also potentially related
with age, we immunohistochemically labeled ipRGCs using Journal of Ophthalmology 3 2.5. Counting of ipRGCs. When counting cell number, we
chose 8 visual fields for each flat-mounted retina as
described in our previous study [25]. The melanopsin-
positive cells (ipRGCs) were counted at 200x magnification
using a confocal microscope (Leica TCS SP2, Leica Microsys-
tems, Heidelberg, Germany). Each retina was counted in a
double-blind manner. The ipRGC survival rate was defined
as (ipRGC number of experimental eye)/(ipRGC number of
control eye) × 100%. 2.5. Counting of ipRGCs. When counting cell number, we
chose 8 visual fields for each flat-mounted retina as
described in our previous study [25]. The melanopsin-
positive cells (ipRGCs) were counted at 200x magnification
using a confocal microscope (Leica TCS SP2, Leica Microsys-
tems, Heidelberg, Germany). Each retina was counted in a
double-blind manner. The ipRGC survival rate was defined
as (ipRGC number of experimental eye)/(ipRGC number of
control eye) × 100%. that the ipRGCs, having a diameter of about 20μm, were
diffusely distributed throughout the retina, with 3–5 den-
dritic branches per cell, a dendritic field diameter of about
300 μm, manifesting a bead-like structure. 2. Materials and Methods No significant dif-
ferences were noted in the distribution and morphology of
ipRGCs among the rd mice at different ages. The numbers
of ipRGCs per visual field at 200x magnification were counted
to be 18.66 ± 1.10 in 1-month-old rd mice, 17.92 ±1.00 in 3-
month-old rd mice, and 18.51 ±0.78 in 6-month-old rd
mice. The differences in the number of ipRGCs were not
statistically significant among the rd mice at different ages
(all P > 0 05) (Figure 1(b)). 2.6. Wheel-Running Experiments. A wheel-running system
(Mini Mitter Co. Inc., Sunriver, OR, USA) was used to eval-
uate the NIF visual function of the rd mice in the PBS control
group (n = 3), 100 ng/μl group (n = 5), and 200 ng/μl group
(n = 5), respectively. C57 mice were involved as wild-type
control (n = 3). Since the survival rate of ipRGCs in the
400 ng/μl group was similar to that in the 200 ng/μl group,
the rd mice in the 400 ng/μl group were not included in
the wheel-running experiments. 3.2. Survival Rate of ipRGCs after Immunotoxin Injection. When the flat-mounted retinas of different dose groups were
compared, we observed a dose-dependent cell death of
ipRGCs following intravitreal injection of immunotoxin
melanopsin-SAP in rd mice. As the dose of melanopsin-
SAP increased, the number of melanopsin-positive cells in
the experimental eye decreased, while that in the control
eye remained the same (Figure 2(a)). The survival rates of
ipRGCs in the 100 ng/μl, 200 ng/μl, and 400 ng/μl groups
were 74.14% ±4.15%, 39.25%± 2.29%, and 38.38%± 3.74%,
respectively. The survival rate of ipRGCs was significantly
reduced in the 200 ng/μl and 400 ng/μl groups when
compared with the 100 ng/μl group (both P < 0 01). But
the difference
between
the 200 ng/μl group
and
the
400 ng/μl group was not statistically significant (P = 0 933)
(Figure 2(b)). When rhythms were stable, each mouse was deeply
anesthetized and unilaterally enucleated, and then the
remaining eye was injected intravitreally with different
doses of immonotoxin as mentioned above. After the injec-
tions, animals were allowed to recover in 12 h light: 12 h
dark conditions. Each mouse was placed in a cage with a running wheel
inside an enclosure in whichwhiteLED lightingwascomputer
controlled and the irradiance was about 100 lux. Each enclo-
sure contained 8 mouse cages. 2. Materials and Methods The results of the wheel-
running experiments showed that when the light/dark cycle
was delayed by 8 h, the rd mice in the PBS control group
(n = 3) were capable of reentraining to the light/dark cycle,
and they took 4.67 ±0.79 days to complete the synchroni-
zation with the shifted cycle (Figure 4(a)); while in the
100 ng/μl group (n = 5) and the 200 ng/μl group (n = 5),
the mice were also able to reentrain but characterized by
a delay, and they required 7.90 ±0.55 days and 11.00
± 0.79 days to complete the synchronization with the
new light/dark cycle, respectively (Figures 4(b) and 4(c)). The differences in the number of days needed for reen-
trainment were statistically significant in all pairwise 3. Results 3.1. Number of ipRGCs in rd Mice at Different Ages. The ipRGCs were immunohistochemically labeled using
antibodies against melanopsin (Figure 1(a)). It was observed 4 Journal of Ophthalmology (a)
1
2
Age (month)
0
mcRGC number/200x
visual field
5
10
15
20
25
3
(b)
Figure 1: The ipRGCs could be immunohistochemically labeled using antibodies against melanopsin. (a) The laser scanning confocal view of
ipRGCs in flat-mounted retina, showing melanopsin immunoreactivity and cell morphology. Bar = 75 μm. (b) The bar graph comparing the
numbers of ipRGCs per visual field (200x magnification) among the rd mice at different ages. The differences in the number of ipRGCs were
not statistically significant (one-way ANOVA with Tukey’s multiple comparison test, P > 0 05). (a) 1
2
Age (month)
0
mcRGC number/200x
visual field
5
10
15
20
25
3 (b) (a) Figure 1: The ipRGCs could be immunohistochemically labeled using antibodies against melanopsin. (a) The laser scanning confocal view of
ipRGCs in flat-mounted retina, showing melanopsin immunoreactivity and cell morphology. Bar = 75 μm. (b) The bar graph comparing the
numbers of ipRGCs per visual field (200x magnification) among the rd mice at different ages. The differences in the number of ipRGCs were
not statistically significant (one-way ANOVA with Tukey’s multiple comparison test, P > 0 05). (A)
(B)
(C)
(D)
(E)
(F)
(a)
100 ng
Melanopsin-SAP (ng/휇l)
200 ng
400 ng
0
10
20
30
40
50
60
70
80
90
100
mcRGC number/200x visual field
expressed as % of controls
⁎
⁎
(b)
Figure 2: Intravitreal
injection of
melanopsin-SAP led to partial
ablation of
ipRGCs in a dose-dependent
manner. (a)
Immunohistochemically labeled ipRGCs using antibodies against melanopsin and comparison of the number of ipRGCs on flat-mounted
retina following intravitreal injection of immunotoxin melanopsin-SAP in rd mice. (A–C) The number of ipRGCs per visual field (200x
magnification) on flat-mounted retinas in different dose groups; (D–F) the results of the control eyes (PBS injection group). As the dose of
melanopsin-SAP increased, the number of melanopsin-positive cells in the experimental eye decreased, while that in the control eye
remained the same. Bar = 250 μm. (b) Analysis of the survival rate of ipRGCs after immunotoxin injection. The ipRGC survival rate was
defined as (ipRGC number of experimental eye)/(ipRGC number of control eye) × 100%. 3. Results The survival rate of ipRGCs was significantly
reduced in the 200 ng/μl and 400 ng/μl groups when compared with the 100 ng/μl group (one-way ANOVA with Tukey’s multiple
comparison test, P < 0 01). But the difference between the 200 ng/μl group and the 400 ng/μl group was not statistically significant (one-
way ANOVA with Tukey’s multiple comparison test, P = 0 933). ∗P < 0 01. 100 ng
Melanopsin-SAP (ng/휇l)
200 ng
400 ng
0
10
20
30
40
50
60
70
80
90
100
mcRGC number/200x visual field
expressed as % of controls
⁎
⁎
(b) (A) (C) (B) (B) (C) (A) (D) (E)
(a) (F) (D) (F) (E) (b) (a) Figure 2: Intravitreal
injection of
melanopsin-SAP led to partial
ablation of
ipRGCs in a dose-dependent
manner. (a)
Immunohistochemically labeled ipRGCs using antibodies against melanopsin and comparison of the number of ipRGCs on flat-mounted
retina following intravitreal injection of immunotoxin melanopsin-SAP in rd mice. (A–C) The number of ipRGCs per visual field (200x
magnification) on flat-mounted retinas in different dose groups; (D–F) the results of the control eyes (PBS injection group). As the dose of
melanopsin-SAP increased, the number of melanopsin-positive cells in the experimental eye decreased, while that in the control eye
remained the same. Bar = 250 μm. (b) Analysis of the survival rate of ipRGCs after immunotoxin injection. The ipRGC survival rate was
defined as (ipRGC number of experimental eye)/(ipRGC number of control eye) × 100%. The survival rate of ipRGCs was significantly
reduced in the 200 ng/μl and 400 ng/μl groups when compared with the 100 ng/μl group (one-way ANOVA with Tukey’s multiple
comparison test, P < 0 01). But the difference between the 200 ng/μl group and the 400 ng/μl group was not statistically significant (one-
way ANOVA with Tukey’s multiple comparison test, P = 0 933). ∗P < 0 01. 4. Discussion was increased from 100 ng/μl to 400 ng/μl, suggesting that
the administration of melanopsin-SAP is associated with
a dose-dependent targeted ablation of ipRGCs, at least in
rd mice. were destructed via specific binding to melanopsin. But
another study revealed that there were at least 5 subtypes
(M1–M5) of ipRGCs with distinct morphological and physi-
ological characteristics; although being intrinsically photo-
sensitive to drive photic reactions, some subtypes might
have no or leaky expression of melanopsin or have tran-
siently expressed melanopsin during development [10]. Therefore, the method of specific binding to melanopsin
may be inadequate to ablate all subtypes of ipRGCs. In the
presence of rods and cones, some subtypes of ipRGCs may
still function to combine and convey the signals derived from
the ONL; thus, we used the rd mice lacking rods and cones so
as to evaluate the effect of partial ablation of ipRGCs on NIF
functions in the absence of rods and cones. In order to verify whether all damages to ipRGCs were
caused by the injection of melanopsin-SAP, we immuno-
histochemically labeled ipRGCs using antibodies against
melanopsin and compared the number of ipRGCs on
flat-mounted retina among 1-, 3-, and 6-month-old C3H/
HeJ mice. We found that in adult C3H/HeJ mice, the number
of ipRGCs did not alter with age, indicating that the injection
of the immunotoxin induced all damages to ipRGCs. We hypothesized that the injection of melanopsin-SAP
would not result in major changes to the retinal structure
other than a decrease in the number of ipRGCs. To test this,
we had to assess the retinal conditions after the injection. Despite the availability of several measurement tools, the
INL and ONL thickness is considered as a direct indicator
reflecting the cellular state in each layer of the retina. Due
to the almost complete loss of ONL in rd mice, we only
measured and analyzed the INL thickness using IPP soft-
ware. We did not observe any significant difference in
the INL thickness between the highest dose (400 ng/μl)
group and the control group, suggesting that the exposure
to melanopsin-SAP did not alter the morphology and struc-
ture of the retina. Since the highest dose of melanopsin-SAP
did not result in any changes to the retinal structure, it is spec-
ulated that lower doses should not cause such changes, and
thus,theINLthicknesswasnotmeasuredinotherdosegroups. 4. Discussion comparisons (all P < 0 01) (Figure 4(e)). In addition, the
locomotor activity of the rd mice was less robust than that
of the wild-type mice (Figure 4(d)). We also found that in
comparison to the controls, the mice injected with immu-
notoxin were less able to precisely synchronize their activ-
ity to the shifted cycle, expressed as a significantly greater
variability in activity onsets with respect to the beginning
of the dark phase. It has been proven in many studies that ipRGCs are not only
intrinsically photosensitive but also capable of transmitting
the photic signals from rods and cones to the brain areas to
mediate NIF functions. In order to clearly establish the roles
of ipRGCs in NIF visual functions, ablation of the whole cell
is required. In some recent studies, the whole cells of ipRGCs 5 Journal of Ophthalmology 5 Control
GCL
IPL
INL
Melanopsin-SAP injection
(a)
Control
0
20
40
60
80
100
120
INL thickness (pixel)
140
160
400 ng/휇l
(b)
Figure 3: The morphological examination of retinal sections after injection of melanopsin-SAP immunotoxin in rd mice. (a)
Photomicrographs of 8 μm paraffin sections of retina were used for H&E staining. No significant morphological differences were observed
between the highest dose group and the control group. Bar = 100 μm. (IPL = inner plexiform layer; INL = inner nucleus layer;
GCL = ganglion cell layer). (b) The thickness of the INL was measured using IPP software (pixel). The difference in the INL thickness
between the highest dose group and the control group was not statistically significant (independent sample t-test, P > 0 05). Control
0
20
40
60
80
100
120
INL thickness (pixel)
140
160
400 ng/휇l
(b) Control
G
I
IN Control (a) (b) Figure 3: The morphological examination of retinal sections after injection of melanopsin-SAP immunotoxin in rd mice. (a)
Photomicrographs of 8 μm paraffin sections of retina were used for H&E staining. No significant morphological differences were observed
between the highest dose group and the control group. Bar = 100 μm. (IPL = inner plexiform layer; INL = inner nucleus layer;
GCL = ganglion cell layer). (b) The thickness of the INL was measured using IPP software (pixel). The difference in the INL thickness
between the highest dose group and the control group was not statistically significant (independent sample t-test, P > 0 05). 4. Discussion Saporin (SAP) is a ribosome-inactivating protein of type
I, which can irreversibly inhibit protein synthesis during
transcription cycle. But binding of SAP to specific targets to
enter into the cell is required for such inhibition. Goz et al. [23] developed a specific immunotoxin consisting of an
antimelanopsin antibody conjugated to saporin, called
melanopsin-SAP. Intravitreal injection of this immunotoxin
could result in targeted destruction of ipRGCs in a dose-
dependent manner, and this is the reason why we chose
it for partial ablation of ipRGCs in our study. But this
immunotoxin also has some limitations; as the targeted
ablation of ipRGCs is also based on the binding of
melanopsin-SAP to melanopsin, it raises the possibility that
some nonmelanopsin-expressing subtypes of ipRGCs still
survive after the injection of this immunotoxin. Thus, in
our study, we used the rd mice lacking rods and cones to
exclude the signals from photoreceptors. g
In our study, the effect of melanopsin-SAP on visual acu-
ity was not assessed, because the rd mice used in this study
had already lost nearly all of their rods and cones, thereby
leading to almost complete visual loss. Prior studies [23] reported that the loss of ipRGCs
became stable at 3 weeks after intravitreal injection of
melanopsin-SAP. So we decided to immunohistochemically
label ipRGCs to count and compare the number of ipRGCs
on flat-mounted retina at 1 month after the immunotoxin
injection. Our findings revealed that the ipRGCs were par-
tially ablated following intravitreal injection of immunotoxin
melanopsin-SAP in rd mice. The survival rate of ipRGCs
decreased from 75% to 40% as the melanopsin-SAP dose As for the assessment of NIF visual functions, we focused
on the reentrainment response, because it has been proven
that this function is mainly regulated by ipRGCs [26]. Our
study also demonstrated that the number of days needed
for reentrainment was similar between rd mice (control
group) and wild-type mice, indicating that rods and cones
might play an insignificant role in this function. 4. Discussion 00:00
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18
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12
Days to reentrain
10
08
06
04
02
0
WT-PBS
rd-PBS
rd-100 ng
rd-200 ng
(e) 4: Entrainment and wheel-running periods of rd mice after injection of melanopsin-SAP immunotoxin. The bar b Figure 4: Entrainment and wheel-running periods of rd mice after injection of melanopsin-SAP immunotoxin. The bar below the actograms
indicates the light (white) and dark (black) periods, and the light and dark periods are indicated by white and gray backgrounds, respectively. When the light/dark cycle was delayed by 8 h, (a) the rd mice in the PBS control group were capable of reentraining to the light/dark cycle, and
they took 4.67 ± 0.79 days to complete the synchronization with the shifted cycle. (b) The rd mice in the 100 ng/μl group and (c) the rd mice in
the 200 ng/μl group required 7.90 ± 0.55 days and 11.00 ± 0.79 days to complete the synchronization with the new light/dark cycle,
respectively, indicating slower reentrainment. 4. Discussion Journal of Ophthalmology 6 00:00
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16
14
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Days to reentrain
10
08
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0
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rd-PBS
rd-100 ng
rd-200 ng
(e)
Figure 4: Entrainment and wheel-running periods of rd mice after injection of melanopsin-SAP immunotoxin. The bar below the actograms
indicates the light (white) and dark (black) periods, and the light and dark periods are indicated by white and gray backgrounds, respectively. When the light/dark cycle was delayed by 8 h, (a) the rd mice in the PBS control group were capable of reentraining to the light/dark cycle, and
they took 4.67 ± 0.79 days to complete the synchronization with the shifted cycle. (b) The rd mice in the 100 ng/μl group and (c) the rd mice in
the 200 ng/μl group required 7.90 ± 0.55 days and 11.00 ± 0.79 days to complete the synchronization with the new light/dark cycle,
respectively, indicating slower reentrainment. (d) The wild-type mice took 3.67 ± 0.29 days to complete the synchronization with the
shifted cycle, and the locomotor activity of the wild-type mice was more robust than that of the rd mice. ∗represents the day delay in the
time of lights on and lights off. (e) The differences in the number of days needed for reentrainment were not statistically significant
between the rd mice in the PBS control group and the wild-type group (two-way ANOVA, followed by Fisher’s LSD post hoc test, P > 0 05). The number of days required for reentrainment were significantly increased in the 100 ng/μl and 200 ng/μl groups when compared with
the PBS control group (two-way ANOVA, followed by Fisher’s LSD post hoc test, P < 0 01). ∗P < 0 01. 4. Discussion [27]
Our findings
Reentrainment
Absent (150 lx)
Most animals unable
to entrain (700 lx)
Advanced onset
Others had light
responses, but
no stable
circadian rhythms
Half able to entrain,
more than 16 days
required (100 lx)
Advanced onset
Half failed to entrain
Able to entrain, more
than 12 days required (15 lx)
Normal (300 lx)
Able to entrain, at least
8–11 days required (100 lx)
Advanced onset
Survival rate
<10%
3–17%
18–40%
63%
40–75% Table 1: Effect of partial ablation of ipRGCs on reentrainment: comparison of study results. Able to entrain, more
than 12 days required (15 lx)
Normal (300 lx)
Able to entrain, at least
8–11 days required (100 lx)
Advanced onset 40–75% more accurate and reliable information for understanding
the mechanism of circadian rhythm-related disorders and
offer insights into the potential treatment of these disorders. (more than 16 days) was required for such reentrainment
[23]. In our study, when the survival rate of ipRGCs ranged
from 40% to 75%, a greater number of days (at least 8–11
days) were needed for reentrainment as compared with the
control group, though all animals were able to resynchronize
with the new light/dark cycle. These results consistently indi-
cate that the regulation of the reentrainment function is
dependent on a certain minimal number of ipRGCs. In addition, as shown in Table 1, the experimental con-
ditions varied among the published studies. Thus, we need
to use a uniform design to optimize the experimental condi-
tions including animal species, irradiance level, and inter-
vention factors, so as to accurately define the association
between the number of ipRGCs and NIF visual functions. Furthermore, as ipRGCs consist of several subtypes project-
ing to different brain areas, future studies may need to clearly
establish the individual roles of each subtype in regulating
NIF functions. Taken together, we believe that a minimal density of
ipRGCs is required to maintain the NIF visual functions. Similarly, a threshold relationship has also been identified
between the lesion extent of the cholinergic basal forebrain
(CBF) and working memory impairment in rats; only when
the density of CBF was lower than 25%, impaired working
memory could be observed [28]. Also, impairment of IF
functions, such as visual field changes, could only be clini-
cally detected when the loss rate of RGCs reached >40%
[29]. 4. Discussion (d) The wild-type mice took 3.67 ± 0.29 days to complete the synchronization with the
shifted cycle, and the locomotor activity of the wild-type mice was more robust than that of the rd mice. ∗represents the day delay in the
time of lights on and lights off. (e) The differences in the number of days needed for reentrainment were not statistically significant
between the rd mice in the PBS control group and the wild-type group (two-way ANOVA, followed by Fisher’s LSD post hoc test, P > 0 05). The number of days required for reentrainment were significantly increased in the 100 ng/μl and 200 ng/μl groups when compared with
the PBS control group (two-way ANOVA, followed by Fisher’s LSD post hoc test, P < 0 01). ∗P < 0 01. rd mice would be somewhat affected. More severe ipRGCs
loss would result in a longer time needed to reentrain. After targeted destruction of ipRGCs via intravitreal
injection of melanopsin-SAP, wheel-running experiments
showed that the rd mice in the 100 ng/μl dose group spent
about 8 days to complete the synchronization with the new
light/dark cycle and those in the 200 ng/μl dose group
required about 11 days to reentrain; the survival rates
of ipRGCs were calculated to be 74.14%± 4.15% and
39.25% ±2.29% in the two groups, respectively. In compari-
son, the rd mice in the PBS control group and the wild-type
mice only took around 4 days to resynchronize with the
shifted cycle. These findings revealed that when the loss rate
of ipRGCs reached about 25%, the reentrainment response of According to published literatures, a lower survival rate
of ipRGCs was associated with a greater impairment of the
reentrainment function in a cell number-dependent fashion,
which was consistent with our findings (Table 1). Hatori et al. reported that, when the survival rate of ipRGCs was less than
10%, all animals seemed unable to reentrain [24]. In another
study, with a survival rate of ipRGCs ranging from 18% to
40% after the injection of melanopsin-SAP, half of the
animals failed to reentrain to the shifted light/dark cycle;
the other half were capable of reentraining, but a longer time 7 Journal of Ophthalmology Journal of Ophthalmology Table 1: Effect of partial ablation of ipRGCs on reentrainment: comparison of study results. Hatori et al. [24]
Guler et al. [22]
Goz et al. [23]
Boudard et al. Acknowledgments The authors thank Dr. Lan Zhou for her help in the
wheel-running experiments. This study was supported by
the Beijing Natural Science Foundation (7164243) and
Beijing Young Talents Project (2014000021469G262). Our study provides evidence that a lower survival rate of
ipRGCs is associated with a greater impairment of the reen-
trainment function in a cell number-dependent manner. Fur-
ther validation in larger animals and clinical patients (wrist
watch for circadian rhythm monitoring is now clinically
available) is still required, which may help to quantify such
association and set standards for clinical examinations. For
instance, our prior study and other published studies consis-
tently showed that chronic ocular hypertension resulted in
damages to ipRGCs and hence the reentrainment function
[25, 31], indicating that we may assess the effect of glaucoma
on ipRGCs by measuring the reentrainment response. 4. Discussion The same is true for NIF functions; a recent study
revealed that in patients with Leber hereditary optic neurop-
athy (LHON) and dominant optic atrophy (DOA), despite a
moderate loss of ipRGCs, the NIF functions were well main-
tained including the photoentrainment of circadian rhythms,
light-induced suppression of melatonin secretion, and pupil-
lary light reflex [30]. This further supports that only when the
loss of ipRGCs reaches a certain threshold can the NIF func-
tions be affected. The authors declare that they have no conflicts of interest. The authors declare that they have no conflicts of interest. Disclosure The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the
manuscript. Journal of Ophthalmology [21] S. Hattar, R. J. Lucas, N. Mrosovsky et al., “Melanopsin and
rod-cone photoreceptive systems account for all major acces-
sory visual functions in mice,” Nature, vol. 424, no. 6944,
pp. 76–81, 2003. [6] P. Somasundaram, G. R. Wyrick, D. C. Fernandez et al.,
“C-terminal phosphorylation regulates the kinetics of a subset
of melanopsin-mediated behaviors in mice,” Proceedings of the
National Academy of Sciences of the United States of America,
vol. 114, no. 10, pp. 2741–2746, 2017. [22] A. D. Guler, J. L. Ecker, G. S. Lall et al., “Melanopsin cells are
the principal conduits for rod-cone input to non-image-
forming vision,” Nature, vol. 453, no. 7191, pp. 102–105, 2008. [7] B. Yasin, E. Kohn, M. Peters et al., “Ectopic expression of
mouse melanopsin in drosophila photoreceptors reveals fast
response kinetics and persistent dark excitation,” The Journal
of Biological Chemistry, vol. 292, no. 9, pp. 3624–3636, 2017. [23] D. Goz, K. Studholme, D. A. Lappi, M. D. Rollag, I. Provencio,
and L. P. Morin, “Targeted destruction of photosensitive reti-
nal ganglion cells with a saporin conjugate alters the effects
of light on mouse circadian rhythms,” PLoS One, vol. 3,
no. 9, article e3153, 2008. [8] C. M. Altimus, A. D. Guler, N. M. Alam et al., “Rod photore-
ceptors drive circadian photoentrainment across a wide range
of light intensities,” Nature Neuroscience, vol. 13, no. 9,
pp. 1107–1112, 2010. [24] M. Hatori, H. Le, C. Vollmers et al., “Inducible ablation of
melanopsin-expressing retinal ganglion cells reveals their cen-
tral role in non-image forming visual responses,” PLoS One,
vol. 3, no. 6, article e2451, 2008. [9] M. T. Do, S. H. Kang, T. Xue et al., “Photon capture and signal-
ling by melanopsin retinal ganglion cells,” Nature, vol. 457,
no. 7227, pp. 281–287, 2008. [10] J. L. Ecker, O. N. Dumitrescu, K. Y. Wong et al., “Melanopsin-
expressing
retinal
ganglion-cell
photoreceptors:
cellular
diversity and role in pattern vision,” Neuron, vol. 67, no. 1,
pp. 49–60, 2010. [25] H. Z. Wang, L. QJ, N. L. Wang, H. Liu, L. Zhang, and G. L. Zhan, “Loss of melanopsin-containing retinal ganglion cells
in a rat glaucoma model,” Chinese Medical Journal, vol. 121,
no. 11, pp. 1015–1019, 2008. [26] S. K. Nayak, T. Jegla, and S. Panda, “Role of a novel photopig-
ment, melanopsin, in behavioral adaptation to light,” Cellular
and Molecular Life Sciences, vol. 64, no. 2, pp. 144–154, 2007. [11] D. Q. Zhang, K. Journal of Ophthalmology Y. Wong, P. J. Sollars, D. M. Berson, G. E. Pickard, and D. G. McMahon, “Intraretinal signaling by gan-
glion cell photoreceptors to dopaminergic amacrine neurons,”
Proceedings of the National Academy of Sciences of the United
States of America, vol. 105, no. 37, pp. 14181–14186, 2008. [27] D. L. Boudard, J. Mendoza, and D. Hicks, “Loss of photic
entrainment at low illuminances in rats with acute photore-
ceptor degeneration,” The European Journal of Neuroscience,
vol. 30, no. 8, pp. 1527–1536, 2009. [12] T. M. Schmidt, S. K. Chen, and S. Hattar, “Intrinsically
photosensitive retinal ganglion cells: many subtypes, diverse
functions,” Trends in Neurosciences, vol. 34, no. 11, pp. 572–
580, 2011. [28] C. C. Wrenn, D. A. Lappi, and R. G. Wiley, “Threshold rela-
tionship between lesion extent of the cholinergic basal fore-
brain in the rat and working memory impairment in the
radial maze,” Brain Research, vol. 847, no. 2, pp. 284–298,
1999. [13] J. Hong, Q. Zeng, H. Wang, D. S. Kuo, W. H. Baldridge, and
N. Wang,
“Controlling
the
number
of
melanopsin-
containing retinal ganglion cells by early light exposure,”
Experimental Eye Research, vol. 111, pp. 17–26, 2013. [29] R. S. Harwerth and H. A. Quigley, “Visual field defects and ret-
inal ganglion cell losses in patients with glaucoma,” Archives of
Ophthalmology, vol. 124, no. 6, pp. 853–859, 2006. [14] A. D. Guler, C. M. Altimus, J. L. Ecker, and S. Hattar, “Multiple
photoreceptors contribute to nonimage-forming visual func-
tions predominantly through melanopsin-containing retinal
ganglion cells,” Cold Spring Harbor Symposia on Quantitative
Biology, vol. 72, no. 1, pp. 509–515, 2007. [30] C. La Morgia, F. N. Ross-Cisneros, A. A. Sadun et al.,
“Melanopsin retinal ganglion cells are resistant to neurodegen-
eration in mitochondrial optic neuropathies,” Brain, vol. 133,
no. 8, pp. 2426–2438, 2010. [15] L. P. Morin and K. M. Studholme, “Separation of function for
classical and ganglion cell photoreceptors with respect to
circadian rhythm entrainment and induction of photosomno-
lence,” Neuroscience, vol. 199, pp. 213–224, 2011. [31] E. Drouyer, O. Dkhissi-Benyahya, C. Chiquet et al., “Glaucoma
alters the circadian timing system,” PLoS One, vol. 3, no. 12,
article e3931, 2008. [16] H. J. Bailes and R. J. Lucas, “Melanopsin and inner retinal
photoreception,” Cellular and Molecular Life Sciences, vol. 67,
no. 1, pp. 99–111, 2010. [32] E. S. Ingham, E. Gunhan, P. M. Fuller, and C. A. References [1] S. Hattar, H. W. Liao, M. Takao, D. M. Berson, and K. W. Yau,
“Melanopsin-containing retinal ganglion cells: architecture,
projections, and intrinsic photosensitivity,” Science, vol. 295,
no. 5557, pp. 1065–1070, 2002. [2] D. M. Berson, F. A. Dunn, and M. Takao, “Phototransduction
by retinal ganglion cells that set the circadian clock,” Science,
vol. 295, no. 5557, pp. 1070–1073, 2002. Moreover, this technique of specifically ablating mela-
nopsin cells potentially has a wide range of applications,
which may also be used in larger animals. A recent study in
rat models also proved that melanopsin-SAP could specifi-
cally deplete ipRGCs in a dose-dependent fashion [32]. In
order to obtain data more pertinent to humans, future studies
should evaluate the effectiveness and specificity of this
ipRGC immunotoxin in larger animal models where genetic
modification seems inappropriate. This will help to provide [3] J. J. Gooley, J. Lu, D. Fischer, and C. B. Saper, “A broad role for
melanopsin in nonvisual photoreception,” The Journal of
Neuroscience, vol. 23, no. 18, pp. 7093–7106, 2003. [4] N. Shirzad-Wasei and W. J. DeGrip, “Heterologous expression
of melanopsin: present, problems and prospects,” Progress in
Retinal and Eye Research, vol. 52, pp. 1–21, 2016. [5] R. N. Van Gelder and E. D. Buhr, “Melanopsin: the tale of the
tail,” Neuron, vol. 90, no. 5, pp. 909–911, 2016. Journal of Ophthalmology 8 Journal of Ophthalmology Fuller,
“Immunotoxin-induced ablation of melanopsin retinal gan-
glion cells in a non-murine mammalian model,” The Journal
of Comparative Neurology, vol. 516, no. 2, pp. 125–140, 2009. [17] G. S. Lall, V. L. Revell, H. Momiji et al., “Distinct contributions
of rod, cone, and melanopsin photoreceptors to encoding irra-
diance,” Neuron, vol. 66, no. 3, pp. 417–428, 2010. [18] R. J. Lucas, S. Hattar, M. Takao, D. M. Berson, R. G. Foster, and
K. W. Yau, “Diminished pupillary light reflex at high irradi-
ances in melanopsin-knockout mice,” Science, vol. 299,
no. 5604, pp. 245–247, 2003. [19] S. Panda, I. Provencio, T. DC et al., “Melanopsin is required for
non-image-forming photic responses in blind mice,” Science,
vol. 301, no. 5632, pp. 525–527, 2003. [20] S. Panda, T. K. Sato, A. M. Castrucci et al., “Melanopsin
(Opn4) requirement for normal light-induced circadian phase
shifting,” Science, vol. 298, no. 5601, pp. 2213–2216, 2002.
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Application of a multi-species bio-economic modelling approach to explore fishing traits within eligible cetacean conservation areas in the Northern Ionian Sea (Central Mediterranean Sea)
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Frontiers in marine science
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OPEN ACCESS OPEN ACCESS
EDITED BY
Ibon Galparsoro,
Technological Center Expert in Marine
and Food Innovation (AZTI), Spain
REVIEWED BY
Stefanos G. Kavadas,
Hellenic Centre for Marine Research
(HCMR), Greece
Satoshi Yamazaki,
University of Tasmania, Australia
*CORRESPONDENCE
Giulia Cipriano
giulia.cipriano@uniba.it
SPECIALTY SECTION
This article was submitted to
Marine Megafauna,
a section of the journal
Frontiers in Marine Science
RECEIVED 28 July 2022
ACCEPTED 27 September 2022
PUBLISHED 17 October 2022
CITATION
Carlucci R, Cipriano G, Cascione D,
Ingrosso M, Russo T, Sbrana A,
Fanizza C and Ricci P (2022)
Application of a multi-species bio-
economic modelling approach to
explore fishing traits within eligible
cetacean conservation areas in the
Northern Ionian Sea (Central
Mediterranean Sea). Front. Mar. Sci. 9:1005649. doi: 10.3389/fmars.2022.1005649 Roberto Carlucci 1,2, Giulia Cipriano 1,2*, Daniela Cascione 2,
Maurizio Ingrosso 1, Tommaso Russo 2,3, Alice Sbrana 3,
Carmelo Fanizza 4 and Pasquale Ricci 1,2 Roberto Carlucci 1,2, Giulia Cipriano 1,2*, Daniela Cascione 2,
Maurizio Ingrosso 1, Tommaso Russo 2,3, Alice Sbrana 3,
Carmelo Fanizza 4 and Pasquale Ricci 1,2 1Department of Biology, University of Bari, Bari, Italy, 2Consorzio Nazionale Interuniversitario per le
Scienze del Mare (CoNISMa), Roma, Italy, 3Department of Biology, University of Rome Tor Vergata,
Rome, Italy, 4Jonian Dolphin Conservation, Taranto, Italy Carlucci R, Cipriano G, Cascione D,
Ingrosso M, Russo T, Sbrana A,
Fanizza C and Ricci P (2022)
Application of a multi-species bio-
economic modelling approach to
explore fishing traits within eligible
cetacean conservation areas in the
Northern Ionian Sea (Central
Mediterranean Sea). Front. Mar. Sci. 9:1005649. doi: 10.3389/fmars.2022.1005649 The assessment of the spatial overlap between eligible cetacean conservation
areas (CCAs) and fishing grounds could be a strategic element in the
implementation of effective conservation measures in the pelagic offshore
areas. A multi-species bio-economic modelling approach has been applied to
estimate the fishing traits in eligible CCAs in the Northern Ionian Sea (NIS,
Central Mediterranean Sea) between 10-800 m of depth, adopting the Spatial
MAnagement of demersal Resources for Trawl fisheries model (SMART). Four
possible CCAs were defined according to the distribution of cetacean species,
their bio-ecological needs, as well as socio-economic needs of human
activities, identifying a Blue, Red, Orange and Green CCAs in the NIS. SMART
spatial domain was a grid with 500 square cells (15×15 NM). The analysis was
conducted for the period 2016-2019, considering the Otter Trawl Bottom
(OTB) fleet activities in the study areas through the Vessel Monitoring System. TYPE Original Research
PUBLISHED 17 October 2022
DOI 10.3389/fmars.2022.1005649 TYPE Original Research
PUBLISHED 17 October 2022
DOI 10.3389/fmars.2022.1005649 TYPE Original Research
PUBLISHED 17 October 2022
DOI 10.3389/fmars.2022.1005649 Application of a multi-species
bio-economic modelling
approach to explore fishing
traits within eligible cetacean
conservation areas in the
Northern Ionian Sea (Central
Mediterranean Sea) © 2022 Carlucci, Cipriano, Cascione,
Ingrosso, Russo, Sbrana, Fanizza and
Ricci. 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. Introduction conservation areas, as the restriction of fishing over large areas
could result in minimal economic losses for the sector, but with
the advantage of ensuring a more effective remedy against the
processes of extinction and loss of diversity and key ecosystem
services (Sumaila et al., 2007). In this regard, cetaceans have
proven to be of maximum importance in the stability and
resiliency of the marine ecosystems (Tromeur and Loeuille,
2017) and in the support of several ecosystem services (Pace
et al., 2015), with positive reflection even on climate change
(Sergio et al., 2008; Hooker et al., 2011; Roman et al., 2014;
Mazzoldi et al., 2019). Therefore, due to threats to and risk of
degradation in their status in the Mediterranean Sea
(ACCOBAMS, 2020) there is a very urgent need to provide
action favouring the maintenance of their critical habitat. However, the planning of cetacean conservation areas
represents a real challenge mostly because the spatial overlap
between the distribution of cetacean critical habitat and fishing
activities is wide. In fact, the feeding preferences of cetaceans and
their behavioural strategies could cause conflicts with the fishing
activities that are classified as a competition for food resources
(Bearzi, 2002; Jusufovski et al., 2019). Spatial analysis of the distribution of key species and their
interaction with human activities at sea is a key aspect of any
ecosystem-based marine spatial planning (MSP, Foley et al.,
2010). Moreover, the MSP approach emphasizes the importance
of including both direct and indirect relationships with the legal,
socio-economic and ecological complexity of governance when
assigning a marine area to a specific use, as there is often a space
of overlap between conflicting components that can and must be
buffered in advance through measures of appropriate sizing,
mitigation and compensation, ensuring greater acceptance and
above all a real effectiveness in the conservation of marine
biodiversity (Ehler and Douvere, 2009). Being able to
harmonize these aspects is crucial if Blue Growth (https://
s3platform.jrc.ec.europa.eu/blue-growth) is to be effectively
supported. For their bio-ecological traits, the effectiveness of
protection measures for cetaceans can be represented by the
establishment of conservation areas that encompass large
portions of the pelagic domain. OPEN ACCESS The spatial extension of fishing activities, hourly fishing effort (h), landings (tons)
and economic value (euros) for each CCA and the NIS were estimated as yearly
median values. Fishing activities were absent in the Blue CCA, where the
presence of the submarine canyon head does not offer accessible fishing
grounds. The hourly fishing effort in the Green area accounted for about 22%
(3443 h) of the total hourly effort of the NIS, while the Orange and Red areas
were about 8% (1226 h) and 2% (295 h), respectively. The Green CCA
corresponded to about 14% (36 tons) of the total landings in the NIS,
whereas the Orange and Red areas represented about 9% (22 tons) and 6%
(16 tons), respectively. The Green CCA accounted for about 13% (156 thousand
euros) of the total economic value of the NIS, while the Orange and Red areas
represented about 6% (69 thousand euros) and 4% (44thousand euros), © 2022 Carlucci, Cipriano, Cascione,
Ingrosso, Russo, Sbrana, Fanizza and
Ricci. This is an open-access article
distributed under the terms of the © 2022 Carlucci, Cipriano, Cascione,
Ingrosso, Russo, Sbrana, Fanizza and
Ricci. 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 Marine Science Frontiers in Marine Science 01 frontiersin.org Carlucci et al. 10.3389/fmars.2022.1005649 10.3389/fmars.2022.1005649 respectively. Results showed no or negligible negative effects on trawl
activities by potential spatial restrictions due to the establishment of CCAs
highlighting the importance to consider spatially integrated information
during the establishment process of conservation areas for cetacean
biodiversity according to the principles of Ecosystem Based Management. KEYWORDS
SMART model, fishing effort, fishing production, conservation, MPA, dolphins
and whales KEYWORDS
SMART model, fishing effort, fishing production, conservation, MPA, dolphins
and whales Introduction In addition, given that is a spatial bio-economic model,
SMART allows to estimate the economic indicators associated to
different patterns of fishing effort, at the scales of both single
vessels and fleets. In this way, SMART can be used to assess the
economic and the biological value of a given fishing area,
supporting quantitative analyses and evaluation in the
framework of marine spatial planning. In this paper, SMART
has been applied on the harbour-specific fleets of bottom
trawlers operating in the study area in order to obtain an
assessment of the potential bio-economic impacts of different
spatial management actions involving the CCAs. According to
Carlucci et al. (2021a), bottom otter trawling (OTB) represents
by far the main fishery in the Northern Ionian Sea, both in terms
of landings and profits and impacts. In the Northern Ionian Sea (NIS, Central Mediterranean
Sea), cetaceans represent key elements in the ecosystem
functioning supporting trophic regulations of the entire food
web (Ricci et al., 2020a; Carlucci et al., 2021a). Several bio-
ecological traits of cetacean species distributed in the NIS
(Carlucci et al., 2018a; Carlucci et al., 2018b; Carlucci et al.,
2020a; Carlucci et al., 2020b; Cipriano et al., 2022) have been
investigated, as well as the potential competition with local
fishing activities (Ricci et al., 2020b; Ricci et al., 2021a). This
ecological knowledge acquired in the last decade is a focal point
in the assessment of cetacean distribution and the interaction
with anthropogenic impacts in the Gulf of Taranto, the
northernmost part of the NIS (Carlucci et al., 2021b). This
information supports the possibility to propose area-based
management tools (ABMTs) for cetacean conservation
(Notarbartolo di Sciara et al., 2016), such as Cetacean
Conservation Areas (CCAs, Carlucci et al., 2021c), aimed at
protecting these species and their critical habitats, mitigating
anthropogenic impacts and promoting the sustainable
development of human maritime activities. In particular, the
NIS can be considered an eligible area for the implementation of
ABMTs and CCAs, where underwater noise, marine litter, ship
collision, and competition for prey by fishery are the main
disturbances involved in interacting with cetaceans (Carlucci
et al., 2021b). Although direct fishing impacts on the cetaceans
(e.g. by-catches) are not recorded in the area (Ricci et al., 2021a),
potential competition for food resources could arise with local
fishing activities (Carlucci et al., 2021a). Introduction However, an
investigation into the potential spatial interactions between
fishing activities and eligible CCAs has never been explored,
although long time series of data are available on species
distribution and their life history traits (Maiorano et al., 2010;
Capezzuto et al., 2010; Carlucci et al., 2018c; Ricci et al., 2021b)
and the characterization of fishing grounds (Russo et al., 2017). Therefore, the main objective of this study is to set up an
assessment of the spatial overlap between eligible CCAs and
fishing grounds in the NIS using a multi-species bio-economic
modelling approach. In particular, the assessment was
conducted in the period 2016-2019 using the Spatial
MAnagement of demersal Resources for Trawl fisheries model
(SMART, Russo et al., 2014; D’Andrea et al., 2020). SMART was
selected since it allows reconstruct, using a combination of
different data sources, the spatial and temporal origin of CCAs were described through fishing traits inherent to the
otter bottom trawl fleet, by using several indicators, such as
fishing effort, landing, economic incomes, and the landing flows
from the fishing grounds included within the CCAs towards the
main harbours of the study area. Introduction Although Marine Protected
Areas (MPAs) have been widely adopted in several marine
ecosystems (Claudet, 2011), the institution of spatial
conservation measures dedicated to the protection of the
pelagic domain on a large scale is poorly applied (Wood et al.,
2008; Game et al., 2010; Kaplan et al., 2010). On the contrary, the
protection of coastal and pelagic offshore areas seems to be a
fundamental corner for effective biodiversity conservation,
because pelagic MPAs can ensure ecological connectivity
between different coastal protected areas, such as those
distributed in gulfs and bays (Guidetti et al., 2013). In
addition, the ecological benefits derived from coastal MPAs
are also often accompanied by positive effects on the
rebuilding of fishing stocks, which have economic fallouts on
the fishery and other associated activities, such as tourism
(Stelzenmüller et al., 2007; Russo et al., 2019). Even more
relevant could be the contribution of deep-sea pelagic Under the umbrella of the European Common fisheries
policy (CFP), the management of fishery resources in the
Mediterranean Sea is largely based on the regulation of the
spatial fishing effort distribution, that is the identification of
areas in which to prohibit some or all types of fishing to protect
the environment and resources (https://oceans-and-fisheries.ec. europa.eu/policy/common-fisheries-policy-cfp_en). However,
the establishment of Fishery-Restricted Area leads to
reallocation of fishing effort (displacement from closed areas to
adjacent or new ones) that can significantly influence the final
effects of this kind of management measures, both in biological
and economic aspects (Bastardie et al., 2018; Russo et al., 2019;
D’Andrea et al., 2020). Therefore, understanding the allocation
of fishing effort displacement is an information to be taken into
account when management regulations, which are characterized Frontiers in Marine Science 02 frontiersin.org 10.3389/fmars.2022.1005649 Carlucci et al. by temporal and/or spatial banning of fishery, need to be
implemented. Several studies have adopted spatial modelling
approaches to investigate the fishing exploitation pattern in
terms of effort distribution, catches and economic production
(Russo et al., 2014; Quijano Quiñones et al., 2021), to simulate
possible spatial management scenarios of trawl fishery (Russo
et al., 2019) or to investigate the risks of interaction between
cetaceans and the fishery (Breen et al., 2017). catches or landings and their final faith in terms of landing
harbour. Study area The study area extends from Punta Alice to Santa Maria di
Leuca covering a surface of about 14000 km² and reaching 1500
m in depth in the Northern Ionian Sea (NIS) (Central
Mediterranean Sea). The hydrographic features of the area are
characterized by up-welling systems (Bakun and Agostini, 2001)
and decadal processes of deep-water circulation inversion with
effects on the energy exchanges between benthic and pelagic
domain (Ricci et al., 2022). The NIS includes several important
habitats from a conservation point of view in shallow (including
seagrass meadows and coralligenous outcrops), pelagic
(upwelling sites) and deep-sea areas (including submarine
canyon and cold-water coral banks) (Capezzuto et al., 2010;
Bo et al., 2011; D’Onghia et al., 2016; Carlucci et al., 2018c;
Castellan et al., 2019; Chimienti et al., 2019) ensuring favourable
conditions for the support of a high biological diversity and
providing diverse ecological services (Carlucci et al., 2021a). Moreover, the study area has been widely recognized as a critical
area for the day-to-day life of striped dolphin Stenella
coeruleoalba and common bottlenose Tursiops truncatus
(Carlucci et al., 2016a; Carlucci et al., 2017; Carlucci et al.,
2018b; Carlucci et al., 2018d; Ciccarese et al., 2019; Santacesaria
et al., 2019; Azzolin et al., 2020). In particular, the spatial
distribution and areas where these dolphins realize feeding,
resting, socializing, and traveling activities have been identified
(Carlucci et al., 2018b; Papale et al., 2020). In addition, other Frontiers in Marine Science 03 frontiersin.org 10.3389/fmars.2022.1005649 Carlucci et al. a wide bathymetric range; as regards the bathyal grounds, the
shrimps (Aristeus antennatus and Aristaeomorpha foliacea) are
the most important resources (Carlucci et al., 2018c; Maiorano
et al., 2010; Carlucci et al., 2016b; Russo et al., 2017). cetacean species occur in the NIS, such as the Risso’s dolphin
(Grampus griseus, Maglietta et al., 2020; Maglietta et al., 2022;
Maglietta et al., 2018; Renò et al., 2019; Carlucci et al., 2020a), the
sperm whale (Physeter macrocephalus, Bellomo et al., 2019), the
Cuvier’s beaked whale (Ziphius cavirostris, Podestà et al., 2016;
Carlucci et al., 2020b) and the fin whale (Balaenoptera physalus,
Dimatteo et al., 2011; Fanizza et al., 2014). Information and data on the cetofauna occurring in the NIS
have been available since 2009. Study area This knowledge has led to the
hypothesis of a delimitation of four possible CCAs based on
different assumptions related to the distribution of cetacean
species according to their bio-ecological needs as well as socio-
economic constraints (Figure 1). The former area, hereafter
called the “Red area”, has an extension of approximately 715
km2 and includes the persistent critical habitats of the striped
dolphin (Carlucci et al., 2018d). The second, hereafter called the
“Orange area”, encompasses approximately 1530 km2 being
enlarged to include all the areas where behavioural activities of
the striped dolphin population were observed from 2009 to 2017
(Carlucci et al., 2018d). The third, hereafter called the “Green
area”, covers approximately 3170 km2 and includes areas where
the highest abundances of both striped and common bottlenose
dolphins were estimated through habitat modelling techniques
(Carlucci et al., 2018a), together with all the sightings recorded
up to 2020 for both the Risso’s dolphin and the sperm whale. The latter CCA, hereafter called the “Blue area”, covers 615 km2
and has been delimited according to the specific spatial needs
indicated by the main stakeholders (e.g. maritime authority,
navy, municipality, NGOs, research institutions) operating in
the study area. The habitat complexity of this NIS is accompanied by several
anthropogenic pressures, which are represented by fishing
activity and marine traffic, as well as the occurrence of navy
exercises areas, and industrial activities (Carlucci et al., 2021b;
Carlucci et al., 2016a). In particular, the geo-morphological and
biological heterogeneity described so far, strongly influences the
distribution of the fishing effort, distribution and the typologies
of fishing gears adopted in the area. Fishing boats are frequently
registered as polyvalent fishing vessels, often changing type of
fishing according to the season and sea/weather conditions as
well as to the variability in the availability of resources and
market demand (Carlucci et al., 2016b). In particular, fishing
occurs from coastal waters to about 800 m in depth and it is
mainly characterized by the bottom otter trawls, that mostly
exploit the shelf break and slope, and the small-scale fishery
operating on coastal grounds (Russo et al., 2017). The most
important fishing resources are the red mullet (Mullus barbatus)
on the continental shelf, the European hake (Merluccius
merluccius), the deep-water rose shrimp (Parapenaeus
longirostris) and the Norway lobster (Nephrops norvegicus) on FIGURE 1
Map of the Northern Ionian Sea (Central Mediterranean Sea) with sightings distribution of S. coeruleoalba (SC), D. SMART Modelling approach and
fishing data the workflow of the smartR R package, can be summarized in the
following logical steps: the workflow of the smartR R package, can be summarized in the
following logical steps: 1. Analysing VMS data to assess the fishing effort by vessel/
cell/time; The assessment of fishing traits of each CCA and the
Northern Ionian Sea was carried out through the SMART
modelling approach, a method able to reconstruct the spatial
and temporal fluxes of landings coming from well-defined areas
(fishing grounds) and times to harbors to which they are
delivered for sale (Russo et al., 2018; Russo et al., 2014;
D’Andrea et al., 2020). The modelling of spatial fishing effort
is based on the use of information obtained by the Vessel
Monitoring System (VMS), which is applied to the remote
control of fishing vessels with length overall (LOA) ≥15 m in
European waters (EC, 2011). The VMS data are combined with
information on landings acquired from fishing logbooks, where
information on landing by species and harbors are reported by
the fishers (Gerritsen and Lordan, 2011). All these data are
collected within the Data Collection Framework since 2006 and
they are provided by the Italian “Ministry of the Agricultural,
Alimentary and Forestry Politics” (Russo et al., 2014). Starting
from this information on the spatial effort and landings, it is
possible to estimate the Landing Per Unit of Effort (LPUE, kg h-1
km-2) for each vessel length (D’Andrea et al., 2020). In addition,
a reconstruction of the effort and production data for vessels
with a LOA < 15 m was carried out using the European
Common Fleet Register (EC, 2010) according to the method
reported in Russo et al. (2018). 2. Processing landings data, combined with VMS data, to
estimate the spatial/temporal productivity of each cell (or spatial
unit), in terms of mean monthly LPUE by species, according to
the method described and applied in Russo et al. (2018); 3. Estimating the cost per vessel/time associated with a given
effort pattern and the related revenues, as a function of the
landings by vessel/species/length class/time; 4. Combining costs and revenues by vessel, on the yearly
scale, to obtain the profit, which is the proxy of the
vessel performance. Each of these steps corresponds to a different module of the
smartR package (D’Andrea et al., 2020). A detailed description
of the SMART workflow is reported in Russo et al. (2019) and
D’Andrea et al. Study area delphis (DD), G. griseus (GG),
T. truncatus (TT), P. macrocephalus (PM) and the spatial limits of CCAs (the black line indicates the border of the gulf). FIGURE 1 FIGURE 1
Map of the Northern Ionian Sea (Central Mediterranean Sea) with sightings distribution of S. coeruleoalba (SC), D. delphis (DD), G. gr
T. truncatus (TT), P. macrocephalus (PM) and the spatial limits of CCAs (the black line indicates the border of the gulf). 04 04 Frontiers in Marine Science frontiersin.org 10.3389/fmars.2022.1005649 Carlucci et al. Carlucci et al. Assessment of the fishing activities and
production within CCAs and in the
Northern Ionian Sea eastern zones, respectively (Rossi and Gabbianelli, 1978). This
choice is due to the difference in bottom trawling fleets in the
two areas in terms of capacity and effort (Maiorano et al., 2022;
Maiorano et al., 2010; Russo et al., 2017), as well as to the
structure of the demersal assemblage (Carlucci et al., 2018c) and
the food webs (Ricci et al., 2019). eastern zones, respectively (Rossi and Gabbianelli, 1978). This
choice is due to the difference in bottom trawling fleets in the
two areas in terms of capacity and effort (Maiorano et al., 2022;
Maiorano et al., 2010; Russo et al., 2017), as well as to the
structure of the demersal assemblage (Carlucci et al., 2018c) and
the food webs (Ricci et al., 2019). The assessment of the OTB fleet fishing exploitation in the
CCAs and the NIS was carried out considering the spatial
distribution of fishing activities, the hourly fishing effort, and
the production in terms of landing and economic revenue
during the period 2016-2019. The spatial extension of fishing
activities (expressed as km2) was calculated as the median of
annual values estimated through VMS data for all OTB LOA
segments. OTB swept areas by LOA segment were estimated in
each year in the range of depth between 10-800 m. In addition,
annual trends of the spatial coverage (%) of each VL segment
were analysed. Therefore, median, minimum, and maximum, interquartile
range (IR) and the percentage values (% calculated on the
median value) of each production indicator for CCAs and the
NIS were analysed. A statistical comparison of the fishing and
production indicators (hourly effort, landing and economic
values) among all CCAs and the NIS was carried out using the
multiple non-parametric Mann–Whitney (U) post hoc test,
based on the Bonferroni correction (McDonald, 2014). The
selection of the KW test was due to the non-normal
distribution of the data tested by the Shapiro–Wilk test
(Shapiro and Wilk, 1965) (Table S1). The statistical analysis
was carried out using PAST 4.03 (Hammer et al., 2001). The hourly fishing effort and the yield (landings) were
estimated using data provided by the SMART model
considering available OTB LOA segments during the
investigated period. SMART Modelling approach and
fishing data (2020), while a diagram of the approach used in
this paper is represented in Figure 2. The analysis was conducted for the period 2016-2019 (48
months), considering the fleets of vessel performing bottom
otter trawling (OTB) in the Northern Ionian Sea and belonging
to three fleet segments defined by vessel length-over-all (namely
LOA <12, LOA between 12-18 and LOA between18-24 m),
being the main segments operating in the study areas (Maiorano
et al., 2019; Maiorano et al., 2010; Russo et al., 2017). To avoid
anomalies in the fishing production induced by fishing effort
variations at the national scale which occurred during the SARS-
CoV-2 coronavirus pandemic period (Russo et al., 2022) the
years 2020-2021 were excluded from the analysis. The spatial domain of the SMART model for the
investigated area was defined as a grid with 500 square cells
(15 × 15 nautical miles). The rationale of the model, as well as FIGURE 2
Diagram of the workflow, from input data to final output, applied in this study to assess the value of CCAs for the trawl fisheries operating in the
area of study. Diagram of the workflow, from input data to final output, applied in this study to assess the value of CCAs for the trawl fisheries operating in the
area of study. 05 Frontiers in Marine Science frontiersin.org Carlucci et al. 10.3389/fmars.2022.1005649 TABLE 1
Mean sales prices for target species indicated by FAO
3alpha code considered in the analysis. TABLE 1
Mean sales prices for target species indicated by FAO
3alpha code considered in the analysis. The combination of the different data sources (i.e. VMS data,
Logbooks data, Common Fleet register and Price at market by
species) allowed to estimate the Fishing effort and the related
landings and revenues for the different CCAs. In addition, being
known the harbour of departure/landing of each trawler, it was
possible to estimate fishing effort, landings and revenues by
harbour-specific fleets. In this way, the results of this modelling
approaches represent estimates of the real values but
simulations. Moreover, considering that the displacement of
effort potentially determined by the CCAs was not predicted,
the results of this study represent an assessment of the status quo
(i.e. the present values of CCAs for trawl fishing). SMART Modelling approach and
fishing data Species
FAO Code
Price (€/kg)
Aristeus antennatus
ARA
18.5
Aristaeomorpha foliacea
ARS
14.0
Boops boops
BOG
0.5
Parapenaeus longirostris
DPS
3.75
Eledone cirrhosa
EOI
4.0
Merluccius merluccius
HKE
6.0
Trachurus trachurus
HOM
1.0
Lophius piscatorius
MON
7.0
Mullus surmuletus
MUR
12.0
Mullus barbatus
MUT
4.0
Nephrops norvegicus
NEP
20.0
Illex coindetii
SQM
4.0 Assessment of the fishing activities and
production within CCAs and in the
Northern Ionian Sea Therefore, monthly landings were
combined with VMS data (using the fishing vessel and
temporal range of the fishing activity as references) to estimate
the monthly LPUE for each species and cell in the grid (see
Russo et al., 2018, for an extensive description of this procedure). The LPUEs obtained were aggregated by the target species of the
trawling (Table 1). In addition, data provided by the SMART
model were used to calculate several indicators, such as the
hourly effort (hours) by OTB LOA segment, the landing, the
spatial LPUE and the economic value for each CCA and the NIS. Economic incomes of fishing landing were calculated by
multiplying the landing value (kg) of main target species of
trawl segment by their price (expressed as mean value in euros
kg-1 per species) (Table 1). Frontiers in Marine Science frontiersin.org Spatial and temporal interactions of
fishing activities within CCAs Also in this area, percentage values decreased over
time with the lowest value in 2018 (36.5%) (Figure S1). In the
NIS, the spatial extension of the OTB fleet showed a median
value of 3469 km2 (IR=522; 36.0% of the total NIS area)
(Figure 3). The OTB VL 18-24 segment showed the highest
percentage values in 2016, with values of 44.1%, while the lowest
was detected in 2019 (28.5%) (Figure S1). In addition, OTB VL
12-18 vessel showed a spatial extension lower than
approximately 36% in all years. Considering the hourly effort by VL segments, fishing
activities in the Red CCA were almost exclusively performed
by OTB 18-24 vessels (median value of 295 hours, IR=197;
91.4% of the total hourly effort) (Figure 6B), Differently, other
VL segments were absent, as VL <12, or characterized by very
negligible activities (less than 30 fishing hours estimated for VL
12-18). Similarly, the hourly effort in the Orange CCA showed
the highest median value of 1102 hours (IR=865) for the OTB
18-24 segment, representing 52.7% of the total hourly effort in
the CCA (Figure 6C). Lower median values were detected for
other segments, where OTB<12 and OTB 12-18 vessels
accounted for 18.2% (median value of 380 hours; IR=36) and
29.1% (median value of 609 hours; IR=725), respectively. In the
Green CCA, the division offishing effort by VL segments showed
the highest median value for OTB VL 18-24 (2499 hours;
IR=3205) accounting for 56.5% of the total fishing hourly
effort of the CCA (Figure 6D). In the NIS, the OTB VL<12
vessels showed the highest median value of hourly effort (7535
hours, IR=3269) representing 49.0% of the total effort, followed
by OTB 12-18 vessels (median value 4900 hours, IR=3761,
31.9%) and the OTB18-24 fleet (median value 2938,
IR=5642, 19.1%). In the NIS, the mean total yearly effort of the whole trawl
fleet showed a median value of 15925 hours (IR= 5174)
(Figure 6A, Table S3). Considering the CC areas, the highest
was estimated for the Green area (median value of 3443 hours,
IR=3070), followed by the Orange area (median value of 1226
hours, IR=1385) and the Red area (median value of 295 hours;
IR=199), which were significantly different between them
(p<0.001; Table S4). In addition, the fishing effort in the Green
area accounted for 21.6% of the total hourly effort of the NIS,
and the Orange and Red areas were 7.7% and 1.9%, respectively. Spatial and temporal interactions of
fishing activities within CCAs The aggregated fishing footprint distributed in potential
fishable areas between 10-800 m of depth is reported, together
with the different CCAs of interest, in Figures 3–5 and Supp. Materials (Table S2; Figure S1). Further analysis involved the estimation of production flows
from the study areas to the main fishing harbours (Crotone,
Cariati, Corigliano, Taranto, Gallipoli, Otranto) of the NIS,
according to the method reported in Russo et al. (2018). Fishing harbours were aggregated at regional level (Calabrian
and Apulian), which are precisely divided by the Taranto valley
in the Gulf of Taranto, occupying the southwestern and north- In the Blue CCA, fishing activity was detected for the OTB
VL 15-18 only in 2018, with an absolute extent of 4 km2
(Figure 3). This indicated that the fishing activities are
substantially absent in this area. In the Red CCA, the median
spatial extent of the entire OTB fleet showed a value of 8 km2
(IR=8), representing a percentage of spatial extension of 0.9% Frontiers in Marine Science 06 frontiersin.org frontiersin.org Carlucci et al. 10.3389/fmars.2022.1005649 FIGURE 3
The total yearly spatial extension (km2 in Log scale) of the OTB fleet within CCAs and the NIS during the investigated period (2016-2019). Boxplots report the median (midline), quartiles (box limits); minimum and maximum values (whiskers out of boxes). FIGURE 3
The total yearly spatial extension (km2 in Log scale) of the OTB fleet within CCAs and the NIS during the investigated period (2016-2019). Boxplots report the median (midline), quartiles (box limits); minimum and maximum values (whiskers out of boxes). with respect to the whole potential fishable area (Figure 3). In
addition, the maximum percentage value of spatial extension
was estimated for the OTB VL_18-24 in 2016 (32 km2, 3.7%),
while other values in the remaining years were lower than 1.5%
(Figure S1). In the Orange CCA, the spatial extension of the OTB
fleet showed a median value of 237 km2 (IR=380; 17.0% of the
Orange area total) (Figure 3). The highest spatial extension was
estimated for the OTB VL 18-24 with a percentage value of
64.1% in 2016, while the lowest was estimated in 2018 (26.8%)
(Figure S1). In the Green area, the median spatial extension was
of 704 km2 (IR=992; 26.5% of the Green CCA total) and the
highest value was observed in 2016 (82.9%) for the OTB VL 18-
24 (Figure 3). Fishing production in the Northern
Ionian Sea and CCAs In the NIS, the median estimated production as landings and
landing values corresponded to 254.8 tons (IR=124.6) and 1.253
mln euro (IR=580.1), respectively (Table S3). In the CCAs, the
highest median value of landings was estimated for the Green Frontiers in Marine Science 07 frontiersin.org Carlucci et al. 10.3389/fmars.2022.1005649 A
B
D
C
FIGURE 4
Spatial distribution of the OTB effort (in hours) by VL 12-18 showing the overlap with the (A) Blue, (B) Red, (C) Orange and (D) Green CCAs. Values in hours are calculated as yearly averages for the period 2016-2019. A B C D C D FIGURE 4
Spatial distribution of the OTB effort (in hours) by VL 12-18 showing the overlap with the (A) Blue, (B) Red, (C) Orange and (D) Green CCAs. Values in hours are calculated as yearly averages for the period 2016-2019. (32.4 tons, IR=14.37, 13%), M. barbatus (29.8 tons, IR=16.87,
11.9%), I. coindettii (26.2 tons, IR=20.18, 10.5%) and P. longirostris (25.9 tons, IR=12.34, 10.3%) (Figure 7 left plots;
Table S5). In term of economic yield, A. antennatus was the most
important species, with the median revenue value of 254.7
thousand euros (IR=132.47, 20.7%), followed by M. merluccius
(151.0 tons, IR=67.06, 12.3%) (Figure 7 right plots). In addition,
N. norvegicus, M. barbatus and I. coindetii each account for
between 8 and 10% of the total economic value of the NIS. area (36.1 tons, IR=26), followed by the Orange area (22.4 tons,
IR=13.7) and the Red area (16.1 tons, IR=11.1). Thus, the Green
area corresponded to 14.2% of the total landings in the NIS, and
the Orange and Red areas represented 8.8% and 6.3%,
respectively. Considering the economic revenue, median values
estimated for the CCA were 156.3 thousand euros (IR=107.9) for
the Green area, 69.2 thousand euros (IR=45.2) for the Orange
area and 43.9 thousand euros (IR=25.4) for the Red one. Thus,
the Green CCA accounts for 12.5% of the total economic value
of the NIS, and the Orange and Red areas represented 5.5% and
3.5%, respectively. All median values calculated for the
production indicators were significantly different between the
CCAs and NIS (p<0.01) (Table S4). The production pattern showed some differences in the
CCAs compared to the NIS. In particular, the Red CCA
showed the lowest number of species in the landing. P. Frontiers in Marine Science Fishing production in the Northern
Ionian Sea and CCAs longirostris was the most important landed and economic
species, with median values of 7.2 tons (IR=4; 47.7% of the
total landing in the Red CCA) and 26.8 thousand euros
(IR=15.04; 65.5% of the total economic yield of Red CCA). Other relevant species in the landing were T. mediterraneus (4.0 Considering the composition of landings in the NIS, the
most landed species were B. boops with a median value of 41.2
tons (IR=19.2; 16.5% of the total landing in the NIS), followed by
T. mediterraneus (33.6 tons, IR=22.53, 13.4%), M. merluccius Frontiers in Marine Science frontiersin.org 08 Carlucci et al. 10.3389/fmars.2022.1005649 A
B
D
C
FIGURE 5
Spatial distribution of the OTB effort (in hours) by VL 18-24 showing the overlap with the (A) Blue, (B) Red, (C) Orange and (D) Green CCAs. Values in hours are calculated as yearly averages for the period 2016-2019. A B C D C D FIGURE 5
Spatial distribution of the OTB effort (in hours) by VL 18-24 showing the overlap with the (A) Blue, (B) Red, (C) Orange and (D) Green CCAs. Values in hours are calculated as yearly averages for the period 2016-2019. tons; 26.5%) and B. boops (2.1 tons, 14.1%), but they were
characterized by a very low economic value. CCA (median value 9.19; IR=5.93) and for 22% of the
economic value in the CCA (median value of 34.47 thousand
euros, IR=22.25). In addition, A. antennatus and A. foliacea
showed high median revenue values equal to 25.3 thousand
euros (IR=24.30, 16.3%) and 23.0 thousand euros (IR=19.26;
14.8%), respectively. In the Orange and Green CCAs, the most important species
in the landings were always P. longirostris, T. mediterraneus and
B. boops, but an increase in the number of landed species was
observed. Deep resources (A. foliacea, A. antennatus and N. norvegicus) were found, as well as commercial cephalopods (I. coindettii and E. cirrhosa). In the Orange CCA, P. longirostris
showed the highest median values of landing (7.93 tons, IR=5.28;
36.1% of the total landing in the CCA) and economic yield (29.7
thousand euros, IR=19.8, 37.4% of the total economic value in
the Orange CCA). In addition, A. foliacea and A. antennatus
accounted for 12.4% and 11.2% of the total economic value in
the Orange CCA (median values of 9.87 thousand euros, IR=5.2
and 8.92 thousand euros, IR=10.4, respectively). In the Green
CCA, P. Fishing production in the Northern
Ionian Sea and CCAs longirostris showed the highest median production
values account for 25.8% of the landing in the investigated Landing and economic flows from CC
areas towards fishing harbours The analysis of landing flows by species towards the main
fishing harbours showed differences in the pattern of production
among the CCAs and the NIS (Figures 8, 9; Table S6). Considering landing flows in the NIS, most of the production
was landed in the Apulian region, with the highest median total
landing of 108.93 tons in Gallipoli (43.4% of the total production Frontiers in Marine Science 09 frontiersin.org frontiersin.org Carlucci et al. 10.3389/fmars.2022.1005649 A
B
D
C
FIGURE 6
The total yearly fishing effort (hours in Log scale) of the OTB fleet estimated by SMART model. In (A) the hourly effort is reported for the overall
OTB fleet in the CCAs and Northern Ionian Sea. The hourly effort by LOA segments is reported in (B) for the Red CCA, in (C) for the Orange
CCA and in (D) for the Green CCA. LOA segments are split into vessels lower than 12 m (<12), vessels between 12-18 m (12_18) and those
between 18-24 m (18_24). Boxplots report the median (midline), quartiles (box limits); minimum and maximum values (whiskers out of boxes). A B C D D FIGURE 6
The total yearly fishing effort (hours in Log scale) of the OTB fleet estimated by SMART model. In (A) the hourly effort is reported for the overall
OTB fleet in the CCAs and Northern Ionian Sea. The hourly effort by LOA segments is reported in (B) for the Red CCA, in (C) for the Orange
CCA and in (D) for the Green CCA. LOA segments are split into vessels lower than 12 m (<12), vessels between 12-18 m (12_18) and those
between 18-24 m (18_24). Boxplots report the median (midline), quartiles (box limits); minimum and maximum values (whiskers out of boxes). of the NIS), followed by that of Taranto (median total value
equal to 43.91 tons; 17.5%), and a very small fraction in Otranto
(median total value equal to 6.29 tons; 2.5%) (Figure 9D). In the
Calabria region, the landings flows were directed towards
Corigliano (median total value equal to 65.82 tons; 26.2%) and
Crotone (median total value equal to 26.18 tons; 10.4%). The
main landed species in Gallipoli were B. boops (median value of
20.87 tons, IR=11.86; 8.3%), M. merluccius (median value of
16.22 tons, IR=9.17; 6.5%), M. barbatus (median value of 14.33
tons, IR=6.84; 5.7%) and I. coindetii and T. mediterraneus, each
accounting for about 4-5% (Figure 8). Frontiers in Marine Science Landing and economic flows from CC
areas towards fishing harbours A similar pattern was
observed for the landing species composition in the remaining
harbours with lower percentage values. Economic yields showed
similar percentage values to the landing flows, with the highest
total median value for the landing in Gallipoli (516.35 thousand
euros, 41.6% of the total economic production from the NIS). Considering the species, A. antennatus showed the highest
economic values in all harbours, excepted for Taranto, where
A. foliacea showed the highest median value equal to 49.75
thousand euros (18.95). Other relevant species were M. merluccius, N. norvegicus, M. barbatus and I. coidettii in
all harbours. species landed in Taranto from the Red CCA. In Corigliano
harbour, T. mediterraneus showed the highest fraction in the
landings (median value 3.99 tons, IR= 6.04; 25.6%), followed by
P longirostris (median value of 3.03 tons, IR=2.33; 19.5%) and B. boops (median value 1.63 tons, IR=1.35; 10.5%). Other species
landed in Corigliano were M. merluccius, Lophius spp. and
M. barbatus. Concerning the economic yield, P. longirostris was also the
most important species in both harbours, showing median
values of 16.06 thousand euros (IR=12.73; 38.5% of the total
economic value) in Taranto and 11.36 thousand euros (IR=8.72;
27%) in Corigliano, respectively. Overall, the median total landing from the Red CCA was
higher within Corigliano harbour (66.9%) than Taranto harbour
(33.1%) (Figure 9A). Similarly, the economic production
accounted for 60.5% (25.23 thousand euros) in the
Corigliano harbour and 39.5% (16.50 thousand euros) in
Taranto, respectively. In the Orange area, the most important species in the
landing flows towards Corigliano harbour were T. mediterraneus (median value of 4.65 tons; IR=6.22; 19.8% of
the total production of the area), P. longirostris (median value of
3.13 tons; IR=2.53; 13.3%) and B. boops (median value of 1.84
tons; IR=1.72; 7.8%). In addition, small amounts of A. foliacea
(median value of 0.71 tons; IR=0.37; 3.0%) and A. antennatus
(median value of 0.26 tons; IR=0.35; 1.1%) were detected in the In the Red CCA, P longirostris showed the highest fraction in
the Taranto landings (median value 4.28 tons; IR=3.39)
accounted for 27.5% of the total landing flow from this CCA
(Figure 8). This species together with B. boops represented all Frontiers in Marine Science frontiersin.org 10 Carlucci et al. 10.3389/fmars.2022.1005649 FIGURE 7
Landings (tons) and economic value (mln euro) by commercial species estimated for each CCA and the Northern Ionian Sea (NIS). Code species
are reported in the Table 1. Landing and economic flows from CC
areas towards fishing harbours Boxplots report the median (midline), quartiles (box limits); minimum and maximum values (whiskers out of boxes). FIGURE 7
Landings (tons) and economic value (mln euro) by commercial species estimated for each CCA and the Northern Ionian Sea (NIS). Code species
are reported in the Table 1. Boxplots report the median (midline), quartiles (box limits); minimum and maximum values (whiskers out of boxes). represented 11.5% (9.87 thousand euros) in Corigliano. Overall,
the median total landing from the Orange CCA was split into
13.66 tons in Corigliano (58.2% of the total landing from the
CCA) and 9.80 tons in Taranto (41.8%), respectively (Figure 9B). Median total revenues were slightly higher in the Corigliano
(56.7%) than in Taranto (43.3%). landings (Figure 8). In Taranto harbour, the main landed species
were always P. longirostris (median value of 4.70 tons, IR=3.44;
20.1%) and B. boops (median value of 2.45 tons, IR=2.52; 10.5%). Other relevant species were M. barbatus, M. merluccius and I. coindetii, and A. antennatus was detected in the landing. Concerning the economic revenue, P. longirostris, A. foliacea
and A. antennatus were the main important species in the
production flows of both harbours. The former species
accounted for 20.5% (17.63 thousand euros) in Taranto and
for 13.7% (11.74 thousand euros) in Corigliano harbour,
respectively, as well as A. antennatus being equal to 8.4% (7.20
thousand euros) for the Taranto production and 5.6% (4.84
thousand euros) in that of the Corigliano. Finally, A foliacea The landings flow from the Green area was mainly directed
to the Corigliano harbour (49.5% of the Green CCA total
landing) and to Taranto (43.7%), while the lowest fraction was
landed in Gallipoli (6.8%) (Figure 9C). In all harbours, P. longirostris and B. boops were the main landed species
(Figure 8). In addition, T. mediterraneus showed a high
median value exclusively in Corigliano (4.65 tons, IR=5.78; Frontiers in Marine Science 11 frontiersin.org Carlucci et al. 10.3389/fmars.2022.1005649 FIGURE 8
Landing flows by species (expressed in %) from the CCAs and NIS towards the main fishing harbours. Species codes are reported in Table 1. FIGURE 8
Landing flows by species (expressed in %) from the CCAs and NIS towards the main fishing harbours. Species codes are reported in Table 1. frontiersin.org Discussion 11.3% of the total landing production). Other relevant species
were M. merluccius, M. barbatus and A. foliacea with values
which ranged between 3-4% in both Corigliano and Taranto
harbours. Considering the economic yield, as observed in the
Orange CCA, P. longirostris, A. foliacea and A. antennatus were
the most important species in all harbours, representing overall
about 54% of the total economic revenue (24.2% in Corigliano
harbour, 25.8% in Taranto and 3.9% in Gallipoli). Other relevant
species were M. merluccius and Lophius spp. in the production
Corigliano harbour, with values of 4.0% and 4.5%, respectively. Overall, the median total economic yield from the Green CCA
was slightly higher in Corigliano harbour (92.65 thousand euros,
47.7%), than in Taranto (86.60 thousand euros 44.6%), while
Gallipoli accounted for 7.8% (15.10 thousand euros). 11.3% of the total landing production). Other relevant species
were M. merluccius, M. barbatus and A. foliacea with values
which ranged between 3-4% in both Corigliano and Taranto
harbours. Considering the economic yield, as observed in the
Orange CCA, P. longirostris, A. foliacea and A. antennatus were
the most important species in all harbours, representing overall
about 54% of the total economic revenue (24.2% in Corigliano
harbour, 25.8% in Taranto and 3.9% in Gallipoli). Other relevant
species were M. merluccius and Lophius spp. in the production
Corigliano harbour, with values of 4.0% and 4.5%, respectively. Overall, the median total economic yield from the Green CCA
was slightly higher in Corigliano harbour (92.65 thousand euros,
47.7%), than in Taranto (86.60 thousand euros 44.6%), while
Gallipoli accounted for 7.8% (15.10 thousand euros). The analysis developed in this study through a multi-species
bio-economic modelling approach is the first attempt to quantify
fishing exploitation patterns within eligible CCAs identified with
the explicit purpose of protecting cetacean species in the
Northern Ionian Sea. The SMART approach was used to
obtain a quantitative reconstruction of fishing activities in the
study area and to provide a baseline for the planning of spatial
conservation measures, as well as for sustainable management of
fishery. Indeed, the output obtained could be considered in the
application of measures required for a sustainable management
of the trawl fishery, as required by the Multi-annual Plan for the
Fisheries exploiting demersal stocks (Sánchez Lizaso et al., 2020). Frontiers in Marine Science frontiersin.org frontiersin.org 12 Carlucci et al. Discussion 10.3389/fmars.2022.1005649 FIGURE 9
Landings flows (tons) from (A) the Red CCA, (B) Orange CCA, (C) Green CCA and (D) the Northern Ionian Sea towards the main fishing harbours
aggregated in the Calabrian (yellow) and Apulian region (violet). Thickness of arrows is proportional to the magnitude of flows. FIGURE 9
Landings flows (tons) from (A) the Red CCA, (B) Orange CCA, (C) Green CCA and (D) the Northern Ionian Sea towards the main fishing harbours
aggregated in the Calabrian (yellow) and Apulian region (violet). Thickness of arrows is proportional to the magnitude of flows. In fact, effective management aimed at reducing discards and
mortality of both target and non-target species require
integrated strategies based on several kinds of regulation
(Colloca et al., 2013). Although the main Annual Multiplan
Fishery regulations are based on effort reduction in terms of
fishing days, other restrictions could be planned to adopt spatial
conservation tools able to synthetize multiple targets in the
conservation of marine biodiversity and ecosystems (Pérez-
Ruzafa et al., 2017; Russo et al., 2019). in the offshore area of the NIS (Carlucci et al., 2020b). However,
the level of conservation represented by the Blue CCA could be
very beneficial for the protection of cetaceans and easily
implemented in the area given the involvement of the main
stakeholders. From an operational perspective, an increase in
knowledge regarding other human pressures impacting in the
CCA, such as naval traffic, should be acquired. Indeed, this
pressure is an important source of impact for cetaceans, such as
the accidental strikes (Pennino et al., 2017), which could be
regulated through the adoption of specific spatial measures on
the routes and speeds of the naval traffic (Guzman et al., 2020). The first main relevant output is the absence of fishing
activities in the smallest CCA (blue) where the head of the
submarine canyon does not offer accessible grounds. Thus, the
area can be an interesting space for the planning of spatial
conservation actions, without conflicts with the local fishery. In
addition, the proximity of the Blue CCA to the coast should be
evaluated from the socio-economic perspective, stimulating the
involvement of different stakeholders for the planning of
regulatory measures (Heck et al., 2011). Discussion A
further noteworthy point is that the Red CCA, which defines a
conservation level aimed at protecting the persistent critical
habitats of the striped dolphin (Carlucci et al., 2018d), is
partially overlapping with the blue area, covering the totally
canyon head. Thus, the establishment of this CCA does not seem
to particularly interfere with local fishing activities and the
potential economic losses due to the banning of the area are
very scarce. At same time, this CCA represents an efficient
conservation level for the life cycle of the striped dolphin, as well
as for deep-sea habitats requiring protection actions (Manea
et al., 2020). Considering the importance of deep-sea habitats,
future studies should investigate the occurrence of other
impacts on the area, providing specific regulations of the
human activities. et al. (2007), should be considered within an overall costs and
benefits assessment addressed to planning effective spatial
measures to conserve the cetaceans and biodiversity. Indeed,
in a scenario of low losses for the fishing industry, other incomes
could be acquired by other ecosystem services, such as those
performed by small cetaceans (Kiszka et al., 2022), compensating
and improving the ecological conditions and the sustainability of
the socio-economic systems linked to the marine resources
(Hammershalg et al., 2019). et al. (2007), should be considered within an overall costs and
benefits assessment addressed to planning effective spatial
measures to conserve the cetaceans and biodiversity. Indeed,
in a scenario of low losses for the fishing industry, other incomes
could be acquired by other ecosystem services, such as those
performed by small cetaceans (Kiszka et al., 2022), compensating
and improving the ecological conditions and the sustainability of
the socio-economic systems linked to the marine resources
(Hammershalg et al., 2019). The need to establish conservation areas for cetaceans is a
fundamental goal of several international protocols, such as
Important Marine Mammal Areas (IMMAs, Notarbartolo di
Sciara et al., 2016; Hoyt and Notarbartolo di Sciara, 2021) or the
Cetaceans Critical Habitats (CCHs, Notarbartolo di Sciara,
2002). These protocols required several selection criteria of the
eligible areas. For instance, IMMA selection criteria are focused
on the distribution and abundance of cetacean populations, the
key life cycle activities occurring in the considered areas, as well
as the assessment of vulnerability of the resident cetacean
populations (Tetley et al., 2022). Discussion Similarly, CCHs require
information on the fishery interactions with the cetaceans to
identify suitable habitats for these organisms (ACCOBAMS-
ECS-WK Threats, 2017; IUCN Marine Mammal Protected
Areas Task Force, 2018). In addition, other international
initiatives in the Ionian basin, such as the EU Strategy for the
Adriatic-Ionian Region (EUSAIR, 2014), includes among its
objectives the implementation of MPAs, with particular
attention to the identification of areas to create new MPAs or
areas requiring special measures for the conservation of
biodiversity, as well as the proposal of complementary
measures for sustainable fishing in the conservation areas of
the Adriatic-Ionian ecoregion (EUSAIR, 2021). These proposals
as a whole should be driven by the application of quantitative
methodologies useful to provide both information on the
ecological consequences of the establishment of CCAs and the
socio-economic effects linked to conservation areas. The
quantification of these aspects is important because
conservation plans often conflict with fishing activities and
other uses of the sea (Grip and Blomqvist, 2020). This is
particularly true in such complex exploited systems as the Gulf
of Taranto, with multiple human use of the maritime space,
relevant sensitive habitats, and high biodiversity at all ecosystem
levels (Carlucci et al., 2021b). No less relevant, urgent planning
of spatial conservation measures is required in the Adriatic-
Ionian region because it is one of the least-protected areas in the
Mediterranean Sea (EUSAIR, 2021). Despite all these factors, the
analysis shows no or very negligible negative effects on trawling
due to potential spatial restrictions on the establishment of
CCAs, especially within the Blue and Red area delimitations. At the same time, the ecological benefits for cetofauna diversity
provided by a more extensive protection level, such as that of the
Orange and Green CCAs, could be accompanied by effects on
demersal stock repopulation, reduction of fishing discards, as The Green and Orange CCAs differ from the Red one by a
higher intensity of fishing activities and in the landing species
composition, especially the occurrence of deep commercial
species in the catches, which are target species of the Northern
Ionian Sea (Maiorano et al., 2022). However, P. longirostris is
always the main species in terms of landing amount and sale in
both CCAs, and only in the Green area do deep-water shrimps
seem to achieve an economic yield similar to that of the deep-
water rose shrimp. Discussion Indeed, the growing
interest in citizen science activities can find opportunities to
develop ocean literacy activities in suitable locations such as
marine protected areas and to promote Sustainable
Development Goals in coastal communities (Ferreira et al.,
2021). However, a critical issue could be represented by the
small size of this CCA, which partially covers the head of
canyons and could not provide and exhaustive protection of
some cetacean species, such as Z. cavirostris, which is distributed Considering the eligible CCAs, the results highlighted a
growing fishing exploitation pattern moving from the Red to
the Green CCA. This increase in fishing effort and production is
expected, because the spatial dimensions of CCAs grow from red
to green area, with the consequence of including additional
fishing grounds. Furthermore, small changes in the species
composition of the landing in each CCA were observed,
showing the absence of the exploitation of deep commercial
resources in the red CCA. Indeed, its landings composition is
exclusively characterized by the shallowest species, such as P. longirostris, T. mediterraneus and B. boops. Though not
negligible, the last two species are characterized by a low
economic yield, and a high discard rate from trawl catches
(Maiorano et al., 2019), and at the same time, they are prey of
T. truncatus (Bearzi et al., 2010; Ricci et al., 2020a). Moreover, Frontiers in Marine Science 13 frontiersin.org Carlucci et al. 10.3389/fmars.2022.1005649 the fishing pressure in this CCA showed the lowest level of
spatial coverage and hourly fishing effort, in line with the
knowledge on the fishing effort displacement in the Northern
Ionian Sea. Here, some fishing grounds are in the south-western
zone between Taranto and the Calabrian area and in the south-
eastern zone off Gallipoli (Russo et al., 2017). The western border
of the CCA is located at the end of the former fishing ground,
where the trawl vessels stop their hauls at the head of the canyon
slope. This condition forces the trawl vessels towards the shelf
platform up to 200 m in depth. Thus, potential fishing
interactions in the Red CCA could interest mainly the
common bottlenose dolphin, since the species is distributed in
shallower areas (Carlucci et al., 2018a; Carlucci et al., 2016a)
showing a trophic overlap with commercial species caught by
several fishing gears (Ricci et al., 2020a; Carlucci et al., 2021a). frontiersin.org Data availability statement The quantification of both fishing pressures and production
in terms of economic value from CCAs is an important strength. Indeed, the identification of the level of fishing pressure in space
and time, the amounts of landings and their species
composition, could provide insight into the intensity of fishing
disturbances to the cetaceans, due to competition for food
resources (Kaschner and Pauly, 2005). Furthermore, such
knowledge may provide data required in the processes of
assessing the conservation status of cetaceans and their
habitats (ACCOBAMS ECS‐WK Threats, 2017; Breen et al.,
2017), as well as in the use of indicators that classify the
environmental state of the marine ecosystem through cetacean
biodiversity (Azzellino et al., 2014). On the other hand, the
quantification of economic value represents a way of assessing
the ecosystem service represented by the fishing resources
production (Holmlund and Hammer, 1999; Pope et al., 2016). Such information could make it possible to manage and mitigate
possible conflicts between the need for biodiversity conservation
and fishing exploitation, especially in the case of fishing
restrictions. In this regard, the results obtained from the
SMART model allow for a better understanding of the
dynamics of trawling activity in areas important for cetaceans
living in the NIS. Not less important, future investigations
should be addressed to quantify collateral impacts on
cetaceans and the ecosystem derived from trawling activities,
such as the underwater noise pollution produced by trawling
vessels (Daly and White, 2021) and the spatial redistribution of
the trawling fishing effort in response to the establishment of
spatial closures (Powers and Abeare, 2009). The former impact
represents a critical disturbance for cetaceans, which should be
assessed in the framework of the of Marine Strategy Framework
Directive (Descriptor 11) (EU, 2017) while the latter could lead
to a potential increase in the fishing pressure around the banned
areas and on other grounds (Elhani et al., 2018). However, it
should be noted that the Blue and Red CCAs investigated in this
study should not influence the redistribution of fishing effort,
since trawling activity is almost entirely absent. Moreover,
potential effects of spatial conservation measures adopted for
cetaceans on the population dynamics of demersal resources
(e.g. spill-over effects from CCAs) represent an important
aspects to investigate through fisheries management scenarios. Th
f h
l
f
i
ld b The original contributions presented in the study are
included in the article/Supplementary Material. Discussion This condition could be affected by the
geographic traits and the position of this CCA, which is the
only area that partially overlaps with south-eastern grounds,
where the main exploited species are A. foliacea and A. antennatus (Russo et al., 2017; Maiorano et al., 2022). Observations of landings flows to harbours also support this
explanation, which in the Green area also show the presence of
the Gallipoli fleet, which is known for its exploitation of deep
water shrimp on the south-eastern slope the Gulf (D’Onghia
et al., 2005; Maiorano et al., 2022). Concerning fishery
performances in the Green and Orange CCAs, hourly fishing
effort in the former CCA was more than twice that of the latter
(3342 against 1226 fishing hours, respectively). However, the
landing and economic yield in proportion to the employed effort
was lower in the Green CCA than the Orange one. This
observation, like that observed at the global scale by Sumaila Frontiers in Marine Science 14 frontiersin.org Carlucci et al. 10.3389/fmars.2022.1005649 biodiversity. To provide spatially integrated information on the
fishing effort and the economic value could be a key point in
planning based on the principles of EBM (Essington et al., 2018). well as increased ecotourism activities with positive spill-over
effects on other economic activities in a more sustainable use of
maritime space. Therefore, the planning of spatial conservation
measures for cetaceans could find points of agreement with a
redefinition of the fishing areas in the Northern Ionian Sea
without generating socio-economic conflicts. Publisher’s note All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed
or endorsed by the publisher. Data availability statement Further
inquiries can be directed to the corresponding author. Author contributions RC, PR, TR: Conceptualization. RC, PR, TR: methodology. TR, AS, DC, PR: formal analysis. CF, RC, PR, GC: data
investigation and sampling design. PR, DC, MI, GC: writing—
original draft preparation. RC, PR, TR DC, MI, AS, CF, GC:
writing—review and editing. RC, PR: supervision. All authors
contributed to the article and approved the submitted version. Conflict of interest The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Ethics statement The animal study was reviewed and approved by the Italian
Ministry of Ecological Transition. References ACCOBAMS (2020). “Assessment of the conservation status of cetaceans in the
ACCOBAMS area,” in Thirteenth meeting of the ACCOBAMS scientific committee,
Monaco, ACCOBAMS-SC13/2020/Doc 10Rev1, 9. (Monaco:ACCOBAMS). Carlucci, R., Cipriano, G., Paoli, C., Ricci, P., Fanizza, C., Capezzuto, F., et al. (2018a). Random forest population modelling of striped and common-bottlenose
dolphins in the gulf of taranto (Northern Ionian Sea, central-eastern Mediterranean
Sea). Estuar. Coast. Shelf Sci. 204, 177–192. doi: 10.1016/j.ecss.2018.02.034 ACCOBAMS-ECS-WK Threats (2017)Inputs to the ACCOBAMS ongoing
effort to map human threats on cetaceans in the Mediterranean and black seas
ACCOBAMS-ECS-WK Threats/2017/Report. In: . Available at: https://www. Carlucci, R., Cipriano, G., Santacesaria, F. C., Ricci, P., Maglietta, R., Petrella, A.,
et al. (2020b). Exploring data from an individual stranding of a cuvier's beaked
whale in the gulf of taranto (Editoriale Scientifica: Northern Ionian Sea, central-
eastern Mediterranean Sea). J. Exp. Mar. Biol. Ecol. 553, 151473. doi: 10.1016/
j.jembe.2020.151473 accobams.org/wp-content/uploads/2016/06/Report_workshop_Threats_31_ECS. pdf (Accessed June 3, 2020). Azzellino, A., Fossi, M. C., Gaspari, S., Lanfredi, C., Lauriano, G., Marsili, L.,
et al. (2014). An index based on the biodiversity of cetacean species to assess the
environmental status of marine ecosystems. Mar. Environ. Res. 100, 94–111. doi:
10.1016/j.marenvres.2014.06.003 Carlucci, R., Fanizza, C., Cipriano, G., Paoli, C., Russo, T., and Vassallo, P. (2016a). Modeling the spatial distribution of the striped dolphin (Stenella
coeruleoalba) and common bottlenose dolphin (Tursiops truncatus) in the gulf of
taranto (Editoriale Scientifica: Northern Ionian Sea, central-eastern Mediterranean
sea)”. Ecol. Indic. 69, 707–721. doi: 10.1016/j.ecolind.2016.05.035 Azzolin, M., Arcangeli, A., Cipriano, G., Crosti, R., Maglietta, R., Pietroluongo,
G., et al. (2020). Spatial distribution modelling of striped dolphin (Stenella
coeruleoalba) at different geographical scales within the EU Adriatic and Ionian
Sea region, central-eastern Mediterranean Sea. Aquat. Conserv. Mar. Freshw. Ecosyst. 30, 1194–1207. doi: 10.1002/aqc.3314 sea)”. Ecol. Indic. 69, 707–721. doi: 10.1016/j.ecolind.2016.05.035 Carlucci, R., Maglietta, L., Buscaino, G., Cipriano, G., Milella, A., Pollazzon, V.,
et al. (2017). “Review on research studies and monitoring system applied to
cetaceans in the gulf of taranto (northern Ionian Sea, central-eastern
Mediterranean Sea),” in Proceedings 14th IEEE international conference on
advanced video and signal based surveillance (AVSS)(Lecce:IEEE). doi: 10.1109/
AVSS.2017.8078473 Bakun, A., and Agostini, V. N. (2001). Seasonal patterns of wind-induced
upwelling/downwelling in the Mediterranean Sea. Sci. Mar. 65, 243–257. doi:
10.3989/scimar.2001.65n3243 Carlucci, R., Maiorano, P., Sion, L., D’Onghia, G., and Tursi, A. (2016b). References ““The
sustainability of fishing in the southern Adriatic and northern Ionian seas”,” in
Governance of the Adriatic and Ionian marine space. Ed. A. Caligiuri (Editoriale
Scientifica), 149–159. Bastardie, F., Rufener, M., Nielsen, J., Maina, I., Kavadas, S., Vassilopoulou, C.,
et al. (2018). “Modelling spatial interactions among fish communities, fishers and
other marine activities: comparing five European case-studies,” in Conference of the
international institute of fisheries economics and trade (IIFET)(Seattle, US:
International Institute of Fisheries Economics & Trade)2018. Session on Marine
Spatial Planning and Multiple Use Management. Carlucci, R., Manea, E., Ricci, P., Cipriano, G., Fanizza, C., Maglietta, R., et al. (2021b). Managing multiple pressures for cetaceans’ conservation with an
ecosystem-based marine spatial planning approach. J. Environ. Manage. 287,
112240. doi: 10.1016/j.jenvman.2021.112240 Bearzi, G. (2002). “Interactions between cetaceans and fisheries in the
Mediterranean Sea,” in “Cetaceans of Mediterranean and black seas: State of
knowledge and conservation Strategies2 in a report to the ACCOBAMS
secretariat, vol. 20 . Ed. G. Notarbartolo di Sciara (Monaco: ACCOBAMS). Carlucci, R., Ricci, P., Cipriano, G., and Fanizza, C. (2018d). Abundance, activity
and critical habitat of the striped dolphin Stenella coeruleoalba in the gulf of taranto
(Northern Ionian Sea, central Mediterranean Sea). Aquat. Conserv. Mar. Freshw. Ecosyst. 28, 324–336. doi: 10.1002/aqc.2867 Bearzi, G., Agazzi, S., Gonzalvo, J., Bonizzoni, S., Costa, M., and Petroselli, A. (2010). Biomass removal by dolphins and fisheries in a Mediterranean Sea coastal
area: do dolphins have an ecological impact on fisheries? Aquat. Conserv. Mar. Freshw. Ecosyst. 20, 549–559. doi: 10.1002/aqc.1123 Castellan, G., Angeletti, L., Taviani, M., and Montagna, P. (2019). The yellow
coral Dendrophyllia cornigera in a warming ocean. Front. Mar. Sci. 6. doi: 10.3389/
fmars.2019.00692 Bellomo, S., Santacesaria, F. C., Fanizza, C., Cipriano, G., Renò, V., Carlucci, R.,
et al. (2019). “Photo-identification of physeter macrocephalus in the gulf of taranto
(Northern Ionian Sea, central-eastern Mediterranean Sea),” in Proceedings IEEE
metrology for the Sea 2019 IMEKO TC-19 international workshop on metrology for
the Sea(Genoa, Italy:IMEKO), 2019 33–37. Chimienti, G., Bo, M., Taviani, M., and Mastrototaro, F. (2019). “Occurrence
and biogeography of Mediterranean cold-water corals,” in Mediterranean Cold-
water corals: Past, present and future (Cham, CH: Springer International
Publishing), 213–243. Bo, M., Bavestrello, G., and Canese, S. (2011). Coral assemblage off the calabrian
coast (South Italy) with new observations on living colonies of Antipathes
dichotoma. Ital J. Zool. 78, 231–242. Supplementary material The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/
fmars.2022.1005649/full#supplementary-material The assessment of the value of conservation areas could be
considered as part of the broader framework of assessing the
ecosystem services provided by specific maritime areas with high Frontiers in Marine Science 15 frontiersin.org 10.3389/fmars.2022.1005649 Carlucci et al. 10.3389/fmars.2022.1005649 References doi: 10.1080/11250001003652619 Ciccarese, S., Carlucci, R., Ciani, E., Corcella, E., Cosentino, A., Fanizza, C., et al. (2019). Cytochrome b marker reveals an independent lineage of Stenella
coeruleoalba in the gulf of taranto. PloS One 14 (3), e0213826. doi: 10.1371/
journal.pone.0213826 Breen, P., Brown, S., Reid, D., and Rogan, E. (2017). Where is the risk? integrating a spatial distribution model and a risk assessment to identify areas of
cetacean interaction with fisheries in the northeast Atlantic. Ocean Coast. Manage. 136, 148–155. doi: 10.1016/j.ocecoaman.2016.12.001 Cipriano, G., Carlucci, R., Bellomo, S., Santacesaria, F. C., Fanizza, C., Ricci, P.,
et al. (2022). Behavioral pattern of risso’s dolphin (Grampus griseus) in the gulf of
taranto (Northern Ionian Sea, central-Eastern Mediterranean Sea). J. Mar. Sci. Eng. 10 (2), 175. doi: 10.3390/jmse10020175 Capezzuto, F., Carlucci, R., Maiorano, P., Sion, L., Battista, D., Giove, A., et al. (2010). The bathyal benthopelagic fauna in the north-western Ionian Sea: structure,
patterns and interactions”. Chem. Ecol. 26, 199–217. doi: 10.1080/0275754100-
3639188 Claudet, J. (2011). Marine protected areas - a multidisciplinary approach
(Cambridge: Cambridge University Press). Colloca, F., Cardinale, M., Maynou, F., Giannoulaki, M., Scarcella, G., Jenko, K.,
et al. (2013). Rebuilding Mediterranean fisheries: a new paradigm for ecological
sustainability. Fish Fish. 14, 89–109. doi: 10.1111/j.1467-2979.2011.00453.x Carlucci, R., Akkaya Baş, A., Liebig, P., Renò, V., Santacesaria, F. C., Bellomo, S.,
et al. (2020a). Residency patterns and site fidelity of Grampus griseus (Cuvier 1812)
in the gulf of taranto (Northern Ionian Sea, central eastern Mediterranean Sea). Mamm. Res. 65 (3), 445–455. doi: 10.1007/s13364-02000485-z Daly, E., and White, M. (2021). Bottom trawling noise: Are fishing vessels
polluting to deeper acoustic habitats? Mar. pollut. Bull. 162, 111877. doi: 10.1016/
j.marpolbul.2020.111877 Carlucci, R., Akkaya Baş, A., Maglietta, R., Renò, V., Fanizza, C., Rizzo, A., et al. (2018b). “Site fidelity, residency and habitat use of the risso’s dolphin grampus
griseus in the gulf of taranto (Northern Ionian Sea, central-eastern Mediterranean
Sea) by photo-identification,” in Proceedings IEEE metrology for the Sea, vol. 2018d. (Bari, Italy:IEEE), 173–177. D’Andrea, L., Parisi, A., Fiorentino, F., Garofalo, G., Gristina, M., Russo, T., et al. (2020). smartR: a r package for spatial modelling of fisheries and simulation of
effort management. Environ. Monit. Assess. 192 (12), 1–17. doi: 10.1111/2041-
210X.13394 Carlucci, R., Bandelj, V., Ricci, P., Capezzuto, F., Sion, L., Maiorano, P., et al. (2018c). Exploring spatio-temporal changes of the demersal and benthopelagic
assemblages of the north-western Ionian Sea (Central Mediterranean Sea). Mar. Ecol. References doi: 10.1109/ACCESS.2020.2990427 Maglietta, R., Renò, V., Cipriano, G., Fanizza, C., Milella, A., Stella, E., et al. (2018). DolFin: an innovative digital platform for studying risso’s dolphins from
the northern Ionian Sea (Central-Eastern Mediterranean). Sci. Rep. 8, 17185. doi:
10.1038/s41598-018-35492-3 EUSAIR (2014). Communication from the commission to the European
parliament, the council, the European economic and social committee and the
committee of the regions (Brussels:European Commission). 17.6.2014 SWD, 190
final. Maiorano, P., Capezzuto, F., Carluccio, A., Calculli, C., Cipriano, G., Carlucci, R.,
et al. (2022). Food from the depths of the Mediterranean: The role of habitats,
changes in the Sea-bottom temperature and fishing pressure. Foods 11, 1420. doi:
10.3390/foods11101420 EUSAIR (2021). Analysis of marine (water) protected areas in EUSAIR and
proposals for corrective measures. (Ljubljana, Slovenia:Government Office of the
Republic of Slovenia for Development and European Cohesion Policy). 77. Maiorano, P., Sabatella, R. F., and Marzocchi, B. M. (2019). Annuario sullo stato
delle risorse e sulle strutture produttive dei mari italiani. Biol. Mar. Mediterranea
22 (1 Suppl.), 358. Fanizza, C., Dimatteo, S., Pollazzon, V., Prunella, V., and Carlucci, R. (2014). An
update of cetaceans occurrence in the gulf of taranto (Western-central
Mediterranean Sea). Biol. Mar. Mediterr. 21 (1), 373–374. Maiorano, P., Sion, L., Carlucci, R., Capezzuto, F., Giove, A., Costantino, G.,
et al. (2010). The demersal faunal assemblage of the north-Western Ionian Sea
(Central mediterranean): present knowledge and perspectives. Chem. Ecol. 26 (1),
219–240. doi: 10.1080/02757541003693987 Ferreira, J. C., Vasconcelos, L., Monteiro, R., Silva, F. Z., Duarte, C. M., and
Ferreira, F. (2021). Ocean literacy to promote sustainable development goals and
agenda 2030 in coastal communities. Educ. Sci. 11, 62. doi: 10.3390/educsci11020062 Foley, M. M., Halpern, B. S., Micheli, F., Armsby, M. H., Caldwell, M. R., Crain,
C. M., et al. (2010). Guiding ecological principles for marine spatial planning. Mar. Pol. 34 (5), 955–966. doi: 10.1016/j.marpol.2010.02.001 Manea, E., Bianchelli, S., Fanelli, E., Danovaro, R., and Gissi, E. (2020). Towards
an ecosystem-based marine spatial planning in the deep Mediterranean Sea. Sci. Total Environ. 715, 1–15. doi: 10.1016/j.scitotenv.2020.136884 Game, E. T., Grantham, H. S., Hobday, A. J., Pressey, R. L., Lombard, A. T.,
Beckley, L. E., et al. (2010). Pelagic MPAs: the devil you know. Trends Ecol. Evol. 25,
63–64. doi: 10.1016/j.tree.2009.09.002 Mazzoldi, C., Bearzi, G., Brito, C., Carvalho, I., Desiderà, E., Endrizzi, L., et al. (2019). From sea monsters to charismatic megafauna: changes in perception and
use of large marine animals. References 404/2011 of 8
April 2011 laying down detailed rules for the implementation of Council
Regulation (EC) No 1224/2009 establishing a Community control system for
ensuring compliance with the rules of the Common Fisheries Policy. Off. J. Eur. Union L112, 1–30. Available at: http://eur-lex.europa.eu/legal-content/EN/TXT/
PDF/?uri=CELEX:32011R0404&from=EN IUCN Marine Mammal Protected Areas Task Force (2018). Guidance on the use
of selection criteria for the identification of important marine mammal areas
(IMMAs). version. 82. Jusufovski, D., Saavedra, C., and Kuparinen, A. (2019). Competition between
marine mammals and fisheries in contemporary harvested marine ecosystems. Mar. Ecol. Prog. Ser. 627, 207–232. doi: 10.3354/meps13068 Ehler, C., and Douvere, F. (2009). Marine spatial planning: a step-by-step
approach toward ecosystem-based management (Paris, UNESCO:
Intergovernmental Oceanographic Commission and Man and the Biosphere
Programme). Kaplan, D. M., Chassot, E., Gruss, A., and Fonteneau, A. (2010). Pelagic MPAs: the
devil is in the details. Trends Ecol. Evol. 25, 62–63. doi: 10.1016/j.tree.2009.09.003 Kaschner, K., and Pauly, D. (2005). ““Competition between marine mammals
and fisheries: Food for thought”,” in The state of the animals III, eds. D.J. Salem and
A.N. Rowan (Washington, DC: Humane Society Press), 95–117. Elahi, R., Ferretti, F., Bastari, A., Cerrano, C., Colloca, F., Kowalik, J., et al (2018). Leveraging vessel traffic data and a temporary fishing closure to informmarine
management Front. Ecol. Environ. 16(8):1–7 doi: 10.1002/fee.19 Kiszka, J. J., Woodstock, M. S., and Heithaus, M. R. (2022). Functional roles and
ecological importance of small cetaceans in aquatic ecosystems. Front. Mar. Sci. 9. doi: 10.3389/fmars.2022.803173 Essington, T. E., Sanchirico, J. N., and Baskett, M. L. (2018). Economic value of
ecological information in ecosystem-based natural resource management depends
on exploitation history. Proc. Natl. Acad. Sci. U.S.A. 115, 1658–1663. doi: 10.1073/
pnas.1716858115 Maglietta, R., Carlucci, R., Fanizza, C., and Dimauro, G. (2022). Machine
learning and image processing methods for cetacean photo identification: a
systematic review. IEEE Access 10, 80195–80207. doi: 10.1109/
ACCESS.2022.3195218 EU (2011). Impact assessment of discard reducing policies. Available at: https://ec. europa.eu/fisheries/documentation/studies/discards_en/. EU (2017). Commission decision (EU) 2017/848 of 17 may 2017 laying down
criteria and methodological standards on good environmental status of marine
waters and specifications and standardised methods for monitoring and
assessment, and repealing decision 2010/477/EU. Official Journal of the European
Union L. 125 (43), 32 Maglietta, R., Renò, V., Caccioppoli, R., Seller, E., Bellomo, S., Santacesaria, F. C.,
et al. (2020). Convolutional neural networks for risso’s dolphins identification. IEEE Access 8, 80195–80206. References Progr. Ser. 598, 1–19. doi: 10.3354/meps12613 Dimatteo, S., Siniscalchi, M., Esposito, L., Prunella, V., Bondanese, P., Bearzi, G.,
et al. (2011). Encounters with pelagic and continental slope cetacean species near
the northern shore of the gulf of taranto, Italy. Ital. J. Zool. 78 (1), 130–132. doi:
10.1080/11250003.2010.532161 Carlucci, R., Capezzuto, F., Cipriano, G., D’Onghia, G., Fanizza, C., Libralato, S.,
et al. (2021a). Assessment of cetacean–fishery interactions in the marine food web
of the gulf of taranto (Northern Ionian Sea, central Mediterranean Sea). Rev. Fish
Biol. Fish. 31 (1), 135–156. doi: 10.1007/s11160-020-09623-x Carlucci, R., Capezzuto, F., Cipriano, G., D’Onghia, G., Fanizza, C., Libralato, S.,
et al. (2021a). Assessment of cetacean–fishery interactions in the marine food web
of the gulf of taranto (Northern Ionian Sea, central Mediterranean Sea). Rev. Fish
Biol. Fish. 31 (1), 135–156. doi: 10.1007/s11160-020-09623-x D’Onghia, G., Calculli, E., Capezzuto, F., Carlucci, R., Carluccio, A., and
Maiorano, P. (2016). New records of cold-water coral sites and fish fauna
characterization of a potential network existing in the Mediterranean Sea. Mar. Ecol. 37, 1398–1422. doi: 10.1111/maec.12356 Carlucci, R., Cipriano, G., Fattoruso, G., Oliveri, M. G., Ricci, P., and Squillante,
M. (2021c). “Parsimonious AHP applied to the conservation of cetaceans: a case
study in the gulf of taranto,” in Proceedings IEEE international workshop on
metrology for the Sea: Learning to measure Sea health parameters, (MetroSea: IEEE)
2021., 1–5. Carlucci, R., Cipriano, G., Fattoruso, G., Oliveri, M. G., Ricci, P., and Squillante,
M. (2021c). “Parsimonious AHP applied to the conservation of cetaceans: a case
study in the gulf of taranto,” in Proceedings IEEE international workshop on
metrology for the Sea: Learning to measure Sea health parameters, (MetroSea: IEEE)
2021., 1–5. D’Onghia, G., Capezzuto, F., Mytilineou, C., Maiorano, P., Kapiris, K., Carlucci,
R., et al. (2005). Comparison of the population structure and dynamics of Aristeus
antennatus (Riss between exploited and unexploited areas in the Mediterranean
Sea. Fish. Res. 76 (1), 22–38. doi: 10.1016/j.fishres.2005.05.007 Frontiers in Marine Science 16 frontiersin.org frontiersin.org Carlucci et al. 10.3389/fmars.2022.1005649 EC (2010). Fleet register on the net. Available at: http://ec.europa.eu/fisheries/
fleet/index.cfm/. Hoyt, E., and Notarbartolo di Sciara, G. (2021). Important marine mammal
areas: a spatial tool for marine mammal conservation. Oryx 55, 330. doi: 10.1017/
S0030605321000272 EC. (2011). Commission Implementing Regulation (EU) No. References PloS One 14 (12), e0226810. doi: 10.1371/
journal.pone.0226810 Gerritsen, H., and Lordan, C. (2011). Integrating vessel monitoring systems
(VMS) data with daily catch data from logbooks to explore the spatial distribution
of catch and effort at high resolution. ICES J. Mar. Sci. 68, 245–252. doi: 10.1093/
icesjms/fsq137 McDonald, J. H. (2014). Handbook of biological statistics. 3rd edition (Baltimore,
Maryland, U.S.A: Sparky House Publishing). Notarbartolo di Sciara, G. (2002). Cetaceans of the Mediterranean and black seas:
State of knowledge and conservation strategies. a report to the ACCOBAMS
secretariat (Monaco: ACCOBAMS) Vol. Section 1, 5. Grip, K., and Blomqvist, S. (2020). Marine nature conservation and conflicts
with fisheries. Ambio 49, 1328–1340. doi: 10.1007/s13280-019-01279-7 Guidetti, P., Notarbartolo-Di-Sciara, G., and Agardy, T. (2013). Integrating
pelagic and coastal MPAs into large-scale ecosystem-wide management. Aquat. Conserv: Mar. Freshw. Ecosyst. 23, 179–182. doi: 10.1002/aqc.2314 Notarbartolo di Sciara, G., Hoyt, E., Reeves, R., Ardron, J., Marsh, H.,
Vongraven, D., et al. (2016). Place-based approaches to marine mammal
conservation. Aquat. Conserv. 26, 85–100. doi: 10.1002/aqc.2642 Guzman, H. M., Hinojosa, N., and Kaiser, S. (2020). Ship’s compliance with a
traffic separation scheme and speed limit in the gulf of Panama and implications for
the risk to humpback whales. Mar. Policy 120, 104113. doi: 10.1016/
j.marpol.2020.104113 Pace, D. S., Tizzi, R., and Mussi, B. (2015). Cetaceans value and conservation in
the Mediterranean Sea. J. Biodivers. Endanger. Species S1, S1004. doi: 10.4172/2332-
2543.S1.004 Papale, E., Fanizza, C., Buscaino, G., Maria, C., Cipriano, G., Crugliano, R., et al. (2020). The social role of vocal complexity in striped dolphins. Front. Mar. Sci. 7. doi: 10.3389/fmars.2020.584301 Hammer, Ø., Harper, D. A. T., and Ryan, P. D. (2001). PAST: Paleontological
statistics software package for education and data analysis. Palaeontol. Electron. 4, 4–9. Hammerschlag, N., Schmitz, O. J., Flecker, A. S., Lafferty, K. D., Sih, A., Atwood,
T. B., et al. (2019). Ecosystem function and services of aquatic predators in the
anthropocene. Trends Ecol. Evol. 34 (4), 369–383. doi: 10.1016/j.tree.2019.01.005 Pennino, M. G., Arcangeli, A., Prado Fonseca, V., Campana, I., Pierce, G. J.,
Rotta, A., et al. (2017). A spatially explicit risk assessment approach: Cetaceans and
marine traffic in the pelagos sanctuary (Mediterranean Sea). PloS One 12 (6),
e0179686. doi: 10.1371/journal.pone.0179686 Heck, N., Dearden, P., and McDonald, A. (2011). Stakeholders’ expectations
towards a proposed marine protected area: a multi-criteria analysis of MPA
performance criteria. Ocean Coast. Manage. 54 (9), 687–695. doi: 10.1016/
j.ocecoaman.2011.07.003 Pérez-Ruzafa, A., Garcı́a-Charton, J. References A., and Marcos, C. (2017). North East
Atlantic vs. Mediterranean marine protected areas as fisheries management tool. Front. Mar. Sci. 4. doi: 10.3389/fmars.2017.00245 Holmlund, C. M., and Hammer, M. (1999). Ecosystem services generated by fish
populations. Ecol. Econ. 29, 253–268. doi: 10.1016/S0921-8009(99)00015-4 Podestà, M., Azzellino, A., Cañadas, A., Frantzis, A., Moulins, A., Rosso, M. P., et al. (2016). ““Cuvier's beaked whale, ziphius cavirostris, distribution and occurrence in
the Mediterranean Sea: high-use areas and conservation threats”,” in Mediterranean
Marine mammal ecology and conservation, vol. 75 . Eds. G. Notarbartolo di Sciara, M. Podestà and B. E. Curry (Amsterdam: Elsevier), 103–140. Hooker, S. K., Cañadas, A., Hyrenbach, K. D., Corrigan, C., Polovina, J. J., and
Reeves, R. R. (2011). Making protected area networks effective for marine top
predators. Endang. Species Res. 13, 203–218. doi: 10.3354/esr00322 Frontiers in Marine Science 17 frontiersin.org Carlucci et al. 10.3389/fmars.2022.1005649 10.3389/fmars.2022.1005649 Pope, K. L., Pegg, M. A., Cole, N. W., Siddons, S. F., Fedele, A. D., Harmon, B. S.,
et al. (2016). Fishing for ecosystem services. J. Environ. Manage. 183, 408–417. doi: 10.1016/j.jenvman.2016.04.024 Russo, T., Catucci, E., Franceschini, S., Labanchi, L., Libralato, S., Sabatella, E. C.,
et al. (2022). Defend as you can, react quickly: the effects of the COVID-19 shock
on a large fishery of the Mediterranean Sea. Front. Mar. Sci. 9. doi: 10.3389/
fmars.2022.824857 Powers, J. E., and Abeare, S. M. (2009). Fishing effort redistribution in response
to area closures. Fish. Res. 99, 216–225. doi: 10.1016/j.fishres.2009.06.011 Russo, T., D’Andrea, L., Franceschini, S., Accadia, P., Cucco, A., Garofalo, G.,
et al. (2019). Simulating the effects of alternative management measures of trawl
fisheries in the central Mediterranean Sea: Application of a multi-species bio-
economic modeling approach. Front. Mar. Sci. 6. doi: 10.3389/fmars.2019.00542 Quijano Quiñones, D. R., López-Rocha, J. A., Hernández-Herrera, I., and
Torres-Irineo, E. (2021). Spatial dynamics modeling of small-scale fishing fleets
with a random walk approach. Front. Mar. Sci. 8. doi: 10.3389/fmars.2021.669112 Russo, T., Morello, E. B., Parisi, A., Scarcella, G., Angelini, S., Labanchi, L.,
et al. (2018). A model combining landings and VMS data to estimate landings by
fishing ground and harbor. Fish. Res. 199, 218–230. doi: 10.1016/
j.fishres.2017.11.002 Renó, V., Dimauro, G., Labate, G., Stella, E., Fanizza, C., Cipriano, G., et al. (2019). A SIFT-based software system for the photo-identification of the risso’s
dolphin. Ecol. Inform. 50, 95–101. doi: 10.1016/j.ecoinf.2019.01.006 Ricci, P., Carlucci, R., Capezzuto, F., Carluccio, A., Cipriano, G., D’Onghia, G.,
et al. References (2022). Contribution of intermediate and high trophic level species to benthic-
pelagic coupling: insights from modelling analysis. Front. Mar. Sci. 9. doi: 10.3389/
fmars.2022.887464 Russo, T., Parisi, A., Garofalo, G., Gristina, M., Cataudella, S., and Fiorentino, F. (2014). SMART: A spatially explicit bio-economic model for assessing and
managing demersal fisheries, with an application to Italian trawlers in the strait
of Sicily. PloS One 9 (1), e86222. doi: 10.1371/journal.pone.0086222 Ricci, P., Ingrosso, M., Carlucci, R., Fanizza, C., Maglietta, R., Santacesaria, F. C.,
et al. (2020a). “Quantifying the dolphins-fishery competition in the gulf of taranto
(Northern Ionian Sea, central Mediterranean Sea),” in Proceedings IMEKO Tc-19
metrology for the Sea(Naples, Italy:IMEKO). Sánchez Lizaso, J. L., Sola, I., Guijarro-Garcı́a, E., Bellido, J. M., and
Franquesa, R. (2020). A new management framework for western
Mediterranean demersal fisheries. Mar. Policy 112, 103772. doi: 10.1016/
J.MARPOL.2019.103772 Ricci, P., Ingrosso, M., Cipriano, G., Fanizza, C., Maglietta, R., Renò, V., et al. (2020b). “Top down cascading effects driven by the odontocetes in the gulf of
taranto (Northern Ionian Sea, central Mediterranean Sea),” in Proceedings IMEKO
Tc-19 metrology for the Sea(Naples, Italy:IMEKO). Santacesaria, F. C., Bellomo, S., Fanizza, C., Maglietta, R., Renò, V., Cipriano, G.,
et al. (2019). Long-term residency of Tursiops truncatus in the Gulf of Taranto
(Northern Ionian Sea, Central-eastern Mediterranean Sea). Genoa Italy 2019, 28–32. Sergio, F., Caro, T., Brown, D., Clucas, B., Hunter, J., Ketchum, J., et al. (2008). Top predators as conservation tools: ecological rationale, assumptions, and efficacy. Annu. Rev. Ecol. Evol. 39, 1–19. doi: 10.1146/annurev.ecolsys.39.110707.173545 Ricci, P., Libralato, S., Capezzuto, F., D’Onghia, G., Maiorano, P., Sion, L., et al. (2019). Ecosystem functioning of two marine food webs in the north-Western
Ionian Sea (Central Mediterranean Sea). Ecol. Evol. 9 (18), 10198–10212. doi: 10.1002/ece3.5527 Shapiro, S. S., and Wilk, M. B. (1965). An analysis of variance test for normality
(complete samples). Biometrika 52, 591–611. doi: 10.1093/biomet/52.3-4.591 Ricci, P., Manea, E., Cipriano, G., Cascione, D., D’Onghia, G., Ingrosso, M., et al. (2021a). Addressing cetacean–fishery interactions to inform a deep-sea ecosystem-
based management in the gulf of taranto (Northern Ionian Sea, central
Mediterranean Sea). J. Mar. Sci. Eng. 9, 872. doi: 10.3390/jmse9080872 Stelzenmüller, V., Maynou, F., and Martin, P. (2007). Spatial assessment of
benefits of a coastal Mediterranean marine protected area. Biol. Conserv. 136, 571–
583. doi: 10.1016/j.biocon.2007.01.002 Sumaila, U. R., Zeller, D., Watson, R., Alder, J., and Pauly, D. (2007). References Potential
costs and benefits of marine reserves in the high seas. Mar. Ecol. Prog. Ser. 345,
305–310. doi: 10.3354/meps07065 Ricci, P., Sion, L., Capezzuto, F., Cipriano, G., D'Onghia, G., and Libralato, S. (2021b). Modelling the trophic roles of the demersal Chondrichthyes in the
northern Ionian Sea (Central Mediterranean Sea). Ecol. Modell. 444, 109468. doi: 10.1016/j.ecolmodel.2021.109468 Tetley, M. J., Braulik, G. T., Lanfredi, C., Minton, G., Panigada, S., Politi, E., et al. (2022). The important marine mammal area network: A tool for systematic spatial
planning in response to the marine mammal habitat conservation crisis. Front. Mar. Sci. 9. doi: 10.3389/fmars.2022.841789 Roman, J., Estes, J. A., Morissette, L., Smith, G., Costa, D., McCarthy, J., et al. (2014). Whales as marine ecosystem engineers. Front. Ecol. Environ. 12, 377–385. doi: 10.1890/130220 Tromeur, E., and Loeuille, N. (2017). Balancing yield with resilience and
conservation objectives in harvested predator–prey communities. Oikos 126 (12),
1780–1789. doi: 10.1111/oik.03985 Rossi, S., and Gabbianelli, G. (1978). Geomorfologia del golfo di taranto. Boll. Soc Geol. Ital. 97 (4), 423–437. Russo, T., Bitetto, E., Carbonara, P., Carlucci, R., D'Andrea, L., Facchini, M. T.,
et al. (2017). A holistic approach to fishery management: evidence and insights
from a central Mediterranean case study (Western Ionian Sea). Front. Mar. Sci. 4. doi: 10.3389/fmars.2017.00193 Wood, L. J., Fish, L., Laughren, J., and Pauly, D. (2008). Assessing progress
towards global marine protection targets: shortfalls in information and action. Oryx
42, 340–351. doi: 10.1017/S003060530800046X Frontiers in Marine Science 18 frontiersin.org
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Mechanical Properties of Renewable Polymer with Thermoplastics Endurance to Ultraviolet irradiation Exposure
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Mechanical Properties of Renewable Polymer
with Thermoplastics Endurance to Ultraviolet
irradiation Exposure
Nurul Syamimi M. Salim1, *, Anika Zafiah M. Rus1, and Masnadia Mashuri1
1Faculty of Mechanical and Manufacturing, University Tun Hussein Onn Malaysia (UTHM), Parit
Raja, 86400, Batu Pahat, Malaysia Nurul Syamimi M. Salim1, *, Anika Zafiah M. Rus1, and Masnadia Mashuri1 1Faculty of Mechanical and Manufacturing, University Tun Hussein Onn Malaysia (UTHM), Parit
Raja, 86400, Batu Pahat, Malaysia Abstract. At present the disposal of waste tyre rubber (WTR) has become
a major waste management problem in the world. Therefore in this study,
polymer blended based on Polyethylene which is Low Density
Polyethylene (LDPE) or High Density Polyethylene (HDPE), with
Renewable Polymer (RP) and waste tyre rubber (WTR) is prepared via
injection molding. Blended polymer such as LDPE/RP/WTR and
HDPE/RP/WTR is known as LRT and HRT respectively. The preparation
of polymer blend steps start with the preparation of RP. The RP is prepared
by crosslinking the renewable monomer with Polymethane Polyphenyl
Isocyanate (MDI) at composition ratio of 1:0.5. The second steps involved
by adding 10 gm of liquid RP prepared earlier on with fixed amount of
LDPE and HDPE of 100 gm. Then the blended LDPE/RP or HDPE/RP
namely as LR or HR respectively is further added with WTR with different
percentages ratio of 5 %, 10 % and 15 %. The manually blended polymer
mixture and filler is then melt mixing using injection moulding to fabricate
the tensile specimen for mechanical tensile test and physical determination
such as density, distribution of WTR in polymer blend and surface fracture
morphology using scanning electron microscope. The samples were then
exposed to UV irradiation exposure in UV Accelerated Weathering for
500, 1000, 1500, 2000, 2500 and 3000 hours to evaluate the photostability
of the polymer blends. The optimum amount of WTR ratio composition is
at 5 % for both LRT and HRT blends which indicate the stability of
polymer blends towards UV irradiation exposure at 1000hours. 01038 (2016)
MATEC Web of Conferences 7
IConGDM 2016
,8 01038 (2016)
MATEC Web of Conferences 7
IConGDM 2016
,8 DOI: 10.1051/matecconf/20167801038 1 Introduction At present most of industrial uses only synthetic polymer from petroleum and natural gas
resources such as polyethylene, polypropylene and polyvinyl chloride. However, this type
of polymer materials that is not renewable. In addition, the polymer of this type has the
disadvantage because it can cause environmental pollution, congestion landfills and depend
entirely on limited natural resources such as petroleum [1]. Renewable polymer is a
renewable material because its ability to undergo degradation (decomposition) in the
biosphere that is associated with micro-organisms, enzyme or under a state of * Corresponding author : nurulsyamimisalim@gmail.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of
the Creative
Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding author : nurulsyamimisalim@gmail.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of
the Creative
Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 01038 (2016)
MATEC Web of Conferences 7
IConGDM 2016
,8 DOI: 10.1051/matecconf/20167801038 nature[2].While, renewable resources for the conversion of renewable polymer is originated
from vegetable oils. The main constituents of vegetable oils are triacylglycerols, which are
the product of esterification of glycerol with three fatty acids. Fatty acids account for 95%
of the total weight of triglycerides and their content is characteristic for each vegetable oil
[3]. In polymer industry, waste vegetable oils which represent a major potential source of
chemicals have been utilized as an alternative feedstock for sustainable monomers. nature[2].While, renewable resources for the conversion of renewable polymer is originated
from vegetable oils. The main constituents of vegetable oils are triacylglycerols, which are
the product of esterification of glycerol with three fatty acids. Fatty acids account for 95%
of the total weight of triglycerides and their content is characteristic for each vegetable oil
[3]. In polymer industry, waste vegetable oils which represent a major potential source of
chemicals have been utilized as an alternative feedstock for sustainable monomers. New technologies, processes and resources are currently under research due to the
current worldwide energy panorama. Dramatic growth in the number of used tyres around
the globe was recorded due to increasing number of vehicles on the road. Approximately
800 million tyres are discarded around the globe annually [4]. This figure is estimated to
increase by 2% every year. The annual global production of tyres is about 1.4 billion unit,
which corresponds to an estimated 17 million tonnes of used tyres each year [5]. Waste tyre particle (WTP) falls into this kind of solid residues and it is managed as
waste even if it is going to be recycled or re-manufactured. This is a burden that adds
significant cost over disposal and in many cases acts as a barrier to improve resource
efficiency [6]. The scientific community’s effort in finding ways to reduce tyre waste has
leaded to intense research on rubber recycling. The technology of polymer blending has
emerged as a useful tool in tailoring polymers to the needs of the end users. Accordingly,
blending with waste rubber is important both from the point of view of disposal of waste
and the reduction in the product cost [7]. * Corresponding author : nurulsyamimisalim@gmail.com The specimen of renewable polymer (RP) from monomer based on palm oil is
reinforced with synthetic polymer, Low Density Polyethylene (LDPE) or High Density
Polyethylene (HDPE) and mixed with waste tyre rubber (WTR) as filler. The specimen of
HDPE/RP, LDPE/RP, WTP/HDPE and WTP/LDPE were irradiated under ultraviolet (UV)
radiation exposure. The tensile test tested to study the effect of UV irradiation exposure on
the mechanical properties of specimens the LDPE/RP, HDPE/RP, WTR/LDPE and
WTR/HDPE. 2 Experimental The raw materials involved for the experimental study were high density polyethylene
(HDPE) density = 0.957 g/cm3, (melt index = 4 g/10 min, vicat softening point = 124 ℃),
low density polyethylene (LDPE) density = 0.922 g/cm3, (melt index = 5 g/10 min, , vicat
softening point = 93 ℃), flexible and rigid Polymethane Polyphenyl Isocyanate (Modified
polymeric-MDI) (viscosity at 25 ℃= 120-160 cps, specific gravity at 25 ℃= 1.18-1.20
g/ml, NCO content, % wt = 26.3-27.3), renewable monomers and waste tyre rubber (WTR). The monomer conversions from waste vegetable oils are started with in-house catalyst
preparation to generate the epoxies from the unsaturated fatty blends. The liquid monomer
is mixed with crosslinker and with HDPE and LDPE until solidifies. The proportion ratio of
crosslinker and monomer is 1:0.5. This process is repeated with different ratio of
renewable-polymer (RP) and WTR of 5 %, 10 % and 15 %. p y
(
)
LDPE/RP, HDPE/RP, WTR/LDPE and WTR/HDPE were fed in the injection molding
machine (Nissei Horizontal Screw Type Injection Molding NP7) equipped with mold of
tensile specimens according to ISO 527 (5A). The processing temperature for nozzle, front,
middle, rear 1 and rear 2 for LDPE were 185 ℃, 180 ℃, 175 ℃, 170 ℃and 165 ℃while,
for HDPE were 205 ℃, 195 ℃, 180 ℃, 175 ℃and 165 ℃. The injection pressure for LDPE
and HDPE were 43.3 and 96.6 MPa respectively. Dumbbell shape samples produced from
injection moulding was exposed to the UV light in UV Lamp Tested Model HD-703 (Haida
International Equipment Co., LTD) at different exposure time at 50 ℃. Different UV
exposure time was conducted for each group of 5 sample to calculate the average value of
mechanical testing. The UV exposed time set 500, 1000, 1500, 2000, 2500 and 3000 hour. 2 2 DOI: 10.1051/matecconf/20167801038 01038 (2016)
MATEC Web of Conferences 7
IConGDM 2016
,8 3.1 Mechanical Properties of LDPE/RP and HDPE/RP blends. The mechanical properties of the blended were analyzed based on their tensile strength and
strain or elongation at break as shown in Figure 1a and Figure 1b. The HDPE provides
ductility that exhibit brittle behaviour with a subsequent loss of toughness to the blended
materials. The addition of RP generally increases the toughness and decreases the strength
of the blends. This could be due to the soft properties of the RP. Meanwhile, the elongation
at break increased. The higher tensile strain of HDPE/RP blends compared to neat HDPE
may also be due to plasticization effect. Based on these results, when the RP is added with
crosslinker, it may act as filler as it improves the tensile strain values. Fig.1. LDPE/RP and HDPE/RP blends after UV Exposure with (a) Tensile Strength (b) Tensile
Strain. Fig.1. LDPE/RP and HDPE/RP blends after UV Exposure with (a) Tensile Strength (b) Tensile
Strain. Besides that, the tensile strain of the LDPE/RP blends is observed to be higher than the
HDPE/RP blends. The high value of elongation at break for the LDPE/RP blends is due to
the more flexible properties. This allows it to significantly yield before breaking, leading to
a high value of tensile strain. Therefore, LDPE/RP is comparatively more flexible than
HDPE/RP. 3.2 Effects of UV irradiation exposure on tensile strength. Figure 2a and Figure 2b shows the graph of tensile strength of LDPE/RP and HDPE/RP
with consistent ratio of RP of 10% and waste tyre rubber (WTR) of 5%, 10% and 15%. The
samples is namely as, L, LR, LRT5, LRT10, LRT15, H, HR, HRT5, HRT10 and HRT15
blends. Where the abbreviation of L is for LDPE, H for HDPE, R for renewable polymer, T
is for waste tyre rubber with proportion ratio of 5, 10 and 15%. The presence of the WTR
ratio did not improve the strength properties of the LDPE samples. Meanwhile WTR
contributed better strength on the H and HR. It is clearly shows that the HRT5 strength
values were approaching the neat HDPE from the Figure 2a. It is also noted that the UV
irradiation exposure also give influence to the crosslink process between RP and the WTR. 3 3 01038 (2016)
MATEC Web of Conferences 7
IConGDM 2016
,8 DOI: 10.1051/matecconf/20167801038 Fig. 2. Effects of UV irradiation exposure on tensile strength for (a) LDPE and (b) HDPE. Fig. 2. Effects of UV irradiation exposure on tensile strength for (a) LDPE and (b) HDPE. H, HR, HRT5, HRT10 and HRT15 show the highest value of the tensile strength
compare to the L, LR, LRT5, LRT10 and LRT15. However the trend starts to decreases
after HRT5. The increase of the WTR percentages content will decrease the mechanical
properties of HDPE and LDPE blends. H, HR, HRT5, HRT10 and HRT15 show the highest value of the tensile strength
compare to the L, LR, LRT5, LRT10 and LRT15. However the trend starts to decreases
after HRT5. The increase of the WTR percentages content will decrease the mechanical
properties of HDPE and LDPE blends. At the beginning the HR facing the decrement of 23.35 %, then the small increment of
3.35 % from HRT5 and then decrement start to begin from HRT10 and HRT15 with value
of 10.80 % and 17.95 % respectively. After UV irradiation exposure the decrement trend
are much slower than before the UV irradiation exposure of LDPE or HDPE blends. It can be seen that by increasing the WTR content the tensile strength of LR, LRT5,
LRT10, LRT15, HR, HRT5, HRT10 and HRT15 was decrease due to the attributed of
WTR content. The WTR still remain as the dispersed in the blend while the LDPE or
HDPE was bearing the tensile strength alone. 3.2 Effects of UV irradiation exposure on tensile strength. Previous studies [8], stated several reasons
for the decrease in tensile strength due to the decreasing of the blends rigidity. It is well known that the WTR is quite hard to dispose and highly resistance to the
degradation due to the various different chemical components on it. In conjunction with
that, WTR particle induces to the photostability of the LRT5, LRT10, LRT15, HRT5,
HRT10 and HRT15 blend because WTR particle did not give any reactions during the UV
irradiation exposure process. UV irradiation exposures not lead the crosslinking between
RP, WTR and the matrix. However, due to the lack of adhesion and low interfacial
bonding, WTR particle lead to the decreasing on mechanical properties. The addition of
WTR in the polymer blend is an advantages in order to recycle the scrap tyres. However,
according to Pilar [9], WTR use as an additive should be at low amounts which are less
than 10% wt. 3.3 Effects of UV irradiation exposure on tensile strain. Figure 3a and 3b shows the graph of strain (%) for samples of polymer blends. Every group
of polymer blends samples show fluctuate trend. It is noted that the presence of the WTR
influence the strain properties. The fluctuate trend for all the samples of LRT5 show the
highest improvement if compare to the sample of LR, LRT10 and LRT15. 1.54 %
difference from comparison of unirradiated and 3000 hour on UV irradiation exposure. The
LRT5 show the most significant improvement of strain (%). At 500 hour UV irradiation
exposure for LRT10 show the biggest decrement with 13.17 %. However the tensile
strength of HRT5, HRT10 and HRT15 slowly increased as shown in Figure 3b. The strain
trend shows almost the same for all samples. However, lowest strain of H, HR, HRT5,
HRT10 and HRT15 as compared to the L, LR, LRT5, LRT10 and LRT15. 4 01038 (2016)
MATEC Web of Conferences 7
IConGDM 2016
,8 DOI: 10.1051/matecconf/20167801038 Fig. 3. Effects of UV irradiation exposure on tensile strain for (a) LDPE and (b) HDPE. Fig. 3. Effects of UV irradiation exposure on tensile strain for (a) LDPE and (b) HDPE. Neat LDPE still have highest strain compare to those samples added with WTR as
shown in Figure 3a. LRT15 show the lowest value on strain. However, LRT5 shows the
better influence of WTR. 5 % of WTR enhance the strain on LDPE approaching the neat
LDPE value. Figure 3b show the effects of UV irradiation exposure on strain for H, HR, HRT5,
HRT10 and HRT15. The trend shows that there are slight increments of strain for HRT5,
HRT10 and HRT15. Meanwhile, at 1000 hours until 3000 hours, the tensile strain of HR
increases. It is well known that HDPE have high crystallinity that will make it hard to
deform and cause to break in low strain. From the previous study [10], it is also proven that by adding the WTR on HDPE blend
will lead the strain to decrease. However, in this study show that there are some increments
on the strain after UV irradiated exposure. It is believe, this situation happen due to the
increase of adhesion between the interfacial of WTR and the matrix LDPE or HDPE. Thus
this condition lead to WTR and HDPE blends become miscible blends, which reducing two
separate phases on the blend that causing strain to increase slightly. 4 Conclusion The processing conditions of polymer blends of LDPE and HDPE by injection molding
were determined and analyzed. The initial processing conditions of the standard LDPE and
HDPE thermoplastic were used as references. It was concluded that the processing
conditions by injection molding were exactly the same to that of neat LDPE. For the
HDPE/RP blends, increases in the parameters used in injection molding were required. Tensile strength and elongation at break increased with increasing RP content at low
compositions and started to decrease at high RP content for the LDPE/RP blends. However,
the tensile strength and strain of the LDPE/RP blends were generally better than the neat
LDPE. Therefore, not only does the presence of RP in the blends provide sustainable
characteristics, but it also improves the mechanical properties. These results comply with
the properties of pure LDPE and HDPE. Apart from the changing ratios of RP to LDPE or
HDPE, the processing temperature was also considered to have an influence on the
mechanical properties of the blends. The effects of sustainable polymer to the LDPE or
HDPE have become more significant as the polymer blends has been exposed to the UV
irradiation. The author would like to thank the Malaysian Government and University Tun Hussein Onn Malaysia
(UTHM), Johor and Postgraduate Incentive Research Grant Vot U222 and Malaysian Technical
University Centre of Excellence (MTUN CoE) for supporting this research study under research grant
Vot 1481. 5 5 5 01038 (2016)
MATEC Web of Conferences 7
IConGDM 2016
,8 DOI: 10.1051/matecconf/20167801038 References 1. S. Wiwin, G.S. Monica, T. Wega, Triyono, A. Ria, I.F.F. Iip, Procedia Environmental
Sciences , 20, 215 (2014) 1. S. Wiwin, G.S. Monica, T. Wega, Triyono, A. Ria, I.F.F. Iip, Procedia Environmental
Sciences , 20, 215 (2014) 2. R. Chandra, R. Rustgi, Prog. Polym. Sci., 23(7), 1273 (2000) 3. M.D.E. Lucas, M.A.R. Meier, Eur. Polym. J., 47(5), 837 (2010) 3. M.D.E. Lucas, M.A.R. Meier, Eur. Polym. J., 47(5), 837 (2010) 4. P.J.H.V. Beukering, M.A. Janssen, Resour. Conserv. Recycl., 33(4), 235 (2001) 4. P.J.H.V. Beukering, M.A. Janssen, Resour. Conserv. Recycl., 33(4), 235 (2001) 5. M. Sienkiewicz, J.K. Lipka, H. Janik, A. Balas, Waste Manage., 32(10), 1742 (2012)
6. S. Ramarad, M. Khalid, C.T. Ratnam, A.L. Chuah, W. Rashmi, Prog. Mater. Sci., 72, 5. M. Sienkiewicz, J.K. Lipka, H. Janik, A. Balas, Waste Manage., 32(10), 1742 (2012) 5. M. Sienkiewicz, J.K. Lipka, H. Janik, A. Balas, Waste Manage., 32(10), 1742 (2012)
6. S. Ramarad, M. Khalid, C.T. Ratnam, A.L. Chuah, W. Rashmi, Prog. Mater. Sci., 72,
100 (2015) p
g
(
)
(
)
6. S. Ramarad, M. Khalid, C.T. Ratnam, A.L. Chuah, W. Rashmi, Prog. Mater. Sci., 72,
100 (2015) 7. M.M. Hassan, N.A. Badway, A.M. Gamal, M.Y. Elnaggar, E.S.A. Hegazy, Nucl. Instrum. Meth. B., 268(9),1427 (2010) 7. M.M. Hassan, N.A. Badway, A.M. Gamal, M.Y. Elnaggar, E.S.A. Hegazy, Nucl. Instrum. Meth. B., 268(9),1427 (2010) ,
( ),
(
)
8. H.H.R. Cui, M.R. Kessler, Polym. Degrad. Stabil, 98(11), 2357 (2013) y
g
9. M.A. Salem, Egypt J. Sol., 24(2), 141 (2001) 9. M.A. Salem, Egypt J. Sol., 24(2), 141 (2001) ,
gyp
,
( ),
(
)
10. I. Grigoriadou, Compos. Part B-Eng., 55, 407 (2013) 10. I. Grigoriadou, Compos. Part B-Eng., 55, 407 (2013 g
p
g
(
)
11. S.M. Tamboli, S.T. Mhaske, D.D. Kale, Indian J. Chem. Techn., 11(6), 853 (2004) 11. S.M. Tamboli, S.T. Mhaske, D.D. Kale, Indian J. Chem. Techn., 11(6), 85 6 6
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Novel FOXL2 variants in two Chinese families with blepharophimosis, ptosis, and epicanthus inversus syndrome
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KEYWORDS Blepharophimosis-ptosis-epicanthus inversus syndrome, FOXL2 variant, whole exome
sequencing, tansfection, protein model prediction Blepharophimosis-ptosis-epicanthus inversus syndrome, FOXL2 variant, whole exome
sequencing, tansfection, protein model prediction frontiersin.org TYPE Original Research
PUBLISHED 12 February 2024
DOI 10.3389/fgene.2024.1343411 TYPE Original Research
PUBLISHED 12 February 2024
DOI 10.3389/fgene.2024.1343411 COPYRIGHT © 2024 Zhao, Meng, Wang and Wang. This is an
open-access article distributed under the terms
of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction in
other forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in this
journal is cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms. Results: The clinical features of BPES, including small palpebral fissures, ptosis,
telecanthus, and epicanthus inversus, were present in all affected patients. Two novel
mutations were detected, c.292T>A and c.383G>T. Whole-exome sequencing
analysis
and
prediction
software
suggested
that
these
mutations
were
pathogenic. Functional studies showed that these two point mutations decreased
FOXL2 protein expression, resulting in subcellular mislocalization and aberrant
transcriptional activity of the steroidogenic acute regulatory protein gene promoter. Conclusion: Our results add to the current understanding of known FOXL2
variants in, and our in vitro experiments provide reference data and insights
into the etiology of BPES. Further studies are needed to identify the possible
mechanisms underlying the action of this mutation on the development of BPES. OPEN ACCESS OPEN ACCESS
EDITED BY
Yongchu Pan,
Nanjing Medical University, China
REVIEWED BY
Yi Ding,
Xi’an Jiaotong University, China
Baoshan Xu,
Sun Yat-sen University, China
*CORRESPONDENCE
Tailing Wang,
wtlcom@sina.com
RECEIVED 23 November 2023
ACCEPTED 29 January 2024
PUBLISHED 12 February 2024 Mingyu Zhao1, Xiaolu Meng2, Jiaqi Wang1 and Tailing Wang1* 1The Department of Facial and Neck Plastic Surgery, Plastic Surgery Hospital, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing, China, 2Plastic Surgery Hospital, Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing, China Introduction: Blepharophimosis, ptosis, and epicanthus inversus syndrome
(BPES) is a rare inherited disorder. This study was aimed to identify and
functionally validate FOXL2 variants in two Chinese families with BPES. Zhao M, Meng X, Wang J and Wang T (2024),
Novel FOXL2 variants in two Chinese families
with blepharophimosis, ptosis, and epicanthus
inversus syndrome. Methods: The proband and his family members were subjected to whole-exome
sequencing to identify disease-associated variants. Several bioinformatic tools
were used to computationally predict altered proteins. In vitro functional assays
were conducted by transfecting wild-type and mutant FOXL2 cDNAs into HEK-
293 cells, followed by subcellular localization assays, luciferase reporter gene
assays, and quantitative real-time polymerase chain reaction. Front. Genet. 15:1343411. Front. Genet. 15:1343411. doi: 10.3389/fgene.2024.1343411 2.5 Luciferase reporter gene assay We used 24-well plates for transcriptional activity assays. HEK293T
cells
were
transfected
with
the
empty
vector
pcDNA3.1-3xflag, the WT or mutant FOXL2 expression vector,
and
the
above-mentioned
reporter
gene
constructs
using
Lipofectamine 2000 reagent (Invitrogen). Four groups were co-
transfected into HEK293 cells, each containing 500 ng of either
pcDNA3.1-3xflag, pcDNA-WT, pcDNA-MT1, or pcDNA-MT2, in
addition to 500 ng of luciferase reporter plasmid (pGL3-StAR) and
40 ng of pRL-TK plasmid (Promega). The total DNA content of each
well was maintained at 1,040 ng/well. Cells were incubated with
plasmid in DMEM for 8 h and then in complete medium for 48 h. Luciferase intensity was measured using an EnSpire Multiplex
Plate Reader (PerkinElmer, Waltham, MA, United States) with a 2 Materials and methods Human embryonic kidney (HEK)293T cells were obtained from
our laboratory stock. The cells were cultured in complete medium
consisting of Dulbecco’s modified Eagle’s medium (DMEM;
Gibco™, United States) with 10% fetal bovine serum (Gibco™) at
37°C and 5% CO2. Lipofectamine 2000 (Invitrogen, Carlsbad, CA,
United States) was used for transfection. The pEGFP-N1 and
pcDNA3.1-3xflag-N plasmids were gently mixed in 250 μl Opti-
MEM™I Reduced Serum Medium (Gibco™, United States). Lipo-
2000 (10 µL) was gently mixed into 250 µL Opti-MEM™I Reduced
Serum Medium and incubated for 5 min at 20°C–25 °C. The diluted
DNA mixture was combined with the Lipo-2000 mixture and
incubated for 20 min at 20°C–25°C. After 4–6 h, the medium was
replaced with complete medium. 2.3 Protein model prediction The FOXL2 gene coding sequence (NM_023067) was entered into
AlphaFold2 to construct the model, which was imported into PyMOL
(version 2.5; Schrödinger, LLC, New York, NY, United States). After
importing the predictive model file, which was in the pdb format, into
the software, the FOXL2-WT, p.Trp98Arg and p.Trp128Leu protein
prediction models were constructed. 2.4 Plasmid Construction and Transfection The coding sequence of FOXL2 (NM_023067) was cloned into
the pEGFP-N1 and pcDNA3.1-3xflag-N vectors. Mutant plasmids
carrying c.292T>A and c.383G>T were generated using targeted
mutagenesis polymerase chain reaction (PCR), using the wild-type
(WT) FOXL2 expression vector as a template. The WT sequences
were designated as pEGFP-WT and pcDNA-WT, and the two
mutations as pEGFP-MT1 (c.292T>A), pEGFP-MT2 (c.383G>T),
pcDNA-MT1
(c.292T>A)
and
pcDNA-MT2
(c.383G>T)
respectively. The human StAR promoter was constructed using
pGL3-basic (Promega, Madison, WI, United States) as the
luciferase reporter vector. All constructs were sequenced and
validated by Sanger sequencing. Here, we identified and verified two FOXL2 variants in two
Chinese families with BPES. Functional studies of these two
missense mutations, MT1 (c.292T>A) (Nallathambi, et al., 2008)
and MT2 (c.383G>T), revealed significant alterations in both
FOXL2 protein expression and the transcriptional repressive
activity of the steroidogenic acute regulatory protein (StAR)
promoter. Our findings emphasize the importance of these
mutations in the etiology of BPES. 1 Introduction Blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES) is an autosomal
dominant disorder with an incidence of 1 in 50,000 individuals. Two subtypes of BPES have
been described: Type I is characterized by abnormal eyelid development and female
infertility, whereas type II is characterized only by abnormal eyelid development and no
infertility in either sex (De Baere et al., 2003; Nallathambi et al., 2008). Approximately 70% of BPES cases are attributed to heterozygous variants of the FOXL2
gene (De Baere et al., 2003; Kim and Bae, 2014; Bunyan and Thomas, 2019). Till date, over
270 FOXL2 variants have been reported to be associated with BPES, 80% of which are intragenic Frontiers in Genetics 01 frontiersin.org Zhao et al. 10.3389/fgene.2024.1343411 using PolyPhen-2, SIFT (Sorting Intolerant from Tolerant), and
MutationTaster. using PolyPhen-2, SIFT (Sorting Intolerant from Tolerant), and
MutationTaster. mutations. These intragenic mutations are further categorized as shift
(44%), in-frame (33%), nonsense (12%), and missense (11%) mutations. Although some defects are linked to genomic rearrangements involving
FOXL2, most genetic defects in BPES result from intragenic mutations
(Beysen et al., 2009). FOXL2 localizes to the nucleus and functions as a
transcriptional regulator in the early development of eyelids and
differentiation of ovaries, maintaining the genetic program. In
addition, FOXL2 inhibits the components required for testicle
development
(Georges
et
al.,
2014). Based
on
the
clinical
heterogeneity of patients with BPES, different mutations in FOXL2
were suggested to be correlated with various BPES types. (De Baere
et al., 2003; Fokstuen et al., 2003; Dipietromaria et al., 2009; Zhou et al.,
2018). Therefore, identifying novel FOXL2 variants and improving the
understanding of the role of mutations in the pathogenesis of BPES may
lead to the determination of biomarkers for early BPES detection and
provide treatment targets for intervention. Thus, we aimed to identify
and
functionally
validate
FOXL2
variants
in
two
Chinese
families with BPES. 2.1 Patients Patients were recruited from the Department of Facial and Neck
Surgery of our hospital. Whole-exome sequencing (WES) was
performed by the Novogene Bio-informatics Co., Ltd. Beijing, China. The two families comprised a total of eight patients. Typical BPES
manifestations, including palpebral fissures, telecanthus, and epicanthus
inversus, were observed in three patients. Informed consent for
participation in this study was obtained from all participants or their
legal guardians in accordance with the tenets of the Declaration of
Helsinki. Written informed consent was obtained from all participants,
with parental consent secured for minors (those under 16). Written
consent was also obtained for the use of photographs. This study was
approved by the Ethics Committee of Plastic Surgery Hospital of the
Chinese Academy of Medical Sciences (No. 2022-157). Frontiers in Genetics frontiersin.org 2.2 Peripheral blood DNA extraction
and WES Peripheral blood (4 mL) was collected from each family member
into EDTA-K2 anticoagulation tubes. Genomic DNA was extracted
from leukocytes in the peripheral venous blood using a QIAamp
DNA blood kit (Qiagen, Hilden, Germany). After obtaining the raw
sequence reads, the data were analyzed by comparison with a
reference sequence or genome (human_B37). The mutation
nomenclature was in accordance with the guidelines of the
Human Genome Variation Society. Mutations were identified 02 frontiersin.org Zhao et al. 10.3389/fgene.2024.1343411 Zhao et al. 10.3389/fgene.2024.1343411 FIGURE 1
Lineage and genomic analysis of FOXL2 and sequence alignment of seven FOXL2 homologs. (A) Affected individuals are indicated by filled symbols
and preceding witnesses are marked with upward arrows. (B) Comparison of FOXL2 protein sequence around 98 and 128 by using UniProt indicates that
the residues surrounding these two mutations are significantly conserved. FIGURE 1
Lineage and genomic analysis of FOXL2 and sequence alignment of seven FOXL2 homologs. (A) Affected individuals are indicated by filled symbols
and preceding witnesses are marked with upward arrows. (B) Comparison of FOXL2 protein sequence around 98 and 128 by using UniProt indicates that
the residues surrounding these two mutations are significantly conserved. FIGURE 1
Lineage and genomic analysis of FOXL2 and sequence alignment of seven FOXL2 homologs. (A) Affected individuals are indicated by filled symbols
and preceding witnesses are marked with upward arrows. (B) Comparison of FOXL2 protein sequence around 98 and 128 by using UniProt indicates that
the residues surrounding these two mutations are significantly conserved. 3.1 Gene analysis and protein model
construction The results of WES indicated the presence of the c.292T>A
mutation in families III(1) and II(1) in F-1 and that of the c.383G>T
mutation in family II(1) in F-2. The results of the polyPhen-2, SIFT,
and MutationTaster programs revealed that the mutations were
situated in the forkhead structural domain and that their positions
are widely conserved in mammals (Figure 1B). Both mutant amino
acids were predicted to damage the forkhead protein (Table 1). CT-3′; StAR-R: 5′-CCCTTGAGGTCGATGCTGAG-3′; GAPDH-F:
5′-CTGCCAACGTGTCAGTGGTG-3′;
and
GAPDH-R:
5′-TCA
GTGTAGCCCAGGATGCC-3′. CT-3′; StAR-R: 5′-CCCTTGAGGTCGATGCTGAG-3′; GAPDH-F:
5′-CTGCCAACGTGTCAGTGGTG-3′;
and
GAPDH-R:
5′-TCA
GTGTAGCCCAGGATGCC-3′. dual-luciferase
reporter
gene
assay
system
(Promega). All
experiments were performed in triplicate. Frontiers in Genetics 3 Results HEK293T cells were transfected with Lipo 2000 and plasmids
containing the empty vectors pEGFP-N1, pEGFP-FOXL2, pEGFP-
FOXL2-MT1, and pEGFP-FOXL2-MT2(Plasmid and Lipo-2000
were used at the dosages described in the Plasmid Construction
and Transfection section). At 24 h after transfection, cell nuclei were
re-stained
with
Hoechst
33,342
(Beyotime
Institute
of
Biotechnology,
Jiangsu,
China)
and
observed
under
a
fluorescence microscope (Olympus, Tokyo, Japan). frontiersin.org 3.2 Subcellular localization using luciferase-based reporter gene assays. The results indicated
that WT FOXL2 inhibited StAR promoter activity, which was not
significantly inhibited in cells transfected with equivalent amounts
of mutant FOXL2 constructs (c.292T>A and c.383G>T) (p < 0.05). These results indicate that FOXL2 with c.292T>A and c.383G>T
mutations lost its function (Figure 4A). Localization studies in HEK293T cells revealed the effect of the
mutations (c.292T>A and c.383G>T) on the subcellular localization
of FOXL2. As shown in Figure 3, WT FOXL2 was localized to the
nucleus, which is consistent with its function as a transcription
factor. In contrast, cells transfected with mutant constructs
(c.292T>A
and
c.383G>T)
showed
a
non-WT
distribution (Figure 3). 2.7 Real-time PCR models constructed using the PyMOL software. (A, C) Normal gene conformation. (B) MT1 (p.Trp98Arg) shows a novel hydrogen bond between
nd TYR-91. (D) MT2 (p.Trp128Leu) shows stable primary conformation but altered secondary structure with the amino acid changes to leuci 3.4 Real-time qPCR We further evaluated the effect of the novel FOXL2 variants
(c.292T>A and c.383G>T) on transactivation capacity of StAR by
measuring the endogenous StAR mRNA expression using real-time
PCR. StAR mRNA expression was significantly higher in cells
transfected with c.292T>A and c.383G>T than that in WT- Frontiers in Genetics 2.7 Real-time PCR Protein structure analysis showed that for c.292T>A(p.Trp98Arg),
the tryptophan isopotential was 5.89. The mutation of tryptophan (a
non-polar amino acid) to arginine (a basic amino acid) resulted in an
isopotential of 10.76; thus, the mutation increased the potential
difference between amino acids. Simultaneously, the hydrogen bonds
between the amino acids increased, resulting in a change in the protein
structure
and
alterations
in
the
protein
function. c.383G>T(p.Trp128Leu) has a leucine isopotential of 6.01 compared
to the WT. Although the potential difference remained mostly
unchanged, changes in the local spatial structure of the forkhead
structural domain were observed (Figure 2). Thus, mutant amino
acids may have important effects on polarity, such as disrupting
protein–DNA interactions. To assess whether the mutations affected the expression of the
downstream target gene StAR, SYBR Green real-time quantitative (q)
PCR was performed using LightCycler® 96 Instrument (Roche, Basel,
Switzerland). The pcDNA3.1-3xflag expression vector (4 µg) containing
WT or mutant FOXL2 cDNA and empty pcDNA3.1-3xflag vector were
transfected into HEK293T cells in 12-well plates using Lipo 2000. After
48 h, total mRNA was extracted from the cells using TRIzol
(Invitrogen). mRNA was reverse transcribed into cDNA using a
Reverse Transcription Kit (Takara). The cDNA (2 μg) was subjected
to real-time PCR using FastStart Universal SYBR Green Master (Rox). GAPDH was used as an endogenous control to standardize data. The
following primers were used StAR-F: 5′-CAGACTTCGGGAACATGC 03 frontiersin.org Zhao et al. 10.3389/fgene.2024.1343411 TABLE 1 Prediction results of PolyPhen-2, SIFT and MutationTaster programs. Mutation
SIFT and score
Polyphen2 and score
MutationTaster and score
c.292T>A
Deleterious, 0.001
Probably damaging, 0.995
Disease causing, 1
c.383G>T
Deleterious, 0.001
Probably damaging, 0.988
Disease causing, 1
FIGURE 2
Prediction models constructed using the PyMOL software. (A, C) Normal gene conformation. (B) MT1 (p.Trp98Arg) shows a novel hydrogen bond between
ARG-98 and TYR-91. (D) MT2 (p.Trp128Leu) shows stable primary conformation but altered secondary structure with the amino acid changes to leucine. TABLE 1 Prediction results of PolyPhen-2, SIFT and MutationTaster programs. Mutation
SIFT and score
Polyphen2 and score
MutationTaster and score
c.292T>A
Deleterious, 0.001
Probably damaging, 0.995
Disease causing, 1
c.383G>T
Deleterious, 0.001
Probably damaging, 0.988
Disease causing, 1 FIGURE 2
Prediction models constructed using the PyMOL software. (A, C) Normal gene conformation. (B) MT1 (p.Trp98Arg) shows a novel hydrogen bond between
ARG-98 and TYR-91. (D) MT2 (p.Trp128Leu) shows stable primary conformation but altered secondary structure with the amino acid changes to leucine. frontiersin.org 3.3 Dual luciferase reporter gene assay We assessed the ability of mutant constructs to transactivate the
StAR promoter, a target of FOXL2 (Zhou et al., 2018; Li et al., 2021) 04 frontiersin.org Zhao et al. 10.3389/fgene.2024.1343411 FOXL2 cells. These results are consistent with those of the dual
luciferase reporter gene assay (Figure 4B). reproductive tumors (Nakashima et al., 2010). Mutations in a single
FOXL2 allele result in reduced levels of functional FOXL2 expression,
FIGURE 3
Protein expression and distribution. Subcellular localization of EGFP, FOXL2, FOXL2-MT1, and FOXL2-MT2. The first column shows the subcellular
localisation of EGFP as a marker for FOXL2 protein; the second column shows the nuclei stained with Hoechst33342. The third column shows the
combined images of the above images (400× magnification). FIGURE 4
Transcriptional activity of c.292T>A mutant FOXL2 and c.383G>T mutant FOXL2. (A) The plasmids pcDNA3.1 (vector), pcDNA3.1 - FOXL2 (WT),
pcDNA3.1 - FOXL2 - (MT1) or FOXL2 - (MT2) were co-transfected with the luciferase vectors driven by the StAR promoter. (B) The relative expression level of StAR,
as measured by RT-PCR, wascompared between the cells transfected with empty vectors and those transfected with WT and MTplasmids. ****p < 0.01, **p< 0.05. FIGURE 3
Protein expression and distribution. Subcellular localization of EGFP, FOXL2, FOXL2-MT1, and FOXL2-MT2. The first column shows the subcellular
localisation of EGFP as a marker for FOXL2 protein; the second column shows the nuclei stained with Hoechst33342. The third column shows the
combined images of the above images (400× magnification). FIGURE 3
Protein expression and distribution. Subcellular localization of EGFP, FOXL2, FOXL2-MT1, and FOXL2-MT2. The first column shows the subcellular
localisation of EGFP as a marker for FOXL2 protein; the second column shows the nuclei stained with Hoechst33342. The third column shows the
combined images of the above images (400× magnification). FIGURE 3
Protein expression and distribution. Subcellular localization of EGFP, FOXL2, FOXL2-MT1, and FOXL2-MT2. The first column shows the subcellular
localisation of EGFP as a marker for FOXL2 protein; the second column shows the nuclei stained with Hoechst33342. The third column shows the
combined images of the above images (400× magnification). FIGURE 4
Transcriptional activity of c.292T>A mutant FOXL2 and c.383G>T mutant FOXL2. (A) The plasmids pcDNA3.1 (vector), pcDNA3.1 - FOXL2 (WT),
pcDNA3.1 - FOXL2 - (MT1) or FOXL2 - (MT2) were co-transfected with the luciferase vectors driven by the StAR promoter. 3.3 Dual luciferase reporter gene assay (B) The relative expression level of StAR,
as measured by RT-PCR, wascompared between the cells transfected with empty vectors and those transfected with WT and MTplasmids. ****p < 0.01, **p< 0.05. FIGURE 4
Transcriptional activity of c.292T>A mutant FOXL2 and c.383G>T mutant FOXL2. (A) The plasmids pcDNA3.1 (vector), pcDNA3.1 - FOXL2 (WT),
pcDNA3.1 - FOXL2 - (MT1) or FOXL2 - (MT2) were co-transfected with the luciferase vectors driven by the StAR promoter. (B) The relative expression level of StAR,
as measured by RT-PCR, wascompared between the cells transfected with empty vectors and those transfected with WT and MTplasmids. ****p < 0.01, **p< 0.05. FOXL2 cells. These results are consistent with those of the dual
luciferase reporter gene assay (Figure 4B). reproductive tumors (Nakashima et al., 2010). Mutations in a single
FOXL2 allele result in reduced levels of functional FOXL2 expression,
and mutations in two FOXL2 alleles can be lethal (Chai et al., 2017). Thus, patients with BPES typically have heterozygous mutations. Frontiers in Genetics frontiersin.org 4 Discussion As reported previously, nearly 90% of patients with BPES have
genetic defects involving FOXL2 (Verdin and De Baere, 2012). In the
present study, we identified and characterized two missense
mutations, MT1 and MT2, which are located in the forkhead
structural domain and alter evolutionarily conserved amino acids. The spatial conformation of the protein was altered, and the
properties of the amino acids at the mutated sites were changed, FOXL2 is an evolutionarily highly conserved transcription factor
that was first identified as a BPES susceptibility gene. It plays important
roles in sex determination, reproduction, metabolism, and tumor
formation. It has also been implicated in the pathogenesis of several
diseases,
including
polycystic
ovarian
syndrome,
keloids,
and 05 frontiersin.org Zhao et al. 10.3389/fgene.2024.1343411 of Medical Sciences (No. 2022-157). 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. Written
informed consent was obtained from the individual(s) for the
publication of any potentially identifiable images or data included
in this article. of Medical Sciences (No. 2022-157). 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. Written
informed consent was obtained from the individual(s) for the
publication of any potentially identifiable images or data included
in this article. resulting in impaired protein function. Both mutations were
predicted to be deleterious. The subcellular localization results
indicated that the mutations led to gene expression in the
cytoplasm outside the nucleus. These results strongly suggest that
the two novel mutations, MT1 and MT2, are pathogenic and
contributed to the pathogenesis of BPES in our patients. MT1 and MT2 were less capable of downstream promoter
activation than the WT, confirming that the c.292T>A and
c.383G>T mutants caused loss of function of the FOXL2 gene. This
may have occurred because of decreased levels of FOXL2 in the nucleus,
where the mutation reduced the rate of FOXL2 translocation. In
addition, soluble molecules with misfolded proteins may not
recognize the binding sites in the target promoter. c.292T>A and
c.383G>T attenuated the transactivation of downstream StAR,
possibly because of the subcellular mislocalization of mutant FOXL2,
loss-of-function of the mutant protein, or other reasons related to
downstream gene activation. Publisher’s note All claims expressed in this article are solely those of the authors
and
do
not
necessarily
represent
those
of
their
affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher. Data availability statement The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest. The data uploaded to the SRA is open access at https://www. ncbi.nlm.nih.gov/sra/. The
numbers
are:
SRR27500370,
SRR27500369,
SRR27500368,
SRR27500367,
SRR27407349,
SRR27407348,
SRR27319410,
SRR27319409,
SRR27319408,
SRR27319407. Funding The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. The article
was supported by Peking Union Medical College Fund, “The
diagnosis and treatment strategy of mild ptosis”, YS202010. In conclusion, we report two pathogenic FOXL2 variants
(c.292T>A and c.383G>T) and confirm for the first time that both
missense mutations lead to reduced expression and activity of FOXL2
protein. This study extends the range of known FOXL2 variants and
contributes to the understanding of the etiology of BPES. 4 Discussion This was consistent with the results of the
previously discovered mutant c.383G > A (Li, et al., 2021). Additionally,
eyelid development is highly sensitive to changes in the amount of
FOXL2 (Yan et al., 2023). Based on the above evidence, FOXL2 variants
may contribute to developmental malformations of the eyelid. Author contributions MZ: Data curation, Formal Analysis, Writing–original draft,
Writing–review
and
editing. XM:
Methodology,
Software,
Writing–review and editing. JW: Funding acquisition, Resources,
Supervision, Writing–review and editing. TW: Funding acquisition,
Project administration, Supervision, Writing–review and editing. Ethics statement The studies involving humans were approved by Ethics
Committee of Plastic Surgery Hospital of the Chinese Academy References Beysen, D., De Paepe, A., and De Baere, E. (2009). Foxl2 mutations and genomic
rearrangements in Bpes. Hum. Mutat. 30, 158–169. doi:10.1002/humu.20807 transactivation reporter systems. Hum. Mol. Genet. 18, 3324–3333. doi:10.1093/hmg/
ddp273 transactivation reporter systems. Hum. Mol. Genet. 18, 3324–3333. doi:10.1093/hmg/
ddp273 Fokstuen, S., Antonarakis, S. E., and Blouin, J. L. (2003). Foxl2-mutations in
blepharophimosis-ptosis-epicanthus
inversus
syndrome
(Bpes);
challenges
for
genetic counseling in female patients. Am. J. Med. Genet. A 117A, 143–146. doi:10. 1002/ajmg.a.10024 Fokstuen, S., Antonarakis, S. E., and Blouin, J. L. (2003). Foxl2-mutations in
blepharophimosis-ptosis-epicanthus
inversus
syndrome
(Bpes);
challenges
for
genetic counseling in female patients. Am. J. Med. Genet. A 117A, 143–146. doi:10. 1002/ajmg.a.10024 Bunyan, D. J., and Thomas, N. S. (2019). Screening of a large cohort of
blepharophimosis, ptosis, and epicanthus inversus syndrome patients reveals a very
strong paternal inheritance bias and a wide spectrum of novel Foxl2 mutations. Eur. J. Med. Genet. 62, 103668. doi:10.1016/j.ejmg.2019.05.007 Chai, P., Li, F., Fan, J., Jia, R., Zhang, H., and Fan, X. (2017). Functional analysis of a
novel Foxl2 indel mutation in Chinese families with blepharophimosis-ptosis-
epicanthus inversus syndrome Type I. Int. J. Biol. Sci. 13, 1019–1028. doi:10.7150/
ijbs.19532 Georges, A., Auguste, A., Bessière, L., Vanet, A., Todeschini, A. L., and Veitia, R. A. (2014). Foxl2: a central transcription factor of the ovary. J. Mol. Endocrinol. 52,
R17–R33. doi:10.1530/JME-13-0159 Chai, P., Li, F., Fan, J., Jia, R., Zhang, H., and Fan, X. (2017). Functional analysis of a
novel Foxl2 indel mutation in Chinese families with blepharophimosis-ptosis-
epicanthus inversus syndrome Type I. Int. J. Biol. Sci. 13, 1019–1028. doi:10.7150/
ijbs.19532 Georges, A., Auguste, A., Bessière, L., Vanet, A., Todeschini, A. L., and Veitia, R. A. (2014). Foxl2: a central transcription factor of the ovary. J. Mol. Endocrinol. 52,
R17–R33. doi:10.1530/JME-13-0159 Kim, J. H., and Bae, J. (2014). Differential apoptotic and proliferative activities of
wild-type Foxl2 and blepharophimosis-ptosis-epicanthus inversus syndrome
(Bpes)-associated mutant Foxl2 proteins. J. Reprod. Dev. 60, 14–20. doi:10. 1262/jrd.2013-090 Kim, J. H., and Bae, J. (2014). Differential apoptotic and proliferative activities of
wild-type Foxl2 and blepharophimosis-ptosis-epicanthus inversus syndrome
(Bpes)-associated mutant Foxl2 proteins. J. Reprod. Dev. 60, 14–20. doi:10. 1262/jrd.2013-090 De Baere, E., Beysen, D., Oley, C., Lorenz, B., Cocquet, J., De Sutter, P., et al. (2003). Foxl2 and Bpes: mutational hotspots, phenotypic variability, and revision
of the genotype-phenotype correlation. Am. J. Hum. Genet. 72, 478–487. doi:10. Frontiers in Genetics Nakashima, M., Chung, S., Takahashi, A., Kamatani, N., Kawaguchi, T.,
Tsunoda, T., et al. (2010). A genome-wide association study identifies four
susceptibility loci for keloid in the Japanese population. Nat. Genet. 42,
768–771. doi:10.1038/ng.645 Nallathambi, J., Laissue, P., Batista, F., Benayoun, B. A., Lesaffre, C., Moumné, L.,
et al. (2008). Differential functional effects of novel mutations of the transcription
factor Foxl2 in Bpes patients. Hum. Mutat. 29, E123–E131. doi:10.1002/humu.
20809 Zhou,
L.,
Wang,
J.,
and
Wang,
T.
(2018).
Functional
study
on
new
Foxl2 mutations found in Chinese patients with Blepharophimosis, Ptosis,
Epicanthus Inversus syndrome. BMC Med. Genet. 19, 121. doi:10.1186/s12881-
018-0631-8 Shendure, J., and Ji, H. (2008). Next-generation DNA sequencing. Nat. Biotechnol. 26,
1135–1145. doi:10.1038/nbt1486 References 1086/346118 De Baere, E., Beysen, D., Oley, C., Lorenz, B., Cocquet, J., De Sutter, P., et al. (2003). Foxl2 and Bpes: mutational hotspots, phenotypic variability, and revision
of the genotype-phenotype correlation. Am. J. Hum. Genet. 72, 478–487. doi:10. 1086/346118 Li, F., Chen, H., Wang, Y., Yang, J., Zhou, Y., Song, X., et al. (2021). Functional studies
of novel Foxl2 variants in Chinese families with blepharophimosis-ptosis-epicanthus
inversus syndrome. Front. Genet. 12, 616112. doi:10.3389/fgene.2021.616112 Dipietromaria, A., Benayoun, B. A., Todeschini, A. L., Rivals, I., Bazin, C., and Veitia,
R. A. (2009). Towards a functional classification of pathogenic Foxl2 mutations using Dipietromaria, A., Benayoun, B. A., Todeschini, A. L., Rivals, I., Bazin, C., and Veitia,
R. A. (2009). Towards a functional classification of pathogenic Foxl2 mutations using 06 frontiersin.org Zhao et al. 10.3389/fgene.2024.1343411 10.3389/fgene.2024.1343411 Verdin, H., and De Baere, E. (2012). Foxl2 impairment in human disease. Horm. Res. Paediatr. 77, 2–11. doi:10.1159/000335236 Verdin, H., and De Baere, E. (2012). Foxl2 impairment in human disease. Horm. Res. Paediatr. 77, 2–11. doi:10.1159/000335236 Yan, Y. C., Zhou, L., and Fan, J. C. (2023). Identification and functional analyses
of a novel Foxl2 pathogenic variant causing blepharophimosis, ptosis, and
epicanthus inversus syndrome. Int. J. Ophthalmol. 16, 680–686. doi:10.18240/
ijo.2023.05.02 Nallathambi, J., Laissue, P., Batista, F., Benayoun, B. A., Lesaffre, C., Moumné, L.,
et al. (2008). Differential functional effects of novel mutations of the transcription
factor Foxl2 in Bpes patients. Hum. Mutat. 29, E123–E131. doi:10.1002/humu. 20809 Zhou,
L.,
Wang,
J.,
and
Wang,
T. (2018). Functional
study
on
new
Foxl2 mutations found in Chinese patients with Blepharophimosis, Ptosis,
Epicanthus Inversus syndrome. BMC Med. Genet. 19, 121. doi:10.1186/s12881-
018-0631-8 Shendure, J., and Ji, H. (2008). Next-generation DNA sequencing. Nat. Biotechnol. 26,
1135–1145. doi:10.1038/nbt1486 07 07 Frontiers in Genetics Frontiers in Genetics frontiersin.org
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Efetividade do ensaio Trad-MCN para avaliação de contaminantes atmosféricos em regiões brasileiras
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Revista Ambiente & Água
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Juliana Caroline Vivian Sposito; Luiza Flávia Veiga Francisco;
Alexeia Barufatti Grisolia* Universidade Federal da Grande Dourados (UFGD), Dourados, MS, Brasil
*Autor correspondente: e-mail: alexeiagrisolia@ufgd.edu.br,
juliana_sposito@hotmail.com, luizaveiga10@hotmail.com RESUMO A planta Tradescantia pallida (Rose) D.R. Hunt var. purpurea tem sido utilizada em
diversos modelos experimentais para detecção de danos morfofisiológicos e genéticos, por ser
uma planta sensível a poluentes atmosféricos. No entanto, dentre os biotestes utilizados
para avaliação do potencial mutagênico de contaminantes ambientais, o bioensaio de
micronúcleo em Tradescantia (Trad-MCN) é um dos testes mais utilizados em estudos de
monitoramento das condições atmosféricas. Diante do exposto, foi realizada uma revisão de
literatura, identificando regiões brasileiras que utilizaram o bioensaio de micronúcleo em
Tradescantia para monitorar alterações genéticas advindas da contaminação atmosférica. O
levantamento de trabalhos indexados foi realizado em bancos de referência, como Portal de
Períodicos CAPES, SciELO, ScienceDirect e PubMed, sendo os mesmos investigados quanto
às regiões brasileiras biomonitoradas, período, área, exposição, monitoramento ativo ou passivo
e planta natural ou clone. Os resultados indicaram que pesquisas baseadas no bioensaio
Trad-MCN foram realizadas em diversas regiões brasileiras. Verificou-se que a maioria dos
trabalhos são oriundos da região sudeste, especialmente do estado de São Paulo. Esse fato pode
ser atribuído ao maior número de indústrias e automóveis em circulação existentes neste estado
quando comparado aos demais, podendo interferir diretamente na qualidade do ar, despertando
assim o interesse no meio científico. Com base nesta investigação, o bioensaio Trad-MCN
revelou eficácia, e a planta Tradescantia demonstrou ser importante indicadora biológica de
danos genéticos ocasionados pela exposição aos poluentes atmosféricos, e é amplamente
utilizada na região sudeste do país. Palavras-chave: bioindicador, micronúcleos, Tradescantia. Ambiente & Água - An Interdisciplinary Journal of Applied Science
ISSN 1980-993X – doi:10.4136/1980-993X
www.ambi-agua.net
E-mail: ambi.agua@gmail.com Ambiente & Água - An Interdisciplinary Journal of Applied Science
ISSN 1980-993X – doi:10.4136/1980-993X
www.ambi-agua.net
E-mail: ambi.agua@gmail.com Ambiente & Água - An Interdisciplinary Journal of Applied Science
ISSN 1980-993X – doi:10.4136/1980-993X
www.ambi-agua.net
E-mail: ambi.agua@gmail.com Rev. Ambient. Água vol. 12 n. 3 Taubaté – May. / Jun. 2017 Palavras-chave: bioindicador, micronúcleos, Tradescantia. 1. INTRODUÇÃO O biomonitoramento corresponde a um método experimental que possibilita detectar
impactos ocasionados por poluentes nos organismos vivos, conhecidos como bioindicadores
(Carneiro e Takayanagui, 2009). Estes organismos fornecem informações sobre alterações
morfológicas e/ou fisiológicas que ocorrem nos mesmos, quando expostos a ambientes que
passaram por mudanças físico-químicas e se encontram degradados (Klumpp et al., 2004). As plantas têm sido frequentemente utilizadas por apresentarem vantagens em relação aos
métodos convencionais, como: avaliação de efeitos em linhagens germinativas, visualização de
danos genéticos em células individuais, sensibilidade, baixo custo operacional, rápida
execução, maior número de amostragens, além de fácil adaptação podendo se desenvolver
durante todo o ano, apresentando-se como um instrumento favorável para estudos de
biomonitoramento (Carvalho, 2005; Catinon et al., 2008; De Souza et al., 2016). As plantas bioindicadoras possuem sensibilidade às alterações do ambiente, fornecendo
informações importantes no monitoramento de poluentes atmosféricos. Essa sensibilidade pode
ser atribuída à principal via de entrada de poluentes, os estômatos, por onde são realizadas
trocas gasosas, verificando-se danos macroscópicos como aparecimento de cloroses, necroses,
queda de folhas ou diminuição no seu crescimento, bem como em nível genético, fisiológico
ou bioquímico (Alves et al., 2001; Savóia, 2007). Algumas plantas do gênero Tradescantia são conhecidas como importantes ferramentas
em estudo de biomonitoramento, como a Tradescantia pallida (Rose) Hunt var. purpurea
Boom e o clone 4430 (Meireles e Cerqueira, 2011). ABSTRACT The Tradescantia pallida (Rose) D.R. Hunt var. purpurea plant has been used in several
experimental models to detect morphophysiological and genetic damage due its susceptibility
to atmospheric pollutants. However, among the bioassays used to evaluate the mutagenic
potential of environmental contaminants, the Tradescantia-Micronucleus (Trad-MCN) Rev. Ambient. Água vol. 12 n. 3 Taubaté – May. / Jun. 2017 501 Efetividade do ensaio Trad-MCN … bioassay is one of the most-used tests in studies of atmospheric conditions monitoring. A review
of literature was therefore carried out, identifying Brazilian regions where the micronucleus
bioassay in Tradescantia was used to monitor genetic alterations caused by atmospheric
contamination. Reference banks were used to survey the indexed studies, such as portal CAPES,
SciELO, ScienceDirect and PubMed, which were investigated regarding the Brazilian bio-
monitored region, period, area, exposure, active or passive monitoring and natural plant or
clone. The results indicated that research based on the Trad-MCN bioassay was performed in
several Brazilian regions. The review verified that the majority of the studies come from the
southeast region, especially from the state of São Paulo. This fact can be explained by the
greater number of industries and automobiles in circulation in this state as compared to others,
which can directly interfere with air quality thus arousing the interest of the scientific
community. Based on this review, the Trad-MCN bioassay has been demonstrated to be
effective and the Tradescantia plant has been shown to be an important biological indicator of
genetic damage caused by exposure to air pollutants and is widely used in the southeastern
region of the country. bioassay is one of the most-used tests in studies of atmospheric conditions monitoring. A review
of literature was therefore carried out, identifying Brazilian regions where the micronucleus
bioassay in Tradescantia was used to monitor genetic alterations caused by atmospheric
contamination. Reference banks were used to survey the indexed studies, such as portal CAPES,
SciELO, ScienceDirect and PubMed, which were investigated regarding the Brazilian bio-
monitored region, period, area, exposure, active or passive monitoring and natural plant or
clone. The results indicated that research based on the Trad-MCN bioassay was performed in
several Brazilian regions. The review verified that the majority of the studies come from the
southeast region, especially from the state of São Paulo. Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 ABSTRACT This fact can be explained by the
greater number of industries and automobiles in circulation in this state as compared to others,
which can directly interfere with air quality thus arousing the interest of the scientific
community. Based on this review, the Trad-MCN bioassay has been demonstrated to be
effective and the Tradescantia plant has been shown to be an important biological indicator of
genetic damage caused by exposure to air pollutants and is widely used in the southeastern
region of the country. Keywords: bioindicator, micronuclei, Tradescantia. 1.1. Clone 4430 x Tradescantia pallida Em 1978, Ma e colaboradores padronizaram teste utilizando Clone BNL 4430, um híbrido
estéril (Tradescantia hirsutiflora × Tradescantia subcaulis) do gênero Tradescantia, contendo
2n=12 cromossomos (todos metacêntricos). Esta esterilidade certifica a homogeneidade
genética do clone na ausência de efeitos mutagênicos (White e Claxton, 2004). Desde então, o
referido clone tem sido utilizado como bioindicador no monitoramento de poluentes
atmosféricos. Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 502 Juliana Caroline Vivian Sposito et al. Entretanto, o Clone 4430 tem aplicações limitadas em países tropicais, dada sua
sensibilidade ao clima (temperatura e umidade do ar elevadas) e aos ataques de insetos quando
expostas por períodos prolongados em ambiente aberto, limitando seu uso para estudos de
biomonitoramento (Sant’Anna, 2003). Neste contexto, a espécie Tradescantia pallida tem sido largamente usada como planta
bioindicadora apresentando resistência natural às intempéries da natureza, além de fácil
propagação (Suyama et al., 2002). A Tradescantia pallida (Rose) D.R Hunt var. purpurea
(Commelinaceae), originária do México, corresponde a uma espécie de planta herbácea
indicadora biológica, suculenta, de 15-25 cm de altura, anual ou perene, raramente epífita, ereta
a decumbente. Planta decorativa de cor roxa, com folhas alternas inteiras, com bainhas
envolvendo o caule, pubescentes, possui inflorescência cimosa, racemosa ou capitata, com
brácteas pequenas a grandes, frequentemente espatáceas (Ribeiro et al., 1999; Lorenzi e Souza,
2001). )
Conhecida popularmente como trapoeraba-roxa, coração-roxo, trapoerabão ou trapoeraba,
apresenta ampla distribuição e fácil adaptação, sendo cultivada em regiões tropicais e
subtropicais. Considerando a Tradescantia como planta bioindicadora, o seu uso pode levar a
identificação dos efeitos diretos sobre os organismos sejam eles anatômicos (Alves et al., 2001;
Crispim et al., 2014), ou até mesmo no DNA (Sposito et al., 2017), sob forma de efeitos
genotóxicos e/ou mutagênicos, sendo interpretadas como respostas adaptativas dos vegetais as
condições ambientais. Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 1.2.2. Testes genéticos g
Segundo Ma et al. (1994), o bioensaio de mutação em pelo estaminal (Trad-SHM) é
baseado na detecção de mutações mitóticas. As células de pelos estaminais são heterozigotas,
com um marcador fenotípico visível que muda a cor dos filamentos do estame de azul
(dominante) para rosa (recessivo) quando expostos a poluentes que podem promover mutações
baseadas na mudança de coloração nas células de pelos estaminais. De acordo com Patussi e
Bündchen (2013), o teste Trad-SHM tem sido empregado na avaliação do risco ambiental para
análise de contaminantes ambientais, sendo utilizado como biomarcador da qualidade
ambiental. O ensaio do cometa pode ser utilizado para detectar quebras na dupla fita de DNA em
vários tipos de células utilizando eletroforese, com base na migração dos fragmentos de DNA
livres resultantes de quebras para fora do núcleo. Desta maneira, o nível de dano genético das
células está relacionado com a extensão dos fragmentos de DNA (Collins, 2004; Speit e
Hartmann, 2005). Sposito et al. (2017), demonstraram que plantas expostas ao tráfego veicular
intenso apresentaram maior número de danos genéticos em relação a plantas não sujeitas ao
estresse ambiental por poluentes atmosféricos, indicando a eficácia da técnica na detecção de
danos no DNA associados ao tráfego veicular. O ensaio do cometa pode ser utilizado para detectar quebras na dupla fita de DNA em
vários tipos de células utilizando eletroforese, com base na migração dos fragmentos de DNA
livres resultantes de quebras para fora do núcleo. Desta maneira, o nível de dano genético das
células está relacionado com a extensão dos fragmentos de DNA (Collins, 2004; Speit e
Hartmann, 2005). Sposito et al. (2017), demonstraram que plantas expostas ao tráfego veicular
intenso apresentaram maior número de danos genéticos em relação a plantas não sujeitas ao
estresse ambiental por poluentes atmosféricos, indicando a eficácia da técnica na detecção de
danos no DNA associados ao tráfego veicular. Dentre as espécies vegetais, a Tradescantia pallida (Trad-MCN) tem sido amplamente
empregada na detecção de danos cromossômicos em células-mãe de grãos de pólen na fase de
tétrade. O teste consiste na estimativa da frequência de micronúcleos, sendo o número de
micronúcleos proporcional à concentração de poluentes (De Andrade Júnior et al., 2008). Micronúcleos correspondem a pequenas massas de cromatina derivadas de quebras
cromossômicas (clastogênese) e/ou de aberrações cromossômicas numéricas (aneugênese)
induzidas por agentes que danificam o cromossomo ou o fuso mitótico. 1.2.1. Testes anatômicos A concentração de elementos químicos não essenciais nas folhas está diretamente
relacionada à deposição de partículas provenientes da poluição atmosférica por emissões
veiculares, alterando a composição química foliar natural destas plantas. Sumita et al. (2003)
avaliaram a capacidade de acumulação de oligoelementos provenientes da poluição atmosférica
em folhas de T. pallida e indicaram o uso potencial desta planta no biomonitoramento de
ambientes urbanos altamente poluídos. Santos et al. (2015) também mostraram que plantas
expostas em áreas de alto fluxo veicular apresentaram maiores concentrações de elementos
relacionados às emissões dos veículos, sendo consideradas sensíveis e um instrumento
alternativo no biomonitoramento. A exposição das plantas aos poluentes atmosféricos também pode alterar a frequência
estomática, sendo desfavorável ao desenvolvimento fisiológico normal da planta como
comprometimento da capacidade fotossintética devido a redução das trocas gasosas. Crispim et
al. (2014) observaram que plantas de T. pallida situadas em áreas com alto fluxo veicular
apresentaram maior número de células epidérmicas e densidade estomática. Estas observações
indicaram que alterações estomáticas interferem na quantidade de poluentes gasosos absorvidos
pela planta, sendo essencial para sua adaptação em condições adversas. Segundo Alves et al. (2001) e Crispim et al. (2014) variações anatômicas, como espessura
da folha e quantidade de estômatos, representam uma estratégia adaptativa a ambientes
poluídos, dificultando o deslocamento dos poluentes para o interior da folha. No entanto,
alterações na capacidade de trocas gasosas acarretam no comprometimento da taxa
fotossintética e, consequentemente, do crescimento da planta, constituindo uma das formas
mais sensíveis de medir o estresse em um organismo. Segundo Zeiger (2006), partículas em
suspensão, ao atingirem as folhas, bloqueiam os estômatos, diminuindo a condutância de CO2,
interferindo na taxa de fotossíntese e, consequentemente, no crescimento das plantas. Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 503 Efetividade do ensaio Trad-MCN … Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 2. MATERIAL E MÉTODOS Realizou-se levantamento bibliográfico de trabalhos científicos publicados e indexados,
em sites de busca como: Portal de Períodicos CAPES, SciELO, ScienceDirect e PubMed, que
utilizaram o teste Trad-MCN em Tradescantia como organismo bioindicador para
monitoramento da qualidade do ar no Brasil. Para tanto, foram utilizadas as seguintes
palavras-chave: poluição atmosférica, biomonitoramento, Tradescantia e micronúcleo. Foram
incluídos apenas pesquisas de monitoramento em plantas expostas in situ. Os trabalhos foram avaliados quanto ao período de monitoramento, região brasileira
monitorada (Norte, Nordeste, Sul, Sudeste e Centro-oeste, e respectivos Estados), área de
estudo (urbana/rural), fontes emissoras de poluição atmosférica (tráfego, atividade industrial,
material particulado e queima de biomassa), planta utilizada no monitoramento (Tradescantia
pallida ou clone híbrido 4430) e procedimentos de monitoramento (passivo/ativo). O
monitoramento passivo consiste em utilizar plantas existentes naturalmente na área de estudo. O monitoramento ativo, por sua vez, refere-se às plantas introduzidas por determinado período
ao local de pesquisa. 1.2.2. Testes genéticos Os fragmentos ou
cromossomos inteiros não se ligam às fibras do fuso mitótico e não são incorporados no núcleo
das células filhas durante a meiose. Segundo Carvalho (2005) cromossomos em divisão
meiótica são mais sensíveis à quebra que os cromossomos em divisão mitótica. Desta forma,
essas estruturas permanecem no citoplasma das células interfásicas e formam a própria
membrana nuclear, originando micronúcleos, os quais se assemelham ao núcleo principal
quanto à forma, estrutura e propriedades de coloração, e podem variar quanto ao tamanho (Ma
et al., 1981; Holland et al., 2008; Meireles e Cerqueira, 2011). O teste tem sido empregado em diversos trabalhos científicos apresentando vantagens
como baixo custo financeiro, fácil acesso ao material e alta sensibilidade aos agentes
genotóxicos, permitindo a avaliação do dano cromossômico em preparações citológicas simples
e de rápida execução, sendo considerado ferramenta fundamental para a detecção de efeitos
clastogênicos e aneugênicos (Klumpp et al., 2004; Pereira et al., 2013). Sposito et al. (2017), em estudo de biomonitoramento passivo realizado em cidades do
estado de Mato Grosso do Sul, demonstram que o teste Trad-MCN é uma excelente ferramenta
para detecção de estressores ambientais, como os poluentes atmosféricos. Segundo Lima
(2001), o monitoramento passivo, é baseado na utilização de organismos existentes
naturalmente no local de pesquisa, e o ativo ocorre quando os organismos são introduzidos na
área de estudo. Considerando que o bioensaio Trad-MCN é considerado ferramenta abrangente no sentido
biológico e geográfico, o presente estudo teve como objetivo realizar levantamento
bibliográfico, aferindo regiões brasileiras monitoradas que utilizaram o teste de micronúcleo
em Tradescantia (Trad-MCN). 504 Juliana Caroline Vivian Sposito et al. Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 3. RESULTADOS E DISCUSSÃO Os resultados do levantamento bibliográfico foram apresentados na Tabela 1. O levantamento bibliográfico abrangeu diversas regiões do país. No entanto, verificou-se
que a maioria dos estudos que utilizaram o bioensaio Trad-MCN como ferramenta de
monitoramento da qualidade do ar ocorreram na região sudeste (43%), especialmente no estado
de São Paulo. Outro aspecto relevante foi a relação deste teste com o tráfego veicular e
atividades industriais. De acordo com estatísticas fornecidas pelo Departamento Nacional de Trânsito, o Brasil
possui 90 milhões de veículos motorizados, dos quais 30% (27.332.101) estão concentrados no
Estado de São Paulo (Brasil, 2016), correspondendo a uma parcela significativa da frota
veicular do país (Brasil, 2013). Segundo estimativa da CETESB (2004), veículos automotores
produzem mais poluição atmosférica que qualquer outra atividade humana. Ambientes urbanos
são áreas sob constante atividade humana e, consequentemente, elevada concentração de
poluentes atmosféricos, provenientes de fontes móveis (frota de veículos automotores) e
estacionárias (atividades industriais). Costa e Droste (2012) verificaram frequências de
micronúcleo (MCN) superiores em área urbana com elevada frota veicular em comparação aos
resultados observados na área rural. Nas regiões urbanas, as emissões de poluentes por veículos contribuem para o aumento de
partículas, que podem ser inaladas nos pulmões, aumentando os riscos para a saúde (Song e
Gao, 2011). Desta forma, o maior número de trabalhos que avaliaram danos genéticos em
T. pallida expostas ao tráfego veicular demonstraram a preocupação com essas fontes
poluidoras e os danos por elas ocasionados à saúde da população. Neste cenário, o
biomonitoramento por meio do bioensaio Trad-MCN pode ser utilizado como ferramenta de
gestão prática na detecção e avaliação da poluição do ar, complementando métodos físicos e
químicos (Sposito et al., 2015). Apesar das fontes móveis serem responsáveis pela maior taxa de poluição atmosférica, Mariani et al. (2009), Savóia et al. (2009), Costa e Droste (2012), Teixeira e Barbério (2012), Barbério et al. (2013) e
Campos et al. (2016) consideraram fontes estaciónarias como importantes meios de poluição
atmosférica, e que devem ser monitoradas. O Estado de São Paulo representa o principal centro
financeiro do país com o maior e mais completo parque industrial do Brasil, aproximadamente 63.708
indústrias (IBGE, 2014) Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 505
Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017
do ar realizados em diferentes regiões brasileiras utilizando o bioensaio Trad-MCN. Região/Estado
Área
Fontes emissoras
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de pelo refe
Mariani et a
2013; De B
5; Cassaneg rio et
t al., 2 3. RESULTADOS E DISCUSSÃO Neste contexto, o monitoramento de regiões com intensa
concentração industrial, como o estado de São Paulo, constitui ferramenta importante para
avaliação dos impactos socioambientais da atividade industrial, além daqueles impactos resultantes
da interação com demais atividades antrópicas. Também foi verificada a relação entre frequência de micronúcleos e exposição a partículas
finas oriundas da queima de biomassa. A queima de biomassa é um dos exemplos de atividade
da ação humana relacionada ao aumento significativo da concentração de poluentes, causando
efeitos adversos à qualidade do ar e consequentemente a saúde da população (Cançado et al.,
2006; Magalhães et al., 2007). Sisenando et al. (2011) observaram relação entre o aumento da
frequência de micronúcleos e o aumento da exposição a partículas finas ocasionadas pela
queima da palha da cana-de-açúcar. Os autores ainda sugerem que T. pallida é um importante
indicador biológico a ser incluído na avaliação do risco humano à exposição a agentes tóxicos. O monitoramento pode ser realizado a partir de dois sistemas distintos: passivo, com a
utilização de organismos naturalmente existentes em uma área, ou ativo, com exposição de
organismos na área a ser avaliada por determinado tempo e em condições controladas (Lima,
2001). Estudos realizados por Meireles et al. (2009) e Guimarães et al. (2000), utilizando os
dois sistemas de monitoramento, observaram que o ativo foi mais sensível aos poluentes e
demonstrou maior ocorrência de micronúcleos em relação ao passivo. Verificaram que plantas
existentes naturalmente no local de estudo, monitoramento passivo, podem se adaptar as
condições ambientais as quais estão expostas, considerando assim o monitoramento ativo mais
sensível, eficaz e amplamente utilizado nos estudos da qualidade do ar. Os bioensaios desenvolvidos utilizando plantas e clones do gênero Tradescantia são
considerados ferramentas valiosas para avaliar os efeitos mutagênicos de contaminantes
ambientais. Por esta razão, desde os primórdios da toxicologia genética, várias espécies e clones
do gênero Tradescantia têm sido utilizados como organismos bioindicadores em estudos e
programas de biomonitoramento (Ma e Grant, 1982). No Brasil, a Tradescantia pallida vem sendo utilizada em substituição ao clone 4430,
fornecendo resultados satisfatórios. Resultados baseados em estudos comparativos obtidos por
Mielli et al. (2009) demonstraram que ambas as plantas apresentaram resultados semelhantes. Desta forma a T. pallida pode substituir o clone 4430 no teste de micronúcleo. 3. RESULTADOS E DISCUSSÃO pallida /clone 4430
Sudeste (SP)
Urbana
Tráfego
Ativo e passivo
T. pallida
Nordeste (BA)
Urbana
Tráfego
Ativo
T. pallida
Sudeste (SP)
Urbana
Tráfego e indústria
Ativo
T. pallida
Nordeste (BA)
Urbana
Tráfego
Ativo e passivo
Clone
Sudeste (SP)
Urbana
Tráfego e indústria
Ativo
T. pallida
Centro-Oeste (MT)
Urbana
Queima de biomassa
Ativo e passivo
T. pallida
Sul (RS)
Urbana e rural
Indústria
Ativo
T. pallida
Centro-Oeste (MS)
Urbana
Tráfego
Passivo
T. pallida
Sudeste (SP)
Urbana e rural
Tráfego e indústria
Ativo
T. pallida
Sudeste (SP)
Urbana
Indústria
Ativo
T. pallida
Sul (RS)
Urbana
Material particulado
Ativo
T. pallida
Sudeste (MG)
Urbana
Tráfego
Passivo
T. pallida
Sul (RS)
Urbana
Tráfego
Ativo
T. pallida
Centro-Oeste (MS)
Urbana
Tráfego
Ativo
T. pallida
Sudeste (MG)
Urbana
Tráfego
Passivo
T. pallida
Sudeste (SP)
Urbana
Tráfego
Ativo
T. pallida
Sudeste (SP)
Urbana
Tráfego
Ativo
T. pallida
Centro-Oeste (MS)
Urbana
Tráfego
Ativo
T. pallida
Sul (RS)
Urbana
Tráfego
Ativo
T. pallida
Sul (RS)
Urbana
Tráfego
Ativo
T. pallida
Sul (RS)
Urbana
Tráfego, indústria e agricultura
Ativo
T. pallida
Sudeste (MG)
Urbana
Indústria
Ativo
T. pallida
Sul (RS)
Urbana
Tráfego
Passivo e ativo
T. pallida
mbro (2014)
Centro-Oeste (MS)
Urbana
Tráfego
Passivo
T. pallida
amento. (a) Trabalhos que verificaram associação significativa entre frequência de micronúcleo e
dade pelo referido teste em locais com áreas poluídas. 08; Mariani et al., 2009; Meireles et al., 2009; Savóia et al., 2009; Sisenando et al., 2011; Costa e Droste, 2012;
al., 2013; De Brito et al., 2013; Pereira et al., 2013; Blume et al., 2014; Crispim et al., 2014; Pereira et al., 2014;
015; Cassanego et al., 2015; da Costa et al., 2015; Caon et al., 2016; Campos et al., 2016; da Costa et al., 2016; 2;
4;
6; o ar realizado
R
Su
No
Su
No
Su
Ce
Su
Ce
Su
Su
Su
Su
Su
Ce
Su
Su
Su
Ce
Su
Su
Su
Su
Su
bro (2014)
Ce
mento. (a) Tra
dade pelo refer
8; Mariani et a
., 2013; De B
15; Cassaneg t al., 2 trage
98) alida
mostr
o (1998 Juliana Caroline Vivian Sposito et al. 506 O processo de industrialização aumenta significativamente a contaminação atmosférica
por misturas complexas de poluentes atmosféricos, como partículas, óxidos de nitrogênio e
carbono (Costa e Droste, 2012). Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 5. AGRADECIMENTOS Os autores são gratos à Universidade Federal da Grande Dourados (UFGD) pelo seu apoio
logístico, à Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado
de Mato Grosso do Sul (FUNDECT) e à Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES). 4. CONSIDERAÇÕES FINAIS O levantamento bibliográfico permitiu concluir que a Tradescantia demonstra ser uma
planta altamente sensível e um excelente organismo teste, sendo considerada uma ferramenta
essencial no biomonitoramento de ambientes degradados. Os resultados do levantamento referente ao uso do bioensaio Trad-MCN revelaram que
poluentes atmosféricos oriundos de atividade industrial, tráfego veicular e queima de biomassa,
apresentaram potencial estresse oxidativo em Tradescantia. Desta forma, sugere-se que a
referida técnica seja utilizada como método complementar às análises químicas. 3. RESULTADOS E DISCUSSÃO Considerando
que o clone 4430 tem aplicações limitadas em países tropicais quanto ao clima e ataque de
insetos, o que limita o seu uso para estudos de biomonitoramento (Sant’Anna, 2003), tais
resultados justificam o maior número de trabalhos que utilizam a planta T. pallida ao invés do
clone. A T. pallida tem sido amplamente utilizada por ser considerada tão eficiente para estudos
de biomonitoramento quanto as plantas geneticamente selecionadas. A utilização de plantas
naturais oferece vantagens, como redução de custo, desenvolvimento em países com condição
climática tropical e subtropical e maior resistência a pragas, já que estão biologicamente
adaptadas (Batalha et al., 1999). Diante do levantamento bibliográfico, verificou-se que todos os trabalhos de
monitoramento utilizando o bioensaio Trad-MCN apresentaram relação significativa entre o
número/frequência de micronúcleos e a contaminação atmosférica ocasionada por atividade
industrial, tráfego veicular e queima de biomassa, com exceção do trabalho de De Brito et al. (2013), no qual não foi detectado genotoxicidade pelo referido ensaio em locais com áreas
poluídas. p
Segundo Santos et al. (2015), o bioensaio Trad-MCN pode servir como uma metodologia
de suporte para a adoção de políticas públicas ambientais mais restritivas no Brasil e extensível
a outros países em desenvolvimento. Além do mais, de acordo com Costa e Droste (2012), Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 507 Efetividade do ensaio Trad-MCN … Teixeira e Barbério (2012), Crispim et al. (2014), Sposito et al. (2015) e Da Costa et al. (2016),
propõe-se, que o ensaio Trad-MCN seja incluído como ferramenta adicional no monitoramento
da qualidade do ar e complementar às análises químicas. Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 6. REFERÊNCIAS ALVES, E. S.; GIUSTI, P. M.; DOMINGOS, M.; SALDIVA, P. H. N.; GUIMARÃES, E. T.;
LOBO, D. J. A. Estudo anatômico foliar do clone híbrido 4430 de Tradescantia:
alterações decorrentes da poluição aérea urbana. Revista Brasileira de Botânica, v. 24,
n. 4, p. 567-576, 2001. http://dx.doi.org/10.1590/S0100-84042001000500012 BARBÉRIO, A.; VOLTOLINI, J. C.; RIBEIRO, M. C. L. Efeitos mutagênicos da poluição
atmosférica em Tradescantia pallida no distrito de Moreira César, em Pindamonhangaba,
SP. Revista Biociências, v. 19, n. 1, p. 69-79, 2013. BATALHA, J. R. F.; GUIMARÃES, E. T.; LOBO, D. J. A.; LICHTENFELS, A. J. F. C.;
DEUR, T. et al. Exploring the clastogenic effects of air pollutants in São Paulo (Brazil)
using the Tradescantia micronuclei assay. Mutation Research/Fundamental and
Molecular Mechanisms of Mutagenesis, v. 426, n. 2, p. 229-232, 1999. http://dx.doi.org/10.1016/S0027-5107(99)00073-1 BLUME, K. K.; COSTA, G. M.; CASSANEGO, M. B. B.; DROSTE, A. Genotoxicidade do ar
em área urbana na região metropolitana de Porto Alegre, RS, Brasil. Revista Brasileira
de Biociências, v. 12, n. 3, p. 158-163, 2014. BRASIL. Departamento Nacional de Trânsito - DENATRAN. Frota de veículos no ano de
2013. 2013. Disponível em: http://www.denatran.gov.br/frota.htm. Acesso em: 10 nov. 2014. BRASIL. Departamento Nacional de Trânsito - DENATRAN. Frota de veículos no ano de
2016. 2016. Disponível em: http://www.denatran.gov.br/frota.htm. Acesso em: 10 mar. 2017. 508 Juliana Caroline Vivian Sposito et al. CAMPOS, C. F.; CAMPOS JR., E. O. D.; SOUTO, H. N.; SOUSA, E. D. F.; PEREIRA, B. B. Biomonitoring of the environmental genotoxic potential of emissions from a complex of
ceramic industries in Monte Carmelo, Minas Gerais, Brazil, using Tradescantia pallida. Journal of Toxicology and Environmental Health, v. 79, p. 123-128, 2016. http://dx.doi.org/10.1080/15287394.2015.1118714 CANÇADO, J. E.; SALDIVA, P. H.; PEREIRA, L. A.; LARA, L. B.; ARTAXO, P.;
MARTINELLI, L. A. et al. The impact of sugar cane-burning emissions on the respiratory
system of children and the elderly. Environmental health perspectives, v. 114, n. 5, p. 725-729, 2006. http://www.jstor.org/stable/3651044 CAON, K.; DA COSTA, G. M.; CASSANEGO, M. B. B.; DROSTE, A. Tradescantia pallida
var. purpurea (Commelinaceae) e biomonitoramento da genotoxicidade do ar no
município de Caxias do Sul, RS, Brasil. Pesquisas, Botânica, n. 69, p. 279-291, 2016. CARNEIRO, R. M. A.; TAKAYANAGUI, A. M. M. Estudos sobre bioindicadores vegetais e
poluição atmosférica por meio de revisão sistemática da literatura. Revista Brasileira de
Ciências Ambientais, v. 13, p. 26-44, 2009. CARVALHO, H. A. 6. REFERÊNCIAS The Tradescantia as vegetable bioindicator in monitoring clastogenic
effects of ionizing radiation. Radiologia Brasileira, v. 38, p. 459-462, 2005. CASSANEGO, M. B. B.; SASAMORI, M. H.; PETRY, C. T.; DROSTE, A. Biomonitoring the
genotoxic potential of the air on Tradescantia pallida var. purpurea under climatic
conditions in the Sinos River basin, Rio Grande do Sul, Brazil. Brazilian Journal of
Biology, v. 75, n. 4, p. 79-87, 2015. http://dx.doi.org/10.1590/1519-6984.05514 COLLINS, A. R. O cometa ensaio para danos ao DNA e reparação. Molecular biotechnology,
v. 26, n. 3, p. 249, 2004. CATINON, M.; AYRAULT, S.; DAUDIN, L.; SEVIN, L.; ASTA, J.; TISSUT, M.;
RAVANEL, P. Atmospheric inorganic contaminants and their distribution inside stem
tissues of Fraxinus excelsior L. Atmospheric environment, v. 42, n. 6, p. 1223-1238,
2008. http://dx.doi.org/10.1016/j.atmosenv.2007.10.082 COMPANHIA DE TECNOLOGIA DE SANEAMENTO AMBIENTAL - CETESB. Relatório
anual de qualidade do ar no estado de São Paulo. São Paulo, 2004. COSTA, G. M.; DROSTE, A. Genotoxicity on Tradescantia pallida var. purpurea plants
exposed to urban and rural environments in the metropolitan area of Porto Alegre,
southern Brazil. Brazilian Journal of Biology, v. 72, n. 4, p. 801-806, 2012. http://dx.doi.org/10.1590/S1519-69842012000500004 CRISPIM, B. A.; VAINI, J. O.; GRISOLIA, A. B.; TEIXEIRA, T. Z.; MUSSURY, R. M.;
SENO, L. O. Biomonitoring the genotoxic effects of pollutants on Tradescantia pallida
(Rose) D.R. Hunt in Dourados, Brazil. Environmental Science and Pollution Research,
v. 19, n. 3, p. 718-723, 2012. http://dx.doi.org/10.1007/s11356-011-0612-3 CRISPIM, B. A.; SPOSITO, J. C.; MUSSURY, R. M.; SENO, L. O.; GRISOLIA, A. B. Effects
of atmospheric pollutants on somatic and germ cells of Tradescantia pallida (Rose) DR
HUNT cv. purpurea. Anais da Academia Brasileira de Ciências, v. 86, n. 4, p. 1899-
1906, 2014. http://dx.doi.org/10.1590/0001-3765201420140338 Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 509 Efetividade do ensaio Trad-MCN … DA COSTA, G. M.; CASSANEGO, M. B. B.; PETRY, C. T.; SASAMORI, M. H.; ENDRES-
JÚNIOR, D.; DROSTE, A. Avaliação da influência do tempo de exposição de
Tradescantia pallida var. purpurea para biomonitoramento da genotoxicidade do ar
atmosférico. Revista Brasileira de Biociências, v. 13, n. 4, p. 224-230, 2015. DA COSTA, G. M.; PETRY, C. T.; DROSTE, A. Active versus passive biomonitoring of air
quality: Genetic damage and bioaccumulation of trace elements in flower buds of
Tradescantia pallida. Water, Air, & Soil Pollution, v. 227, n. 7, p. 1-12, 2016. http://dx.doi.org/10.1007/s11270-016-2923-y DE ANDRADE JR., S. J.; SANTOS JR., J. C. 6. REFERÊNCIAS S.; DA LUZ OLIVEIRA, J.; CERQUEIRA, E. D. M. M.; MEIRELES, J. R. C. Micronúcleos em tétrades de Tradescantia pallida (Rose)
Hunt. cv. purpurea Boom: alterações genéticas decorrentes de poluição aérea urbana. Acta Scientiarum Biological Sciences, v. 30, n. 3, p. 295-301, 2008. DE BRITO, K. C. T.; DE LEMOS, C. T.; ROCHA, J. A. V.; MIELLI, A. C.;
MATZENBACHER, C.; VARGAS, V. M. F. Comparative genotoxicity of airborne
particulate matter (PM2. 5) using Salmonella, plants and mammalian cells. Ecotoxicology and Environmental Safety, v. 94, n. 1, p. 14-20, 2013. http://dx.doi.org/10.1016/j.ecoenv.2013.04.014 DE SOUZA, C. P.; DE ANDRADE, G. T.; FONTANETTI, C. S. Evaluation of herbicides
action on plant bioindicators by genetic biomarkers: a review. Environmental
monitoring
and
assessment,
v. 188,
n. 12,
p. 694,
2016. http://dx.doi.org/10.1007/s10661-016-5702-8 GUIMARÃES, E. T.; DOMINGOS, M.; ALVES, E. S.; CALDINI, N.; LOBO, D. J. A.;
LICHTENFELS, A. J. F. C. et al. Detection of the genotoxicity of air pollutants in and
around the city of São Paulo (Brazil) the Tradescantia-micronucleus (Trad-MCN) assay. Environmental and Experimental Botany, v. 44, n. 1, p. 1-8, 2000. http://dx.doi.org/10.1016/S0098-8472(00)00050-2 HOLLAND, N.; BOLOGNESI, C.; KIRSCH-VOLDERS, M.; BONASSI, S.; ZEIGER, E.;
KNASMUELLER, S. et al. The micronucleus assay in human buccal cells as a tool for
biomonitoring DNA damage: the HUMN project perspective on current status and
knowledge gaps. Mutation Research/Reviews in Mutation Research, v. 659, n. 1, p. 93-108, 2008. http://dx.doi.org/10.1016/j.mrrev.2008.03.007 INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA - IBGE. Pesquisa industrial
anual: empresa. 2014. Disponível em: http://www.ibge.gov.br/estadosat/temas.php? sigla=sp&tema=piaempresa2014. Acesso em: 10 mar. 2017. KLUMPP, A.; ANSEL, W.; FOMIM, A.; SCHNIRRING, S.; PICKL, C. Influence of climatic
condition the mutations in pollen mother cells of Tradescantia clone 4430 and
implications for the Trad-MCN bioassay protocol. Hereditas, v. 141, n. 2, p. 142-148,
2004. http://dx.doi.org/10.1111/j.1601-5223.2004.01806.x LIMA, J. S. A. Utilização de bioindicadores de poluição em condições temperadas e tropicais. In: MAIA, N. B. M.; MARTOS, H. L.; BARELLA, W. (Org.). Indicadores ambientais:
conceitos e aplicações. São Paulo: Educ/Comped/Inep, 2001. LORENZI, H.; SOUZA, H. M. Plantas ornamentais no Brasil: arbustivas, herbáceas e
trepadeiras. Nova Odessa: Instituto Plantarum, 2001. Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 510 Juliana Caroline Vivian Sposito et al. MA, T. H.; SPARROW, A. H.; SCHAIRER, L. A.; NAUMAN, A. F. Effect of 1,2-
dibromoethane (DBE) on meiotic chromosomes of Tradescantia. Mutation
Research/Genetic
Toxicology,
v. 58,
n. 2-3,
p. 251-258,
1978. https://doi.org/10.1016/0165-1218(78)90016-2 MA, T. H. Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 6. REFERÊNCIAS Tradescantia micronucleus bioassay and pollen tube chromatid aberration test for
in situ monitoring and mutagen screening. Environmental Health Perspectives, v. 37,
p. 85, 1981. MA, T. H.; GRANT, W. F. The Tradescantias: adventurous plants. Herbarist, n. 48, p. 36-44,
1982. MA, T. H.; CABRERA, G. L.; CHEN, R.; GILL, B. S.; SANDHU, S. S.; VANDENBERG, A. L. et al. Tradescantia micronucleus bioassay. Mutation Research/Fundamental and
Molecular Mechanisms of Mutagenesis, v. 310, n. 2, p. 221-230, 1994. http://dx.doi.org/10.1016/0027-5107(94)90115-5 MAGALHÃES, D.; BRUNS, R. E.; VASCONCELLOS, P. C. Polycyclic aromatic
hydrocarbons as sugarcane burning tracers: a statistical approach. Química Nova, v. 30,
n. 3, p. 577-581, 2007. http://dx.doi.org/10.1590/S0100-40422007000300014 MARIANI, R. L.; JORGE, M. P. M.; PEREIRA, S. S.; MELIONE, L. P.; CARVALHO-
OLIVEIRA, R.; SALDIVA, P. H. N. Association between micronuclei frequency in
pollen mother cells of Tradescantia and mortality due to cancer and cardiovascular
diseases: A preliminary study in Sao Jose dos Campos, Brazil. Environmental Pollution,
v. 157, n. 6, p. 1767-1770, 2009. http://dx.doi.org/10.1016/j.envpol.2009.02.023 MEIRELES, J.; ROCHA, R.; COSTA NETO, A.; CERQUEIRA, E. Genotoxic effects of
vehicle traffic pollution as evaluated by micronuclei test in Tradescantia (Trad-MCN). Mutation Research/Genetic Toxicology and Environmental Mutagenesis, v. 675, n. 1, p. 46-50, 2009. http://dx.doi.org/10.1016/j.mrgentox.2009.02.005 MEIRELES, J. R. C.; CERQUEIRA, E. M. M. Use of the Micronucleus Test on Tradescantia
(Trad-MCN) to Evaluate the Genotoxic Effects of Air Pollution. In: MOLDOVEANU,
A. M. (Ed.). Air
pollution:
new
developments. [S.l.]:
InTech,
2011. http://dx.doi.org/10.5772/17959MIELLI, A. C.; MATTA, M. E. M.; NERSESYAN, A.;
SALDIVA, P. H. N.; UMBUZEIRO, G. A. Comparação entre as Respostas do Clone
4430 e Tradescantia pallida no Teste de Micronúcleos (Trad-MN). Journal of the
Brazilian Society of Ecotoxicology, v. 4, n. 1-3, 49-54, 2009. PATUSSI, C.; BÜNDCHEN, M. Avaliação in situ da genotoxicidade de triazinas utilizando o
bioensaio Trad-SHM de Tradescantia clone 4430. Revista Ciência & Saúde Coletiva,
v. 18, n. 4, p. 1173-1178, 2013. PEREIRA, B. B.; DE CAMPOS JÚNIOR, E. O.; MORELLI, S. In situ biomonitoring of the
genotoxic effects of vehicular pollution in Uberlândia, Brazil, using a Tradescantia
micronucleus assay. Ecotoxicology and environmental safety, v. 87, p. 17-22, 2013. http://dx.doi.org/10.1016/j.ecoenv.2012.10.003 Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 511 Efetividade do ensaio Trad-MCN … PEREIRA, B. B.; DE CAMPOS JR, E. O.; DE LIMA, E. A. P.; BARROZO, M. A. S.;
MORELLI, S. Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 6. REFERÊNCIAS Biomonitoring air quality during and after a public transportation strike in
the center of Uberlândia, Minas Gerais, Brazil by Tradescantia micronucleus bioassay. Environmental Science and Pollution Research, v. 21, n. 5, p. 3680-3685, 2014. http://dx.doi.org/10.1007/s11356-013-2335-0 RIBEIRO, J. E. L. S.; HOPKINS, M. J. G.; VICENTINI, A.; SOTHERS, C. A.; COSTA, M. A. S.; BRITO, J. M. et al. Flora da Reserva Ducke: Guia de identificação das plantas
vasculares de uma floresta de terra firme na Amazônia Central. Manaus: INPA-DFID,
1999. SANT’ANNA, E. T. G. Poluição atmosférica urbana na cidade de São Paulo e mutagênese:
avaliação de riscos utilizando-se bioindicadores vegetais do gênero Tradescantia,
2003. Tese (Doutorado em Fisiopatologia Experimental) - Universidade de São Paulo,
São Paulo, 2003. SANTOS, A. P. M.; SEGURA-MUÑOZ, S. I.; NADAL, M.; SCHUHMACHER, M.;
DOMINGO, J. L.; MARTINEZ, C. A. et al. Traffic-related air pollution biomonitoring
with Tradescantia pallida (Rose) Hunt. cv. purpurea Boom in Brazil. Environmental
monitoring and assessment, v. 187, n. 2, p. 1-10, 2015. http://dx.doi. org/10.1007/s10661-014-4234-3 SAVÓIA, E. J. L. Potencial de Trandescantia pallida cv. purpurea para biomonitoramento
da poluição aérea de Santo André. 2007. Tese (Doutorado em Fisiopatologia
Experimental) - Faculdade de Medicina, Universidade de São Paulo, São Paulo, 2007. SAVÓIA, E. J. L.; DOMINGOS, M.; GUIMARÃES, E. T.; BRUMATI, F.; SALDIVA, P. H. N. Biomonitoring genotoxic risks under the urban weather conditions and polluted
atmosphere in Santo André, SP, Brazil, through Trad-MCN bioassay. Ecotoxicology and
Environmental
Safety,
v. 72,
n. 1,
p. 255-260,
2009. http://dx.doi. org/10.1016/j.ecoenv.2008.03.019 SISENANDO, H. A.; DE MEDEIROS, S. R. B.; SALDIVA, P. H.; ARTAXO, P.; HACON, S. S. Genotoxic potential generated by biomass burning in the Brazilian Legal Amazon by
Tradescantia micronucleus bioassay: a toxicity assessment study. Environmental
Health, v. 10, n. 1, p. 1, 2011. http://dx.doi.org/10.1186/1476-069X-10-41 SONG, F.; GAO, Y. Size distributions of trace elements associated with ambient particular
matter in the affinity of a major highway in the New Jersey–New York metropolitan area. Atmospheric Environment, v. 45, n. 37, p. 6714-6723, 2011. http://dx.doi. org/10.1016/j.atmosenv.2011.08.031 SPEIT, G.; HARTMANN, A. The comet assay: a sensitive genotoxicity test for the detection
of DNA damage. Molecular toxicology protocols, v. 291, p. 85-95, 2005. http://dx.doi.org/10.1385/1-59259-840-4:085 SPOSITO, J. C. V.; CRISPIM, B. A.; MUSSURY, R. M.; GRISOLIA, A. B. Genetic instability
in plants associated with vehicular traffic and climatic variables. Ecotoxicology and
environmental
safety,
v. 120,
p. 445-448,
2015. http://dx.doi.org/10. 1016/j.ecoenv.2015.06.031 Rev. Ambient. Água vol. 12 n. Rev. Ambient. Água vol. 12 n. 3 Taubaté – May / Jun. 2017 6. REFERÊNCIAS 3 Taubaté – May / Jun. 2017 512 Juliana Caroline Vivian Sposito et al. SPOSITO, J. C. V.; CRISPIM, B. A.; ROMÃN, A. I.; MUSSURY, R. M.; PEREIRA, J. G.;
SENO, L. O. et al. Evaluation the urban atmospheric conditions in different cities using
comet and micronuclei assay in Tradescantia pallida. Chemosphere, v. 175, p. 108-113,
2017. http://dx.doi.org/10.1016/j.chemosphere.2017.01.136 SUMITA, N. M.; MENDES, M. E.; MACCHIONE, M.; GUIMARAES, E. T.;
LICHTENFELS, A. J. D. F. C.; LOBO, D. J. D. A. et al. Tradescantia pallida cv. purpurea boom in the characterization of air pollution by accumulation of trace elements. Journal of the Air & Waste Management Association, v. 53, n. 5, p. 574-579, 2003. http://dx.doi.org/10.1080/10473289.2003.10466197 SUYAMA, F.; GUIMARÃES, E. T.; LOBO, D. J.; RODRIGUES, G. S.; DOMINGOS, M.;
ALVES, E. S. et al. Pollen mother cells of Tradescantia clone 4430 and Tradescantia
pallida var. purpurea are equally sensitive to the clastogenic effects of X-rays. Brazilian
Journal of Medical and Biological Research, v. 35, n. 1, p. 127-129, 2002. http://dx.doi.org/10.1590/S0100-879X2002000100018 TEIXEIRA, M. C. V.; BARBÉRIO, A. Biomonitoramento do ar com Tradescantia pallida
(Rose) DR Hunt var purpurea Boom (Commelinaceae). Revista Ambiente & Água, v. 7, n. 3, 2012. http://dx.doi.org/10.4136/ambi-agua.982 WHITE, P. A.; CLAXTON, L. D. Mutagens in contaminated soil: a review. Mutation
Research/Reviews in Mutation Research, v. 567, n. 2, p. 227-345, 2004. http://dx.doi.org/10.1016/j.mrrev.2004.09.003 YAGUINUMA, D. H.; BRITO, L. G. L.; FLUMINHAN, A. Avaliação dos danos genéticos
provocados por radiação solar e poluição aérea através da análise de micronúcleos em
Tradescantia pallida cv purpurea. Fórum Ambiental da Alta Paulista, v. 10, n. 12, p. 255-269, 2014. http://dx.doi.org/10.17271/1980082710122014919 ZEIGER, E. The effect of air pollution on plants. In: TAIZ; L.; ZEIGER, E. Plant Physiology. 5th ed. Sunderland: Sinauer, 2006.
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Evolution of folate biosynthesis and metabolism across algae and land plant lineages
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Scientific reports
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Evolution of folate biosynthesis and
metabolism across algae and land
plant lineages Received: 28 August 2018
Accepted: 25 March 2019
Published: xx xx xxxx Received: 28 August 2018
Accepted: 25 March 2019
Published: xx xx xxxx V. Gorelova1,4, O. Bastien3, O. De Clerck2, S. Lespinats3, F. Rébeillé3 & D. Van Der S Tetrahydrofolate and its derivatives, commonly known as folates, are essential for almost all living
organisms. Besides acting as one-carbon donors and acceptors in reactions producing various
important biomolecules such as nucleic and amino acids, as well as pantothenate, they also supply
one-carbon units for methylation reactions. Plants along with bacteria, yeast and fungi synthesize
folates de novo and therefore constitute a very important dietary source of folates for animals. All
the major steps of folate biosynthesis and metabolism have been identified but only few have been
genetically characterized in a handful of model plant species. The possible differences in the folate
pathway between various plant and algal species have never been explored. In this study we present
a comprehensive comparative study of folate biosynthesis and metabolism of all major land plant
lineages as well as green and red algae. The study identifies new features of plant folate metabolism
that might open new directions to folate research in plants. Tetrahydrofolate (THF) and its derivatives, known as folates or B9 vitamins, are essential elements in the metab-
olism of all living organisms, except methanogenic and sulfate-reducing Archaea that use tetrahydromethano-
pterin (H4MPT) or its derivative tetrahydrosarcinapterin (H4SPT) in their C1 metabolism1. The THF molecule
is composed of a pterin moiety, para-aminobenzoic acid (pABA) and a glutamate tail. To the N5 and N10 posi-
tions of THF, one-carbon (C1) units of various oxidation states are attached (Fig. 1A). Essentially, folates act
as donors and acceptors of C1 units in C1 transfer reactions that are involved in many major metabolic pro-
cesses. Folates are used in synthesis of nucleic acids (with the exception of cytosine), amino acids, pantothenate,
formyl-methionyl-tRNA, and S-adenosyl-methionine (SAM). While animals are mainly depend on their dietary
sources for folate supply, bacteria, fungi and plants synthesize folates de novo. pp y
g
p
y
In plants, folate biosynthesis localizes to three subcellular compartments. Pterin and pABA are synthesized in
cytosol and plastids, respectively. Subsequently, the THF molecule is assembled in mitochondria, and the gluta-
mate tail is added (Fig. 1B).hi Like other folate-producing organisms, plants synthesize p-ABA in two steps. The first step is catalysed by
aminodeoxychorismate synthase (ADCS) (Fig. 1B). www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 28 August 2018
Accepted: 25 March 2019
Published: xx xx xxxx Evolution of folate biosynthesis and
metabolism across algae and land
plant lineages Precursors: GTP, guanosine triphosphate; DHN-P3, dihydroneopterin
triphosphate; DHN-P, dihydroneopterin monophosphate; DHN, dihydroneopterin; DHM, dihydromonapterin;
HMDHP, 6-hydroxymethyldihydropterin; HMDHP-P2, 6-hydroxymethyldihydropterin pyrophosphate; DHP,
dihydropteroate; DHF, dihydrofolate; THF, tetrahydrofolate; THF-Glu(n), tetrahydrofolate polyglutamate; ADC,
aminodeoxychorismate; pABA, para-aminobenzoic acid. Enzymes: GTPCHI, GTP cyclohydrolase I; DHN-P3-
diphosphatase, dihydroneopterin triphosphate pyrophosphatase; DHNA, dihydroneopterine aldolase; HPPK,
HMDHP pyrophosphokinase; DHPS, dihydropteroate synthase; DHFS, dihydrofolate synthetase; DHFR,
dihydrofolate reductase; FPGS, folylpolyglutamate synthetase; ADCS, aminodeoxychorismate synthase; ADCL,
aminodeoxychorismate lyase; GGH, gamma-glutamyl hydrolase. Figure 1. Structure of the THF molecule and folate biosynthesis. (A) Structure of THF molecule. (B)
Folate biosynthesis in higher plants. Precursors: GTP, guanosine triphosphate; DHN-P3, dihydroneopterin
triphosphate; DHN-P, dihydroneopterin monophosphate; DHN, dihydroneopterin; DHM, dihydromonapterin;
HMDHP, 6-hydroxymethyldihydropterin; HMDHP-P2, 6-hydroxymethyldihydropterin pyrophosphate; DHP,
dihydropteroate; DHF, dihydrofolate; THF, tetrahydrofolate; THF-Glu(n), tetrahydrofolate polyglutamate; ADC,
aminodeoxychorismate; pABA, para-aminobenzoic acid. Enzymes: GTPCHI, GTP cyclohydrolase I; DHN-P3-
diphosphatase, dihydroneopterin triphosphate pyrophosphatase; DHNA, dihydroneopterine aldolase; HPPK,
HMDHP pyrophosphokinase; DHPS, dihydropteroate synthase; DHFS, dihydrofolate synthetase; DHFR,
dihydrofolate reductase; FPGS, folylpolyglutamate synthetase; ADCS, aminodeoxychorismate synthase; ADCL,
aminodeoxychorismate lyase; GGH, gamma-glutamyl hydrolase. cytosolic Nudix hydrolase12. The second step is catalysed by a non-specific phosphatase13. The final step of the
synthesis of the pterin moiety is mediated by dihydroneopterin aldolase (DHNA), which cleaves the lateral side
chain of dihydroneopterin, releasing glycolaldehyde and 6-hydroxymethyldihydropterin (HMDHP)14. y
g g y
y
y
y
y
y
Synthesis of THF in mitochondria starts with pyrophosphorylation of HMDHP and its subsequent coupling
with pABA that results in the formation of dihydropteroate (DHP). These two reactions are mediated by HMDHP
pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) enzymatic activities. These two enzymes were
found to be coupled on a single polypeptide in several plant species, such as pea15,16, Arabidopsis17, rice and
wheat18. DHP is further converted to dihydrofolate (DHF) in the reaction mediated by dihydrofolate synthetase
(DHFS), which attaches the first glutamate residue to the carboxyl moiety of pABA in DHP (Fig. 1B). The penul-
timate step of folate biosynthesis is catalysed by dihydrofolate reductase (DHFR) that reduces DHF into THF. DHFR activity can be performed by a monofunctional enzyme as in mammals, or by a bifunctional protein,
coupled with thymidylate synthase (TS), as in protozoa. Among plant species, bifunctional DHFR-TS genes were
described in carrot19, pea20, maize21 and Arabidopsis22.hi In the pathway leading to THF-Glun synthesis, two reactions mediate the attachment of glutamate. The first
reaction is catalysed by DHFS. Evolution of folate biosynthesis and
metabolism across algae and land
plant lineages Similar to their fungal2,3 and protozoan4 counterparts,
Arabidopsis and tomato ADCSs exist as a bifunctional protein with two functional domains: the glutamine ami-
dotransferase domain (GAT) homologous to E. coli PabA and the chorismate binding domain homologous to E. coli PabB5,6.h The second step in the biosynthesis of pABA is mediated by aminodeoxychorismate lyase (ADCL) that catal-
yses the beta-elimination of pyruvate and aromatization of the ADC ring to produce pABA7,8. Synthesis of the pterin moiety starts with the conversion of GTP into dihydroneopterin triphosphate and
formate, catalysed by GTP cyclohydrolase I (GTPCHI) (Fig. 1B). Studies on spinach, tomato and Arabidopsis
demonstrated that plant GTPCHI has two functional domains that are essential for the enzymatic activity, as nei-
ther domain has a complete set of the residues required for substrate binding and catalysis9–11. Dihydroneopterin
undergoes dephosphorylation that proceeds in two steps. The first step is the removal of pyrophosphate by 1Department of Biology, Laboratory of Functional Plant Biology, Ghent University, K.L Ledeganckstraat 35, 9000,
Ghent, Belgium. 2Department of Biology, Phycology Research Group, Ghent University, Krijgslaan 281, 9000, Gent,
Belgium. 3Laboratoire de Physiologie Cellulaire Vegetale, UMR168 CNRS-CEA-INRA-Universite Joseph Fourier
Grenoble I, Bioscience and Biotechnologies Institute of Grenoble, CEA-Grenoble, 17 rue des Martyrs, 38054, Grenoble,
Cedex 9, France. 4Present address: Department of Botany and Plant Biology, Laboratory of Plant Biochemistry and
Physiology, University of Geneva, Quai E. Ansermet 30, 1211, Geneva, Switzerland. Correspondence and requests for
materials should be addressed to D.V.D.S. (email: Dominique.VanDerStraeten@UGent.be) 1 Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 www.nature.com/scientificreports/ Figure 1. Structure of the THF molecule and folate biosynthesis. (A) Structure of THF molecule. (B)
Folate biosynthesis in higher plants. Precursors: GTP, guanosine triphosphate; DHN-P3, dihydroneopterin
triphosphate; DHN-P, dihydroneopterin monophosphate; DHN, dihydroneopterin; DHM, dihydromonapterin;
HMDHP, 6-hydroxymethyldihydropterin; HMDHP-P2, 6-hydroxymethyldihydropterin pyrophosphate; DHP,
dihydropteroate; DHF, dihydrofolate; THF, tetrahydrofolate; THF-Glu(n), tetrahydrofolate polyglutamate; ADC,
aminodeoxychorismate; pABA, para-aminobenzoic acid. Enzymes: GTPCHI, GTP cyclohydrolase I; DHN-P3-
diphosphatase, dihydroneopterin triphosphate pyrophosphatase; DHNA, dihydroneopterine aldolase; HPPK,
HMDHP pyrophosphokinase; DHPS, dihydropteroate synthase; DHFS, dihydrofolate synthetase; DHFR,
dihydrofolate reductase; FPGS, folylpolyglutamate synthetase; ADCS, aminodeoxychorismate synthase; ADCL,
aminodeoxychorismate lyase; GGH, gamma-glutamyl hydrolase. Figure 1. Structure of the THF molecule and folate biosynthesis. (A) Structure of THF molecule. (B)
l
b
h
h h
l
T
h
h
d h d Figure 1. Structure of the THF molecule and folate biosynthesis. (A) Structure of THF molecule. (B)
Folate biosynthesis in higher plants. Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 Evolution of folate biosynthesis and
metabolism across algae and land
plant lineages SHMT is assumed to operate in the cytosol, plastids, and mitochondria29. In the latter compart-
ment, the production of 5,10-CH2-THF is mainly mediated by Gly cleavage through the action of the T-protein
of glycine decarboxylase (GDC), a reaction of paramount importance in photorespiration, typical for C3
plants. (Fig. 2). Another source of one-carbon units for folate metabolism is formate. Formate is converted to
10-formyl-tetrahydrofolate (10-CHO-THF) in the reaction catalysed by 10-CHO-THF synthetase (FTHFS)30. Subsequently, 10-CHO-THF can be metabolized in purine biosynthesis or converted to 5,10-methenyl-THF
(5,10-CH+-THF) via 5,10-CH2-tetrahydrofolate dehydrogenase/5,10-CH+-tetrahydrofolate cyclohydrolase
(MTHFD-MTHFC) (Fig. 2)31. MTHFC and MTHFD activities were found to be coupled on a bifunctional poly-
peptide in pea31 and Arabidopsis32. The reverse reaction resulting in hydrolysis of 10-formyl-THF into THF and formate is carried out by
10-CHO-THF deformylase (10-FDF) (Fig. 2). Two 10-FDF genes were characterized in Arabidopsis and found
to be crucial for photorespiration32.hhi There are two alternative ways to generate 5,10-CH+-THF. The first one involves 5-formyltetrahydrofolate
cycloligase (5-FCL) that catalyses the conversion of 5-formyltetrahydrofolate (5-CHO-THF) into 5,10-CH+-THF
(Fig. 2). Unlike other folate species, 5-CHO-THF is not used as a one-carbon (C1) donor but acts as a potent
inhibitor of SHMT and several other enzymes of C1 metabolism33. Therefore, 5-CHO-THF is considered to be a
regulator of C1 metabolism. The second way to generate 5,10-CH+-THF involves glutamate formiminotransferase
(GFT), a folate-dependent enzyme that takes part in histidine degradation in mammals and some bacteria. GFT
mediates the transfer of a formimino group from formiminoglutamate to THF, producing 5-formimino-THF
which is then converted to 5,10-CH+-THF by the action of formiminotetrahydrofolate cyclodeaminase (FTHFC)
(Fig. 2)34. Biochemical and genetic characterization of plant GFT is still poor35.hl g
g
p
p
The flux of one-carbon units from folate metabolism demands the activity of methylenetetrahydrofolate
reductase (MTHFR) that catalyses the reduction of 5,10-CH2-THF to 5-methyltetrahydrofolate (5-CH3-THF)
which enters the methyl cycle (Fig. 2). y
y
g
While biochemistry of plant folate biosynthesis and metabolism is considered well characterized, their com-
partmentalisation and regulation await further studies. The task could become utmost challenging considering
that the regulation of folate metabolism might differ between species. Examples showing that biofortification
strategies are not equally efficient for all crop species corroborate this notion36. Unfortunately, genes of folate
biosynthesis and metabolism were characterised only in few model plant species. Evolution of folate biosynthesis and
metabolism across algae and land
plant lineages In the second reaction catalysed by folylpolyglutamate synthetase (FPGS), the
polyglutamate tail of THF-Glun is formed by the sequential addition of glutamate residues to THF-Glu1 (Fig. 1B). The polyglutamate tail can be shortened or removed by the activity of vacuolar gamma-glutamyl hydrolases
(GGH) that play an important role in regulation of folate homeostasis23–25. y
g
Enzymes involved in the interconversion of various folate species are important components of folate metabo-
lism not only for folate-producing organisms but also for those that rely on their diet for the folate supply26. These
enzymes orchestrate folate metabolism and therefore regulate the flux of C1 units in response to environmental
or developmental cues. Therefore, their activities have to be tightly regulated.h ph
g
y
g
There are several sources of one-carbon units for folate metabolism in plants. Serine is an important entry
point to 5,10-methylene-tetrahydrofolate (5,10-CH2-THF) in all eukaryotes. Loading of THF occurs upon con-
version of Ser into Gly, mediated by SHMT, which hooks a methylene group onto THF, in a reversible reaction Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 2 www.nature.com/scientificreports/ Figure 2. Interconversion of folate species. THF, tetrahydrofolate; 5-CH3-THF, 5-methyltetrahydrofolate;
5,10-CH2-THF, 5,10-methylenetetrahydrofolate; 5,10-CH+-THF, 5,10-methenyltetrahydrofolate; 5-CHO-
THF, 5-formyltetrahydrofolate; 10-CHO-THF, 10-formyltetrahydrofolate. SHMT, serine hydroxymethyl
transferase; GDC, glycine decarboxylase complex, FTHFC, formiminotetrahydrofolate cyclodeaminase;
5-FCL, 5-formyltetrahydrofolate cycloligase; GFT, glutamate formiminotransferase; FTHFS, 10-CHO-THF
synthetase; MTHFD-MTHFC, 5,10- CH2-THF dehydrogenase/5,10-CH+-THF cyclohydrolase; MTHFR,
methylenetetrahydrofolate reductase; 10-FDF, 10-CHO-THF deformylase. Figure 2. Interconversion of folate species. THF, tetrahydrofolate; 5-CH3-THF, 5-methyltetrahydrofolate;
5,10-CH2-THF, 5,10-methylenetetrahydrofolate; 5,10-CH+-THF, 5,10-methenyltetrahydrofolate; 5-CHO-
THF, 5-formyltetrahydrofolate; 10-CHO-THF, 10-formyltetrahydrofolate. SHMT, serine hydroxymethyl
transferase; GDC, glycine decarboxylase complex, FTHFC, formiminotetrahydrofolate cyclodeaminase;
5-FCL, 5-formyltetrahydrofolate cycloligase; GFT, glutamate formiminotransferase; FTHFS, 10-CHO-THF
synthetase; MTHFD-MTHFC, 5,10- CH2-THF dehydrogenase/5,10-CH+-THF cyclohydrolase; MTHFR,
methylenetetrahydrofolate reductase; 10-FDF, 10-CHO-THF deformylase. (Fig. 2)27,28. SHMT is assumed to operate in the cytosol, plastids, and mitochondria29. In the latter compart-
ment, the production of 5,10-CH2-THF is mainly mediated by Gly cleavage through the action of the T-protein
of glycine decarboxylase (GDC), a reaction of paramount importance in photorespiration, typical for C3
plants. (Fig. 2). Another source of one-carbon units for folate metabolism is formate. Formate is converted to
10-formyl-tetrahydrofolate (10-CHO-THF) in the reaction catalysed by 10-CHO-THF synthetase (FTHFS)30. Subsequently, 10-CHO-THF can be metabolized in purine biosynthesis or converted to 5,10-methenyl-THF
(5,10-CH+-THF) via 5,10-CH2-tetrahydrofolate dehydrogenase/5,10-CH+-tetrahydrofolate cyclohydrolase
(MTHFD-MTHFC) (Fig. 2)31. MTHFC and MTHFD activities were found to be coupled on a bifunctional poly-
peptide in pea31 and Arabidopsis32. (Fig. 2)27,28. Results
T Taxon sampling for the analysis and identification of putative orthologs in folate metabo-
lism. Our comparative study is largely focused on land plant species. A moss Physcomitrella patens, a lycophyte
Selaginella moellendorfii and an ancient angiosperm species Amborella trichopoda were included in the study. Four monocotyledonous genomes, namely, Musa acuminata, Oryza sativa, Sorghum bicolor, Triticum aestivum
and Zea mays and sixteen dicotyledonous genomes, namely, Arabidopsis thaliana, Brassica rapa, Capsella rubella,
Carica papaya, Citrus sinensis, Cucumis sativus, Fragaria vesca, Glycine max, Gossipium raimondii, Linum usitatis-
simum, Medicago truncatula, Phaseolus vulgaris, Populus trichocarpa, Solanum lycopersicum, Solanum tuberosum
and Vitis vinifera were analysed. f
y
Several green algae members of Charophyta, namely, Ostreococcus tauri, Ostreococcum lucimarinus and
Micromonas pusilla (Prasinophyceae), Chlamydomonas reinhardtii and Volvox carteri (Chlorophyceae), Chlorella
vulgaris and Chlorella variabilis (Trebouxiophyceae), Ulva mutabilis (Ulvophyceae), and Klebsormidium flaccidum
(Klebsormidiophyceae) were used in the study. Two red algae, namely, Chondrus crispus (Florideophyceae) and
Cyanidioschyzon merolae (Cyanidiophyceae) were also covered. To identify putative orthologs of folate pathway genes in the species listed above, full-length protein sequences
from Arabidopsis thaliana were used as a query to run a BLAST protein sequence alignment37. To infer putative
orthology the selected protein sequences were scrutinized for the presence of functional protein domains using
the Pfam database38. The selected protein sequences were also surveyed for the presence of subcellular targeting
signals and for the pattern of conserved protein motifs. ADCS. Our comparative analysis shows that ADCS is encoded by a single copy gene in genomes of all analysed
algal species as well as in most analysed higher plant species (Fig. 3). Being in line with reported targeting of
Arabidopsis ADCS to chloroplasts7 our prediction of the subcellular localization suggested that ADCS homologs
in algae and higher plants possess chloroplast targeting signals (Fig. 3). Examination of selected protein sequences for the presence of functional domains revealed that besides glu-
tamine amidotransferase domain (GAT) and chorismate binding domains, algal and higher plant ADCS bear
homology to the N terminal region domain of anthranilate synthase component I (alpha subunit) (Fig. 3). ADCS
proteins from P. patens, P. trichocarpa, F. vesca, A. thaliana and C. rubella contain one or two additional domains
(Fig. 3), possibly manifesting a process of neofunctionalization of ADCS within these species. The motif pat-
tern (sequence within which motifs appear) is conserved throughout algal and land plant species (Supplemental
Fig. 1). ADCL. Results
T While algae possess a single copy of ADCL, the majority of higher plant species contains two or three
ADCL genes (Fig. 4). Like those of algae, the genomes of A. thaliana, C. rubella and B. rapa also encode a single
ADCL gene, indicating loss of the second ADCL gene during evolution of Brassicaceae. Our phylogenetic study
revealed that ADCL genes of higher plants fall into two clades (Fig. 4). Interestingly, each clade contains an
ADCL homolog from every analysed higher plant species. The presence of an ADCL gene from the A. trichopoda
genome in both clades suggests that the divergence of ADCL genes occurred before diversification of flower-
ing plants. The prediction of the subcellular localization revealed that all proteins from the clade containing A. trichopoda_XP_006833333.1 have a chloroplast localization signal, whereas proteins from the clade containing
the A. trichopoda_XP_011623669.2 isoform are exclusively cytosolic (Fig. 4). Moreover, the P. patens genome also
encodes two cytosolic and one plastidial ADCL isoform, further confirming that duplication and diversification
of ADCL genes occurred during early evolution of land plants (Fig. 4). The plastidial localization of GFP-tagged
ADCL from Arabidopsis has been previously demonstrated7, while a confirmation for cytosolic ADCL remains
unreported. All analysed ADCL polypeptides contain a single Aminotransferase class I and II (PF00155.20) domain
(Fig. 4). The motif pattern is well conserved among the analysed algal and higher plant species (Supplemental
Fig. 2). GTPCHI. In silico analysis demonstrates that while algae have a single GTPCHI isoform, the majority of ana-
lysed higher plant species possesses two or more GTPCHI isoforms (Supplemental Fig. 3). Prediction of sub-
cellular targeting suggested cytosolic localization of all analysed proteins, supporting the experimental data for
Arabidopsis and tomato GTPCHI proteins11.i p
p
Our findings reveal the presence of two GTPCHI domains throughout algae and higher plant lineages, with
the exception of L. usitatissimum GTPCHI that possesses three domains, the first being duplicated (Supplemental
Fig. 3). Remarkably, the two GTPCHI domains show very different conserved motif composition, that further
suggests their strong divergence (Supplemental Fig. 4). HPPK-DHPS. Genomes of the majority of analysed algae and higher plant species contain a single
HPPK-DHPS gene (Supplemental Fig. 5). Prediction of their subcellular localization suggested mitochondrial
localization of the majority of studied HPPK-DHPS isoforms (Supplemental Fig. 5). This finding is in line with
experimentally determined mitochondrial targeting of HPPK-DHPS proteins in pea15 and Arabidopsis39. Evolution of folate biosynthesis and
metabolism across algae and land
plant lineages Increasing availability of
genome-scale data provides an excellent opportunity to trace the origin of the pathway components in algae, to
identify possible differences in regulation and compartmentalisation of folate metabolism in various plant species
and to plan future biofortification approaches that will take into account individual features of folate metabolism
of a given species. To identify the possible differences and to trace their emergence during evolution, we ventured
into a comparative genomic and phylogenetic study including all major land plant lineages as well as red and
green algae. Our study provides a comprehensive view on the folate biosynthesis and metabolism throughout the
plant kingdom and points out novel aspects of folate metabolism. We demonstrate that bifunctionality of enzymes
in folate biosynthesis and enzymes involved in interconversion of folate species is a common feature for algae
and higher plant species. Our comparative study shows that the number of genes, localization and the structure
of the isoforms they encode is highly conserved across algae and land plants. Moreover, our findings underscore Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 3 www.nature.com/scientificreports/ the notion that folate metabolism of different subcellular compartments is characterized by specific sources of
one-carbon units. Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 Results
T Our comparative study shows that the bifunctionality is conserved across algae and land plant species, as
HPPK and two DHPS domains (DHPS1 and DHPS2) were identified in most analysed proteins (Supplemental
Fig. 5). While all higher plant species possess two DHPS domains, some algae and lower land plant species (S. moellendorfii and P. patens) have only one DHPS domain. P. trichocarpa, C. rubella and S. bicolor proteins contain Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Phylogenetic analysis, subcellular localization and domain composition of ADCS proteins. Species
names are followed by protein identifiers. The bar indicates the mean distance of 2.0 changes per amino acid
residue. The numbers at the branching points indicate the percentage of times that each branch topology was
found during bootstrap analysis (n = 1000). The box contains predicted functional domains. Schemes on the
right represent domain organisation of analysed proteins (colored boxes represent functional domains, lengths
of black lines correspond to lengths of proteins. The scale bar below shows protein containing 500 amino acids). Cyt, cytosolic localization; chl, plastidial localization. Indication of double localization (e.g. cyt chl) for a single
protein implies its probable localization to both compartments. Figure 3. Phylogenetic analysis, subcellular localization and domain composition of ADCS proteins. Species
names are followed by protein identifiers. The bar indicates the mean distance of 2.0 changes per amino acid
residue. The numbers at the branching points indicate the percentage of times that each branch topology was
found during bootstrap analysis (n = 1000). The box contains predicted functional domains. Schemes on the
right represent domain organisation of analysed proteins (colored boxes represent functional domains, lengths
of black lines correspond to lengths of proteins. The scale bar below shows protein containing 500 amino acids). Cyt, cytosolic localization; chl, plastidial localization. Indication of double localization (e.g. cyt chl) for a single
protein implies its probable localization to both compartments. additional domains (Supplemental Fig. 5). Our in silico protein analysis indicates that the motif pattern is well
conserved (Supplemental Fig. 6). DHFS. Phylogenetic analysis revealed that DHFS is encoded by a single copy gene in genomes of most ana-
lysed species, with the exception of G. max and M. truncatula genomes which are characterized by high poly-
ploidy (Supplemental Fig. 7). Results
T Being in line with experimental evidence for Arabidopsis DHFS39, our analysis
suggested that DHFS in higher plant species localizes to mitochondria, while in algae it can also localize to cytosol
or plastids (Supplemental Fig. 7). p
(
pp
g
)
In silico analysis revealed that DHFS proteins contain the same set of functional domains as FPGS polypep-
tides (see below): FPGS1, FPGS2 and muramyl ligase (Supplemental Fig. 7). Although DHFS and FPGS share
similar biochemical function, sequences of these two proteins diverged immensely. The divergence is further Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 5 www.nature.com/scientificreports/ Figure 4. Phylogenetic analysis, subcellular localization and domain composition of ADCL proteins. Species
names are followed by protein identifiers. The bar indicates the mean distance of 0.6 changes per amino acid
residue. The numbers at the branching points indicate the percentage of times that each branch topology was
found during bootstrap analysis (n = 1000). Schemes on the right represent domain organisation of analysed
proteins (color boxes represent functional domains, lengths of black lines correspond to lengths of proteins. The
scale bar below shows protein containing 500 amino acids). The box contains predicted functional domains. Cyt, cytosolic localization; chl, plastidial localization. Figure 4. Phylogenetic analysis, subcellular localization and domain composition of ADCL proteins. Species
names are followed by protein identifiers. The bar indicates the mean distance of 0.6 changes per amino acid
residue. The numbers at the branching points indicate the percentage of times that each branch topology was
found during bootstrap analysis (n = 1000). Schemes on the right represent domain organisation of analysed
proteins (color boxes represent functional domains, lengths of black lines correspond to lengths of proteins. The
scale bar below shows protein containing 500 amino acids). The box contains predicted functional domains. Cyt, cytosolic localization; chl, plastidial localization. illustrated by the protein sequence analysis which demonstrates that DHFS and FPGS proteins have very dif-
ferent patterns of conserved motifs (compare Supplemental Figs 8 and 11). It has been previously reported that
Arabidopsis DHFS is more similar to the DHFS-FPGS gene from E. coli than to Arabidopsis FPGS genes39. DHFR. Our phylogenetic analysis demonstrates that single-copy DHFR-TS genes are present in algal genomes,
while DHFR-TS in higher plant species is encoded by two or more genes (Supplemental Fig. 9). Prediction
of subcellular localization suggested that DHFR-TS isoforms can be targeted to multiple compartments
(Supplemental Fig. Results
T 9), corroborating our previous finding of multiple subcellular targeting of DHFR-TS isoforms
in Arabidopsis40. The data in Supplemental Fig. 9 demonstrate that genomes of all analysed higher plant and algal
species encode bifunctional DHFR-TS enzymes. p
y
A previous study of Arabidopsis DHFR-TS demonstrated that one of the isoforms lacks enzymatic activity and
acts as an inhibitor of its family members. Phylogenetic analysis suggested that such inhibitory isoforms may be
also present in Arabidopsis’ closest relatives, B. rapa and C. rubella40. The present study shows that the inhibitory
isoforms lack the last protein motif, while all other analysed DHFR-TS proteins retain it (Supplemental Fig. 10). It is plausible that this motif is important for the DHFR and TS activities, possibly being essential for a proper
protein conformation. FPGS. The present study shows that, unlike algae, land plant species possess two or more FPGS isoforms. Being in line with experimental data39,41 our prediction of subcellular localization suggests that the isoforms Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 6 www.nature.com/scientificreports/ Figure 5. Phylogenetic analysis, subcellular localization and domain composition of FPGS proteins. Species
names are followed by protein identifiers. The bar indicates the mean distance of 1.0 change per amino acid
residue. The numbers at the branching points indicate the percentage of times that each branch topology was
found during bootstrap analysis (n = 1000). Schemes on the right represent domain organisation of analysed
proteins (color boxes represent functional domains, lengths of black lines correspond to lengths of proteins. The
scale bar below shows protein containing 500 amino acids). The box contains predicted functional domains. Cyt, cytosolic localization; chl, plastidial localization; mit, mitochondrial localization. Indication of double
localization (e.g. cyt chl) for a single protein implies its probable localization to both compartments. Figure 5. Phylogenetic analysis, subcellular localization and domain composition of FPGS proteins. Species
names are followed by protein identifiers. The bar indicates the mean distance of 1.0 change per amino acid
residue. The numbers at the branching points indicate the percentage of times that each branch topology was
found during bootstrap analysis (n = 1000). Schemes on the right represent domain organisation of analysed
proteins (color boxes represent functional domains, lengths of black lines correspond to lengths of proteins. The
scale bar below shows protein containing 500 amino acids). The box contains predicted functional domains. Cyt, cytosolic localization; chl, plastidial localization; mit, mitochondrial localization. Indication of double
localization (e.g. Results
T usitatissimum plastidial
MTHFD-MTHFC isoforms that bear an additional domain identified as ACT (Supplemental Figs 20–22). Our
study shows that the motif pattern is well conserved (Supplemental Figs 23–25). MTHFD-MTHFC. In silico analysis indicates that higher plant species contain multiple MTHFD-MTHFC
isoforms that fall into three clades, according to their predicted subcellular localization in cytosol, mitochon-
dria and plastids, each comprising a gene from A. trichopoda (Supplemental Figs 20–22). Each clade includes a
MTHFD-MTHFC homolog from A. trichopoda, suggesting their emergence early during speciation of flowering
plants. Analysis of protein sequences showed that all analysed proteins possess the NAD(P)-binding (PF02882.18)
and MTHFD-MTHFC catalytic (PF00763.22) domains, with the exception of two L. usitatissimum plastidial
MTHFD-MTHFC isoforms that bear an additional domain identified as ACT (Supplemental Figs 20–22). Our
study shows that the motif pattern is well conserved (Supplemental Figs 23–25). 5-FCL. Supplemental Fig. 26 illustrates a comparative analysis for 5-FCL. It is encoded by a single gene in algal
genomes, in contrast to some higher plant species, which contain two 5-FCL coding genes. As P. patens genome
contains two 5-FCL genes, its duplication presumably occurred during speciation of land plants or earlier. Prediction of subcellular localization has shown that the analysed 5-FCL proteins are mainly targeted to mito-
chondria but can also reside in plastids and in the cytosol (Supplemental Fig. 26). The prediction is in line with
the experimentally determined mitochondrial localization of 5-FCL activity in pea43. All analysed proteins have a
similar motif pattern (Supplemental Fig. 27) and bear a single 5-formyltetrahydrofolate cycloligase (PF01812.19)
functional domain (Supplemental Fig. 26); no other domains could be identified. 5-FCL. Supplemental Fig. 26 illustrates a comparative analysis for 5-FCL. It is encoded by a single gene in algal
genomes, in contrast to some higher plant species, which contain two 5-FCL coding genes. As P. patens genome
contains two 5-FCL genes, its duplication presumably occurred during speciation of land plants or earlier. Prediction of subcellular localization has shown that the analysed 5-FCL proteins are mainly targeted to mito-
chondria but can also reside in plastids and in the cytosol (Supplemental Fig. 26). The prediction is in line with
the experimentally determined mitochondrial localization of 5-FCL activity in pea43. All analysed proteins have a
similar motif pattern (Supplemental Fig. 27) and bear a single 5-formyltetrahydrofolate cycloligase (PF01812.19)
functional domain (Supplemental Fig. 26); no other domains could be identified. GFT. Results
T In previous work it was shown that mit AtFPGS, lacking the FPGS1 domain, could complement a yeast FPGS
mutant39, indicating that the absence of this domain did not impair the activity. Prediction of targeting signals
suggests that subcellular localization of FPGS proteins varies within each clade (Fig. 5). This indicates that local-
ization signals of FPGS were gained in each lineage individually. In previous work it was shown that mit AtFPGS, lacking the FPGS1 domain, could complement a yeast FPGS
mutant39, indicating that the absence of this domain did not impair the activity. Prediction of targeting signals
suggests that subcellular localization of FPGS proteins varies within each clade (Fig. 5). This indicates that local-
ization signals of FPGS were gained in each lineage individually. GGH. Our analysis shows that GGH is encoded by multiple genes in several algal and land plant genomes
(Supplemental Fig. 12). Interestingly, GGH is the only enzyme of the folate pathway that is encoded by several
genes in genomes of algae species. This observation corroborates the notion that the enzyme plays an important
role in regulation of folate pool24 and highlights the demand of a more profound investigation of its function. Being in line with previous studies of GGH enzymes from Arabidopsis and tomato23,25 our subcellular localization
analysis predicted existence of secretory signals in all analysed GGH proteins (Supplemental Fig. 12). Protein
analysis revealed that all analysed GGH proteins possess a single peptidase C26 (PF07722.13) functional domain
(Supplemental Fig. 12) and show a highly conserved motif pattern (Supplemental Fig. 13). GDC and SHMT. The in silico analysis revealed that the GDC T-protein is encoded by a single gene in algal
genomes, while land plant species possess single or multiple GDC T-protein genes (Supplemental Fig. 14). The
predicted mitochondrial localization of the analysed GDC isoforms is in line with its role in photorespiration42. Protein analysis suggested the presence of two functional domains: an aminomethyltransferase folate-binding
domain (PF01571.20) and a glycine cleavage T-protein C-terminal barrel domain (PF08669.10). Phylogenetic
analysis suggested that genomes of both algae and land plants encode multiple SHMT isoforms that fall into
three big clades. According to our prediction of subcellular localization, each clade comprises isoforms with
either mitochondrial, cytosolic or plastidial localization, confirming previous findings in pea and potato20,28
(Supplemental Fig. 15). GDC and SHMT. Results
T The in silico analysis revealed that the GDC T-protein is encoded by a single gene in algal
genomes, while land plant species possess single or multiple GDC T-protein genes (Supplemental Fig. 14). The
predicted mitochondrial localization of the analysed GDC isoforms is in line with its role in photorespiration42. Protein analysis suggested the presence of two functional domains: an aminomethyltransferase folate-binding
domain (PF01571.20) and a glycine cleavage T-protein C-terminal barrel domain (PF08669.10). Phylogenetic
analysis suggested that genomes of both algae and land plants encode multiple SHMT isoforms that fall into
three big clades. According to our prediction of subcellular localization, each clade comprises isoforms with
either mitochondrial, cytosolic or plastidial localization, confirming previous findings in pea and potato20,28
(Supplemental Fig. 15). FTHFS. Our comparative study demonstrates that FTHFS is encoded by a single gene in all examined species,
with the exception of G. max, L. usitatissimum and P. trichocarpa that have two FTHFS genes (Supplemental
Fig. 16). In agreement with the finding that FTHFS activity is associated with the cytosolic fraction of pea cot-
yledons, our prediction of subcellular localization suggested that FTHFS resides exclusively in the cytosol of all
analysed algae and land plant species (Supplemental Fig. 16). Protein analysis revealed that all analysed FTHFS
proteins possess a single formate-tetrahydrofolate ligase (PF01268.18) functional domain (Supplemental Fig. 16)
and show a highly conserved motif pattern (Supplemental Fig. 17). 10-FDF. Phylogenetic analysis reveals the presence of single genes encoding 10-FDF in algal genomes, whereas
land plant species might possess several 10-FDF genes (Supplemental Fig. 18). Being in agreement with reported
mitochondrial targeting of Arabidopsis 10-FDF isoforms and their role in photorespiration32, our subcellular
localization prediction suggests mitochondrial localization of the analysed 10-FDF proteins (Supplemental
Fig. 18). Our protein analysis demonstrates that all analysed 10-FDF bear a single formate-tetrahydrofolate ligase
domain (PF01268.18) and have a highly similar motif pattern (Supplemental Figs 18 and 19). MTHFD-MTHFC. In silico analysis indicates that higher plant species contain multiple MTHFD-MTHFC
isoforms that fall into three clades, according to their predicted subcellular localization in cytosol, mitochon-
dria and plastids, each comprising a gene from A. trichopoda (Supplemental Figs 20–22). Each clade includes a
MTHFD-MTHFC homolog from A. trichopoda, suggesting their emergence early during speciation of flowering
plants. Analysis of protein sequences showed that all analysed proteins possess the NAD(P)-binding (PF02882.18)
and MTHFD-MTHFC catalytic (PF00763.22) domains, with the exception of two L. Results
T cyt chl) for a single protein implies its probable localization to both compartments. Figure 5. Phylogenetic analysis, subcellular localization and domain composition of FPGS proteins. Species
names are followed by protein identifiers. The bar indicates the mean distance of 1.0 change per amino acid
residue. The numbers at the branching points indicate the percentage of times that each branch topology was
found during bootstrap analysis (n = 1000). Schemes on the right represent domain organisation of analysed
proteins (color boxes represent functional domains, lengths of black lines correspond to lengths of proteins. The
scale bar below shows protein containing 500 amino acids). The box contains predicted functional domains. Cyt, cytosolic localization; chl, plastidial localization; mit, mitochondrial localization. Indication of double
localization (e.g. cyt chl) for a single protein implies its probable localization to both compartments. localize to multiple subcellular compartments, namely, mitochondria, cytosol and plastids (Fig. 5). Analysis of
protein sequences revealed the presence of three functional domains in FPGS polypeptides: a muramyl ligase
domain and two FPGS domains (FPGS1 and FPGS2). Our phylogenetic analysis demonstrated that FPGS of
higher plants branch into two clades, each containing a gene from A. trichopoda (Fig. 5). The two clades differ by
their domain composition. The clade containing the A. trichopoda_ERN08073.1 isoform retains both FPGS func-
tional domains, while the clade comprising the A. trichopoda_XP_020522267 isoform lacks the FPGS1 domain. localize to multiple subcellular compartments, namely, mitochondria, cytosol and plastids (Fig. 5). Analysis of
protein sequences revealed the presence of three functional domains in FPGS polypeptides: a muramyl ligase
domain and two FPGS domains (FPGS1 and FPGS2). Our phylogenetic analysis demonstrated that FPGS of
higher plants branch into two clades, each containing a gene from A. trichopoda (Fig. 5). The two clades differ by
their domain composition. The clade containing the A. trichopoda_ERN08073.1 isoform retains both FPGS func-
tional domains, while the clade comprising the A. trichopoda_XP_020522267 isoform lacks the FPGS1 domain. Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 7 www.nature.com/scientificreports/ In previous work it was shown that mit AtFPGS, lacking the FPGS1 domain, could complement a yeast FPGS
mutant39, indicating that the absence of this domain did not impair the activity. Prediction of targeting signals
suggests that subcellular localization of FPGS proteins varies within each clade (Fig. 5). This indicates that local-
ization signals of FPGS were gained in each lineage individually. Results
T GFT appears to be encoded by a single gene in algae, whereas genomes of land plants can contain single
or multiple GFT genes (Supplemental Fig. 28). Subcellular localization prediction suggested that GFT isoforms
predominantly reside in the cytosol in higher plants, but can also be targeted to both mitochondria and plastids,
as for instance, in A. trichopoda, C. sinensis, L. usitatissimum, O. sativa, P. trichocarpa and Z. mays (Supplemental
Fig. 28). Protein analysis demonstrated that GFT proteins possess a single formiminotransferase domain
(PF07837.11) and show a well conserved motif pattern throughout algal and land plant lineages (Supplemental
Fig. 29). G. raimondii, L. usitatissimum, C. sinensis and C. sativus bear an additional domain. GFT. GFT appears to be encoded by a single gene in algae, whereas genomes of land plants can contain single
or multiple GFT genes (Supplemental Fig. 28). Subcellular localization prediction suggested that GFT isoforms
predominantly reside in the cytosol in higher plants, but can also be targeted to both mitochondria and plastids,
as for instance, in A. trichopoda, C. sinensis, L. usitatissimum, O. sativa, P. trichocarpa and Z. mays (Supplemental
Fig. 28). Protein analysis demonstrated that GFT proteins possess a single formiminotransferase domain
(PF07837.11) and show a well conserved motif pattern throughout algal and land plant lineages (Supplemental
Fig. 29). G. raimondii, L. usitatissimum, C. sinensis and C. sativus bear an additional domain. MTHFR. Our comparative analysis illustrates that unlike those of algal species, genomes of most land plant
species encode several MTHFR isoforms (Supplemental Fig. 30). Prediction of subcellular localization suggested
that MTHFR of algae and land plant species reside in the cytosol (Supplemental Fig. 30). The cytosolic locali-
zation of MTHFR is in line with the assumption that the cytosol is the predominant location of the one-carbon
flux from folate metabolism toward methionine production44. Analysis of protein sequences demonstrated that MTHFR. Our comparative analysis illustrates that unlike those of algal species, genomes of most land plant
species encode several MTHFR isoforms (Supplemental Fig. 30). Prediction of subcellular localization suggested
that MTHFR of algae and land plant species reside in the cytosol (Supplemental Fig. 30). The cytosolic locali-
zation of MTHFR is in line with the assumption that the cytosol is the predominant location of the one-carbon
flux from folate metabolism toward methionine production44. Analysis of protein sequences demonstrated that Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 8 www.nature.com/scientificreports/ Figure 6. Results
T Phylogenetic trees of the protein sequences of the TS domain constructed using maximum likelihood
method Phyml (see text). The selected model using Bayesian information criterion was LG + G + I with
gamma shape parameter estimate = 1.117 and the proportion of invariable site estimate = 0.207. Nodes values
represents the Bayesian posterior probabilities branch support. The species colour code corresponds to the type
of plastid pigments, as follows: purple, chlorophyll a; green, chlorophyll a and b; red, chlorophyll a and c. Figure 6. Phylogenetic trees of the protein sequences of the TS domain constructed using maximum likelihood
method Phyml (see text). The selected model using Bayesian information criterion was LG + G + I with
gamma shape parameter estimate = 1.117 and the proportion of invariable site estimate = 0.207. Nodes values
represents the Bayesian posterior probabilities branch support. The species colour code corresponds to the type
of plastid pigments, as follows: purple, chlorophyll a; green, chlorophyll a and b; red, chlorophyll a and c. MTHFR of algae and land plants contain a single MTHFR functional domain (Supplemental Fig. 28) and show a
well conserved motif pattern (Supplemental Fig. 31). Evolution of DHFR-TS and HPPK-DHPS in plants versus other organisms. In the green lineage, the
assembly of the folate molecule occurs within the mitochondria (Fig. 1) and involves two bifunctional enzymes,
DHFR-TS and HPPK-DHPS. This is not always the case in other organisms where these activities can be driven
by monofunctional enzymes. It is well recognized that the fusion between DHFR and TS is a milestone in evolu-
tion, illustrating the separation between unikonts and bikonts45. To test whether the other bifunctional enzyme
of the folate biosynthesis pathway follows the same evolutionary history as DHFR-TS, we compared the phyloge-
netic evolution of the TS and DHPS domains in wide range of organisms, including bacteria, protists (Alveolata,
fungi, red algae (Rhodophyta), Rhizaria, stramenopiles), plants and animals. TS. The phylogeny based on the maximum likelihood (ML) for the TS domain from 22 species representative
of the various kingdoms clearly illustrates the evolutionary separation between organisms having monofunctional
TS (unikonts) versus those having bifunctional DHFR-TS (bikonts) (Fig. 6).h g
g
Thus, the phylogeny of the TS domain clearly revealed a monophyletic origin of the bikont clade. To obtain
a better idea of the relative distance between these various species, we represented the ML phylogeny in a
three-dimensional space (Fig. Results
T In this figure, the distance between two data points on the figure tends to display the distance between species. Links between points show the ML phylogenic tree presented in the Fig. 6. Blue branches: monofunctional
TS, red branches: bifunctional TS. Brown letters, bikont cluster; brown letters in bold, the green lineage; black
letters, unikont cluster. Abbreviations for genus names: Arab_tha1, Arabidopsis thaliana 1; Ory_sat1, Oryza
sativa 1; Chla_rei, Chlamydomonas reinhardtii; Chla_eug, Chlamydomonas eustigma; Ostr_tau, Ostreococcus
tauri; Plas_ber, Plasmodium berghei; Plas_cha, Plasmodium chabaudi; Toxo_gon, Toxoplasma gondii; Neos_can,
Neospora caninum; Cyan_mer, Cyanidioschyzon merolae; Leis_maj, Leishmania major strain Friedlin; Phae_tri,
Phaeodactylum tricornutum; Thal_pse, Thalassiosira pseudonana; Phyt_soj, Phytophthora sojae; Mus_musc, Mus
musculus; Rat_nor, Rattus norvegicus; Neur_cra, Neurospora crassa; Neur_tet, Neurospora tetrasperma; Sach_
cer, Saccharomyces cerevisiae; Metha_ja, Methanocaldococcus jannaschii; Baci_sub, Bacillus subtilis; Baci_hal,
Bacillus halodurans. Figure 7. Phylogenetic representation in the protein sequence space for TS domains. Non-linear mapping
methods are designed to offer a configuration of points in this multidimensional space that is representative of
the observed distances. With this method, axes become arbitrary and every rotation or symmetry is admissible. In this figure, the distance between two data points on the figure tends to display the distance between species. Links between points show the ML phylogenic tree presented in the Fig. 6. Blue branches: monofunctional
TS, red branches: bifunctional TS. Brown letters, bikont cluster; brown letters in bold, the green lineage; black
letters, unikont cluster. Abbreviations for genus names: Arab_tha1, Arabidopsis thaliana 1; Ory_sat1, Oryza
sativa 1; Chla_rei, Chlamydomonas reinhardtii; Chla_eug, Chlamydomonas eustigma; Ostr_tau, Ostreococcus
tauri; Plas_ber, Plasmodium berghei; Plas_cha, Plasmodium chabaudi; Toxo_gon, Toxoplasma gondii; Neos_can,
Neospora caninum; Cyan_mer, Cyanidioschyzon merolae; Leis_maj, Leishmania major strain Friedlin; Phae_tri,
Phaeodactylum tricornutum; Thal_pse, Thalassiosira pseudonana; Phyt_soj, Phytophthora sojae; Mus_musc, Mus
musculus; Rat_nor, Rattus norvegicus; Neur_cra, Neurospora crassa; Neur_tet, Neurospora tetrasperma; Sach_
cer, Saccharomyces cerevisiae; Metha_ja, Methanocaldococcus jannaschii; Baci_sub, Bacillus subtilis; Baci_hal,
Bacillus halodurans. DHPS. The maximum likelihood phylogenetic tree obtained for the DHPS domain appears very different
from that of TS. In this case also, we identified organisms having a monofunctional DHPS and others having a
bifunctional HPPK-DHPS (Fig. 8). DHPS. The maximum likelihood phylogenetic tree obtained for the DHPS domain appears very different
from that of TS. In this case also, we identified organisms having a monofunctional DHPS and others having a
bifunctional HPPK-DHPS (Fig. 8). ( g
)
Animals, which depend on their diet for folate supply, do not possess DHPS activity. Results
T 7).hi p
g
This figure represents a ‘sequence space’. It is the projection in 3D of the highly multidimensional space con-
taining all the possible sequences of the TS domain46,47. To assign a position to each sequence in this three dimen-
sional space, non-linear mapping methods are used to offer a configuration of points preserving as much as
possible the distances observed between the various sequences in the original space (see Material and Methods). With such methods, axes become arbitrary and every rotation or symmetry is admissible48. The meaning of this
representation is therefore essentially carried by distance: the distance between two points on the figure tends
to display the distance between species. Links between points show the ML phylogenic tree, and thus the figure
combines distance-based and ML-based phylogenies. p y g
Such representation can provide additional information compared to the previous one. For example,
Toxoplasma gondii, an apicomplexan having a bifunctional DHFR-TS, was positioned far from other apicom-
plexa but close to organisms such as Leishmania major, an excavate, a situation different from that seen in Fig. 6. This illustrates that two points relatively close in the sequence space are not necessarily close from an evolutionary
point of view. For example, Bigelowiella natans and Mus musculus, or Toxoplasma gondii and Saccharomyces
cerevisiae may appear close in the sequence space whereas they are not related in the ML tree. In other words, the
evolutionary distances (as shown in the ML tree) can be large, despite relative similarities between the sequences. It is also clear that monofunctional (in blue) and bifunctional (in red) branches occupy a different space in this
three-dimensional representation, indicating a totally independent evolution of the TS domain after its fusion
with DHFR. In addition, the location of the Toxoplasma/Neospora group, far from that of the Plasmodium group
in the sequence space could explain the relatively low support value of 0.57 (Fig. 6) calculated for the branch sep-
aration leading to Chlamydomonas on one hand and alveolates on the other hand. Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 9 9 www.nature.com/scientificreports/ Figure 7. Phylogenetic representation in the protein sequence space for TS domains. Non-linear mapping
methods are designed to offer a configuration of points in this multidimensional space that is representative of
the observed distances. With this method, axes become arbitrary and every rotation or symmetry is admissible. Results
T Most of the mono-
functional DHPS are found in bacteria (unikonts), but some bacteria display also a bifunctional enzyme. From
this point of view, it is interesting to note that within gamma proteobacteria there are species displaying mono-
functional DHPS (Escherichia coli, Salmonella typhi) whereas others (Francisella philomiragia, Ricketsellia grylli)
display a bifunctional HPPK-DHPS. This is also true for the alpha proteobacteria (containing Rickettsia bellii,
Wolbachia), while beta proteobacteria (not represented here) and firmicutes (Bacillus subtilis, Staphylococcus
aureus) contain mostly monofunctional DHPS. These data raise the question of the origin of the fusion of the two
domains. Likewise, fungi (unikonts) also display a bifunctional HPPK-DHPS, as found in plants (bikonts). Thus,
the separation unikonts/bikonts does not apply for this enzyme, suggesting that the evolutionary events leading
to the two bifunctional enzymes HPPK-DHPS and DHFR-TS were completely different. The 3D representation
(Fig. 9) also shows a different situation compared with the one obtained with the TS domain. In this case, mono-
functional and bifunctional DHPS do not occupy a different space. Monofunctional enzymes (blue branches)
occupy a small space compared to the bifunctional HPPK-DHPS (red branches) which is widely spread within
the entire sequence space. This might suggest more constraint on the monofunctional enzyme, which evolved
less than the bifunctional one. It is interesting to note that in Fig. 7 as well as in Fig. 9, the green lineage is always
compacted in a rather small space, whereas the apicomplexa are quite widely distributed. This is indicative of a
much higher mutational rate in the latter compared with the former. Discussion Folate biosynthesis and metabolism in higher plants have been studied for almost two decades. Despite the wealth
of genetic and biochemical evidence, many questions remain to be addressed. Particularly, the regulation and
compartmentalisation of folate biosynthesis and metabolism in various plant lineages require scrutiny. Our com-
parative study encompasses all steps of folate biosynthesis and interconversion of folate species in land plants, as
well as green and red algae and reveals novel aspects of folate metabolism that can support further experimental
studies in the field and help to design effective biofortification strategies. An earlier comparative genomic analysis
of bacterial and plant folate metabolism identified new bacterial GTPCHI and FPGS gene families and predicted
a bacterial folate transporter49. p
Our comparative study revealed that while algae possess single isoforms of the studied genes, plant species
tend to have multiple isoforms regulating the same steps in folate metabolism. The multiple isoforms could derive
from whole genome duplication or duplication of certain genomic regions, contributing to the development of
organelle-specific features of folate metabolism of land plants. Interestingly, three steps of folate biosynthesis,
namely ADCS, HPPK-DHPS and DHFS, are catalysed by single isoforms in most analysed land plant species. The restriction of the number of these genes to a single copy in land plant genomes might reflect the necessity
for a tight regulation of the abundance of folate intermediates. Being in line with the known inhibition by folate Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 10 www.nature.com/scientificreports/ Figure 8. Phylogenetic trees of the protein sequences of the DHPS domain constructed using maximum
likelihood method Phyml (see text). The selected model using Bayesian information criterion was LG + G + I
with gamma shape parameter estimate = 1.058 and the proportion of invariable site estimate = 0.047. Nodes
values represents the Bayesian posterior probabilities branch support. The species colour code corresponds to
the type of plastid pigments, as follows: purple, chlorophyll a; green, chlorophyll a and b; and red, chlorophyll a
and c. Figure 8. Phylogenetic trees of the protein sequences of the DHPS domain constructed using maximum
likelihood method Phyml (see text). The selected model using Bayesian information criterion was LG + G + I
with gamma shape parameter estimate = 1.058 and the proportion of invariable site estimate = 0.047. Nodes
values represents the Bayesian posterior probabilities branch support. Discussion The species colour code corresponds to
the type of plastid pigments, as follows: purple, chlorophyll a; green, chlorophyll a and b; and red, chlorophyll a
and c. Figure 9. Phylogenetic representation in the protein sequence space for DHPS domains. Non-linear mapping
methods are designed to offer a configuration of points in this multidimensional space that is representative of
the observed distances. With this method, axes become arbitrary and every rotation or symmetry is admissible. In this figure, the distance between two data points on the figure tends to display the distances between species. Links between points show the ML phylogenic tree presented in the Fig. 8. Blue branches: monofunctional
enzyme, red branches: bifunctional enzyme. Brown letters, bikont cluster; brown letters in bold, the green
lineage; black letters, unikont cluster. Abbreviations for genus names: Arab_thM, Arabidopsis thaliana
Mitochondrial; Arab_thC, Arabidopsis thaliana Cytosolic; Popu_tri, Populus trichocarpa; Pisu_sat, Pisum
sativum; Oryz_sat, Oryza sativa; Phys_pat, Physcomitrella patens; Chla_rei, Chlamydomonas reinhardtii; Ostr_
tau, Ostreococcus tauri; Plas_yoe, Plasmodium yoelii; Plas_ber, Plasmodium berghei; Toxo_gon, Toxoplasma
gondii; Neos_can, Neospora caninum; Cyan_mer, Cyanidioschyzon merolae; Phae_tri, Phaeodactylum
tricornutum; Thal_pse, Thalassiosira pseudonana; Aura_lim, Aurantiochytrium limacinum; Phyt_soj,
Phytophthora sojae; Aspe_fum, Aspergillus fumigatus; Aspe_ory, Aspergillus oryzae; Neur_cra, Neurospora
crassa; Pneu_car, Pneumocystis carinii; Ashb_gos, Ashbya gossypii; Cand_gla, Candida glabrata; Bige_nat,
Bigelowiella natans; Rick_gry, Rickettsiella grylli; Chla_pne, Chlamydia pneumonia; Rick_bel, Rickettsia bellii;
Wolb_dro, Wolbachia Drosophila simulans; Fran_phi, Francisella philomiragia; Esch_col, Escherichia coli; Salm_
typ, Salmonella typhi; Baci_sub, Bacillus subtilis; Stap_aur, Staphylococcus aureus; Syne_cho, Synechococcus sp.;
Anab_var, Anabaena variabilis; Micr_cht, Microcoleus. Figure 9. Phylogenetic representation in the protein sequence space for DHPS domains. Non-linear mapping
methods are designed to offer a configuration of points in this multidimensional space that is representative of
the observed distances. With this method, axes become arbitrary and every rotation or symmetry is admissible. In this figure, the distance between two data points on the figure tends to display the distances between species. Links between points show the ML phylogenic tree presented in the Fig. 8. Blue branches: monofunctional
enzyme, red branches: bifunctional enzyme. Brown letters, bikont cluster; brown letters in bold, the green
lineage; black letters, unikont cluster. www.nature.com/scientificreports/ www.nature.com/scientificreports/ intermediates both at the transcript and the protein levels50, this notion suggests that biofortification strategies
aiming to enhance folate content should involve elevation of the expression of the downstream genes of folate
biosynthesis pathway, namely, ADCS, HPPK-DHPS and DHFS. Such an approach has been already successfully
employed to elevate folate content in potato51. p y
p
It remains to be addressed why certain enzymes of folate biosynthesis are encoded by single isoforms
in duplication-prone land plant genomes. Exceptions with two ADCS (in B. rapa, G. max and V. vinifera),
HPPK-DHPS (G. max and L. usitatissimum) and DHFS (G. max and M. truncatula) are also intriguing. On the
one hand, one can ascribe the existence of duplicated isoforms to genetic redundancy inherent to plant genomes. On the other hand, it is possible that the duplicated isoforms might implement a different function or reflect a
difference in the regulation of folate biosynthesis. In the future, it will also be important to address whether the
duplicated genes encode functional enzymes. Our analysis of conserved amino acids for several folate pathway
enzymes, namely, GTPCHI, ADCS, HPPK-DHPS, DHFS and MTHFR, suggests that the isoforms included in
our study bear enzymatic activity (Supplemental Figs 32–36). Interestingly, two out the five plant species that
bear two copies of ADCS and HPPK-DHPS are naturally rich in folates, namely, B. rapa and G. max. Apart from
ADCS and HPPK-DHPS, B. rapa genome bears several copies of GTPCHI. The B. rapa case echoes the bioforti-
fication strategy applied for potato, where ADCS, GTPCHI and HPPK-DHPS genes have been overexpressed51. It is tempting to speculate that such an enhancement approach might prove efficient for species that bear a single
copy of the abovementioned genes. However versatile, Arabidopsis is not a flawless model to be used to study
regulation of plant metabolism. As highlighted by the study of bifunctional DHFR-TS40 and by the present study,
Arabidopsis and other species from Brassicaceae, namely, C. rubella and B. rapa have unique features that are not
shared with species from other land plant lineages. Thus, species from Brassicaceae bear enzymatically inactive
DHFR-TS homologs that inhibit activity of active homologs, thereby regulating availability of THF40. This regu-
lation seems not to be operating in other plant lineages. Brassicaceae members also exceptionally lack cytosolic
ADCL isoform (Fig. 4). Moreover, Arabidopsis bears a cytosolic HPPK-DHPS isoform that might be involved in
stress response17. www.nature.com/scientificreports/ Interestingly, this feature is unique to Arabidopsis and not shared even with other Brassicaceae
species (Supplemental Fig. 5). It is tempting to assume that Arabidopsis might differ from species of other plant
lineages in its regulation of folate biosynthesis. Taking this into consideration, drawing parallels from studies
using Arabidopsis should be done with caution. g
p
Prediction of subcellular localization allowed to identify novel aspects of folate biosynthesis and metabo-
lism. Although it must be kept in mind that these in silico predictions have to be experimentally confirmed, our
study suggests that ADCL can localize to the cytosol. The presence of the cytosolic isoform in every analysed
land plant species, except species from Brassicaceae, might indicate an important function different from that in
folate biosynthesis. To date, the cytosolic role of the enzyme remains unknown. It is tempting to speculate that
cytosolic ADCL contributes to pABA production that so far has been only reported to occur in plastids in the
plant cell. This assumption is in line with studies showing cytosolic localization of certain enzymes operating in
the shikimate pathway that provides chorismate for pABA synthesis. If this scenario is operative, there should be
a cytosolic conversion of chorismate to aminodeoxychorismate (ADC) similar to that performed by ADCS in
plastids; alternatively, ADC can be exported from plastids. As a folate independent, stress-related function for the
cytosolic isoform of HPPK-DHPS, an enzyme catalysing the successive step in the folate biosynthesis, has been
suggested previously52, it is possible that the cytosolic ADCL might also fulfil a role in stress response. Generally
speaking, the intracellular compartmentalization of folate biosynthesis established for Arabidopsis (depicted in
Fig. 1) seems to be a general feature for land plants. However, the situation might be more complex in algae since
the predicted localization of the enzymes appeared more fluctuating from one species to another. For example,
there is no putative DHFR activity located in the mitochondria of most algae, and in K. flaccidum the entire folate
biosynthesis pathway (except for the ADCS activity) could take place in the cytosol. y
p
y (
p
y)
p
y
Analysis of subcellular compartmentalization of genes involved in interconversion of folate species sug-
gests that the sources of one-carbon units in plant cells are specific for different subcellular compartments and
this specificity is highly conserved across higher plant lineages (Fig. 10). Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 Discussion Abbreviations for genus names: Arab_thM, Arabidopsis thaliana
Mitochondrial; Arab_thC, Arabidopsis thaliana Cytosolic; Popu_tri, Populus trichocarpa; Pisu_sat, Pisum
sativum; Oryz_sat, Oryza sativa; Phys_pat, Physcomitrella patens; Chla_rei, Chlamydomonas reinhardtii; Ostr_
tau, Ostreococcus tauri; Plas_yoe, Plasmodium yoelii; Plas_ber, Plasmodium berghei; Toxo_gon, Toxoplasma
gondii; Neos_can, Neospora caninum; Cyan_mer, Cyanidioschyzon merolae; Phae_tri, Phaeodactylum
tricornutum; Thal_pse, Thalassiosira pseudonana; Aura_lim, Aurantiochytrium limacinum; Phyt_soj,
Phytophthora sojae; Aspe_fum, Aspergillus fumigatus; Aspe_ory, Aspergillus oryzae; Neur_cra, Neurospora
crassa; Pneu_car, Pneumocystis carinii; Ashb_gos, Ashbya gossypii; Cand_gla, Candida glabrata; Bige_nat,
Bigelowiella natans; Rick_gry, Rickettsiella grylli; Chla_pne, Chlamydia pneumonia; Rick_bel, Rickettsia bellii;
Wolb_dro, Wolbachia Drosophila simulans; Fran_phi, Francisella philomiragia; Esch_col, Escherichia coli; Salm_
typ, Salmonella typhi; Baci_sub, Bacillus subtilis; Stap_aur, Staphylococcus aureus; Syne_cho, Synechococcus sp.;
Anab_var, Anabaena variabilis; Micr_cht, Microcoleus. Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 11 www.nature.com/scientificreports/ THF, tetrahydrofolate; 5-CH3-THF, 5-methyltetrahydrofolate; 5,10-CH2-THF, 5,10-methylenetetrahydrofolate;
5,10-CH+-THF, 5,10-methenyltetrahydrofolate; 5-CHO-THF, 5-formyltetrahydrofolate; 10-CHO-THF,
10-formyltetrahydrofolate; Ser, serine; Gly, glycine. SHMT, serine hydroxymethyltransferase; GDC, glycine
decarboxylase complex, FTHFC, formiminotetrahydrofolate cyclodeaminase; 5-FCL, 5-formyltetrahydrofolate
cycloligase; GFT, glutamate formiminotransferase; FTHFS, 10-CHO-THF synthetase; MTHFD-MTHFC, 5,10-
CH2-THF dehydrogenase/5,10-CH+-THF cyclohydrolase; MTHFR, methylenetetrahydrofolate reductase; 10-
FDF, 10-CHO-THF deformylase. Figure 10. Sources of one-carbon units in the plant cell. Arrows in bold indicate sources of one-carbon units. THF, tetrahydrofolate; 5-CH3-THF, 5-methyltetrahydrofolate; 5,10-CH2-THF, 5,10-methylenetetrahydrofolate;
5,10-CH+-THF, 5,10-methenyltetrahydrofolate; 5-CHO-THF, 5-formyltetrahydrofolate; 10-CHO-THF,
10-formyltetrahydrofolate; Ser, serine; Gly, glycine. SHMT, serine hydroxymethyltransferase; GDC, glycine
decarboxylase complex, FTHFC, formiminotetrahydrofolate cyclodeaminase; 5-FCL, 5-formyltetrahydrofolate
cycloligase; GFT, glutamate formiminotransferase; FTHFS, 10-CHO-THF synthetase; MTHFD-MTHFC, 5,10-
CH2-THF dehydrogenase/5,10-CH+-THF cyclohydrolase; MTHFR, methylenetetrahydrofolate reductase; 10-
FDF, 10-CHO-THF deformylase. enzyme was thereafter transferred to the nucleus. It was proposed that the ancient bacterium likely at the origin of
mitochondria belonged to the α-proteobacteria class53. Since α-proteobacteria contain species displaying either
monofunctional DHPS or bifunctional HPPK-DHPS such as those presented here (Rickettsia, Wolbachia), it
would be interesting to know whích α-proteobacterial lineage provided the mitochondrial ancestor. Several stud-
ies hint towards the group of Rickettsiales, but this conclusion is today highly challenged54,55. g
p
y
g y
g
Furthermore, our study reveals that all analysed ADCS homologs, in addition to GAT and chorismate binding
domains, possess an anthranilate synthase (AS) domain. Anthranilate synthase (AS) catalyses the first step in
the biosynthesis of tryptophan from chorismate56. It is thus tempting to speculate that ADCS of algae and higher
plant species are capable of anthranilate synthesis. If the AS domain of ADCS is functional, the protein might play
a role in tryptophan synthesis that is known to occur in plastids of higher plants57 and subsequently contribute to
auxin biosynthesis, known to use Trp as a major precursor58. Alternatively, it may play a regulatory role therein. A possible contribution of ADCS to auxin synthesis might link the folate and auxin pathways. Interestingly,
folate has previously been reported to influence auxin distribution during seedling development, together with
sucrose59. Furthermore, it was recently shown that root gravitropism is regulated by a crosstalk between PABA
(product of the ADCS + ADCL reaction), ethylene and auxin in Arabidopsis60. (p
)
y
p
In several higher plant species some folate pathway enzymes, namely, ADCS, HPPK-DHPS, plastidial
MTHFD-MTHFC and GFT, bear additional functional domains. The ACT domain residing on the polypeptide
chain of bifunctional MTHFD-MTHFC of L. usitatissimum is often found in proteins regulated by amino acids. www.nature.com/scientificreports/ Thus, conversion of 5-CHO-THF, a
putative folate storage form, by 5-FCL and the flux of one-carbon units from 10-CHO-THF to 5,10-CH2-THF
via the sequential action of 10-FDF and GDC are assumed to mainly occur in mitochondria. In the cytosol,
one-carbon units appear to be mainly derived from the conversion of Ser to Gly catalysed by SHMT. However,
the conversion offormate to 10-CHO-THF mediated by FTHFS and the conversion of N-formiminoglutamate
into 5-formimino-THF catalysed by GFT can also serve as a source of one-carbon units. As plastids seem to lack
the abovementioned enzymes, they are assumed to mainly obtain their C1-THF derivatives from the activity of
SHMT and the transport of metabolically active folate forms from mitochondria and cytosol. Because of the pres-
ent lack of reliable in silico tools for prediction of subcellular localization of algal proteins, localization of folate
pathway enzymes in the algae species analysed cannot be unequivocally concluded. Presently, only experimental
approaches are able to reveal the localization of enzymatic steps of folate biosynthesis and metabolism. pp
y
p
y
Our comparative analysis shows that HPPK and DHPS as well as DHFR and TS activities are coupled on a
single bifunctional polypeptide across algae and land plant lineages. This is not the case in all organisms, and, as
a matter of fact, the fusion between the DHFR and TS genes coincides with the appearance of biflagellate organ-
isms. Indeed, unikonts, with one cilium, show monofunctional enzymes, while bikonts, with two cilia, possess
bifunctional enzymes. Our results confirm this well-recognised milestone in the phylogenetic tree of evolution45. However, this is not the case for the fusion of the HPPK and DHPS genes which have a completely different
evolutionary history compared with DHFR and TS. Indeed, the clade for the bifunctional HPPK-DHPS contains
bacteria and fungi (unikonts) as well as plants and alveolates (bikonts). It is interesting to note that all eukary-
otes analysed here were grouped in the clade containing the bifunctional enzyme (Fig. 8). Since HPPK-DHPS
is a mitochondrial enzyme, it is possible that the bacteria at the origin of the endosymbiotic event that resulted
in the advent of mitochondria already contained a bifunctional HPPK-DHPS, and that the gene coding for this Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 12 www.nature.com/scientificreports/ Figure 10. Sources of one-carbon units in the plant cell. Arrows in bold indicate sources of one-carbon units. Materials and Methodsi Identification of putative orthologs and protein analysis. To identify putative orthologs of folate
pathway genes, full-length protein sequences from Arabidopsis thaliana were used as a query to run a blastp
with E-value ≤ 1e-10 cutoff 37. To infer putative orthology the selected protein sequences were scrutinized for the
presence of functional protein domains using the Pfam database38 and ScanProsite61. The conserved motifs were
identified using the MEME suite62. Analysis of conserved amino acids was conducted using BLAST Conserved
Domain Database tool63. Phylogenetic analysis. Full-length amino acid sequences were aligned and manually corrected and edited
in MEGA 764 using MUSCLE software65. The phylogenetic relationship was inferred using the maximum likeli-
hood method implemented in RaxML66 using WAG + G + I model that was selected by ProtTest67. The maximum
likelihood tree was evaluated with 1000 bootstrap replicates. Phylogenetic trees were visualized using FigTree. The method described above was used to build phylogenetic trees for Figs 3–5 and Supplemental Figs 3, 5, 7, 9,
12, 14–16, 18, 20–22, 26, 28, 30. For the comparison of TS and DHPS domains from plants and other organisms, amino acid sequences
were retrieved from NCBI resources, JGI genome portal or specific databases such as, plasmoDB and TAIR. Phylogenetic studies of the bifunctional enzymes DHFR-TS and HPPK-DHPS were performed with the largest
domain in each case, i.e. TS and DHPS which have been identified using Conserved Domain Search Service
v3.1563 Parameters: Expect Value threshold = 0.01, Composition based statistics adjustment. Amino acid domains
sequences were aligned in an iterative process using ClustalX software68. During the process, alignments were
curated using both ClustalX 2.0 and Jalview 269. The phylogenetic relationship was inferred using the maximum
likelihood method PhyML70 with the Smart Model Selection71 under the Bayesian information criterion (BIC)72. The maximum likelihood tree was evaluated with aBayes73 and phylogenetic trees were visualized using FigTree.h h
y
p y g
g
g
The phylogenetic trees obtained from the maximum likelihood method PhyML were represented in the pro-
tein sequence space, where each residue in the protein is represented by a dimension with 20 possible positions
along that axis, corresponding to the possible amino acids47,74,75. This space is high-dimensional since its dimen-
sion is 20 to the power of the number of residues in the sequence. www.nature.com/scientificreports/ It is tempting to speculate that this domain might be involved in regulation of the protein by Ser and Gly abun-
dance. However, the domain is absent in all other analysed species, even in the closest relatives of L. usitatissi-
mum. Apparently, this domain appeared in MTHFD-MTHFC as a result of a random insertion during genome
rearrangement of L. usitatissimum. Presence of additional domains in ADCS, HPPK-DHPS and GFT cannot be
immediately linked to a specific function. Moreover, the rare occurrence of the additional domains suggests that
they occurred in result of random insertions. y
In conclusion, the present comparative study revealed novel features of folate metabolism and emphasized the
need for further understanding of its regulation in different plant species. Our study suggests that the subcellular
localization of folate biosynthesis might be different between algae and land plants, indicating that the localiza-
tion established in Arabidopsis might not be an absolute physiological requirement. Our phylogenetic analysis
implies that duplication of genes controlling certain steps in the folate biosynthesis pathway might have served
as a way to increase folate production in certain species. Consequently, the information on the gene numbers
could be informative for planning biofortification strategies for related species. The analyses of DHFR/TS and
HPPK/DHPS (Figs 6–9) suggest that different steps in the biosynthetic pathway might have a unique evolutionary
history.iiii y
Once experimentally verified, our findings will be useful for development of species-specific biofortification
strategies and better understanding of roles of folate metabolism in plant physiology. Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 13 www.nature.com/scientificreports/ Materials and Methodsi Nevertheless, it can be represented in a 3D
space by using Multidimensional scaling methods which consider the distances between data in the original
high dimensional space and associate a configuration of points in a lower dimensional space (the so called rep-
resentation space, which is here a 3D Euclidean space)76,77. Obviously, distances cannot be always preserved and
distortions appear78. Most often, the preservation of short distances is favored so as to preserve local properties. Consequently, axes have no specific meaning and maps must be considered as invariant by translation, rotation
and symmetry. The information in maps is carried by distances between points. Here we used the Sammon’s
mapping79. It should be emphasized that multidimensional scaling offers an intuitive representation of the orig-
inal data structure (the similarity between sequences). Distances between sequences were computed using the
PAM250 matrix and the Phylip prodist program80. The method described above was used to build phylogenetic
trees for Figs 6 and 8. Protein subcellular localization. Subcellular localization of analysed proteins was predicted using
TargetP81, SherLoc282, MultiLoc83, PredAlgo84. Double localizations originated from predictions by different tools. Protein subcellular localization. Subcellular localization of analysed proteins was predicted using
T
tP81 Sh L
282 M ltiL
83 P dAl
84 D
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di ti
b diff
t t
l Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 References 1. Chistoserdova, L., Vorholt, J. A., Thauer, R. K. & Lidstrom, M. E. C1 transfer enzymes and coenzymes linking methylotrophic
bacteria and methanogenic Archaea. Science 281, 99–102 (1998). g
2. Edman, J. C., Goldstein, A. L. & Erbe, J. G. Para‐aminobenzoate synthase gene of Saccharomyces cerevisiae encodes a bifunctiona
enzyme. Yeast 9, 669–675 (1993).h y
(
)
3. James, T. Y. et al. The pab1 gene of Coprinus cinereus encodes a bifunctional protein for para aminobenzoic acid (PABA) synthesis:
implications for the evolution of fused PABA synthases. Journal of Basic Microbiology 42, 91–103 (2002). l
l
d
f l
h
l
f
b
d
h
l
l y
(
)
3. James, T. Y. et al. The pab1 gene of Coprinus cinereus encodes a bifunctional protein for para aminobenzoic acid (PABA) synthesis
implications for the evolution of fused PABA synthases. Journal of Basic Microbiology 42, 91–103 (2002). 4. Triglia, T. & Cowman, A. F. Plasmodium falciparum: a homologue of p-aminobenzoic acid synthetase. Experimental Parasitology
92, 154–158 (1999). ,
(
)
5. Basset, G. J. et al. Folate synthesis in plants: the p-aminobenzoate branch is initiated by a bifunctional PabAPabB protein that is
targeted to plastids. Proceedings of the National Academy of Sciences of the United States of America 101, 1496–1501 (2004). 5. Basset, G. J. et al. Folate synthesis in plants: the p-aminobenzoate branch is initiated by a bifunctional PabAPabB protein that i
targeted to plastids. Proceedings of the National Academy of Sciences of the United States of America 101, 1496–1501 (2004). g
p
g
f
y f
f
f
6. Camara, D., Richefeu-Contesto, C., Gambonnet, B., Dumas, R. & Rébeillé, F. The synthesis of pABA: Coupling between the
glutamine amidotransferase and aminodeoxychorismate synthase domains of the bifunctional aminodeoxychorismate synthase
from Arabidopsis thaliana. Archives of Biochemistry and Biophysics 505, 83–90 (2011). p
f
y
p y
(
)
7. Basset, G. J. et al. Folate synthesis in plants: the last step of the p‐aminobenzoate branch is catalyzed by a plastidia
aminodeoxychorismate lyase. The Plant Journal 40, 453–461 (2004). y
yh
8. Green, J. M., Merkel, W. K. & Nichols, B. P. Characterization and sequence of Escherichia coli pabC, the gene encoding
aminodeoxychorismate lyase, a pyridoxal phosphate-containing enzyme. Journal of Bacteriology 174, 5317–5323 (1992). 8. G ee , J. .,
e ke , W. K. & N c o s,
. . www.nature.com/scientificreports/ S
bb
S
S
l
b f
fi
f
d
C
O
h
l
(
) f
36. Strobbe, S. & Van Der Straeten, D. Folate biofortification in food crops. Current Opinion in Biotechnology 44, 202–211 (2017). 36. Strobbe, S. & Van Der Straeten, D. Folate biofortification in food crops. Current Opinion in Biot 36. Strobbe, S. & Van Der Straeten, D. Folate biofortification in i
37. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. Journal of Molecular Biolog
403–410 (1990).h A. et al. The Pfam protein families database. Nucleic Acids Research 38. Bateman, A. et al. The Pfam protein families database. Nucleic Acids Research 30, 276–280 (2002). h
p
39. Ravanel, S. et al. Tetrahydrofolate biosynthesis in plants: molecular and functional characterization of dihydrofolate synthetase and
three isoforms of folylpolyglutamate synthetase in Arabidopsis thaliana. Proceedings of the National Academy of Sciences 98,
15360–15365 (2001).i 40. Gorelova, V. et al. Dihydrofolate reductase/thymidylate synthase fine-tunes the folate status and controls redox homeostasis in
plants. The Plant Cell 29, 2831–2853 (2017).h h
1. Mehrshahi, P. et al. Functional analysis of folate polyglutamylation and its essential role in plant metabolism and development. T
Plant Journal 64, 267–279 (2010).h 42. Oliver, D. J. The glycine decarboxylase complex from plant mitochondria. Annual Review of Plant Biology 45, 323–337 (1994). 43. Roje, S., Janave, M. T., Ziemak, M. J. & Hanson, A. D. Cloning and characterization of mitochondrial 5-formyltetrahydrof
cycloligase from higher plants. Journal of Biological Chemistry 277, 42748–42754 (2002). 4. Isegawa, Y., Watanabe, F., Kitaoka, S. & Nakano, Y. Subcellular distribution of cobalamin-dependent methionine synthase in Euglen
gracilis Z. Phytochemistry 35, 59–61 (1993). g
y
y
45. Stechmann, A. & Cavalier-Smith, T. Rooting the eukaryote tree by using a derived gene fusion. Science 297, 89–91 (2002). l
l
f h
l b
l
d
f l
d
b d
h 6. Bastien, O. et al. Analysis of the compositional biases in Plasmodium falciparum genome and proteome using Arabidopsis thalian
as a reference. Gene 336, 163–173 (2004).i 7. Bastien, O., Ortet, P., Roy, S. & Maréchal, E. A configuration space of homologous proteins conserving mutual information and
allowing a phylogeny inference based on pair-wise Z-score probabilities. BMC Bioinformatics 6, 49 (2005). Lespinats, S. In MATEC Web of Conferences. 10002 (EDP Sciences g
p
f
f
49. www.nature.com/scientificreports/ de Crécy-Lagard, V., El Yacoubi, B., de la Garza, R. D., Noiriel, A. & Hanson, A. D. Comparative genomics of bacterial and plant
folate synthesis and salvage: predictions and validations. BMC Genomics 8, 245 (2007). 49. de Crécy-Lagard, V., El Yacoubi, B., de la Garza, R. D., Noiriel, A. & Hanson, A. D. Co
folate synthesis and salvage: predictions and validations. BMC Genomics 8, 245 (2007). 0. Gorelova, V., Ambach, L., Rébeillé, F., Stove, C. & Van Der Straeten, D. Folates in Plants: Research Advances and Progress in Crop
Biofortification. Frontiers in Chemistry 5 (2017).ii 50. Gorelova, V., Ambach, L., Rébeillé, F., Stove, C. & Van Der Straeten, D. Folates in Plants: Res
Biofortification. Frontiers in Chemistry 5 (2017).ii i
51. De Lepeleire, J. et al. Folate biofortification of potato by tuber-specific expression of four folate biosynthesis genes. Molecular Plant
11, 175–188 (2018). 52. Navarrete, O. et al. A folate independent role for cytosolic HPPK/DHPS upon stress in Arabidopsis thaliana. Phytochemistry 73,
23–33 (2012). 3. Yang, D., Oyaizu, Y., Oyaizu, H., Olsen, G. J. & Woese, C. R. Mitochondrial origins. Proceedings of the National Academy of Sciences
82, 4443–4447 (1985). 54. Fitzpatrick, D. A., Creevey, C. J. & McInerney, J. O. Genome phylogenies indicate a meaningful α-proteobacterial phylogeny and
support a grouping of the mitochondria with the Rickettsiales. Molecular Biology and Evolution 23, 74–85 (2005).f 55. Martijn, J., Vosseberg, J., Guy, L., Offre, P. & Ettema, T. J. Deep mitochondrial origin outside the sampled alphaproteobacteria. Nature 557, 101 (2018). 56. Kawamura, M., Keim, P. S., Goto, Y., Zalkin, H. & Heinrikson, R. L. Anthranilate synthetase component II from Pseudomonas
putida. Covalent structure and identification of the cysteine residue involved in catalysis. Journal of Biological Chemistry 253,
4659–4668 (1978).h 57. Radwanski, E. R. & Last, R. L. Tryptophan biosynthesis and metabolism: biochemical and molecular genetics. The Plant Cell 7
(1995).h (
)
58. Mano, Y. & Nemoto, K. The pathway of auxin biosynthesis in plants. Journal of Experimental Botany 63, 2853–2872 (2012). Y. & Nemoto, K. The pathway of auxin biosynthesis in plants. Journ h
y
y
f
p
y
9. Stokes, M. E., Chattopadhyay, A., Wilkins, O., Nambara, E. & Campbell, M. M. Interplay between sucrose and folate modula
auxin signaling in Arabidopsis. Plant Physiology 162, 1552–1565 (2013). l
l
d b
lk b
b
d
h l
d
l g
g
p
y
gy
(
)
60. Nziengui, H. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 16. Mouillon, J.-M., Ravanel, S., Douce, R. & Rébeillé, F. Folate synthesis in higher-plant mitochondria: coupling between
dihydropterin pyrophosphokinase and the dihydropteroate synthase activities. Biochemical Journal 363, 313–319 (2002). y
p
py
p
p
y
p
y
(
)
17. Storozhenko, S. et al. Cytosolic Hydroxymethyldihydropterin Pyrophosphokinase/Dihydropteroate Synthase from Arabid
thaliana A Specific Role in Early Development and Stress Response. Journal of Biological Chemistry 282, 10749–10761 (2007). pi
y
p
p
f
g
y
18. Gillies, S. A., McIntosh, S. R. & Henry, R. J. A cereal crop with enhanced folate: Rice transgenic for wheat HPPK/DHPS (2008) y
p
g
19. Luo, M., Orsi, R., Patrucco, E., Pancaldi, S. & Cella, R. Multiple transcription start sites of the carrot dihydrofolate reduc
thymidylate synthase gene, and sub-cellular localization of the bifunctional protein. Plant Molecular Biology 33, 709–722 (199 y
y
y
g
p
gy
0. Neuburger, M., Rébeillé, F., Jourdain, A., Nakamura, S. & Douce, R. Mitochondria are a major site for folate and thymidylate
synthesis in plants. Journal of Biological Chemistry 271, 9466–9472 (1996). synthesis in plants. Journal of Biological Chemistry 271, 946 y
p
f
g
y
1. Cox, K., Robertson, D. & Fites, R. Mapping and expression of a bifunctional thymidylate synthase, dihydrofolate reductase gene
from maize. Plant Molecular Biology 41, 733–739 (1999).h y
f
g
y
21. Cox, K., Robertson, D. & Fites, R. Mapping and expressi g
from maize. Plant Molecular Biology 41, 733–739 (1999)h gy
2. Lazar, G., Zhang, H. & Goodman, H. M. The origin of the bifunctional dihydrofolate reductasethymidylate synthase isogenes o
Arabidopsis thaliana. The Plant Journal 3, 657–668 (1993). ph
3. Akhtar, T. A. et al. Tomato γ-glutamylhydrolases: expression, characterization, and evidence for heterodimer formation. Plan
Physiology 148, 775–785 (2008).h y
gy
(
)
4. Akhtar, T. A. et al. A central role for gamma‐glutamyl hydrolases in plant folate homeostasis. The Plant Journal 64, 256–266 (2010) 24. Akhtar, T. A. et al. A central role for gamma‐glutamyl hydrolases in plant folate homeostasis. The Plant Journal 64, 256–266 (2010). 25. Orsomando, G. et al. Plant γ-Glutamyl Hydrolases and Folate Polyglutamates Characterization, Compartmentation, and Co-
Occurrence in Vacuoles. Journal of Biological Chemistry 280, 28877–28884 (2005). 4. Akhtar, T. A. et al. A central role for gamma‐glutamyl hydrolases in plant folate homeostasis. The Plant Journal 64, 256–266 (2010)
5. Orsomando, G. et al. www.nature.com/scientificreports/ Plant γ-Glutamyl Hydrolases and Folate Polyglutamates Characterization, Compartmentation, and Co
l
l f
l
l
h
(
) g
g
y
y
ph
(
25. Orsomando, G. et al. Plant γ-Glutamyl Hydrolases and Folate Polyglutamates Characterization, Compartmentation, and
O
i V
l
J
l f Bi l
i
l Ch
i t
280 28877 28884 (2005) h
25. Orsomando, G. et al. Plant γ-Glutamyl Hydrolases and Folate Polyglutamates Characterization, Compartmentation
Occurrence in Vacuoles Journal of Biological Chemistry 280 28877–28884 (2005) 25. Orsomando, G. et al. Plant γ-Glutamyl Hydrolases and Folate Polyglutamates Characterization, Compartm
Occurrence in Vacuoles. Journal of Biological Chemistry 280, 28877–28884 (2005). 25. Orsomando, G. et al. Plant γ Glutamyl Hydrolases and Folate Polyglutamates Characterization, Compartmentation, and
Occurrence in Vacuoles. Journal of Biological Chemistry 280, 28877–28884 (2005). n Vacuoles. Journal of Biological Chemistry 280, 28877–28884 (2005 26. Ravanel, S., Douce, R. & Rebeille, F. In Advances in Botanical Research Vol. 59, 67–106 (Elsevier, 2011). 27. Hanson, A. D. & Roje, S. One-carbon metabolism in higher plants. Annual Review of Plant Biology 52, 119–137 (2001). M
ll
J M
l Gl
d
b l
h
h
h h
l
ll
h
l
C
b l 28. Mouillon, J. M. et al. Glycine and serine catabolism in non‐photosynthetic higher plant cells: their role in C1 metabolism. The Plant
Journal 20, 197–205 (1999). 29. Besson, V., Neuburger, M., Rébeillé, F. & Douce, R. Evidence for three serine hydroxymethyltransferases in green. Plant Physiol. Biochem 33, 665–673 (1995).hfi 0. Kirk, C. D., Imeson, H. C., Zheng, L.-L. & Cossins, E. A. The affinity of pea cotyledon 10-formyltetrahydrofolate synthetase for
polyglutamate substrates. Phytochemistry 35, 291–296 (1994). p yg
y
y
31. Kirk, C. D., Chen, L., Imeson, H. C. & Cossins, E. A. A 5, 10-methylenetetrahydrofolate dehydrogenase: 5,
10-methenyltetrahydrofolate cyclohydrolase protein from Pisum sativum. Phytochemistry 39, 1309–1317 (1995).h 32. Collakova, E. et al. Arabidopsis 10-formyl tetrahydrofolate deformylases are essential for photorespiration. The Plant Cell 20,
1818–1832 (2008).h 33. Stover, P. & Schirch, V. The metabolic role of leucovorin. Trends in Biochemical Sciences 18, 102–106 (1993). h
34. Jeanguenin, L. et al. Moonlighting glutamate formiminotransferases can functionally replace 5-formyltetrahydrofolate cycloligase. Journal of Biological Chemistry 285, 41557–41566 (2010). 35. Guo, W. et al. In ICPM conference Dalian, China (2017).i 35. Guo, W. et al. In ICPM conference Dalian, China (2017). References C a acte
at o
a d seque ce o
sc e c
a co
pabC, t e ge e e cod
g
aminodeoxychorismate lyase, a pyridoxal phosphate-containing enzyme. Journal of Bacteriology 174, 5317–5323 (1992). 9. Auerbach, G. et al. Zinc plays a key role in human and bacterial GTP cyclohydrolase I. Proceedings of the National Academy of
Sciences 97, 13567–13572 (2000). y
y
py
p
p
g
y
f
gy
9. Auerbach, G. et al. Zinc plays a key role in human and bacterial GTP cyclohydrolase I. Proceedings of the National Academy of
Sciences 97, 13567–13572 (2000). 0. Nar, H. et al. Active site topology and reaction mechanism of GTP cyclohydrolase I. Proceedings of the National Academy of Science
92, 12120–12125 (1995).i 1. Basset, G. et al. Folate synthesis in plants: the first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase
I. Proceedings of the National Academy of Sciences 99, 12489–12494 (2002). g
f
y f
12. Klaus, S. M. et al. A nudix enzyme removes pyrophosphate from dihydroneopterin triphosphate in the folate synthesis pathway of
bacteria and plants. Journal of Biological Chemistry 280, 5274–5280 (2005). f
g
y
13. Suzuki, Y. & Brown, G. M. The biosynthesis of folic acid XII. Purification and properties of dihydroneopterin triphosphate
pyrophosphohydrolase. Journal of Biological Chemistry 249, 2405–2410 (1974). py
p
p
y
f
g
y
4. Goyer, A. et al. Folate biosynthesis in higher plants. cDNA cloning, heterologous expression, and characterization o
dihydroneopterin aldolases. Plant Physiology 135, 103–111 (2004).i y
p
y
gy
(
)
15. Rébeillé, F., Macherel, D., Mouillon, J. M., Garin, J. & Douce, R. Folate biosynthesis in higher plants: purification and molecular
cloning of a bifunctional 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase/7,8‐dihydropteroate synthase localized in
mitochondria. The EMBO Journal 16, 947–957 (1997). Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 14 www.nature.com/scientificreports/ The Bayesian information criterion: background, derivation, and applications. Wiley Interdisciplinary
Reviews: Computational Statistics 4, 199–203 (2012). p
73. Anisimova, M., Gil, M., Dufayard, J.-F., Dessimoz, C. & Gascuel, O. Survey of branch support methods demonstrates accuracy,
power, and robustness of fast likelihood-based approximation schemes. Systematic Biology 60, 685–699 (2011). 74. Bornberg-Bauer, E. & Chan, H. S. Modeling evolutionary landscapes: mutational stability, topology, and superfunnels in sequence
space. Proceedings of the National Academy of Sciences 96, 10689–10694 (1999).h p
g
f
y f
5. Dryden, D. T., Thomson, A. R. & White, J. H. How much of protein sequence space has been explored by life on Earth? Journal o
The Royal Society Interface 5, 953–956 (2008). h
76. France, S. L. & Carroll, J. D. Two-way multidimensional scaling: A review. IEEE Transactions on Systems, Man, and Cybernetics, Part
C (Applications and Reviews) 41, 644–661 (2011). pp
77. Lee, J. A. & Verleysen, M. Nonlinear Dimensionality Reduction. (Springer Science & Business Media, 2007). y
y
p
g
78. Lespinats, S. & Aupetit, M. In Computer Graphics Forum. 113–125 (Wiley Online Library, 2010). . & Aupetit, M. In Computer Graphics Forum. 113–125 (Wiley Onli Lespinats, S. & Aupetit, M. In Computer Graphics Forum. 113–125 ( p
p
y
y
79. Sammon, J. W. A nonlinear mapping for data structure analysis. IEEE Transactions on Computers 100, 401–409 (1969). F l
J E
l
f
DNA
l k l h
d
h J
l f M l
l
E
l 80. Felsenstein, J. Evolutionary trees from DNA sequences: a maximum likelihood approach. Journal of Molecular Evolution 17, 368
(1981). 1. Emanuelsson, O., Brunak, S., Von Heijne, G. & Nielsen, H. Locating proteins in the cell using TargetP, SignalP and related tools
Nature Protocols 2, 953–971 (2007). 2. Briesemeister, S. et al. SherLoc2: a high-accuracy hybrid method for predicting subcellular localization of proteins. Journal o
Proteome Research 8, 5363–5366 (2009). 3. Blum, T., Briesemeister, S. & Kohlbacher, O. MultiLoc2: integrating phylogeny and Gene Ontology terms improves subcellula
protein localization prediction. BMC Bioinformatics 10, 274 (2009). f
4. Tardif, M. et al. PredAlgo: a new subcellular localization prediction tool dedicated to green algae. Molecular Biology and Evolution
29, 3625–3639 (2012). www.nature.com/scientificreports/ et al. Root gravitropism is regulated by a crosstalk between para-aminobenzoic acid, ethylene, and auxin. Plant
Physiology 178, 1370–1389 (2018). Scientific Reports | (2019) 9:5731 | https://doi.org/10.1038/s41598-019-42146-5 15 www.nature.com/scientificreports/ 61. Gattiker, A., Gasteiger, E. & Bairoch, A. M. ScanProsite: a reference implementation of a PROSITE scanning tool. Ap
Bioinformatics 1, 107–108 (2002). f
(
)
62. Bailey, T. L. et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Research 37, W202–W208 (2009). 3. Marchler-Bauer, A. et al. CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Research 39
D225–D229 (2010). (
)
64. Kumar, S., Stecher, G. & Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular
Biology and Evolution 33, 1870–1874 (2016). gy
(
)
65. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32, 1792–1797
(2004). (
)
6. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30
1312–1313 (2014).i (
)
67. Abascal, F., Zardoya, R. & Posada, D. ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21, 2104–2105 (2005). 68. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007). (
)
67. Abascal, F., Zardoya, R. & Posada, D. ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21, 2104–2105 (2005). i
al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–29 i
. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinform f
9. Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M. & Barton, G. J. Jalview Version 2—a multiple sequence alignment edito
and analysis workbench. Bioinformatics 25, 1189–1191 (2009). y
f
0. Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML
3.0. Systematic Biology 59, 307–321 (2010). y
gy
(
)
71. Lefort, V., Longueville, J.-E. & Gascuel, O. SMS: Smart Model Selection in PhyML. Molecular Biology and Evolution, msx149 (2017).h y
gy
71. Lefort, V., Longueville, J.-E. & Gascuel, O. SMS: Smart Model Selection in PhyML. Molecular Biology and Evolution, msx149 (2
72 Neath A A & Cavanaugh J E The Bayesian information criterion: background derivation and applications Wiley Interdiscipl y
gy
71. Lefort, V., Longueville, J.-E. & Gascuel, O. SMS: Smart Model Sh 2. Neath, A. A. & Cavanaugh, J. E. Author Contributions V.G. conducted all analyses (with the exception of analysis Evolution of DHFR-TS and HPPK-DHPS in plants
versus other organisms), designed experiments, analyzed and interpreted data and drafted the manuscript;
O.B., S.L. and F.R. conducted analysis of the evolution of DHFR-TS and HPPK-DHPS, designed experiments,
analyzed and interpreted data and wrote the corresponding part of the manuscript; O.D.C. and D.V.D.S. designed
experiments, interpreted the data and corrected the draft manuscript. D.V.D.S. coordinated the project. All
authors commented on the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-42146-5.h Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-4 Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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A Patient Registry for the Management of Uterine Fibroids in Canada: Protocol for a Multicenter, Prospective, Noninterventional Study
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JMIR research protocols
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A Patient Registry for the Management of Uterine Fibroids in
A Patient Registry for the Management of Uterine Fibroids in
Canada: Protocol for a Multicenter, Prospective, Noninterventional
Canada: Protocol for a Multicenter, Prospective, Noninterventional
Study
Study Mohamed A. Bedaiwy
The University of British Columbia Follow this and additional works at: https://ir.lib.uwo.ca/paedpub Western University
Western University
Scholarship@Western
Scholarship@Western Western University
Western University
Scholarship@Western
Scholarship@Western Paediatrics Publications Paediatrics Department Paediatrics Department p p
p p
Bedaiwy, Mohamed A.; Janiszewski, Peter; Singh, Sukhbir S.; Laberge, Philippe; Kives, Sari; Leyland,
Nicholas; Vilos, George; Polsky, Joshua; and Belland, Liane, "A Patient Registry for the Management of
Uterine Fibroids in Canada: Protocol for a Multicenter, Prospective, Noninterventional Study" (2018).
Paediatrics Publications. 1482. Mohamed A. Bedaiwy, Peter Janiszewski, Sukhbir S. Singh, Philippe Laberge, Sari Kives, Nicholas Leyland,
George Vilos, Joshua Polsky, and Liane Belland Citation of this paper:
Citation of this paper: Bedaiwy, Mohamed A.; Janiszewski, Peter; Singh, Sukhbir S.; Laberge, Philippe; Kives, Sari; Leyland,
Nicholas; Vilos, George; Polsky, Joshua; and Belland, Liane, "A Patient Registry for the Management of
Uterine Fibroids in Canada: Protocol for a Multicenter, Prospective, Noninterventional Study" (2018). Paediatrics Publications. 1482. https://ir.lib.uwo.ca/paedpub/1482 Authors
Authors
Mohamed A. Bedaiwy, Peter Janiszewski, Sukhbir S. Singh, Philippe Laberge, Sari Kives, Nicholas Leyland,
George Vilos, Joshua Polsky, and Liane Belland This article is available at Scholarship@Western: https://ir.lib.uwo.ca/paedpub/1482 A Patient Registry for the Management of Uterine Fibroids in
Canada: Protocol for a Multicenter, Prospective, Noninterventional
Study iwy1, MD, PhD; Peter Janiszewski2, PhD; Sukhbir S Singh3, MD; CAPTURE Steering Committe Corresponding Author:
Mohamed A Bedaiwy, MD, PhD
Department of Obstetrics and Gynecology
The University of British Columbia
Children’s & Women’s Hospital
Room D415A, 4500 Oak Street
Vancouver, BC, V6H 3N1
Canada Phone: 1 604 875 2000 ext 4310
Email: mohamed.bedaiwy@cw.bc.ca Phone: 1 604 875 2000 ext 4310
Email: mohamed.bedaiwy@cw.bc.ca Abstract Background: Uterine fibroids are the most common benign tumor in women. Among those with fibroids, approximately 30%
become symptomatic, with abnormal uterine bleeding, pelvic pain, and bulk symptoms. Despite the high prevalence of fibroids,
little information is available regarding symptoms, treatment choices, and outcomes for patients. Objective: A Canada-wide patient registry was established to understand the real-world practice. This registry included patient
presentation and treatment preferences, health care provider attitudes, and clinical outcomes in the management of symptomatic
uterine fibroids. Methods: This study is a prospective, noninterventional, observational patient registry. It will include women diagnosed with
uterine fibroids and being managed for symptoms. Participant inclusion criteria were (1) at least 18 years of age, (2) premenopausal
with a confirmed diagnosis of uterine fibroids, and associated symptoms, and (3) initiating treatment (drug intervention, procedure
intervention, or a combination of both) or watchful waiting. Patients (or legal representative) must understand the nature of the
project and provide written informed consent before enrollment. Participant exclusion criteria were (1) they have known or
suspected clinically significant pelvic pathology not associated with uterine fibroids, and (2) they are undergoing an emergency
hysterectomy at the initial visit. Outcomes will be evaluated in the context of routine clinical practice. Results: Participant recruitment of this registry began in July 2015. This study currently has a total sample of 1500 patients. C
l
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i t
fi t i C
d
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id
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d b
fit
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t hf l
iti
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l Results: Participant recruitment of this registry began in July 2015. This study currently has a total sample of 1500 patients. Conclusions: This registry, a first in Canada, will accumulate evidence on the risks and benefits of watchful waiting, and medical
and procedural interventions. It will contribute to enhancing access to treatment options for patients. nt recruitment of this registry began in July 2015. This study currently has a total sample of 1500 patients. Conclusions: This registry, a first in Canada, will accumulate evidence on the risks and benefits of watchful waiting, and medical
and procedural interventions. It will contribute to enhancing access to treatment options for patients. Trial Registration: ClinicalTrials.gov NCT02580578; https://clinicaltrials.gov/ct2/show/NCT02580578 (Archived by WebCite
at http://www.webcitation.org/6yax4Hpvr) International Registered Report Identifier (IRRID): RR1-10.2196/10926 International Registered Report Identifier (IRRID): RR1-10.2196/10926 (JMIR Res Protoc 2018;7(11):e10926) doi: 10.2196/10926 JMIR Res Protoc 2018 | vol. 7 | iss. 11 | e10926 | p. 1
(page number not for citation purposes) Protocol A Patient Registry for the Management of Uterine Fibroids in
Canada: Protocol for a Multicenter, Prospective, Noninterventional
Study Protocol
A Patient Registry for the Management of Uterine Fibroids in
Canada: Protocol for a Multicenter, Prospective, Noninterventional
Study
Mohamed A Bedaiwy1, MD, PhD; Peter Janiszewski2, PhD; Sukhbir S Singh3, MD; CAPTURE Steering Committee
1Department of Obstetrics and Gynecology, The University of British Columbia, Vancouver, BC, Canada
2Department of Medical Affairs, Allergan Inc, Markham, ON, Canada
3Department of Obstetrics, Gynecology and Newborn Care, Ottawa Hospital Research Institute, The University of Ottawa, Ottawa, ON, Canada
C
di
A th
Bedaiwy et al
JMIR RESEARCH PROTOCOLS JMIR RESEARCH PROTOCOLS Bedaiwy et al Mohamed A Bedaiwy1, MD, PhD; Peter Janiszewski2, PhD; Sukhbir S Singh3, MD; CAPTURE Steering Committee
1Department of Obstetrics and Gynecology, The University of British Columbia, Vancouver, BC, Canada
2Department of Medical Affairs, Allergan Inc, Markham, ON, Canada
3Department of Obstetrics, Gynecology and Newborn Care, Ottawa Hospital Research Institute, The University of Ottawa, Ottawa, ON, Canada Authors
Authors This article is available at Scholarship@Western: https://ir.lib.uwo.ca/paedpub/1482 KEYWORDS Canada; leiomyoma; registries http://www.researchprotocols.org/2018/11/e10926/ http://www.researchprotocols.org/2018/11/e10926/ JMIR RESEARCH PROTOCOLS Bedaiwy et al In the past 15 years, there has been only 1 completed registry
for patients with UF who have undergone uterine artery
embolization [14]. In the United States, the COMPARE-UF
trial is underway [15]. A Canadian prospective patient registry
provides the opportunity to address many unanswered questions,
particularly the management patterns and outcomes associated
with different treatments for UF and patients’ preferences for,
and satisfaction with, managing UF symptoms. Introduction Uterine
fibroids
(UF)
are
benign,
monoclonal,
hormone-sensitive, smooth muscle tumors that represent the
most common solid pelvic tumor in women [1-3]. They affect
20% to 40% of women during their reproductive years [4]. Despite their prevalence, very little is known about the etiology
of the disease, burden of illness, and quality of life associated
with the condition. Although most patients with UF are
asymptomatic, UF can cause heavy and prolonged menstrual
bleeding, pelvic pressure or pain, bulk symptoms, and
reproductive dysfunction. Patients with UF show increased use
of analgesics, sanitary products, and health care resources. There
are more physician visits, increased absenteeism, and a decline
in energy, and overall activities, including sexual activity [5]. Apart from epidemiologic data, the natural history of UF is
poorly understood. With this background in mind, the objectives of this study are
to (1) enhance the understanding of the natural history of
symptomatic UF, (2) understand Canadian provider and patient
preferences for managing symptomatic UF, (3) examine factors
that have an impact on the uptake of UF treatment options and
compliance with therapy, (4) examine the effectiveness and
efficiency of UF treatment options across ethnicities in
real-world practice in Canada, and (5) provide real-world data
characterizing long-term clinical and patient-reported outcomes,
including effectiveness and safety, associated with different UF
management options. In addition to the palpation of an enlarged irregular uterine
contour on pelvic examination, ultrasonography is used to
confirm the diagnosis of UF [6]. Recently magnetic resonance
imaging (MRI) has gained popularity, but the cost and access
to this technology prohibit routine screening. In a uterus with
fewer than 5 lesions, ultrasonography appears to be equally
effective as detection by MRI and virtually identical for
assessing their size and location [7]. However, when the number
of lesions is higher, MRI exceeds the technical limitations of
ultrasound in precise fibroid mapping and characterization. Participants Approximately 1000 patients will be enrolled from 20 Canadian
investigational sites, including both academic and community
practices. Inclusion criteria for the study are premenopausal
women at least 18 years of age with confirmed diagnosis and
symptoms associated with UF that are being observed (watchful
waiting), currently being treated, or initiating treatment (drug
intervention, procedure intervention, or a combination of both). All patients (and the patient’s authorized legal representative)
should understand the nature of the project and provide written
informed consent before or at the initial study visit. Exclusion
criteria for the study are known or suspected significant pelvic
pathology not associated with UF and patients undergoing an
emergency hysterectomy at the initial visit. UPA has shown to be more effective than placebo [11] and as
effective as the injectable gonadotropin-releasing hormone
agonist leuprolide acetate [12] for treating UF symptoms. Leuprolide acetate received Health Canada indication for
preoperative improvement of anemia in symptomatic UF women
in 2017 [13]. The uptake, long-term clinical and patient-reported
outcomes, efficacy, safety, as well as the pharmacoeconomics
of indicated and off-label treatment options for these patients
in Canada, have yet to be determined. Overall Study Design and Plan The CAnadian women wiTh Uterine fibroids REgistry
(CAPTURE) is a prospective, observational cohort study
(NCT02580578) that will include women who have been
diagnosed with symptomatic UF. Outcomes will be evaluated
as part of routine clinical practice. The target study population
will include patients with a diagnosis of UF from 20 study sites
across Canada. Participating physicians will not be required to
perform any medical procedure that is outside their normal
clinical practice. Serious and nonserious adverse drug reactions
(ADRs) will be followed up as per local standard of care and
as expected in an observational research study. Similarly, both
serious and nonserious adverse events (AEs) during and
following procedural intervention will be reported. Each study
site was responsible for local ethics review board approval as
per their institutional policy. A significant issue in the field of fibroid identification is the
necessity for differentiating fibroids from other pathologies
such as adenomyomas and uterine neoplasms. Although
uncommon in women less than 40 years old [8], these conditions
require the differential diagnosis with fibroids because the
treatment options and prognosis may differ [9]. Patterns of use
and accuracy of diagnostic imaging of UF have not been studied
systematically in the publicly funded Canadian health care
system. Treatment options for UF patients should ideally address 3
goals: (1) relief of signs and symptoms, (2) sustained reduction
in fibroid size, and (3) retention or enhancement of fertility if
desired. At the same time, treatment should also aim to improve
quality of life, minimize side effects, be convenient for patients,
and target UF directly [10]. Medical, procedural, or combined
treatment options are available for patients with symptomatic
UF. However, there were no medications to treat these women
in Canada until 2013 when ulipristal acetate (UPA) was
approved. http://www.researchprotocols.org/2018/11/e10926/ JMIR Res Protoc 2018 | vol. 7 | iss. 11 | e10926 | p. 2
(page number not for citation purposes) Study Visits The visit schedule is comprised of 4 study visits (baseline, 3-
to 6-month, 12-month, and 24-month; Table 1) and pre-, intra-,
and postoperative visits for patients undergoing procedural
interventions (Table 2). Participants Lost to Follow Up Efforts will be made to keep patients in the registry for at least
24 months. A patient will be considered to have missed a study
visit after all efforts have been made by site personnel to contact
the patient to reschedule within 1 month of the missed
appointment. If a patient has missed a follow-up visit, all efforts
should be made to reach the patient to ensure that subsequent
follow-up visits are completed. The EQ-5D-5L [16] is a validated 5-question survey with a
visual analog scale used to measure health outcomes that
provides a simple descriptive profile and a single index value
for health status. The UFS-QOL [17] is a validated 37-question
disease-specific symptom and health-related quality of life
questionnaire for UF. It has been used extensively in studies to
assess the impact of the disease in patients and treatment options
in randomized clinical trials. The AMSS questionnaire is a
validated 15-question survey that evaluates the severity and
effect of bleeding that occurs during and between menses. There
are 7 baseline questions for UF patients asking about the purpose
of their visit and what outcome they are hoping to achieve. Withdrawal of Participants Participation in this registry is voluntary, and patients may
withdraw at any time and for any reason. Once the patient
informs the site staff that she wishes to withdraw her
participation, a “Withdrawal from registry” form will be http://www.researchprotocols.org/2018/11/e10926/ JMIR Res Protoc 2018 | vol. 7 | iss. 11 | e10926 | p. 2
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RenderX JMIR RESEARCH PROTOCOLS Bedaiwy et al demographic information, (4) obtaining a medical history, and
(5) assessing patient current health status. This will be followed
by patient assessments that involve questions on UFs using the
EuroQol 5-Dimensions 5-Levels (EQ-5D-5L) survey, the
Uterine Fibroid Symptom and Quality of Life (UFS-QOL)
questionnaire, the Aberdeen Menorrhagia Severity Scale
(AMSS) questionnaire, and evaluation for concomitant
medications and history of previous procedural interventions. demographic information, (4) obtaining a medical history, and
(5) assessing patient current health status. This will be followed
by patient assessments that involve questions on UFs using the
EuroQol 5-Dimensions 5-Levels (EQ-5D-5L) survey, the
Uterine Fibroid Symptom and Quality of Life (UFS-QOL)
questionnaire, the Aberdeen Menorrhagia Severity Scale
(AMSS) questionnaire, and evaluation for concomitant
medications and history of previous procedural interventions. completed with the date and reason. The patient will be informed
that all data collected up to that point will be used in the
analysis. From that point on, the patient will no longer be
contacted for the registry, and no further data on that participant
will be collected. completed with the date and reason. The patient will be informed
that all data collected up to that point will be used in the
analysis. From that point on, the patient will no longer be
contacted for the registry, and no further data on that participant
will be collected. JMIR Res Protoc 2018 | vol. 7 | iss. 11 | e10926 | p. 3
(page number not for citation purposes) http://www.researchprotocols.org/2018/11/e10926/ Baseline Visit If deemed eligible for registry participation, the patient will
return for a visit in 3 to 6 months. When available, imaging and
other test results (biopsies, blood work) will also be documented. The baseline visit will involve (1) obtaining informed consent,
(2) checking for inclusion/exclusion criteria, (3) collecting Table 1. Schedule of events of the CAPTURE study at baseline and scheduled visits. Scheduled follow-up visits
Examinations
24-montha
12-montha
3- to 6-montha
Baseline
—
—
—
✓
Signed informed consent formb
—
—
—
✓
Inclusion/exclusion criteria
—
—
—
✓
Demographics
—
—
—
✓
Medical history
✓
✓
✓
✓
Fertility wishes
✓
✓
✓
✓
Blood work (hemoglobin)c
✓
✓
✓
✓
Gynecologic symptoms
✓
✓
✓
✓
Ultrasound of fibroids
—
—
—
✓
Type and location of fibroids
—
—
—
✓
Endometrial biopsyc
✓
✓
✓
✓
Planned/updated management plan
✓
✓
✓
✓
UFS-QOLd
✓
✓
✓
✓
Bleeding assessmente
✓
✓
✓
✓
EQ-5D-5Lf
✓
✓
✓
✓
Concomitant medications
✓
✓
✓
—
Adverse events Table 1. Schedule of events of the CAPTURE study at baseline and scheduled visits. Table 1. Schedule of events of the CAPTURE study at baseline and scheduled visits. aVisits can be performed by telephone or in clinic; however, an in-person visit is preferred. bMust be obtained from patient prior to study enrollment. dUFS-QOL: Uterine Fibroid Symptom and Quality of Life questionnaire. dUFS-QOL: Uterine Fibroid Symptom and Quality of Life questionnaire. eTool for assessment is the Aberdeen Menorrhagia Severity Scale questionnaire. eTool for assessment is the Aberdeen Menorrhagia Severity Scale questionnaire. fEQ-5D-5L: EuroQol 5-dimensions 5-levels survey used to measure health status. fEQ-5D-5L: EuroQol 5-dimensions 5-levels survey used to measure health status. JMIR Res Protoc 2018 | vol. 7 | iss. 11 | e10926 | p. 3
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RenderX XSL•FO
RenderX JMIR RESEARCH PROTOCOLS Bedaiwy et al Table 2. Schedule of events of the CAPTURE study at procedural intervention and hysterectomy. Baseline Visit Hysterectomy
Procedural interventiona
Examinations
1-year follow up
Postoperative
Intraoperative
Preoperative
—
—
—
—
Signed informed consent form
—
—
—
—
Inclusion/exclusion criteria
—
—
—
—
Demographics
—
—
—
—
Medical history
—
—
—
—
Fertility wishes
—
—
—
✓
Blood work (hemoglobin)b
✓
—
—
—
Gynecologic symptoms
—
—
—
✓
Ultrasound of fibroids
—
—
—
—
Type and location of fibroids
—
✓
—
—
Endometrial biopsy
—
—
—
✓
Planned/updated management plan
✓
✓
—
✓
UFS-QOLc
—
✓
—
✓
Bleeding assessmentd
✓
✓
—
✓
EQ-5D-5Le
—
—
—
✓
Concomitant medications
—
✓
✓
✓
Adverse event
aProcedural intervention is not mandatory in the registry, and data will be collected if physician and patient decide on a procedural intervention to
address symptoms associated with uterine fibroids. Table 2. Schedule of events of the CAPTURE study at procedural intervention and hysterectomy. Table 2. Schedule of events of the CAPTURE study at proced aProcedural intervention is not mandatory in the registry, and data will be collected if physician and patient decide on a procedural intervention to
address symptoms associated with uterine fibroids. b not mandatory in the registry, and data will be collected if physician and patient decide on a procedural intervention to
ed with uterine fibroids. bTo be captured if available. cUFS-QOL: Uterine Fibroid Symptom and Quality of Life questionnaire. dNot required if patient undergoes hysterectomy procedure. Otherwise the tool for assessment is the Aberdeen Menorrhagia Severity Scale questionnaire. eEQ-5D-5L: EuroQol 5-dimensions 5-levels survey used to measure health status. dNot required if patient undergoes hysterectomy procedure. Otherwise the tool for assessment is the Aberdeen Menorrhagia Severity Scale questionnaire. eEQ-5D-5L: EuroQol 5-dimensions 5-levels survey used to measure health status. morcellation (if performed), will be documented. In the case of
myomectomy, fibroid cleavage planes and density (subjective)
of the fibroids will be obtained as well. Preoperative Visit If a patient undergoes a hysterectomy, a follow-up visit will
occur 1 year after the hysterectomy (in addition to the
postoperative visit). This follow up will replace all remaining
scheduled follow-up study visits. Information on postoperative
symptoms, overall satisfaction, UFS-QOL-Hysterectomy, and
EQ-5D-5L will be obtained. Once this follow up is completed,
the patient will be deemed to have completed all study visits. This visit will only occur if the patient and physician agree on
a procedural intervention. During this visit, participants will be
evaluated for updates and an assessment of the management
plan, and for ADRs (if medication was prescribed at the previous
visit). Also, patient questionnaires (EQ-5D-5L, UFS-QOL, and
AMSS) will be administered. Postoperative Visit This visit will only occur if the patient and physician agree on
a procedural intervention. During this visit, information on AEs
following the procedure, pathology, and patient questionnaires
(EQ-5D-5L, UFS-QOL, and AMSS) will be recorded. Visits at Three to Six Months, Twelve Months, and
Twenty-Four Months These visits can be performed in the clinic or via telephone;
however, an in-person visit is preferred. Participants will be
evaluated for updates and an assessment of the management
plan, and for ADRs (if medication was prescribed at the previous
visit). Also, patient questionnaires (EQ-5D-5L, UFS-QOL, and
AMSS) will be administered. Follow up will occur after the
baseline meeting at 3 to 6 months, 12 months, and 24 months. http://www.researchprotocols.org/2018/11/e10926/ JMIR Res Protoc 2018 | vol. 7 | iss. 11 | e10926 | p. 4
(page number not for citation purposes) Assessment of Clinical Efficacy and Safety This is a patient registry that tracks the routine practice and
management of patients with UF in Canada. The physician will
collect data at the initial and follow-up visits between 3 to 6
months, 12 months, and 24 months. Also, if a procedural
intervention is performed, pre-, intra-, and postoperative data
will be captured. Patients will be asked to complete the
EQ-5D-5L, UFS-QOL, and the brief bleeding questionnaires
as per the visit schedule outlined in Tables 1 and 2. Intraoperative Visit Unscheduled visits (other than the prescribed appointments)
are any unexpected visits, including emergency assessments or
changes to care plans. Participants will be assessed for the reason
of the visit, update and evaluation of the management plan, and
ADRs (if medication was previously prescribed). This visit will only occur if the patient and physician agree on
a procedural intervention. During this visit, information on the
procedure performed and the AEs will be obtained. Specific
details on the type of procedure, route, and surgical times will
be recorded. Intraoperative description of outcomes, including JMIR Res Protoc 2018 | vol. 7 | iss. 11 | e10926 | p. 4
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RenderX JMIR RESEARCH PROTOCOLS Bedaiwy et al Additionally, where imputation techniques are not adequate,
sensitivity analysis will be employed. All parameter estimates,
confidence intervals, and P values will be reported from the
model, and a 2-sided significance level of .05 will be used. Pregnancy Follow Up If an unplanned pregnancy occurs with hormonal medical
therapy during this study, a serious AE will be recorded. Also,
the “Pregnancy follow up” form will be completed to capture
baseline pregnancy information. If the patient consents to be
followed up on the outcome of the pregnancy, then postdelivery
follow-up information will be collected once available. No study
visits will be completed during the pregnancy. Following
delivery, the patient will resume the study visit schedule until
completion. The original terms used by participating physicians to identify
ADRs will be coded using the Medical Dictionary for
Regulatory Activities (MedDRA). The MedDRA system organ
class and preferred term will be used to present the number and
percentage of patients with ADRs, the drug relationship,
seriousness, and intensity of ADRs. Chi-square tests will be
employed to compare the number of ADRs between groups of
interest. Data Management Process Overall coordination of the registry will be led by a steering
committee that includes 6 academic gynecologists and 1
community gynecologist. Committee responsibilities include
establishing the registry database and protocol, appointing a
scientific committee for data analysis and interpretation, and
governing the dissemination of the registry findings through a
publication plan. All members of the steering committee have
a vote on decision making, with the Chair providing a deciding
vote in the event of a tie. The study sponsor, Allergan Canada,
does not have a vote or seat on the steering committee but will
facilitate meetings. Statistical Methods The analysis will include all patients enrolled in the study who
have been entered into the database. Duration of Study The duration of the study will be 2 years from the recruitment
of the final patient, which occurred in February 2018. It is
expected that the last visit of the final patient will occur in
February 2020. All study data will be captured on HealthDiary, which is a
Web-based electronic data-capture system. Data will be
managed by the Applied Health Research Center (AHRC), a
service unit within the HUB Health Research Solutions and
fully integrated with the Li Ka Shing Knowledge Institute of
St. Michael’s Hospital, Toronto, Ontario, Canada. The AHRC
is a full-service clinical research coordinating center staffed
with highly trained individuals including statisticians,
methodologists, research coordinators, contract and finance
managers, and research informatics specialists. The facility will
serve as the data management center. It has managed large
randomized trials, cohort studies, and patient registries across
provincial and international borders, ensuring privacy
regulations using state-of-the-art, Web-based, data management
software. The center will be responsible for developing the
electronic case report form (eCRF), conducting data
management, and issuing and clarifying queries with the
participating sites. The research database includes automated
edit and logic checks to assist with the data management
activities. Data entry screens and edit checks will be tested with
mock patient data and removed from the database before
beginning the study. In addition to programmed edit and logic
checks, manual data verification, and consistency checks will
be performed to ensure valid, relevant, and complete data for
analysis. http://www.researchprotocols.org/2018/11/e10926/ Acknowledgments This study is funded by Allergan Inc. Editorial support was provided by Charlene Rivera, PhD, from Complete HealthVizion,
and was funded by Allergan plc (Dublin, Ireland). All authors have contributed to the preparation and development of this
manuscript, and have read and approved the final manuscript. This manuscript was reviewed by the CAPTURE Steering Committee,
which also contributed to the development of the study goals. The authors also greatly acknowledge the CAPTURE Publications
Committee. Results In Canada, there is a lack of documented data characterizing
the UF patient population, treatment options, and outcomes
associated with women who have UF. Also, no data exist
regarding patient preference and satisfaction related to their
management. Moreover, with the introduction of UPA—the
first Health Canada-approved medication for the treatment of
UF—an evaluation of effectiveness and safety is desirable. It
should be noted that UPA is undergoing a clinical investigation
in the United States and is currently not approved. In Europe,
a prospective, observational, noninterventional study is ongoing
to document real-world data related to current treatment patterns
and outcomes associated with UPA in patients with symptoms
associated with UF. This registry is collecting valuable
real-world data in Canada on the medical and procedural
management of UF to address these issues. Participant recruitment for this registry began in July 2015. The
registry has recruited 1500 patients, with the first patient
enrolled in the second half of 2015. Patient follow up will occur
for up to 2 years, with the last patient visit occurring in 2020. It is anticipated that national/industrial funding will be available
to continue this initiative beyond 2020 based on the information
generated from this registry. Importance of the CAPTURE Registry Despite the prominence of UF and their detrimental effects on
patient health, little is known about many aspects of the
condition, including the etiology of the disease, the natural
history of UF, the impact of UF on fertility and pregnancy
outcomes, and the effectiveness of UF treatments in real-world
clinical practice. There are virtually no well-designed, long-term,
epidemiologic studies published in this disease area. The
evidence on the comparative long-term effectiveness of
treatment options is also of poor quality. The Agency for
Healthcare Research and Quality (AHRQ) has published reviews
on the state of the evidence for the management of UF in 2001
and 2007. A more recent review was published by Singh and Statistical Analysis The goal of this study is to establish a registry of Canadian
patients with symptomatic UF. We have recruited a sample of
1500 patients to help describe the management of UF in this
population. Initially, exploratory data analysis will summarize all variables
of interest by reporting means and standard deviations for
continuous variables, and frequencies (percentages) for
categorical variables. Analysis of variance will be used to
compare continuous variables between treatment groups of
interest. Chi-square tests will be used to compare categorical
variables between the 2 groups. A linear mixed model will be
employed to examine the association between treatment groups
while adjusting for other covariates of clinical interest. These
variables include demographics, UF history, comorbidities,
medications, and medical tests (both current and previous). The model will include a time-by-treatment-interaction term to
determine whether treatments have improved quality of life
relative to other treatment groups. Furthermore, a random effect
will be included at the patient level to incorporate correlation
arising from the repeated measures design of the study. Medical history and concomitant medical conditions/diseases
will be coded according to MedDRA. Data handling procedures,
including edit check specifications, data entry guide, authorized
corrections, coding instructions, and locking procedure, will be
detailed in the study data management plan. Since this is an observational registry, missing data and
drop-outs (ie, early study termination) are expected. Statistical
techniques will be used to examine the missing data (including
logistic regression) and, if feasible, multiple imputations will
be used to make any adjustments. JMIR Res Protoc 2018 | vol. 7 | iss. 11 | e10926 | p. 5
(page number not for citation purposes) JMIR Res Protoc 2018 | vol. 7 | iss. 11 | e10926 | p. 5
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RenderX JMIR RESEARCH PROTOCOLS Bedaiwy et al Belland [10] outlining clinical data on the medical and surgical
management of UF along with a treatment algorithm to assist
health care professionals. Data Quality Assurance The database will be secured and enable quality control to be
performed at the time of data entry. Quality control will focus
on value ranges and missing data. Any discrepancies that occur
during eCRF data entry will be flagged in real time by the
system and will be saved until addressed by the data entry
personnel. Our literature search for established UF registries revealed only
1 in the past 15 years called the FIBROID Registry. This was
a multicenter, prospective, longitudinal study of the short- and
long-term outcomes of uterine artery embolization [14]. Despite
the lack of data on the management of UF, the future of
obtaining real-world data in this area appears promising. In
addition to this registry in Canada, registries in both the United
States and Europe have been announced or are currently
underway. In the second half of 2014, the Patient-Centered
Outcomes Research Institute and AHRQ announced that the
Duke University School of Medicine’s Department of Obstetrics
and Gynecology and the Duke Clinical Research Institute will
lead a 5-year patient registry to evaluate the effectiveness of
different treatment strategies for UF patients. In compliance with Good Clinical Practice and regulatory
requirements, the sponsor, a third party on behalf of the sponsor,
health regulatory agencies, or research ethics boards may
conduct quality assurance audits at any time during or following
the study. The participating investigator must agree to allow
auditors direct access to all project-related documents, including
source documents, and must agree to allocate their time and the
time of their project staff to the auditors to discuss findings and
issues. Potential Limitations This registry has some limitations. The instruments used at
different visits are self-reported, which may result in reporting
bias. The noninterventional design can lead to selection bias as
well as confounding. Finally, failure to follow up and the
expected attrition of the participants at subsequent visits
following recruitment could impact on the sample size, thus
limiting the external validity. JMIR Res Protoc 2018 | vol. 7 | iss. 11 | e10926 | p. 6
(page number not for citation purposes) References 1. Baird DD, Dunson DB, Hill MC, Cousins D, Schectman JM. High cumulative incidence of uterine leiomyoma in black
and white women: ultrasound evidence. Am J Obstet Gynecol 2003 Jan;188(1):100-107. [Medline: 12548202]
2. Bulun SE. Uterine fibroids. N Engl J Med 2013 Oct 03;369(14):1344-1355. [doi: 10.1056/NEJMra1209993] [Medline: 1. Baird DD, Dunson DB, Hill MC, Cousins D, Schectman JM. High cumulative incidence of uterine leiomyoma in black
and white women: ultrasound evidence. Am J Obstet Gynecol 2003 Jan;188(1):100-107. [Medline: 12548202] 1. Baird DD, Dunson DB, Hill MC, Cousins D, Schectman JM. High cumulative incidence of uterine leiomyoma in black
and white women: ultrasound evidence. Am J Obstet Gynecol 2003 Jan;188(1):100-107. [Medline: 12548202] 2. Bulun SE. Uterine fibroids. N Engl J Med 2013 Oct 03;369(14):1344-1355. [doi: 10.1056/NEJMra1209993] [Medline:
24088094] 3. Zimmermann A, Bernuit D, Gerlinger C, Schaefers M, Geppert K. Prevalence, symptoms and management of uterine
fibroids: an international internet-based survey of 21,746 women. BMC Womens Health 2012 Mar 26;12:6 [FREE Full
text] [doi: 10.1186/1472-6874-12-6] [Medline: 22448610] 3. Zimmermann A, Bernuit D, Gerlinger C, Schaefers M, Geppert K. Prevalence, symptoms and management of uterine
fibroids: an international internet-based survey of 21,746 women. BMC Womens Health 2012 Mar 26;12:6 [FREE Full
text] [doi: 10.1186/1472-6874-12-6] [Medline: 22448610] 4. Wallach EE, Vlahos NF. Uterine myomas: an overview of development, clinical features, and management. Obstet Gynecol
2004 Aug;104(2):393-406. [doi: 10.1097/01.AOG.0000136079.62513.39] [Medline: 15292018] 4. Wallach EE, Vlahos NF. Uterine myomas: an overview of development, clinical features, and management. Obstet Gynecol
2004 Aug;104(2):393-406. [doi: 10.1097/01.AOG.0000136079.62513.39] [Medline: 15292018] 5. Vilos G, Vilos A, Ferrazzi S. Symptomatic uterine fibroids (UFs) are common in premenopausal Canadian women and
lead to reduced quality of life (QoL) across multiple domains. J Obstet Gynecol Can 2014;36(Suppl 1):S26. 6. Vilos GA, Allaire C, Laberge P, Leyland N. The management of uterine leiomyomas. J Obstet Gynaecol Can 2015 5. Vilos G, Vilos A, Ferrazzi S. Symptomatic uterine fibroids (UFs) are common in premenopausal Canadian women and
lead to reduced quality of life (QoL) across multiple domains. J Obstet Gynecol Can 2014;36(Suppl 1):S26. 6. Vilos GA, Allaire C, Laberge P, Leyland N. The management of uterine leiomyomas. J Obstet Gynaecol Can 2015
Feb;37(2):157-178. [doi: 10.1016/S1701-2163(15)30338-8] [Medline: 25767949] 7. Dueholm M, Lundorf E, Hansen ES, Ledertoug S, Olesen F. Accuracy of magnetic resonance imaging and transvaginal
ultrasonography in the diagnosis, mapping, and measurement of uterine myomas. Am J Obstet Gynecol 2002
Mar;186(3):409-415. [Medline: 11904599] 7. Authors' Contributions The members of the CAPTURE Registry Steering Committee are as follows: Dr Philippe Laberge (Chair), Dr Sari Kives, Dr
Nicholas Leyland, Dr George Vilos, Dr Joshua Polsky, and Dr Liane Belland. The members of the CAPTURE Publications
Committee are the first author (MAB), Dr Ally Murji, and Dr Olga Bougie. http://www.researchprotocols.org/2018/11/e10926/ http://www.researchprotocols.org/2018/11/e10926/ XSL•FO
RenderX JMIR RESEARCH PROTOCOLS Bedaiwy et al Conflicts of Interest MAB has participated in advisory boards for Allergan Canada and AbbVie. PJ is an employee of Allergan. SSS has participated
in advisory boards, as a primary investigator, and presented continuing medical education events sponsored by Allergan Canada,
Bayer Pharma, and AbbVie. References Dueholm M, Lundorf E, Hansen ES, Ledertoug S, Olesen F. Accuracy of magnetic resonance imaging and transvaginal
ultrasonography in the diagnosis, mapping, and measurement of uterine myomas. Am J Obstet Gynecol 2002
Mar;186(3):409-415. [Medline: 11904599] 8. Vercellini P, Viganò P, Somigliana E, Daguati R, Abbiati A, Fedele L. Adenomyosis: epidemiological factors. Best Pract
Res Clin Obstet Gynaecol 2006 Aug;20(4):465-477. [doi: 10.1016/j.bpobgyn.2006.01.017] [Medline: 16563868] 8. Vercellini P, Viganò P, Somigliana E, Daguati R, Abbiati A, Fedele L. Adenomyosis: epidemiological factors. Best Pract
Res Clin Obstet Gynaecol 2006 Aug;20(4):465-477. [doi: 10.1016/j.bpobgyn.2006.01.017] [Medline: 16563868] 8. Vercellini P, Viganò P, Somigliana E, Daguati R, Abbiati A, Fedele L. Adenomyosis: epidemiological factors. Best Pract
Res Clin Obstet Gynaecol 2006 Aug;20(4):465-477. [doi: 10.1016/j.bpobgyn.2006.01.017] [Medline: 16563868]
9. Farquhar C, Brosens I. Medical and surgical management of adenomyosis. Best Pract Res Clin Obstet Gynaecol 2006
Aug;20(4):603 616 [doi: 10 1016/j bpobgyn 2006 01 012] [Medline: 16563872] Res Clin Obstet Gynaecol 2006 Aug;20(4):465-477. [doi: 10.1016/j.bpobgyn.2006.01.017] [Medline: 16563868]
9. Farquhar C, Brosens I. Medical and surgical management of adenomyosis. Best Pract Res Clin Obstet Gynaecol 2006
Aug;20(4):603-616. [doi: 10.1016/j.bpobgyn.2006.01.012] [Medline: 16563872] 9. Farquhar C, Brosens I. Medical and surgical management of adenomyosis. Best Pract Res Clin Obstet Gynaecol 2006
Aug;20(4):603-616. [doi: 10.1016/j.bpobgyn.2006.01.012] [Medline: 16563872] 9. Farquhar C, Brosens I. Medical and surgical management of adenomyosis. Best Pract Res Cli
Aug;20(4):603-616. [doi: 10.1016/j.bpobgyn.2006.01.012] [Medline: 16563872] 10. Singh SS, Belland L. Contemporary management of uterine fibroids: focus on emerging medical treatments. Curr Med Res
Opin 2015 Jan;31(1):1-12. [doi: 10.1185/03007995.2014.982246] [Medline: 25365466] 11. Donnez J, Tatarchuk TF, Bouchard P, Puscasiu L, Zakharenko NF, Ivanova T, PEARL I Study Group. Ulipristal acetate
versus placebo for fibroid treatment before surgery. N Engl J Med 2012 Feb 02;366(5):409-420. [doi:
10.1056/NEJMoa1103182] [Medline: 22296075] 12. Donnez J, Tomaszewski J, Vázquez F, Bouchard P, Lemieszczuk B, Baró F, PEARL II Study Group. Ulipristal acetate
versus leuprolide acetate for uterine fibroids. N Engl J Med 2012 Feb 02;366(5):421-432. [doi: 10.1056/NEJMoa1103180]
[Medline: 22296076] 13. AbbVie Corporation. Product Monograph. Lupron Depot. Leuprolide acetate for depot suspension. 2017. URL: http://www. abbvie.ca/content/dam/abbviecorp/ca/en/docs/LUPRON_DEPOT_ENDO_PM_EN.pdf[WebCite Cache ID 6yZhWmsSd] 13. AbbVie Corporation. Product Monograph. Lupron Depot. Leuprolide acetate for depot suspension. 2017. URL: http://www. abbvie.ca/content/dam/abbviecorp/ca/en/docs/LUPRON_DEPOT_ENDO_PM_EN.pdf[WebCite Cache ID 6yZhWmsSd] 14. Myers ER, Goodwin S, Landow W, Mauro M, Peterson E, Pron G, FIBROID Investigators. Prospective data collection of
a new procedure by a specialty society: the FIBROID registry. Obstet Gynecol 2005 Jul;106(1):44-51. [doi:
10.1097/01.AOG.0000165827.66541.dd] [Medline: 15994616] 14. References Myers ER, Goodwin S, Landow W, Mauro M, Peterson E, Pron G, FIBROID Investigators. Prospective data collection of
a new procedure by a specialty society: the FIBROID registry. Obstet Gynecol 2005 Jul;106(1):44-51. [doi:
10.1097/01.AOG.0000165827.66541.dd] [Medline: 15994616] 15. Stewart EA. Uterine fibroids. Lancet 2001 Jan 27;357(9252):293-298. [doi: 10.1016/S0140-6736(00)03622-9] [Medline:
11214143] 15. Stewart EA. Uterine fibroids. Lancet 2001 Jan 27;357(9252):293-298. [doi: 10.1016/S0140-6736(00)03622-9] [Medline:
11214143] 16. EuroQol. EQ-5D-5L. 2017. URL: https://euroqol.org/eq-5d-instruments/eq-5d-5l-about/ [accessed 2018-04-10] [WebCite
Cache ID 6yZhfVbqO] 16. EuroQol. EQ-5D-5L. 2017. URL: https://euroqol.org/eq-5d-instruments/eq-5d-5l-about/ [accessed 2018-04-10] [WebCite
Cache ID 6yZhfVbqO] 17. SIRFoundation. Uterine Fibroid Symptom Health-Related Quality of Life Questionnaire (UFS-QOL). 2017. URL: https:/
/www.sirfoundation.org/clinical-research2/clinical-research3/ufs-qol/ [accessed 2018-04-08] [WebCite Cache ID 6yZhrtmu5] 17. SIRFoundation. Uterine Fibroid Symptom Health-Related Quality of Life Questionnaire (UFS-QOL). 2017. URL: https:/
/www.sirfoundation.org/clinical-research2/clinical-research3/ufs-qol/ [accessed 2018-04-08] [WebCite Cache ID 6yZhrtmu5] JMIR Res Protoc 2018 | vol. 7 | iss. 11 | e10926 | p. 7
(page number not for citation purposes) http://www.researchprotocols.org/2018/11/e10926/ JMIR Res Protoc 2018 | vol. 7 | iss. 11 | e10926 | p. 8
(page number not for citation purposes) Abbreviations JMIR Res Protoc 2018 | vol. 7 | iss. 11 | e10926 | p. 7
(page number not for citation purposes) Bedaiwy et al JMIR RESEARCH PROTOCOLS UFS-QOL: Uterine Fibroid Symptom and Quality of Life questionnaire
UPA: ulipristal acetate Edited by G Eysenbach; submitted 04.05.18; peer-reviewed by B Ansa; comments to author 19.06.18; accepted 06.08.18; published
20.11.18 Please cite as:
Bedaiwy MA, Janiszewski P, Singh SS, CAPTURE Steering Committee
A Patient Registry for the Management of Uterine Fibroids in Canada: Protocol for a Multicenter, Prospective, Noninterventional
Study
JMIR Res Protoc 2018;7(11):e10926
URL: http://www.researchprotocols.org/2018/11/e10926/
doi: 10.2196/10926
PMID: 30459144 ©Mohamed A Bedaiwy, Peter Janiszewski, Sukhbir S Singh, CAPTURE Steering Committee. Originally published in JMIR
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Feno de maniçoba em dietas para ovinos: consumo de nutrientes, digestibilidade aparente e balanço nitrogenado
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Revista Brasileira de Zootecnia
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Correspondências devem ser enviadas para: divan@cca.ufpb.br Maniçoba hay in diets for sheep: intake, apparent digestibility and nitrogen balance ABSTRACT - The objective was to determine the voluntary intake and digestibility of DM, OM and nutrients (CP, GE,
NDF, ADF, CT, NSC and EE). And nitrogen balance of diets containing maniçoba maniçoba (Manihot glaziovii Muell. Arg.)
hay Twenty-four Santa Ines lambs, average 28.62 kg LW were allotted to a completely randomized design and fed diets
containing 20, 40, 60 e 80% of maniçoba hay.The mean intakes of DM, OM, CP and CT were 1.24, 1.12; 0.20; and 0.827
kg/d, respectively, and the mean intakes of ME, NDF, ADF, EE and NSC were, respectively, 2.78 mcal/kg, 401.01, 215.50,
83.42, 426.62, and 126.10 g/d. The apparent digestibility for DM, CP, and CE were respectively, 60.22, 61.28, 56.16, 61.90,
and for NDF, ADF CT and NSC were, respectively, 44.73, 54.63, 61.50, and 78.41. The nitrogen balance was positive, with
mean of 7.20 g N/kg DM/d, and the apparently retained nitrogen represented 21.45% of ingested N. The inclusion of 80%
of maniçoba hay in the diet did not affect the DM intake, however, the nutrient apparent digestibility in the diet decreased
as maniçoba hay increased in the diet. Key Words: digestibility coefficient, Manihot glaziovii Muell. Arg., native forage, sheep, voluntary intake Key Words: digestibility coefficient, Manihot glaziovii Muell. Arg., native forage, sheep, voluntary inta Divan Soares da Silva2, Jacilene Maria da Cunha Castro3, Ariosvaldo Nunes de Medeiros2,
Edgard Cavalcanti Pimenta Filho2, Daerson Dantas Barroso4 1 Parte da tese apresentada pelo segundo autor ao curso de Doutorado Integrado em Zootecnia da UFPB/UFRPE/UFC. P
pelo CNPq. resentada pelo segundo autor ao curso de Doutorado Integrado em Zootecnia da UFPB/UFRPE/UFC. Pesquisa financiada p
q
2 DZ/CCA/UFPB, CEP: 58397-000 - Areia, PB. ,
,
3 Zootecnista formada pelo DZ/CCA/UFPB - CEP: 58397-000 - Areia, PB. 3 Zootecnista formada pelo DZ/CCA/UFPB - CE 4 Programa de Pós-Graduação em Zootecnia/UFPB - Areia, PB. RESUMO - Objetivou-se determinar o consumo voluntário, a digestibilidade da MS, MO e dos nutrientes (PB, EB, FDN,
FDA, CT, CNF e EE) e o balanço de nitrogênio de dietas contendo feno de maniçoba (Manihot glaziovii Muell. Arg.). Vinte
quatro cordeiros Santa Inês com média de 28,62 kg PV foram distribuídos em um delinemento completamente casualizado e
alimentados com rações contendo 20, 40, 60 ou 80% de feno de maniçoba. Os consumos médios de MS, MO, PB e CT foram
1,24; 1,12; 0,20 e 0,82 kg/dia, respectivamente, e os consumos médios de EM, FDN, FDA, EE e CNF foram, respectivamente,
2,78 mcal/dia; 401,01; 215,50; 83,42; 426,62; e 126,10 g/dia. Os coeficientes de digestibilidade de MS, PB e EB foram,
respectivamente, 60,22; 56,16 e 61,90%, e os de FDN, FDA, CT e CNF, 44,73; 54,63; 61,50 e 78,41%, respectivamente. O balanço nitrogenado foi positivo, com média de 7,20 g N/kg MS/dia, e o nitrogênio aparentemente retido representou 21,45%
do N ingerido. A inclusão de 80% de feno de maniçoba na dieta não afetou o consumo de MS, no entanto, reduziu a digestibilidade
aparente dos nutrientes da dieta. Revista Brasileira de Zootecnia
© 2007 Sociedade Brasileira de Zootecnia
ISSN impresso: 1516-3598
ISSN on-line: 1806-9290
www.sbz.org.br R. Bras. Zootec., v.36, n.5, p.1685-1690, 2007 (supl.) Introdução pelo trato digestivo. A digestibilidade é influenciada por
fatores relacionados ao animal ou inerentes ao alimento,
como composição, relação entre os nutrientes, forma de
preparo das rações e densidade energética da ração. O valor nutritivo de um alimento está condicionado ao
consumo voluntário, à digestibilidade e à eficiência
energética. Segundo Coelho da Silva & Leão (1979), a
digestibilidade de um alimento é a capacidade de permitir a
utilização de seus nutrientes pelo animal, expressa pelo
coeficiente de digestibilidade do nutriente. Cochran & Galyean
(1994) definiram a digestibilidade como a fração de determi-
nado alimento ou constituinte da dieta perdida na passagem O consumo voluntário é determinante na produção animal,
pois constitui o aporte de nutrientes para atender às exigências
de mantença e produção (Noller & Moe, 1995). O consumo
depende do animal, do alimento, das condições de alimentação
e do meio ambiente (Mertens, 1994) e pode ser regulado por
fatores físicos, psicogênicos e fisiológicos (Forbes, 1995). 1686 Silva et al. gaveta, com separador para coleta total de fezes e urina,
providas de comedouro e bebedouro. O ensaio teve dura-
ção de 21 dias, 14 de adaptação e sete de coleta de dados
e os animais foram pesados no início e no final do período
experimental. De acordo com Mertens (1994) e Forbes (1995), quando
a densidade energética da dieta é alta ou o teor de fibra é
baixo, a ingestão é regulada pela demanda fisiológica de
energia. Todavia, no caso de dietas com elevado conteúdo
de fibra ou baixa densidade energética, o consumo é regu-
lado por mecanismos físicos, como o efeito de enchimento
do rúmen-retículo. No entanto, a ingestão de fibra é impor-
tante, especificamente a FDN, para manter em equilíbrio a
ruminação e salivação, o pH e a atividade ruminal. As dietas experimentais foram constituídas de 20, 40, 60
e 80% de feno de maniçoba (Manihot glaziovii Muell. Arg.), na dieta total, e concentrado composto de milho,
farelo de soja, óleo de soja, melaço e núcleo mineral. O consumo e a digestibilidade são altamente
correlacionados à qualidade do alimento. Para rações ricas
em concentrado, com teor de FDN abaixo de 25% e
digestibilidade acima de 66%, o consumo é menor quanto
mais digestivo o alimento, enquanto, em rações de baixa
qualidade, com teor de FDN acima de 75%, o consumo é
maior quanto melhor a digestibilidade do alimento (Mertens,
1994; Cardoso et al., 2000). Introdução O feno foi confeccionado com ramas de maniçoba com
folhas e hastes de aproximadamente 1,0 cm de diâmetro. As
plantas encontravam-se no estádio vegetativo de floração
e início de frutificação. O material coletado foi triturado,
espalhado sobre lonas plásticas e revirado freqüentemente
para desidratação até o ponto de feno. Para confecção das
rações experimentais, o feno foi triturado em forma de farelo. As dietas foram fornecidas à vontade, duas vezes ao
dia, sempre às 7h e às 16h, procedendo-se à pesagem do
alimento fornecido e das sobras para estimativa do consumo
diário individual. A quantidade fornecida foi ajustada
diariamente, considerando 20% de sobras. Do mesmo modo,
foi estimado o consumo de água. Segundo Mertens (1992), a redução drástica nos níveis
de fibra em dietas para ruminantes pode ser prejudicial para
a digestibilidade total dos alimentos, uma vez que a fibra é
fundamental para manutenção das condições ótimas do
rúmen. Portanto, o teor de FDN da ração não deve ser
inferior a 25% de MS e 70 a 75% do teor de MS deve ser
proveniente do volumoso. Segundo Cardoso et al. (2000),
a quantidade ideal de FDN na dieta não está definida e pode
variar de acordo com o nível de produção animal e do tipo
de forragem utilizada. Amostras das rações oferecidas foram coletadas e
encaminhadas a análises químicas, segundo Silva & Queiroz
(2002), e os resultados são descritos na Tabela 1. Das sobras de ração coletou-se uma alíquota diária de
20% e formaram-se amostras compostas individuais, que
foram acondicionadas em sacos plásticos e armazenadas
para análises. Para a coleta de fezes e de urina, utilizaram-se
baldes plásticos. Nos coletores de urina, foram colocados
diariamente 20 mL de uma solução de ácido clorídrico a 50%. A coleta de dados para análises de fezes e urina foi realizada
diariamente, sempre às 8h30, registrando-se as quantidades
excretadas por animal. Após homogeneização do material,
retirou-se uma alíquota diária de 20% para confecção de
amostras compostas por animal, tanto de fezes como de
urina, as quais foram acondicionadas em sacos plásticos
(para fezes) e tubos plásticos (para urina) e armazenadas em
freezer até o momento das análises. Silva (2000), utilizando feno de Cynodon spp. Cv. Tifton-85 em dietas com 38,5; 52,6 e 66,4% de FDN para ovinos
Santa Inês, observou redução no consumo e na digestibilidade
da MS com o aumento no teor de FDN da ração. Introdução Estudando a composição química e o valor nutritivo do
feno de maniçoba, Barros et al. (1990) encontraram valores
93,3% de MS; 12,0% de PB; 5,2% de proteína digestível;
58,6% de FDN; 17,1% de lignina, energia digestível de 2,0
mcal/kg MS e coeficientes de digestibilidade de MS e PB de
47,4 e 46,4%, respectivamente. Araújo et al. (1996) encon-
traram valores de 89,88% MS, 9,46% PB, 62,77% NDT e
digestibilidade de MS e PB de 66,8 e 54,57%, respectiva-
mente, para o feno de maniçoba. Os carboidratos totais (CT) e os nutrientes digestíveis
totais (NDT) foram obtidos segundo Sniffen et al (1992),
enquanto os carboidratos não fibrosos (CNF) foram calcu-
lados por meio de adaptação da equação de Weiss (1999). Foram estimados o consumo de nutrientes, o coeficiente de
digestibilidade de MS, MO, PB, FDN, FDA, EB, CT e CNF,
o consumo de NDT e o balanço nitrogenado. O balanço
nitrogenado foi determinado pela diferença entre o N consu-
mido e o eliminado nas fezes e na urina. Objetivou-se neste estudo determinar o consumo, a
digestibilidade dos nutrientes e o balanço nitrogenado de
rações completas com diferentes níveis de feno de maniçoba
para ovinos Santa Inês em fase de recria. © 2007 Sociedade Brasileira de Zootecnia Material e Métodos Foram utilizados 24 cordeiros Santa Inês, machos não-
castrados, com peso médio de 28,62 kg e 112 dias de idade,
alojados em gaiolas para estudos de digestibilidade, do tipo O delineamento experimental utilizado foi inteiramente
ao acaso, com quatro tratamentos e seis repetições. As Feno de maniçoba em dietas para ovinos: consumo de nutrientes, digestibilidade aparente e balanço nitrogenado 1687 Tabela 1 - Composição químico-bromatológica das dietas expe-
rimentais
Table 1 -
Chemical composition of the experimental diets
Ingrediente
Feno maniçoba na dieta (%)
Ingredient
Maniçoba hay in the diet
20
40
60
80
Milho (Corn grain)
57,08
39,07
21,49
4,35
Farelo de soja (Soybean meal)
15,22
13,13
11,07
8,06
Feno de maniçoba (Maniçoba hay) 20,49
40,58
60,28
80,59
Melaço (Molasses)
3,76
3,72
3,68
3,65
Óleo de soja (Soybean oil)
0,88
0,88
0,87
0,86
Calcário calcítico (Limestone)
0,80
0,88
0,87
0,77
Núcleo mineral (Mineral salts)
1,77
1,75
1,73
1,72
MS (%) (DM)
85,71
85,91
86,07
86,23
MO (%) (OM)
91,50
90,52
89,66
88,88
PB (%) (CP)
16,37
16,67
16,96
16,92
EM (ME) (mcal/kg MS)
2,51
2,30
2,13
1,81
EE (%)
4,81
5,58
6,34
7,13
FDN (%) (NDF)
27,46
30,42
33,34
36,46
FDA (%) (ADF)
10,10
15,17
20,15
25,24
CT (%) (TC)
70,29
68,25
66,31
64,80
CNF (%) (NFC)
42,86
37,86
33,01
28,37
Lignina (%) (Lignin)
3,96
5,38
6,77
8,21
Celulose (%) (Cellulose)
7,47
11,49
15,42
19,42
NIDN (%) (NDIN)
1,86
1,87
1,88
1,87
NIDA (%) (ADIN)
2,33
1,77
1,23
0,70
Cinzas (%) (Ash)
8,50
9,48
10,34
11,12
Ca (g/kg)
9,00
9,57
9,80
9,91
P (g/kg)
4,89
4,74
4,60
4,44
NIDN - nitrogênio insolúvel em detergente neutro, NIDA - nitrogênio
insolúvel em detergente ácido (NDIN- Neutral detergent insoluble nitrogen, ADIN
- Acid detergent insoluble nitrogen). para ovinos. Resultado semelhante ao obtido neste
estudo foi encontrado por Silva (2000) em dietas com rela-
ções concentrado:volumoso de 60:40, 40:60 e 20%:80%. Segundo Forbes (1995), o consumo voluntário apresen-
ta relação positiva com o peso corporal em animais em fase
de crescimento. Os valores encontrados em %PV foram
próximos aos descritos pelo NRC (1985) e sugerem consumo
entre 4,3 e 5,0 %PV para cordeiros desmamados precoce-
mente e com taxas de crescimento de 200 a 250 g/dia. Material e Métodos Os níveis de feno na dieta total influenciaram (P<0,05)
o consumo de PB expresso em g/kgPV0,75, com média de
16,70 g/kg PV0,75, e a equação ajustada indicou aumento de
0,0537g para cada 1% de feno adicionado à dieta (Tabela 2). As análises de variância mostram efeito linear decrescente
(P<0,01) dos níveis de feno sobre o consumo de EM
(Tabela 2), que apresentou média de 2,78 mcal/kg MS/dia. A equação de regressão descreveu redução em torno de
0,0136 mcal/kg MS/dia para cada unidade percentual de
feno acrescida. Apesar de o consumo de MS não ter
diferido entre os níveis de feno, verificou-se diferença no
consumo de EM, o que pode ser justificado pela compo-
sição da dieta, que não foram isoenergéticas. As dietas
com maior proporção de volumoso apresentaram menor
concentração de EM (Tabela 1), provavelmente em virtude
do teor mais elevado de FDN. NIDN - nitrogênio insolúvel em detergente neutro, NIDA - nitrogênio
insolúvel em detergente ácido (NDIN- Neutral detergent insoluble nitrogen, ADIN
- Acid detergent insoluble nitrogen). Os valores de consumo de EM foram obtidos a partir do
coeficiente de digestibilidade da EB (Tabela 4), ajustado em
82% entre a energia digestível e a metabolizável. Os consumos
de FDN, FDA e EE foram influenciados (P<0,01) pelos níveis
de feno de maniçoba nas rações. Houve efeito linear crescente
dos níveis de feno sobre o consumo desses nutrientes
(Tabela 3), o que está relacionado à composição das dietas
(Tabela 1), que apresentaram níveis crescentes de FDN,
FDA e EE conforme diminuiu a participação de concentrado
e aumentou a de feno. Os valores médios de consumo foram
de 401, 215,5 e 83,42 g/dia, respectivamente, para FDN, FDA
e EE. Em relação ao peso vivo, o consumo médio diário de
FDN foi de 1,4%. análises estatísticas das variáveis estudadas foram inter-
pretadas por meio de análises de variância e de regressão
utilizando-se o SAEG (UFV, 2000), adotando-se os níveis de
1 e 5% de probabilidade. © 2007 Sociedade Brasileira de Zootecnia Resultados e Discussão metabolizable energy (ME) in sheep in the initial raising phase fed diets with maniçoba hay Tabela 3 - Consumo de nutrientes em ovinos em fase de recria alimentados com dietas contendo feno de maniçoba
Table 3 -
Intake of nutrients in sheep in the initial raising phase fed diets with maniçoba hay
Nutriente
Feno maniçoba na dieta (%)
CV (%)
Regressão
R2
Nutrient
Maniçoba hay in the diet
Regression
20
40
60
80
FDN (g) (NDF)
354,00
378,06
399,70
472,31
13,92
Yˆ = 306,87 + 1,8828**X
0,91
FDN(%PV) (NDF, %BW)
1,16
1,26
1,41
1,78
12,10
Yˆ = 0,9055 + 0,0099**X
0,91
FDA (g) (ADF)
127,44
182,27
233,55
318,61
13,87
Yˆ = 59,274 + 3,1239**X
0,98
EE (g)
68,02
79,80
84,24
101,64
14,34
Yˆ = 57,093 + 0,5265**X
0,95
CT (g) (TC)
864,64
832,32
783,24
830,35
14,74
Yˆ = 827,64
-
CNF (g) (NSC)
510,65
454,25
383,55
358,04
15,86
Yˆ = 558,75 - 2,6426**X
0,97
NDT (g) (TDN)
880,70
807,20
702,36
642,53
18,51
Yˆ = 962,98 - 4,096**X
0,99
** (P<0,01). onsumo de nutrientes em ovinos em fase de recria alimentados com dietas contendo feno de maniçoba
take of nutrients in sheep in the initial raising phase fed diets with maniçoba hay FDN. O consumo de NDT, no entanto, foi afetado pelas
dietas (P<0,01) e apresentou média de 758,20 g/dia. Houve
efeito linear decrescente e redução de 4,096 g/dia no con-
sumo NDT a cada unidade percentual de feno acrescida na
ração (Tabela 3), o que pode estar relacionado ao aumento
no consumo de FDN e à redução no consumo de CNF. Os coeficientes de digestibilidade da MS decresceram
de 71,30 para 46,92%, enquanto os da MO reduziram de
72,12 para 48,06% para dietas com 20 e 80% de feno,
respectivamente. A equação de regressão comprovou redu-
ção na digestibilidade da MS de 0,404% a cada 1% de
aumento no nível de feno (Tabela 4). As dietas contendo 20
e 40% de feno de maniçoba apresentaram coeficientes de
digestibilidade superiores a 65%, enquanto aquela com
80% do volumoso apresentou baixo coeficiente de
digestibilidade da MS. Foram observadas diferenças significativas (P<0,01)
na digestibilidade de MS, MO, PB, EB, FDN, FDA, CT, CNF
e cinzas (Tabela 4), porém, os níveis de feno na dieta não
influenciaram a digestibilidade do EE, cujo coeficiente médio
foi 69,56%. Resultados e Discussão ntes e de energia metabolizável (EM) em ovinos em fase de recria alimentados com dietas contendo feno Tabela 2 - Consumo de nutrientes e de energia metabolizável (EM) em ovinos em fase de recria alimentados com dietas contendo feno
de maniçoba
Table 2 -
Intake of nutrients and metabolizable energy (ME) in sheep in the initial raising phase fed diets with maniçoba hay
Nutriente
Feno maniçoba na dieta (%)
CV (%)
Regressão
R2
Nutrient
Maniçoba hay in the diet
Regression
20
40
60
80
MS (kg) (DM)
1,24
1,24
1,20
1,30
14,80
Yˆ = 1,24
-
MS (% PV) (DM, % BW)
4,10
4,12
4,21
4,90
12,85
Yˆ = 3,6985 + 0,0126*X
0,72
MS (g/kg PV0,75) (DM, g/kg BW0.75)
95,72
96,32
97,05
111,12
11,50
Yˆ = 88,323 + 0,2346*X
0,67
MO (kg) (OM)
1,13
1,12
1,10
1,15
14,80
Yˆ = 1,13
-
PB (g) (CP)
200,30
205,42
203,80
220,54
15,55
Yˆ = 207,52
-
PB (g/kgPV0,75) (CP, g/kg BW0.75)
15,44
15,95
16,50
18,84
12,20
Yˆ = 13,996 + 0,0537**X
0,85
EM (mcal/kg) (MSME, mcal/kg DM)
3,21
2,89
2,59
2,41
20,57
Yˆ = 3,4548 - 0,0136**X
0,98
* (P<0,05) ** (P<0,01). Tabela 3 - Consumo de nutrientes em ovinos em fase de recria alimentados com dietas contendo feno de maniçoba
Table 3 -
Intake of nutrients in sheep in the initial raising phase fed diets with maniçoba hay
Nutriente
Feno maniçoba na dieta (%)
CV (%)
Regressão
R2
Nutrient
Maniçoba hay in the diet
Regression
20
40
60
80
FDN (g) (NDF)
354,00
378,06
399,70
472,31
13,92
Yˆ = 306,87 + 1,8828**X
0,91
FDN(%PV) (NDF, %BW)
1,16
1,26
1,41
1,78
12,10
Yˆ = 0,9055 + 0,0099**X
0,91
FDA (g) (ADF)
127,44
182,27
233,55
318,61
13,87
Yˆ = 59,274 + 3,1239**X
0,98
EE (g)
68,02
79,80
84,24
101,64
14,34
Yˆ = 57,093 + 0,5265**X
0,95
CT (g) (TC)
864,64
832,32
783,24
830,35
14,74
Yˆ = 827,64
-
CNF (g) (NSC)
510,65
454,25
383,55
358,04
15,86
Yˆ = 558,75 - 2,6426**X
0,97
NDT (g) (TDN)
880,70
807,20
702,36
642,53
18,51
Yˆ = 962,98 - 4,096**X
0,99
** (P<0,01). Resultados e Discussão As dietas não influenciaram os consumos de MS e MO
expressos em kg/dia e o consumo de PB expresso em g/dia,
os quais apresentaram valores médios de 1,24; 1,12 kg/dia
e 207,51 g/dia, respectivamente (Tabela 2). Os consumos de
MS calculados em %PV e em g/kgPV0,75, no entanto, foram
afetados significativamente (P<0,05) pelas dietas e apre-
sentaram valores médios de 4,33%PV e 100,05 g/kg PV0,75. As equações de regressão revelaram efeito linear crescente
com o aumento dos níveis de feno nas dietas, com ajustes
de 0,0126 %PV e 0,2346 g/kg PV0,75 no CMS para cada 1%
de acréscimo no nível de feno da ração. Os níveis de feno nas dietas não influenciaram o con-
sumo de CT (média de 827,63 g/dia), provavelmente em
virtude de o teor deste nutriente praticamente não diferir,
resultando apenas em pequenas variações entre as dietas
(Tabela 2). Esse consumo foi superior ao de 566,8 g/dia
observado por Araújo et al. (2000) em ovinos alimentados
com dietas contendo feno de maniçoba. Esses valores foram próximos aos encontrados por
Barros et al. (1990) e Barros et al. (1997), que verificaram
consumos de 90,2 a 97,6 g/kg PV0,75/dia e 3,9% PV, no
entanto, superaram a média observada por Araújo et al. (1996), de 75,81 g/kg0,75, ao fornecerem feno de maniçoba O consumo médio de CNF (426,62 g/dia) foi influen-
ciado pela dieta (P<0,01). A análise de regressão indicou
efeito linear decrescente dos níveis de feno na dieta sobre
o consumo de CNF (Tabela 3), com ajustes de -2,6426 g/dia. Esse resultado foi inverso ao observado para consumo de 1688 Silva et al. © 2007 Sociedade Brasileira de Zootecnia Resultados e Discussão Tabela 2 - Consumo de nutrientes e de energia metabolizável (EM) em ovinos em fase de recria alimentados com dietas contendo feno
de maniçoba
Table 2 -
Intake of nutrients and metabolizable energy (ME) in sheep in the initial raising phase fed diets with maniçoba hay Tabela 2 - Consumo de nutrientes e de energia metabolizável (EM) em ovinos em fase de recria alimentados com dietas contendo feno
de maniçoba
Table 2 -
Intake of nutrients and metabolizable energy (ME) in sheep in the initial raising phase fed diets with maniçoba hay
Nutriente
Feno maniçoba na dieta (%)
CV (%)
Regressão
R2
Nutrient
Maniçoba hay in the diet
Regression
20
40
60
80
MS (kg) (DM)
1,24
1,24
1,20
1,30
14,80
Yˆ = 1,24
-
MS (% PV) (DM, % BW)
4,10
4,12
4,21
4,90
12,85
Yˆ = 3,6985 + 0,0126*X
0,72
MS (g/kg PV0,75) (DM, g/kg BW0.75)
95,72
96,32
97,05
111,12
11,50
Yˆ = 88,323 + 0,2346*X
0,67
MO (kg) (OM)
1,13
1,12
1,10
1,15
14,80
Yˆ = 1,13
-
PB (g) (CP)
200,30
205,42
203,80
220,54
15,55
Yˆ = 207,52
-
PB (g/kgPV0,75) (CP, g/kg BW0.75)
15,44
15,95
16,50
18,84
12,20
Yˆ = 13,996 + 0,0537**X
0,85
EM (mcal/kg) (MSME, mcal/kg DM)
3,21
2,89
2,59
2,41
20,57
Yˆ = 3,4548 - 0,0136**X
0,98
* (P<0,05) ** (P<0,01). Resultados e Discussão Tabela 4 - Digestibilidade aparente dos nutrientes em ovinos em fase de recria alimentados com dietas contendo feno de maniçoba
Table 4 -
Apparent nutrient digestibility in sheep in the initial raising phase fed diets with maniçoba hay 0,1217% na digestibilidade desse nutriente. Os coeficientes
variaram de 59,0 a 50,42% e a média foi de 56,16%. Araújo
et al. (2000) também observaram redução na digestibilidade
da PB à medida que aumentaram o nível de feno de maniçoba
nas dietas e obtiveram valores de 62,3 e 58,1% nos níveis de
40 e 60%, respectivamente. A redução na digestibilidade da
PB da dieta com 80% de feno de maniçoba pode estar
relacionada à concentração de N ligado à FDN. carboidratos estruturais, no entanto, são menos digestíveis,
o que justifica os resultados obtidos neste estudo. Nesta pesquisa, dietas com 60 e 80% de feno de
maniçoba apresentaram teores de FND mais elevados, o que
resultou em menor nível energético. Esse menor nível
energético ocasionou redução dos coeficientes de
digestibilidade da PB, EB e da fração fibrosa em comparação
às dietas com 20 e 40% do volumoso, uma vez que a
eficiência na utilização da proteína está positivamente rela-
cionada à disponibilidade de energia que será utilizada
pelos microrganismos do rúmen para transformarem o N da
dieta em proteína microbiana. A digestibilidade da energia bruta foi influenciada
pelas dietas (P<0,01) e apresentou coeficiente médio de
61,90%. A análise de regressão revelou valores decrescen-
tes para a digestibilidade à medida que aumentou a partici-
pação do volumoso na dieta, com ajustes de -0,36% para
cada unidade de feno acrescida. Para a dieta com 80% de
volumoso, obteve-se coeficiente de digestibilidade da EB
de 50,20%, próximo ao de 46,4% observado por Barros et al. (1990) com o feno de maniçoba. Ao aumentar a proporção
de feno, aumentaram as concentrações de FDN e FDA e
diminuiu a densidade energética da dieta, o que levou,
conseqüentemente, à redução na digestibilidade da EB. O consumo médio de nitrogênio (N) foi de 33,20 g/dia
e não foi influenciado pelas dietas (Tabela 5), o que pode
ser explicado pela composição das dietas (isoprotéicas) e
pelo fato de o consumo de MS não ter diferido entre as
dietas. Esse resultado se assemelha ao observado por
Barros et al. (1990) em ovinos alimentados com dietas
contendo feno de maniçoba. Resultados e Discussão Com o aumento dos níveis de feno de maniçoba,
verificou-se redução na digestibilidade dos nutrientes,
confirmada pela análise de regressão, que revelou efeito
linear decrescente, conforme relatado por Ladeira et al. (1999), Cardoso et al. (2000) e Dias et al. (2000), que encon-
traram coeficientes de digestibilidade mais baixos para
dietas com maiores níveis de volumoso. O coeficiente de digestibilidade da MO (média de
61,28%), segundo a análise de regressão, reduziu 0,3976%
a cada incremento do volumoso. A digestibilidade da MO
da dieta com 80% de feno de maniçoba foi 66,6% menor que
a da dieta com 20% de feno (Tabela 4). Esse comportamento
pode estar relacionado à menor concentração de CNF em
relação aos carboidratos totais nas rações com maior pro-
porção de feno de maniçoba. A digestibilidade do alimento é influenciada pelo con-
teúdo e pelo tipo de fibra (Van Soest, 1985). A fração FDN
do concentrado é potencialmente mais digestível que a de
forrageiras. Observou-se efeito linear decrescente dos níveis de
feno sobre a digestibilidade da PB (Tabela 4), de modo que,
a cada 1% de feno acrescentado na dieta, houve redução de © 2007 Sociedade Brasileira de Zootecnia maniçoba em dietas para ovinos: consumo de nutrientes, digestibilidade aparente e balanço nitrogenado 1689 Tabela 4 - Digestibilidade aparente dos nutrientes em ovinos em fase de recria alimentados com dietas contendo feno de maniçoba
Table 4 -
Apparent nutrient digestibility in sheep in the initial raising phase fed diets with maniçoba hay
Nutriente
Feno de maniçoba na dieta (%)
CV (%)
Regressão
R2
Nutrient
Maniçoba hay in the diet
Regression
20
40
60
80
MS (%) (DM)
71,30
65,16
57,51
46,92
10,02
Yˆ = 80,423 - 0,404**X
0,98
MO (%) (OM)
72,12
66,14
58,80
48,06
9,50
Yˆ = 1,159 - 0,3976**X
0,98
PB (%) (CP)
59,00
56,95
58,30
50,42
8,10
Yˆ = 2,249 - 0,1217**X
0,64
EB (%) (GE)
72,21
65,40
59,72
50,20
7,58
Yˆ = 79,808 - 0,3586**X
0,99
FDN (%) (NDF)
63,85
52,21
37,23
25,64
27,90
Yˆ = 77,129 - 0,648**X
0,99
FDA (%) (ADF)
74,10
59,40
49,30
35,80
25,91
Yˆ = 85,909 - 0,6255**X
0,99
EE (%)
70,26
72,00
69,80
66,26
8,60
Yˆ = 69,58
-
CT (%) (CT)
75,30
67,83
57,63
45,14
12,04
Yˆ = 86,641 - 0,5033**X
0,99
CNF (%) (NSC)
83,26
80,90
78,86
70,64
5,70
Yˆ = 88,386 - 0,1994**X
0,88
** (P<0,01). Resultados e Discussão A excreção de N fecal foi afetada pelas dietas (P<0,01),
o que não ocorreu com o N urinário, que apresentou valores
médios de 14,56 e 11,43 g/dia, respectivamente. A dieta com
80% de volumoso resultou em maior excreção de N (Tabela
5), certamente em virtude do teor de N ligado à FDA do feno
de maniçoba, que representa 23,2% do N total da dieta. As frações FDN e FDA apresentaram valores médios de
digestibilidade de 44,73 e 54,63%, respectivamente, e foram
maiores para a dieta com 20% de feno e menores para aquela
com 80% do volumoso (Tabela 4), o que pode estar associado
aos acréscimos dos teores de lignina das dietas. As análises de variância para carboidratos totais e CNF
indicaram redução na digestibilidade com o aumento do
nível de feno na dieta (média de 61,50 e 78,41%, respectiva-
mente). Esses resultados corroboram os encontrados por
Ladeira et al. (1999) e Cardoso et al. (2000) em bovinos
alimentados com feno de Cynodon dactylon e cinco níveis
de concentrado. A concentração de carboidratos não-estru-
turais em alimentos concentrados é maior que em volumosos,
que possuem alto teor de carboidratos estruturais. Os Segundo Barros et al. (1990), esse efeito decorre, em
parte, da alta concentração de lignina na maniçoba, caracte-
rística acentuada das espécies lenhosas, o justifica a baixa
digestibilidade da proteína bruta dessa forrageira. A excreção
média de N representou 78,31% do N total ingerido. O nitrogênio aparentemente retido (N Apar Ret) repre-
sentou em média 21,45% do N ingerido. O balanço de N (em
média de 7,20 g/animal/dia) não foi influenciado pelas dietas
e variou de 8,31 g/animal/dia (dieta com menor participação © 2007 Sociedade Brasileira de Zootecnia 1690 Silva et al. Literatura Citada y
g
y
p
NATIONAL RESEARCH COUNCIL - NRC. Nutrient requirements
of sheep. Washington, D.C.; 1985. 99p. ARAÚJO, E.C.; SILVA, V.M.; PIMENTEL, A.L. et al. Valor nutritivo
e consumo voluntário de forrageiras nativas da região semi-
árida do Estado de Pernambuco – VII Maniçoba (Manihot
epruinosa Pax & Hoffmann). In: SIMPÓSIO NORDESTINO
DE ALIMENTAÇÃO DE RUMINANTES, 6., 1996, Natal. Anais... Natal: Sociedade Nordestina de Produção Animal, 1996. p.194. NOLLER, C.H.; MOE, P.W. Determination of NRC energy and
protein
requirements
for
ruminants. In:
SIMPÓSIO
INTERNACIONAL SOBRE EXIGÊNCIAS NUTRICIONAIS DE
RUMINANTES, 1995, Viçosa, MG. Anais... Viçosa, MG: JARD,
1995. p.53-76. SILVA, A.M.A. Exigências de energia e proteína, composição
corporal e digestibilidade de nutrientes em ovinos. Jaboticabal: Universidade Estadual Paulista, 2000. 93p. Tese
(Doutorado em Zootecnia) - Universidade Estadual Paulista, 2000. ARAÚJO, G.G.L.; MOREIRA, J.N.; GUIMARÃES FILHO, C. et al. Diferentes níveis de feno de maniçoba, na alimentação de ovinos:
digestibilidade e desempenho animal. In: REUNÃO ANUAL DA
SOCIEDADE BRASILEIRA DE ZOOTECNIA, 37., 2000,
Viçosa, MG. Anais... Viçosa, MG: Sociedade Brasileira de
Zootecnia, 2000. p.399. SILVA, D.J. Análises de alimentos: métodos químicos e
biológicos. Viçosa, MG: Editora UFV, 2002. 235p. SNIFFEN, C.J.; O’CONNOR, J.D.; van SOEST, P.J. et al. A net
carbohydrate and protein system for evaluating cattle diets: II. Carbohydrate and protein availability. Journal of Animal BARROS, N.N.; SALVIANO, L.M.C.; KAWAS, J.R. Valor nutritivo
de maniçoba para caprinos e ovinos. Pesquisa Agropecuária
Brasileira, v.25, n.3, p.387-392, 1990. Science, v.70, n.11, p.3562 3577, 1992. UNIVERSIDADE FEDERAL DE VIÇOSA - SAEG. Sistema para
análises estatísticas. Versão 8.0. Viçosa, MG:Fundação Arthur
Bernardes, 2000. (CD-ROM). BARROS, N.N.; SIMPLÍCIO, A.A.; FERNANDES, F.D. Terminação
de borregos em confinamento no Nordeste do Brasil. Sobral: EMBRAPA-CNPC, 1997. 24p. (Circular Técnica, 12). Van SOEST, P.J. Composition, fiber quality, and nutritive value of
forages. In: Forage evaluation and utilization. Madison:
American Society of Agronomy, 1985. p.412-421. CARDOSO, R.C.; VALADARES FILHO, S.C.; COELHO DA SILVA,
J.F. et al. Consumo e digestibilidades aparentes totais e parciais
de rações contendo diferentes níveis de concentrado, em
novilhos F1 Limousin x Nelore. Revista Brasileira de
Zootecnia, v.29, n.6, p.1832-1843, 2000. WEISS, W.P. Energy prediction equations for ruminant feeds. In:
CORNELL
NUTRITION
CONFERENCE
FOR
FEED
MANUFACTURERS, 61., 1999, Ithaca. Proceedings… Ithaca:
Cornell University, 1999. p.176-185. COCHRAN, R.C.; GALYEAN, M.L. Measurement of in vivo forage
digestion by ruminants. In: FAHEEY JR., G.C. (Ed.) Forage
quality, evaluation, and utilization. Madison: American
Society of Agronomy, 1994. p.613-643. Conclusões LADEIRA, M.M.; VALADARES FILHO, S.C.; COELHO DA SILVA,
J.F. et al. Consumo e digestibilidades aparentes totais e parciais
de dietas contendo diferentes níveis de concentrado, em
novilhos Nelore. Revista Brasileira de Zootecnia, v.28, n.2,
p.395-403, 1999. A inclusão de até 80% de feno de maniçoba em subs-
tituição ao milho e ao farelo de soja em dietas para cordeiros
não alterou o consumo de matéria seca e o balanço de
nitrogênio. Todavia, o consumo de NDT e a digestibilidade
da dieta decresceram com o aumento dos níveis de feno. MERTENS, D.R. Análise da fibra e sua utilização na avaliação de
alimentos
e
formulação
de
rações. In:
SIMPÓSIO
INTERNACIONAL DE RUMINANTES, 1992, Lavras. Anais… Lavras: Universidade Federal de Lavras, 1992. p.188. MERTENS, D.R. Regulation of forage intake. In: FAHEY JR., G.C. (Ed.) Forage quality, evaluation, and utilization. Madison:
American Society of Agronomy, 1994. p.450-493. de feno) a 5,84 g/animal/dia (dieta com maior quantidade do
volumoso) (Tabela 5). Limousin x Nelore alimentados com dietas contendo cinco níveis
de concentrado. Revista Brasileira de Zootecnia, v.29, n.2,
p.545-554, 2000. FORBES, J.M. Voluntary food intake and diet selection in
farm animals. Wallingford: CAB International, 1995. 532p. Resultados e Discussão Tabela 5 - Nitrogênio ingerido (NIng), nitrogênio fecal (NFec), nitrogênio excretado (NExcr), balanço nitrogenado (Bal N) e nitrogênio
aparentemente retido (N Apar Ret) de ovinos em fase de recria alimentados com dietas contendo feno de maniçoba
Table 5 -
Nitrogen intake (NI), fecal nitrogen (NF), excreted nitrogen (NE), nitrogen balance (N bal) and apparently retained nitrogen (NApRet) in sheep
in the initial raising phase fed diets with maniçoba hay
Item
Feno maniçoba na dieta (%)
CV (%)
Regressão
R2
Maniçoba hay in the diet
Regression
20
40
60
80
NIng (g/d) (NI)
32,05
32,87
32,61
35,30
15,56
Yˆ = 33,21
-
NFec (g/d) (NF)
13,00
14,20
13,61
17,44
17,17
Yˆ = 11,376 + 0,0637**X
0,69
NExcr (g/d) (NE)
23,74
26,76
24,04
29,45
14,34
Yˆ = 22,392 + 0,0721*X. 0,48
Bal N (N bal)
8,31
6,11
8,57
5,84
42,58
Yˆ = 7,21
-
N apar ret (%) (N ApRet)
25,64
17,90
26,35
15,95
33,42
Yˆ = 20,06
-
* (P<0,05); ** (P<0,01). Tabela 5 - Nitrogênio ingerido (NIng), nitrogênio fecal (NFec), nitrogênio excretado (NExcr), balanço nitrogenado (Bal N) e nitrogênio
aparentemente retido (N Apar Ret) de ovinos em fase de recria alimentados com dietas contendo feno de maniçoba
Table 5 -
Nitrogen intake (NI), fecal nitrogen (NF), excreted nitrogen (NE), nitrogen balance (N bal) and apparently retained nitrogen (NApRet) in sheep
in the initial raising phase fed diets with maniçoba hay Tabela 5 - Nitrogênio ingerido (NIng), nitrogênio fecal (NFec), nitrogênio excretado (NExcr), balanço nitrogenado (Bal N) e nitrogênio
aparentemente retido (N Apar Ret) de ovinos em fase de recria alimentados com dietas contendo feno de maniçoba
Table 5 -
Nitrogen intake (NI), fecal nitrogen (NF), excreted nitrogen (NE), nitrogen balance (N bal) and apparently retained nitrogen (NApRet) in sheep
in the initial raising phase fed diets with maniçoba hay Limousin x Nelore alimentados com dietas contendo cinco níveis
de concentrado. Revista Brasileira de Zootecnia, v.29, n.2,
p.545-554, 2000. de feno) a 5,84 g/animal/dia (dieta com maior quantidade do
volumoso) (Tabela 5). Literatura Citada y
g
y
COELHO DA SILVA, J.F.; LEÃO, M.I. Fundamentos de nutrição
dos ruminantes. Piracicaba: Livroceres, 1979. 380p. Recebido: 15/2/2005
Aprovado: 13/4/2007 DIAS, H.L.C.; VALADARES FILHO, S.C.; COELHO DA SILVA,
J.F. et al. Consumo e digestões totais e parciais em novilhos F1 © 2007 Sociedade Brasileira de Zootecnia
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English
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Selective attention for affiliative and agonistic interactions of dominants and close affiliates in macaques
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Scientific reports
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cc-by
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www.nature.com/scientificreports www.nature.com/scientificreports macaques Oliver Schülke 1,2,3*, Natalie Dumdey1,3 & Julia Ostner1,2,3 1,2,3*, Natalie Dumdey1,3 & Julia Ostner1,2,3 Monitoring conspecifics is a crucial process in social learning and a building block of social cognition. Selective attention to social stimuli results from interactions of subject and stimulus characteristics with
dominance rank often emerging as an important predictor. We extend previous research by providing
as stimuli naturally occurring affiliative interactions between group members instead of pictorial or
auditory representations of conflicts, and by extending to the affiliative relationship, i.e. social bond,
between subject and stimulus instead of just their dominance relations. Our observational data on
adult female rhesus macaques support the prediction that subjects pay more attention to affiliative
interactions of others than to solitary controls. Exceedingly more attention was paid to conflicts
unfolding in the group which can have more prompt and direct consequences than others’ friendly
interactions. The valence of the stimulus (affiliative vs. agonistic) affected biases towards individuals
dominant over the subject, but not the ubiquitous bias towards close affiliates of the subject. Keeping
track of the whereabouts and interactions of key social partners has been proposed as a prerequisite
for behavioral coordination among bonded partners. In groups of socially very active monkeys, social
attention is gated by both social dominance and social bonding. Vertebrates living in social groups evolved a suite of socio-cognitive skills to maneuver their social environment1. These skills include individual recognition, tracking one’s own past interactions, monitoring others’ interactions,
assessing others’ relationships, and attributing mental states to others. Such abilities are thought to place increas-
ingly large demands on the cognitive apparatus1. Processes involved in the use of social knowledge are a) the
acquisition of social information, i.e. information on other individuals and their interactions, b) the application
of such information, and c) its exploitation2. Each of these processes is subject to phenotypic constraints leading
to inter-individual variation in the use of social knowledge2. There exists now ample evidence both for the ability
to individually recognize conspecifics3 and for gregarious animals to take into account past events when making
social decisions4. Therefore, the focus of social cognition research shifted to understand variation in social atten-
tion, in social information acquisition, and in how and when individuals monitor others. Social information can function as a reward and incentive for nonhuman primates in experimental tasks sug-
gesting that the acquisition of social information is important5–7. Scientific Reports | (2020) 10:5962 | https://doi.org/10.1038/s41598-020-62772-8 www.nature.com/scientificreports/ to maintain attention and whether subjects visually followed the stimulus with high-ranking individuals receiving
more attention even if it had to be paid for7,16. Social attention also varies with subject rank; only high-ranking
subjects showed late reactions suggestive of voluntary attention sensitive to stimulus rank16; low ranking subjects
showed a fast, quasi-reflexive reaction that was not modulated by stimulus dominance rank. Subject rank also
affects the relative attention focused on a dominant stimulus versus a stimulus encoding information about a
potential mating partner7. Thus, individual characteristics may interact with traits of the stimulus individual
(dominance and sexual attractiveness). It is less clear whether the affiliative relationship between the subject and
the stimulus affects social attention. f
Apart from paying attention preferentially to high-ranking group mates, individuals may preferentially attend
to close affiliates or kin, yet studies on this topic are relatively scarce. Acoustic playback experiments suggest
that primates attend more to fear screams17 and alarm calls18 from closely affiliated, i.e. bonded compared to
non-bonded stimulus providers. In an observational study, female mandrills glanced more often at their female
kin than at non-kin, and generally attended more to conflicts compared to neutral situations13. Social information
on the agonistic interactions of one’s close affiliates gains salience, because aggression often is redirected to affili-
ates of the original opponents19. Bonded partners are also recruited preferentially for agonistic support to join in
the ongoing conflict20. Therefore, it pays to monitor the behavior of one’s affiliates to be prepared. The agonistic
interactions of one’s close affiliates may directly and promptly affect the subject.hfif fi
y
y
p
p yf
j
The affiliative interactions of group members are less likely to have direct and immediate effects on a subject,
which may partly explain the pronounced imbalance towards aggression and dominance differentials as stimuli
in attention studies (see references above). If a close affiliate is involved in a friendly interaction, salience may
accrue from the social information not only about the specific interaction, but also about the dyadic affiliative
relationship of or immediate exchanges among the observed individuals. Close affiliative relationships, which are
similar to human friendships21,22, are formed and maintained by affiliative interactions and often serve as stable
alliances in within-group conflicts23–25. Thus, monitoring others’ affiliative interactions may serve to keep track
of others’ social relationships and indirectly the state of the alliance network in the group, as has been shown for
ravens26. Scientific Reports | (2020) 10:5962 | https://doi.org/10.1038/s41598-020-62772-8 www.nature.com/scientificreports/ Beyond information about the alliance network, monitoring others also provides information about the
current value of commodities exchanged among partners and relative partner value27–29.h g
g p
p
The studies reviewed above suggest that any selectivity in social monitoring is driven by the salience of the
information gathered (social salience hypothesis). Alternatively, social attention may be focused on close affil-
iates irrespective of their current activity, the type of social interaction they are involved in or their interaction
partners30. The frequent joint activities that characterize social bonds need partners to coordinate behaviorally31
which requires both or at least one partner to modify its behavior to match the partner’s activity. Such adjustment
requires individuals to pay attention to their close affiliates’ whereabouts and behavior at all times30,32 (social
bonding hypothesis). g yp
Here, we assessed in female rhesus macaques (Macaca mulatta) a subject’s selective attention to spontaneous
naturally occurring affiliative and agonistic interactions among her group members, and to solitary individuals
as controls. These stimulus interactions were scored, for each of the bystander subjects, for whether they involved
a) at least one of the subject’s two closest affiliates, and b) at least one individual higher ranking in dominance
than the subject. As main effects, we predicted i) that subjects pay more attention to social interactions than
controls and because of the imminent risk more to agonistic than to affiliative interactions, ii) that subjects pay
more attention to social interactions or controls if they involve at least one of their closest affiliates, and iii) if they
involve a group mate higher ranking than themselves. From the social salience hypothesis, we further predicted
iv) that involvement of higher ranking or closely affiliated stimulus individuals have exceedingly stronger effects
when comparing controls to affiliation and on to agonistic stimulus interactions. To scrutinize this prediction a bit
more, we predicted v) that more attention should be paid to the interaction of a close affiliate with an individual
higher instead of lower ranking than the subject bystander. From the social bonding hypothesis, we predicted that
vi) close affiliates should always be monitored irrespective of whether they are alone, in affiliation or in conflict
with a third party. macaques Not all information is valued to the same degree
however, leading to selective attention varying with traits of the subject, of the stimulus individual or event, or
their combination. Social information is thought to be valued particularly high, if its acquisition leads to direct
benefits for the attentive subject. Selective attention to less immediate, indirect or slowly accruing benefits has
been investigated to a lesser degree. Research on within group variation in social attention established that social attention is gated by dominance
rank in primates, leading to an “attentional structure” reflecting the dominance hierarchy in a social group8–10. This pattern of selective attention towards higher ranking individuals9 along with competition for attractive high
ranking partners11 has since been found in a number of species and using different paradigms from natural obser-
vations to experimental designs7,12–14; a recent study lacked a dominance effect though15. In eye tracking experi-
ments, the rank-class of the stimulus individual determined whether monkeys had to be provided with rewards 1Department Behavioral Ecology, JFB Institute for Zoology and Anthropology, Georg-August University Göttingen,
Kellnerweg 6, 37077, Goettingen, Germany. 2Research Primate Social Evolution, German Primate Center, Goettingen,
Germany. 3Leibniz ScienceCampus Primate Cognition, Goettingen, Germany. *email: oschuel@gwdg.de Scientific Reports | (2020) 10:5962 | https://doi.org/10.1038/s41598-020-62772-8 www.nature.com/scientificreports/ Methods
S
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b During our observations we may have missed gazes that occurred shortly before or after
the group scan, but we consider it unlikely that such effects may have biased our data in relation to our predictions. The
1,359 stimulus interactions were scored for whether they involved noises (vocalizing, grooming-noises, self-scratching,
cage rattling, loud chewing) or movements (any form of locomotion) which might have drawn attention. A total of
1,158 control observations were conducted of attention paid towards solitary stimulus individuals that were noisy or
moved. Data were generated by two observers (813 and 1704 observations balanced between groups). Parallel observa-
tions were conducted for two sessions (12 h) during which 81 stimulus events and 302 bystander reactions were scored. Observers agreed on 86.4% of the bystander reactions and inter-observer reliability for attention scoring was estimated
at Cohen’s Kappa of 0.7 (R package “irr”34) indicating substantial agreement35. Data analysis. Dominance hierarchies were generated from the outcomes of 235 and 355 decided dyadic
agonistic conflicts36 using normalized David’s Scores37 generated with the function “DS” in the R package
“EloRating” version 0.4338 in R version 3.4.439. In testing our predictions, we used normalized David’s Scores as a
continuous measure of dominance.fi Affiliative relationship strength was assessed from the instantaneous group scan data. Relative observation fre-
quencies of grooming, contact sitting, and close proximity were combined from both partners into a dyadic value
per behavior and scaled by dividing by the group mean. We then built a dyadic composite sociality index DSI40
from those affiliative behaviors that correlated in row-wise matrix correlations run on dyadic data in MatMan
1.1 (Noldus, Wageningen, NL). The DSI is one on average and increasingly high values represent dyads spending
exceedingly more time affiliating than the average dyad in the group, whereas lower values indicate dyads that
were rarely seen affiliating41. Close affiliates were classified for each female as the two partners with whom she had
the highest DSI values. Average DSI was 2.3 across all close affiliates, meaning they affiliated more than twice as
long as the average dyad in the group. g
g
y
g
p
All further analyses were performed in R [version 3.4.442], by using the Rstudio interface [version 1.0.15343]. Methods
S
d
b Study subjects and data collection. The study was conducted in the colony of rhesus macaques housed
at the German Primate Center Göttingen, Germany. We observed two groups, one comprising eight adult females
age 9–10 without males or immatures and one with a subadult male and nine adult females age 3–23 without
immatures. The smaller group was housed in a ~24 m² indoor plus ~18 m² outdoor enclosure and the larger group
had access to two of these indoor and outdoor enclosures. The monkeys could move freely between indoor and
outdoor area and between rooms, apart from brief periods during some of our observations (see below).fi To assess the affiliative relationships between all group members, we conducted instantaneous group scan
observations33 at 10 min intervals using handheld computers (HP ipaq 114). Each subject was scored only once
with priority of grooming over contact sitting and contact sitting over being in close proximity of <1 m. If the
individual had more than one partner in the highest priority category, only one partner was chosen alternating
going with or against the direction of the clock. A total of 615 scans were recorded in the larger and 625 in the
smaller group over the course of five weeks. Additionally, and separate from these group scans, we collected data on social attention to agonistic and friendly
interactions or solitary controls in 77 two-hour sessions (38 sessions in the large and 39 in the small group). Attention
was scored only for those subjects that were in the same room as the stimulus individuals and had an unobstructed view
on the scene. The reaction of all females to every affiliative (body contact, grooming) and agonistic (including one or
more of the following behavioral elements: open mouth display, pointing posture, push and pull, head bob, lunge, chase,
slap, bite, ground slap, make room, flee, crouch) interaction between two identified group mates was recorded. Upon
the start of the social interaction, we recorded the nature and participants of the interaction and only once for each Scientific Reports | (2020) 10:5962 | https://doi.org/10.1038/s41598-020-62772-8 www.nature.com/scientificreports/ subject female, like in a quick group scan, whether she directed her attention to the interaction or not, judging from
head orientation and gaze direction. Therefore, the likelihood of recording a case of visual attention was not related to
the duration of the interaction. Methods
S
d
b To test what influenced the probability of a bystander to pay attention to a stimulus or not, we ran two Generalized
Linear Mixed Models (GLMM) with a binomial error structure and logit link function, using the function ‘glmer’ of
the R package lme444. In the first model, we included the two statistical interaction terms i) between the stimulus
type (control, affiliation or aggression, also called stimulus valence in the text) and the engagement of a close affiliate
of the subject bystander in the stimulus interaction and ii) between the stimulus type and the engagement of a higher
ranking individual than the subject bystander in the stimulus interaction, and iii) all three main effects, as well as iv)
the rank of the subject bystander as fixed effects. After we found that the first interaction term was not significant
(see Table S1), we excluded it and reported in the main text results from the reduced model. For the second model,
we used only the subset of the data where only one close affiliate of the subject bystander was present in a social
interaction; the full data set also included stimuli where both interaction partners were close affiliates. In this model,
we included the interaction between the stimulus type (affiliation or aggression) with whether the member of the
social interaction who was not a close affiliate was higher ranking than the subject bystander or not. For both models
we also included the conspicuousness of the stimulus (whether it was noisy, also involved movement or none of the
two) as a control variable, and included event ID, bystander ID and actor and receiver of the social interaction as
random effects. Using a likelihood ratio test, we compared the full model with a null model lacking the fixed effects,
but comprising the same control and random effects to test whether the predictors of interest improved model fit. Model stability was assessed by comparing the model estimates of the full dataset with the estimates of the model
after leaving out data points one by one. No influential cases were detected. We checked whether predictors were
collinear by calculating Variance Inflation Factors (VIF45) for a linear model, excluding the random effects. Methods
S
d
b affiliative
−0.14
0.18
−0.79
0.430
Subject dom. rank (sqrt nDS)
−0.09
0.11
−0.90
0.367
Table 1. Predictors of rhesus macaque social attention to spontaneously occurring natural social stimuli. Results of a logistic model (whether subject gazed at stimulus) controlling for conspicuousness of the stimulus
(whether it was noisy, involved movement or none of the two) and including as random effects stimulus event
ID, subject ID, ID of actor and receiver; 10.162 observations, 2.478 stimulus events, 18 subjects. The full model
was significantly different from the null model including all control and random effects (Chi2 = 362.05, df = 7,
P < 0.0001). Significant predictors are presented in bold font. Significance of the different levels of variable
stimulus type was assessed by releveling the intercept. Table 1. Predictors of rhesus macaque social attention to spontaneously occurring natural social stimuli. Results of a logistic model (whether subject gazed at stimulus) controlling for conspicuousness of the stimulus
(whether it was noisy, involved movement or none of the two) and including as random effects stimulus event
ID, subject ID, ID of actor and receiver; 10.162 observations, 2.478 stimulus events, 18 subjects. The full model
was significantly different from the null model including all control and random effects (Chi2 = 362.05, df = 7,
P < 0.0001). Significant predictors are presented in bold font. Significance of the different levels of variable
stimulus type was assessed by releveling the intercept. Table 1. Predictors of rhesus macaque social attention to spontaneously occurring natural social stimuli. Results of a logistic model (whether subject gazed at stimulus) controlling for conspicuousness of the stimulus
(whether it was noisy, involved movement or none of the two) and including as random effects stimulus event
ID, subject ID, ID of actor and receiver; 10.162 observations, 2.478 stimulus events, 18 subjects. The full model
was significantly different from the null model including all control and random effects (Chi2 = 362.05, df = 7,
P < 0.0001). Significant predictors are presented in bold font. Significance of the different levels of variable
stimulus type was assessed by releveling the intercept. 0.00
0.25
0.50
0.75
1.00
control
affiliation aggression
stimulus interaction type
probability of paying attention
no close affiliate
at least 1 close affiliate
0.00
0.25
0.50
0.75
1.00
control
affiliation aggression
stimulus interaction type
no higher ranking
at least 1 higher ranking
Figure 1. Methods
S
d
b Since
VIFs were close to one for all models, collinearity was not an issue.h This work followed the Animal Behaviour Society’s guidelines for the treatment of animals in behavioral
research and teaching, and adhered to standards as defined by the European Union Council Directive 2010/63/
EU on the protection of animals used for scientific purposes. The study was approved by the Ethics Committee of
the German Primate Center Göttingen (AZ E1-19). g
All data generated and analyzed during this study are included in this published article and its supplemental
information files. Ethical approval. All applicable international, national, and/or institutional guidelines for the care and use
of animals were followed. The Ethics Committee of the German Primate Center approved of this study (AZ
E1-19) that was completely observational. Results. The affiliative and the dominance relationships between the subject and the stimulus individuals
modulated subject attention in different ways. Whether the stimulus situation involved a close affiliate of the
subject or not did not modulate the effect of stimulus type on attention (Table S1). Subjects always were more
attentive to a close affiliate with the size of this effect being independent of whether the stimulus interaction was
agonistic, affiliative or a solitary control (Table 1; Fig. 1a).f fi
In contrast, the dominance relationship between subject and stimulus individual(s) did not affect subject
attention the same way across stimulus types. Involvement of a higher ranking individual only raised more atten-
tion for control or affiliative interactions; surprisingly, no such difference was found for agonistic interactions
perhaps due to a ceiling effect (Table 1; Fig. 1b). Beyond these relative effects, the dominance rank of the subject
had no effect on its attention. In the subset of stimulus events that included only one close affiliate of the subject,
the dominance relation between the second - not affiliated - stimulus individual and the subject did not affect Scientific Reports | (2020) 10:5962 | https://doi.org/10.1038/s41598-020-62772-8 www.nature.com/scientificreports/ Estim. StdErr
z
Pr(>|z|)
Stimulus type/valence
Affiliative vs. control
0.99
0.15
6.44
<0.0001
Agonistic vs. control
3.40
0.16
20.79
<0.0001
Affiliative vs. agonistic
2.41
0.18
13.69
<0.0001
At least one close affiliate (yes)
0.30
0.06
4.69
<0.0001
At least one higher ranking (yes)
0.55
0.14
3.97
<0.0001
Stimuls type:One higher ranking (yes)
One higher ranking (yes)
Affiliative vs. control
−0.29
0.17
−1.71
0.087
One higher ranking (yes)
Agonistic vs. control
−0.42
0.17
−2.54
0.011
One higher ranking (yes)
Agonistic vs. Methods
S
d
b Probability of subjects paying attention to stimuli increased from solitary controls to affiliative
interactions and on to agonistic interactions as stimuli. A similar bias towards stimuli that involved at least
one of the subject’s two closest affiliates is expressed across stimulus types. Stimuli that involved at least one
individual higher ranking than the subject evoked more attention only if they were controls or affiliative
interactions but not if agonistic. See Table 1 for detailed results of GLMM. 0.00
0.25
0.50
0.75
1.00
control
affiliation aggression
stimulus interaction type
no higher ranking
at least 1 higher ranking 0.00
0.25
0.50
0.75
1.00
control
affiliation aggression
stimulus interaction type
probability of paying attention
no close affiliate
at least 1 close affiliate control
affiliation aggressio
stimulus interaction type Figure 1. Probability of subjects paying attention to stimuli increased from solitary controls to affiliative
interactions and on to agonistic interactions as stimuli. A similar bias towards stimuli that involved at least
one of the subject’s two closest affiliates is expressed across stimulus types. Stimuli that involved at least one
individual higher ranking than the subject evoked more attention only if they were controls or affiliative
interactions but not if agonistic. See Table 1 for detailed results of GLMM. ocial attention (Table 2; Fig. 2). Females did not pay more attention to the interaction of their close affiliates with
igher ranking individuals, not even if they were engaged in an agonistic interaction. Overall, the valence of the stimulus/stimulus type was by far the most important predictor of attention. Agonistic interactions raised much more attention than affiliative ones, but both raised more attention than con-
trol situations with a solitary stimulus individual. All analyses were controlled for the conspicuousness of the
stimulus, i.e. whether it involved noise/vocalization and movement. Discussion 0.00
0.25
0.50
0.75
1.00
affiliation
aggression
stimulus interaction type
probability of paying attention
other higher ranking
other not higher ranking
Figure 2. Subset of the data with stimulus interactions that had exactly one of the two closest affiliates of
the subject and the other stimulus individual being dominant over the subject or not, to test whether a close
affiliate in conflict is more likely to be monitored if the opponent is a bigger threat to the subject. The opponent’s
dominance rank relative to the subject did not affect attention. See Table 2 for detailed results of GLMM. 0.00
0.25
0.50
0.75
1.00
affiliation
aggression
stimulus interaction type
probability of paying attention
other higher ranking
other not higher ranking other higher ranking
other not higher ranking Figure 2. Subset of the data with stimulus interactions that had exactly one of the two closest affiliates of
the subject and the other stimulus individual being dominant over the subject or not, to test whether a close
affiliate in conflict is more likely to be monitored if the opponent is a bigger threat to the subject. The opponent’s
dominance rank relative to the subject did not affect attention. See Table 2 for detailed results of GLMM. Figure 2. Subset of the data with stimulus interactions that had exactly one of the two closest affiliates of
the subject and the other stimulus individual being dominant over the subject or not, to test whether a close
affiliate in conflict is more likely to be monitored if the opponent is a bigger threat to the subject. The opponent’s
dominance rank relative to the subject did not affect attention. See Table 2 for detailed results of GLMM. aggressive compared to neutral facial displays of conspecifics46, and Barbary macaques glanced longer at anxious
(i.e. scratching) weakly bonded than strongly bonded group members, possibly as the former may be more likely
to start a fight47. Our findings, that third party conflicts drew far more attention from the subject bystander than
affiliative or solitary control situations corroborate these results.fi fi
An efficient way to avoid threat and resource competition is to generally keep track of the whereabouts of
one’s potential opponents regardless of the opponent’s specific behavior. The widespread primate inclination to
focus attention on higher-ranking individuals supports this idea30. Discussion Arguably, the most likely direct and prompt/immediate benefit of paying attention to others is the avoidance
of danger by monitoring potential threats. To do so, individuals should monitor individuals and situations that
may carry the risk of themselves becoming involved in an aggressive interaction. Indeed, mandrills were more
attentive to conflict situations compared to neutral ones13, rhesus macaques looked more rapidly at pictures of Scientific Reports | (2020) 10:5962 | https://doi.org/10.1038/s41598-020-62772-8 www.nature.com/scientificreports/ Estim. StdErr
z
Pr(>|z|)
Stimulus type (agonistic vs. affiliative)
2.27
0.22
10.12
<0.0001
Other higher ranking (yes)
0.31
0.21
1.51
0.131
Stimulus Type:Other higher ranking
−0.20
0.24
−0.80
0.423
Subject dom. rank (sqrt nDS)
−0.08
0.16
0.52
0.606
Table 2. Model results for subset of stimulus interactions that had exactly one close affiliate and the other
individual being higher ranking than the subject or not. The crucial interaction term stimulus type:other
higher ranking is not significant. The full model is different from the null model with control (stimulus
conspicuousness) and random effects (stimulus event ID, subject ID, actor ID, recipient ID; Chi2 = 292.6, df = 5,
P < 0.0001; 2169 observations, 1123 stimulus events, 18 subjects). Estim. StdErr
z
Pr(>|z|)
Stimulus type (agonistic vs. affiliative)
2.27
0.22
10.12
<0.0001
Other higher ranking (yes)
0.31
0.21
1.51
0.131
Stimulus Type:Other higher ranking
−0.20
0.24
−0.80
0.423
Subject dom. rank (sqrt nDS)
−0.08
0.16
0.52
0.606 Table 2. Model results for subset of stimulus interactions that had exactly one close affiliate and the other
individual being higher ranking than the subject or not. The crucial interaction term stimulus type:other
higher ranking is not significant. The full model is different from the null model with control (stimulus
conspicuousness) and random effects (stimulus event ID, subject ID, actor ID, recipient ID; Chi2 = 292.6, d
P < 0.0001; 2169 observations, 1123 stimulus events, 18 subjects). Table 2. Model results for subset of stimulus interactions that had exactly one close affiliate and the other
individual being higher ranking than the subject or not. The crucial interaction term stimulus type:other
higher ranking is not significant. The full model is different from the null model with control (stimulus
conspicuousness) and random effects (stimulus event ID, subject ID, actor ID, recipient ID; Chi2 = 292.6, df = 5,
P < 0.0001; 2169 observations, 1123 stimulus events, 18 subjects). Scientific Reports | (2020) 10:5962 | https://doi.org/10.1038/s41598-020-62772-8 www.nature.com/scientificreports/ The subjects further exhibited an attentional bias towards the affiliative interactions of their own close affiliates,
but this bias did not exceed the bias towards monitoring solitary close affiliates.ffi g
yfi
Differences in attention towards close affiliates and others were not modulated by stimulus valence but were
equally strong for agonistic, affiliative, and solitary stimulus events. The bias towards close affiliates was not exac-
erbated by increasing the directness and immediacy of the threat involved. Instead, our results can be interpreted
as a general attention bias towards close affiliates independent of the affiliate’s social activity. Such ubiquitous
close monitoring of affiliates has been proposed as a prerequisite for behavioral coordination and adjustment
of own behavior to achieve this goal and therefore may serve as an index of social bond strength30. Investing in
such close monitoring may have opportunity costs in terms of reduced feeding efficiency or reduced attention
to other, non-bonded individuals. Like any visual monitoring it may also attract the attention of those observed
with possible negative effects like redirected aggression. The upside is that social monitoring may also facilitate
social learning which may be particularly important for immatures14. Future studies will explore the immediate
consequences of social monitoring.i q
g
In conclusion, this study partly replicates previous findings on selective attention to social stimuli in nonhu-
man primates, particularly the general attraction to dominant individuals. We extend previous work in several
ways. We show selective social monitoring of a dominant’s affiliative interactions that should not have direct and
prompt effects on the subject’s wellbeing like extending agonistic conflicts do. We show that biases towards mon-
itoring higher ranking individuals disappear when monitoring agonistic interactions even if these involve close
affiliates. We further show that attention to the whereabouts and interactions of one’s own close affiliates is gener-
alized which suggests that it serves in coordinating the affiliative relationship itself. As a side effect, the individual
gains more social information about its closest partners than other group members. Social interest is typically
assessed with pictures7, acoustic playbacks1 and videos46 as stimuli or uses gaze following paradigms with human
observers57. Here, we recorded the reaction of bystanders to spontaneously occurring social interactions among
other group members. The design allowed statistically disentangling the effects of stimulus valence (agonistic,
affiliative and non-social) as well as affiliative and dominance relationships between stimuli and subjects on atten-
tion. www.nature.com/scientificreports/ www.nature.com/scientificreports/ shown that redirection of aggression is preferentially directed towards bond partners of the original opponents19,
closely observing conflicts involving affiliates may increase the chance to avoid becoming target of redirection. Secondly, selective attention towards close affiliates’ aggressive interactions may also enhance behavioral coordi-
nation needed to join in the ongoing fight in case the affiliate calls for coalitionary aid and agonistic support20,30. Thus, there are several direct benefits of monitoring conflicts of group members, which may be amplified if one of
the opponents is a bonded partner. Accordingly, subjects in this study were sensitive to whether a nearby conflict
involved one of their close affiliates, but the bias towards monitoring close affiliates in agonistic interaction did
not exceed the bias towards close affiliates in affiliative or control situations. Subjects did not pay more attention if
the opponent in a conflict of their affiliate was higher ranking than themselves and therefore posed a bigger threat,
which may result from a ceiling effect of very high attention to agonistic interactions.f y
gf
y
g
g
A different useful yet more indirect source of information that is rarely investigated in studies of social atten-
tion are affiliative interactions of third parties. Monitoring friendly interactions of group members provides
the observer with valuable information on others’ social relationships1,30,31 as well as the value of commodities
exchanged among group members48,49. Keeping track of the status quo and dynamics of social ties in one’s group
aids in social decision making, for example in strategic partner choice for coalition formation or in the decision
of whether or not to attack a specific target50,51. Given the importance of bonds and alliances in some social spe-
cies52–54, it should pay to closely track others’ affiliative interactions to possibly impede emerging bonds26,55,56. Sooty mangabeys, chimpanzees, and horses strategically intervene in grooming interactions threatening their
own relationship status and those that involved a close affiliate of the bystander55,56. Female rhesus macaques in
this study paid more attention to third party affiliation if it involved an individual higher ranking than themselves
possibly because social exchanges and relationships of powerful individuals have more far reaching consequences. www.nature.com/scientificreports/ If applied in field studies the design will allow to assess the consequences of inter-individual differences in
social monitoring in terms of efficacy of coalition partner choice and longer-term fitness outcomes. Data availability
ll d
d
d y
All data generated and analyzed during this study are included in this published article and its supplemental
information files. Received: 19 February 2019; Accepted: 13 March 2020;
Published: xx xx xxxx Received: 19 February 2019; Accepted: 13 March 2020;
Published: xx xx xxxx Received: 19 February 2019; Accepted: 13 March 2020;
Published: xx xx xxxx Discussion Female rhesus macaques in our study also paid
more attention to individuals outranking themselves but only if the higher ranking individual was alone or in an
affiliative interaction. Agonistic interactions were almost always monitored no matter whether they involved an
opponent higher ranking than the subject or not. Previous studies also found low ranking subjects to be more
attentive and possibly more unselectively so16,47, whereas high social status may require selective attention only
to other high rankers16. Yet, in mandrills and the rhesus macaque females in our study such an effect of subject
rank on attention for agonism and affiliation of others was not found to go above and beyond the relative effects
described above13. This discrepancy may be explained by rank dependent differences in the time course of gaz-
ing16 that cannot be picked up with the methods employed in the latter studies (but see14). The methods employed
here do not allow discerning which of the interaction partners the subject was gazing at.hlfi g
p
j
g
g
Threats can also be avoided by selectively monitoring third party conflicts that involve a close affiliate of the
subject. Such monitoring may prepare closely affiliated bystanders for upcoming interactions. First, as it has been References L. Social status gates social attention in monkeys. Current Biology 16, R119–120 (2006). 17. Palombit, R. A., Seyfarth, R. M. & Cheney, D. L. The adaptive value of ‘friendships’ to female baboons: experimental and
b
ti
l
id
A i
l B h
i
54 599 614 http //d i
/10 1006/
b 1996 0457 (1997) p
g
y
gy
(
)
17. Palombit, R. A., Seyfarth, R. M. & Cheney, D. L. The adaptive value of ‘friendships’ to female baboons: experimental and
observational evidence. Animal Behaviour 54, 599–614, https://doi.org/10.1006/anbe.1996.0457 (1997). 17. Palombit, R. A., Seyfarth, R. M. & Cheney, D. L. The adaptive value of friendships to female baboons: experiment
observational evidence. Animal Behaviour 54, 599–614, https://doi.org/10.1006/anbe.1996.0457 (1997).f p
g
18. Micheletta, J. et al. Social bonds affect anti-predator behaviour in a tolreant species of macaque, Macaca nigra. Proceedings of the
Royal Society B: Biological Sciences 279, https://doi.org/10.1098/rspb.2012.1470 (2012).l y
y
g
g
9. Watts, D., Colmenares, F. & Arnold, K. In Natural conflict resolution (eds. Aureli, F. & de Waal, F. B. M.) 281–304 (University o
California Press, 2000). 0. Young, C., Majolo, B., Schülke, O. & Ostner, J. Male social bonds predict partner recruitment in cooperative aggression in wil
Barbary macaques. Animal Behaviour 80, 675–682 (2014). y
q
21. Silk, J. B. Using the ‘F’-word in primatology. Behaviour 139, 421–446, https://doi.org/10.1163/156853902760102735 (2002). g
p
gy
p
g
22. Massen, J., Sterck, E. & de Vos, H. Close social associations in animals and humans: functions and mechanisms of friend
Behaviour 147, 1379–1412, https://doi.org/10.1163/000579510X528224 (2010).h 22. Massen, J., Sterck, E. & de Vos, H. Close social associations in animals and humans: fu
Behaviour 147, 1379–1412, https://doi.org/10.1163/000579510X528224 (2010).h g
23. Ostner, J. & Schülke, O. The evolution of social bonds in primate males. Behaviour 151, 871–906, https://doi.org/10.1163/156853
9x-00003191 (2014). 24. Watts, D. P. In Mind the Gap (eds. Kappeler, P. M. & Silk, J. B.) 109–138 (Springer, 2009). 25. De Waal, F. In Coalitions and alliances in humans and other animals (eds. Harcourt, A. H. & de Waal, F. B. M.) 233–258 (Oxford
University Press, 1992).i y
6. Massen, J. J. M., Szipl, G., Spreafico, M. & Bugnyar, T. Ravens interven in others’ bonding attempts. Current Biology 24, 2733–2736
(2014). (
)
7. Barrett, L., Henzi, S., Weingrill, T., Lycett, J. & Hill, R. Market forces predict grooming reciprocity in female baboons. References References
1. Seyfarth, R. M. & Cheney, D. L. Social cognition. Animal Behaviour 103, 191–202, https://doi.org/10.1016/j.anbehav.2015.01.030
(2015). y
y
g
p
g
j
(2015). 2. Carter, A., Torrents Ticó, M. & Cowlishaw, G. Sequential phenotypic constraints on social information use in wild baboons. eLIFE
5, e13125 (2016). (
)
2. Carter, A., Torrents Ticó, M. & Cowlishaw, G. Sequential phenotypic constraints on social information use in wild baboons. eLIFE
5, e13125 (2016).i 2. Carter, A., Torrents Ticó, M. & Cowlishaw, G. Sequential phenotypic constraints on social information use in wild baboons. eLIFE
5, e13125 (2016).i (
)
3. Wiley, R. H. Specificity and multiplicity in the recognition of individuals: implications for the evolution of social behaviour. Biological Reviews 88, 179–195, https://doi.org/10.1111/j.1469-185X.2012.00246.x (2013). 3. Wiley, R. H. Specificity and multiplicity in the recognition of individuals: implications for the evolution of social behaviour
Biological Reviews 88, 179–195, https://doi.org/10.1111/j.1469-185X.2012.00246.x (2013). 3. Wiley, R. H. Specificity and multiplicity in the recognition of individuals: implications for the evolution of social behaviour. Biological Reviews 88, 179–195, https://doi.org/10.1111/j.1469-185X.2012.00246.x (2013). g
p
g
j
(
)
4. Schino, G. & Aureli, F. Reciprocity in group-living animals: partner control versus partner choice. Biological Reviews; https://doi
org/10.1111/brv.12248 (2016). g
5. Chang, S. et al. Neuroethology of primate social behavior. Proceedings of the National Academy of Sciences 110, 10387–10394, https://
doi.org/10.1073/pnas.1301213110 (2013). 5. Chang, S. et al. Neuroethology of primate social behavior. Proceedings of the National Academy of Sciences 110, 10387–10394, https://
doi.org/10.1073/pnas.1301213110 (2013). g
p
(
)
6. Anderson, J. Social stimuli and social rewards in primate learning and cognition. Behavioral Processes 42, 159–175 (1998). 7 D
R O Kh
A V & Pl tt M L M
k
i
d
ti
l
ti
f
i l i
b
h
C , J
p
g
g
,
(
)
7. Deaner, R. O., Khera, A. V. & Platt, M. L. Monkeys pay per view: adaptive valuation of social images by rhesus macaques. Current
Biology 15, 543–548, https://doi.org/10.1016/j.cub.2005.01.044 (2005). Scientific Reports | (2020) 10:5962 | https://doi.org/10.1038/s41598-020-62772-8 www.nature.com/scientificreports/ 8. McNelis, N. L. & Boatright-Horowitz, S. L. Social monitoring in a primate group: the relationship between visual attention and
hierarchical ranks. Animal Cognition 1, 65–69, https://doi.org/10.1007/s100710050008 (1998). g
p
g
9. Chance, M. R. A. Attention structure as the basis of primate rank orders. Man 2, 503–518 (1967). olly, C. J. Social Groups of Monkeys, Apes and Men. References Proceedings o
the Royal Society London B 266, 665–670 (1999). y
y
28. Barrett, L., Henzi, S. P., Weingrill, T., Lycett, J. E. & Hill, R. A. Female baboons do not raise the stakes but they give as good as
get Animal Behaviour 59 763–770 https://doi org/10 1006/anbe 1999 1361 (2000) 28. Barrett, L., Henzi, S. P., Weingrill, T., Lycett, J. E. & Hill, R. A. Female baboons do not raise the stakes but they give as good as they
get. Animal Behaviour 59, 763–770, https://doi.org/10.1006/anbe.1999.1361 (2000). get. Animal Behaviour 59, 763–770, https://doi.org/10.1006/anbe g
g
9. Fruteau, C., Voelkl, B., van Damme, E. & Noe, R. Supply and demand determine the market value of food providers in wild verve
monkeys. Proceedings of the National Academy of Sciences of the United States of America 106, 12007–12012, https://doi.org/10.1073
pnas.0812280106 (2009). p
0. Dunbar, R. I. M. & Shultz, S. Bondedness and sociality. Behaviour 147, 775–803, https://doi.org/10.1163/000579510x501151 (2010) ,
,
y
,
,
p
g
(
)
31. Johnson, C. & Karin-D’Arcy, M. Social attention in nonhuman primates: a behavioral review. Aquatic Mammals 32, 423–442 (2006). 32. Wolf, W., Launay, J. & Dunbar, R. I. M. Joint attention, shared goals and social bonding. British journal of psychology (London,
England: 1953) 107, 322–337, https://doi.org/10.1111/bjop.12144 (2016). y
p
q
32. Wolf, W., Launay, J. & Dunbar, R. I. M. Joint attention, shared goals and social bonding. British journal of psychology (London,
England: 1953) 107, 322–337, https://doi.org/10.1111/bjop.12144 (2016). g
p
g
j p
33. Altmann, J. Observational study of behavior: sampling methods. Behaviour 49, 227–266, https://doi.org/10.1163/156853974x0
(1974).fi (1974). 34 P
k
‘i ’ V i
ffi i
t
f i t
t
li bilit
d
t V
i
0 84 1 (CRAN htt
//
j
t
2019) (1974). 34 Package ‘irr’, Various coefficients of interrater reliability and agreement, Version 0 84 1 (CRAN; https://www r-project org, 2019) 34. Package ‘irr’, Various coefficients of interrater reliability and agreement, Version 0.84.1 (CRAN; https://www.r-project.org, 201 fi
och, G. The measurement of observer agreement for categorical dat fi
Landis, J. & Koch, G. The measurement of observer agreement for c h
6. Ostner, J., Heistermann, M. & Schülke, O. Dominance, aggression and physiological stress in wild male Assamese macaques
(Macaca assamensis). Hormones and Behavior 54, 613–619 (2008). 7. de Vries, H., Stevens, J. & Vervaecke, H. Measuring and testing the steepness of dominance hierarchies. Animal Behaviour 71
585–592 (2006). 38. References (Jonathan Cape y
p
f
y
p
p
11. Seyfarth, R. M. A model of social grooming among adult female monkeys. Journal of Theoretical Biology 65, 671–698 (1977). d
l
l
f
l
k
(
h
l y
p
f
y
p
p
11. Seyfarth, R. M. A model of social grooming among adult female monkeys. Journal of Theoretica 11. Seyfarth, R. M. A model of social grooming among adult female monkeys. Journal of Theoretical Biology 65, 671–698 (1977). 12 Keverne E B Leonard R Scruton D & Young S Visual monitoring in social groups of talapoin monkeys (Miopithecus talapoin) 11. Seyfarth, R. M. A model of social grooming among adult female monkeys. Journal of Theoretical Biology 65, 671–698 (1977). 12. Keverne, E. B., Leonard, R., Scruton, D. & Young, S. Visual monitoring in social groups of talapoin monkeys (Miopithecus talapoin) 11. Seyfarth, R. M. A model of social grooming among adult female monkeys. Journal of Theoretical Biology 65, 671 698 (1977). 12. Keverne, E. B., Leonard, R., Scruton, D. & Young, S. Visual monitoring in social groups of talapoin monkeys (Miopithecus talapoin
Animal Behaviour 26, 933–944 (1978). 12. Keverne, E. B., Leonard, R., Scruton, D. & Young, S. Visual monitoring in social groups of talapoin monkeys (Miopithecus talap
Animal Behaviour 26, 933–944 (1978). 3. Schino, G. & Sciarretta, M. Patterns of social attention in mandrills, Mandrillus sphinx. International Journal of Primatology 37
752–761, https://doi.org/10.1007/s10764-016-9936-7 (2016). p
g
4. Grampp, M., Sueur, C., van de Waal, E. & Botting, J. Social attention biases in juvenile wild vervet monkeys: implications for
socialisation and social learning processes. Primates 60, 261–275, https://doi.org/10.1007/s10329-019-00721-4 (2019). g p
p
g
5. Renevey, N., Bshary, R. & van de Waal, E. Philopatric vervet monkey females are the focus of social attention rather independently
of rank. Behaviour 150, 599–615 (2013). 16. Shepherd, S. V., Deaner, R. O. & Platt, M. L. Social status gates social attention in monkeys. Current
17 P l
bi
R A
S
f
h R M & Ch
D L Th
d
i
l
f ‘f i
d hi
’
f 16. Shepherd, S. V., Deaner, R. O. & Platt, M. L. Social status gates social attention in monkeys. Current Biology 16, R119–120 (200
17. Palombit, R. A., Seyfarth, R. M. & Cheney, D. L. The adaptive value of ‘friendships’ to female baboons: experimenta 16. Shepherd, S. V., Deaner, R. O. & Platt, M. References Package ‘EloRating’ - Animal dominance hierarchies by Elo Rating Version 0.46.8 (CRAN; https://github.com/gobbios/EloRating,
2019). 9. R: A language and environment for statistical computing R Foundation for Statistical Computing; http://www.R-project.org
(Vienna, Austria, 2018). (
)
0. Silk, J. B., Altmann, J. & Alberts, S. C. Social relationships among adult female baboons (Papio cynocephalus) I. Variation in the
strength of social bonds. Behavioral Ecology and Sociobiology 61, 183–195, https://doi.org/10.1007/s00265-006-0249-2 (2006). 41. Silk, J. B., Cheney, D. L. & Seyfarth, R. M. A practical guide to the study of social relationships. Evolutionary Anthropology 22,
213–225, https://doi.org/10.1002/evan.21367 (2013). p
g
42. R Development Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing,
Vienna, Austria, http://www.R-project.org/ (2018). p
p
j
g
43. RStudio Team. RStudio: Integrated development environment for R. RStudio, Inc., Boston, MA, http://www.rstudio.com (2016
hl
lk
lk
l
d ff
d l
l
l f
l
ft 43. RStudio Team. RStudio: Integrated development environment for R. RStudio, Inc., Boston, MA, http://www.rstudio.com (2016). 44. Bates, D., Mächler, M., Bolker, B. & Walker, S. Fitting linear mixed-effects models using lme4. Journal of Statistical Software 67, 1–48
(2015). 4. Bates, D., Mächler, M., Bolker, B. & Walker, S. Fitting linear mixed-effects models using lme4. Journal of Statistical Software 67, 1–48
(2015). 45. Fox, J. & Weisberg, S. An R companion to applied regression. 2nd edn, (Sage, 2011). 46. Bethell, E. J., Holmes, A., MacLarnon, A. & Semple, S. Evidence that emotion mediates social attention in rhesus macaques. PLoS
One 7, e44387, https://doi.org/10.1371/journal.pone.0044387 (2012). p
g
j
p
7. Whitehouse, J., Micheletta, J., Kaminski, J. & Waller, B. M. Macaques attend to scratching in others. Animal Behaviour 122, 169–175
https://doi.org/10.1016/j.anbehav.2016.10.020 (2016). 48. Henzi, S. P. & Barrett, L. Infants as a commodity in a baboon market. Animal Behaviour 63, 915–921, https://doi.org/10.1006/
anbe.2001.1986 (2002). 49. Kaburu, S. S. K. & Newton-Fisher, N. E. Bystanders, parcelling, and an absence of trust in the grooming interactions of wild male
chimpanzees. Scientific Reports 6, 1–10, https://doi.org/10.1038/srep20634 (2016).i zees. Scientific Reports 6, 1–10, https://doi.org/10.1038/srep20634 ( p
fi
p
p
g
p
0. Noë, R. A model of coalition formation among male baboons with fighting ability as the crucial parameter. Animal Behaviour 47
211–213 (1994). 1. Perry, S., Barrett, H. C. & Manson, J. H. White-faced capuchin monkeys show triadic awareness in their choice of allies. Anima
Behaviour 67, 165–170, https://doi.org/10.1016/j.anbehav.2003.04.005 (2004).h 52. Brent, L. J. Scientific Reports | (2020) 10:5962 | https://doi.org/10.1038/s41598-020-62772-8 References N., Chang, S. W. C., Gariépy, J.-F. & Platt, M. L. The neuroethology of friendship. Annals of the New York Academy of
Sciences 1316, 1–17, https://doi.org/10.1111/nyas.12315 (2014). f
h
h
Th
l
f f
d h
l
f
h l
h
d p
g
y
(
)
3. Seyfarth, R. M. & Cheney, D. L. The evolutionary origins of friendship. Annual Review of Psychology 63, 153–177, https://doi
org/10.1146/annurev-psych-120710-100337 (2012). Scientific Reports | (2020) 10:5962 | https://doi.org/10.1038/s41598-020-62772-8 www.nature.com/scientificreports/ 54. Ostner, J. & Schülke, O. In Advances in the Study of Behavior Vol. 50 (eds. Marc Naguib et al.) 127 175 (Academic Press, 2018). 55. Mielke, A. et al. Bystanders intervene to impede grooming in Western chimpanzees and sooty mangabeys. Royal Society Open
Science 4; https://doi.org/10.1098/rsos.171296 (2017). . Ost e , J. & Sc ü ke, O. dvances in the Study of ehavior Vo . 50 (eds. a c Nagu b et al.)
7
75 ( cade
c
ess, 0 8). 5. Mielke, A. et al. Bystanders intervene to impede grooming in Western chimpanzees and sooty mangabeys. Royal Society Open
Science 4; https://doi.org/10.1098/rsos.171296 (2017). p
g
(
)
6. Lesch, K. et al. Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science
274, 1527–1531 (1996). ,
(
)
57. Rosati, A. G. & Santos, L. R. Tolerant barbary macaques maintain juvenile levels of social attention in old age, but despotic rhesus
macaques do not. Animal Behaviour 130, 199–207, https://doi.org/10.1016/j.anbehav.2017.06.019 (2017). ,
(
)
57. Rosati, A. G. & Santos, L. R. Tolerant barbary macaques maintain juvenile levels of social attention in old age, but despotic rhesus
macaques do not. Animal Behaviour 130, 199–207, https://doi.org/10.1016/j.anbehav.2017.06.019 (2017). Acknowledgements g
We thank Uwe Schönmann, Annette Husung, and the animal keepers at the German Primate Center for logistical
support; Johannes Borys for help with data collection and Delphine de Moor for help with statistical modelling. The study was supported by the Leibniz ScienceCampus Primate Cognition and benefitted from discussions in
the DFG-RTG 2070 Understanding Social Relationships (DFG, German Research Foundation Project number
254142454). Competing interestsh g
The authors declare no competing interests. Author contributions O.S. and J.O. designed the study and wrote the manuscript, N.D. collected the data, O.S. and N.D. analyzed the
data. Additional information Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-62772-8. Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-62772-8. Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-62772-8 Correspondence and requests for materials should be addressed to O.S. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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SEAFLOOR POSITIONING MODEL FOR SIMULTANEOUS ESTIMATION OF SOUND VELOCITY
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The international archives of the photogrammetry, remote sensing and spatial information sciences/International archives of the photogrammetry, remote sensing and spatial information sciences
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1. INTRODUCTION solitary underwater point or the transponder array's center
remaining unaffected by default. Due to the severe attenuation of electromagnetic waves
underwater, GNSS-A (Global Navigation Satellite System-
Acoustic Ranging Combined), which extends the observation
network to the seafloor, was proposed and developed(Spiess, F.,
1980; Xu et al., 2005; Ikuta et al., 2008; Chen, 2014; Yang, Qin,
2021). The GNSS-A observation system consists of a surface unit
(usually a vessel or buoy) and a subsurface transponder. The
surface unit is equipped with a GNSS antenna, a vessel attitude
sensor, and a ship bottom transducer, allowing real-time location
of the surface unit based on the International Terrestrial
Reference Frame ITRF and dynamic GNSS. During one
measurement, the transponder receives the signal emitted by the
transducer and sends the sound signal back to the transducer, then
observers will obtain the transmission time by correlating the
emitted and received signals. After that, the subsea transponder's
coordinates can be determined by establishing the ranging
equation with the ocean sound velocity structure. (Yokota et al.,
2018b; Ishikawa et al., 2020). The difference method is a good strategy for reducing systematic
error, and it can be used to obtain the seabed transponder position
with cm-level precision(Xu et al., 2005; Chen et al., 2020). However, as some scholars have pointed out, the ill-conditioned
problem that occurred in the difference equation will result in a
poor estimation of the vertical coordinate(Yang, Qin, 2021),
which is ineffective for solving the three-dimensional location of
the seabed transponder. In contrast to the linear propagation of electromagnetic waves,
the propagation of acoustic signals underwater follows Snell's
law of reflection and curved route, especially when acoustic
incidence angles are considerable. Some researchers attempted to
improve seafloor positioning accuracy by using acoustic ray
tracking algorithm and developed some exact equations for
calculating sound line tracking time(Dosso, Stan, 1998; Yan,
1999; Zielinski, 1999; Lu et al., 2012). This technique based on
the physical properties of acoustic waves can reduce the residual
series of the observation equation to some extent, but it needs
sound velocity profile data and presupposes that the sound
velocity does not fluctuate in the horizontal direction (horizontal
stratification of the sound velocity profile). The acoustic tracking
algorithm still does not effectively tackle the problem of spatial
and temporal variation of sound velocity, and it is incapable of
meeting the high-precision positioning requirements when
processing actual measurement data(Kido et al., 2008b; Tomita
et al., 2019). The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China SEAFLOOR POSITIONING MODEL FOR
SIMULTANEOUS ESTIMATION OF SOUND VELOCITY Shengqiu Zhang 1, Tianhe Xu 2 *, Xianping Qin 3 1 College of Geology Engineering and Geomatics, Chang’an University, Xi’an 710054, Shanxi, China – shengqiuz@163.com
2 Institute of Space Science, Shandong University, Weihai 264209, Shandong, China – thxugfz@163.com
3 Xi’an Research Institute of Surveying and Mapping, Xi’an 710054, Shanxi, China – qxianping@126.com Commission IV, WG IV/5 KEY WORDS: GNSS-A, Seafloor Geodesy, Positioning, Sound velocity, Temporal and spatial variation, B-Spline ABSTRACT: The spatial and temporal change of sound speed has a significant impact on the accuracy of GNSS-A underwater positioning. However,
it is hard to collect enough data to cover all the spatial and temporal changes of sound velocity. We built an acoustic ranging model
that included the sound velocity component, and then we looked at how the model could be used for three-dimensional seabed location
and undersea crust monitoring. Meanwhile, B-spline curves are used to construct a sound velocity model that encompasses temporal
variation as well as two-dimensional spatial gradients. The method was utilized to assess the simulated data and the in-situ data
collected in July 2019, respectively. Simulation results show that the root mean square of the horizontal coordinates solved by the
ranging model is less than 10cm, and the model may meet the demands of subsea crust monitoring under certain situations. In the real
data experiment, the square root of variance of the coordinate was better than 10 cm. The sound velocity model findings indicated that
the variance of sound velocity in the experimental marine region was less than 1m/s, with clear daily patterns and short-period
variations. The algorithm does not need the sound velocity profile information and can save the measurement time of the sound
velocity profile. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License. * Corresponding author This contribution has been peer-reviewed. 1. INTRODUCTION Seasonal fluctuations, daily variations induced by
temperature, and short-period variations caused by internal
waves all contribute to the temporal variability of sound velocity. Spatial differences of sound velocity appear as horizontal
inhomogeneities due to anomalous seawater advection caused by Eliminating systematic errors in acoustic ranging is essential for
achieving greater positioning precision of subsea transponders. Generally, system errors include hardware time delays,
inaccuracies in sound velocity, and other measurement errors
caused by the complicated maritime environment. To reduce or
counteract systematic errors in underwater measurement, SIO
fixed configurations (the surface unit is roughly in the horizontal
center of three or four subsea transponders) and symmetric walk-
away configurations are widely used.(Spiess et al., 1998; Obana
et al., 2000; Osada et al., 2003; Kido et al., 2006; Kido et al.,
2008a; Fujimoto, 2014; Tomita et al., 2017). Both configurations
are based on the premise that the systematic error is approximate
at the symmetry point, with the horizontal coordinates of the 279 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China Section 4 provides the calculation and analysis of the simulated
and measurement data, and the conclusion is given in Section 5. Section 4 provides the calculation and analysis of the simulated
and measurement data, and the conclusion is given in Section 5. internal waves or ocean currents, which are usually found in the
ocean's shallower waters (Honsho et al., 2019). The introduction
of a time-dependent parameter associated with sound velocity in
the ranging equation can significantly improve localization
precision(Fujita et al., 2006; Ikuta et al., 2008; Kido et al., 2008a;
Honsho, Kido, 2017), and all of these studies can derive the
temporal variation of underwater sound velocity concerning a
fixed sound velocity profile. The distance-dependent linear
model has been demonstrated to be useful in solving the
horizontal gradient of the sound velocity while also allowing for
flexible depth control. 1. INTRODUCTION Under specific conditions, the continuous
variation of the horizontal gradient can be derived by modeling
in the time domain. (Yasuda et al., 2017; Yokota et al., 2018a;
Kinugasa et al., 2020; Watanabe et al., 2020). ,
g
2. ACOUSTIC RANGING EQUATION AND
COLLINEARITY PROBLEM 2. ACOUSTIC RANGING EQUATION AND
COLLINEARITY PROBLEM This contribution has been peer-reviewed.
https://doi org/10 5194/isprs archives XLVI 3 W1 2022 279 2022 | © Author(s) 2022 CC BY 4 0 License
280 This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License.
280 This contribution has been peer-reviewed. 2. ACOUSTIC RANGING EQUATION AND
COLLINEARITY PROBLEM g
,
;
,
)
In the multi-sensor experiment for 3d positioning, the horizontal
position of the seabed station is determined by GNSS-A
underwater positioning and the vertical position is determined by
the pressure sensor (Liu et al., 2019), which increases the
difficulty of unifying the seabed station with the global
framework. Meanwhile, algorithms using sound velocity profiles
may take a long time to process a large number of data with the
acoustic ray tracking technique. When only acoustic sensors are
available, we hope to get the transponder's three-dimensional
position by constructing a simple sound velocity model. Therefore, the speed of sound will be solved as an unknown
parameter if the angle of measurement incidence changes
dramatically. The method without sound velocity profile has
been validated in shallow waters for 3d positioning with a
precision better than 5cm(Yang et al., 2011). To prevent frequent
measurements of sound velocity, linear and bilinear models are
proposed to take into account the fluctuation of sound velocity
with depth(Chen, 2014). The model including the offset error of
transceiver lever arms is also utilized to improve positioning
performance with a precision of better than 1cm(Chen et al.,
2019). Subsequently, some scholars extend the equation with
time bias and adopt an elastic approach to describe the system
error more flexibly by modeling the residuals(Yang, Qin, 2021). All of the approaches discussed above can significantly improve
seabed positioning precision; however, few studies on the outer
precision of positioning results have been conducted since
accurate values of seabed transponder coordinates are difficult to
get by other technical means. Figure 1. Acoustic observational model. Acoustic ranging model with sound velocity parameter can be
expressed as: 𝜌𝑖= 𝑓(𝑋𝑠0, 𝑋𝑝) + 𝑓(𝑋𝑠1, 𝑋𝑝) + ∆𝐶𝜏𝑖+ 𝜀𝑖
(1)
𝑓(𝑋𝑠, 𝑋𝑃) = √(𝑥𝑠−𝑥𝑝)
2 + (𝑦𝑠−𝑦𝑝)
2 + (𝑧𝑠−𝑧𝑝)
2
(2) e
𝜌𝑖 = the ranging between the transponder on the
seafloor and the transducer under the ship at epoch 𝑖 where 𝑓(𝑋𝑠, 𝑋𝑝) = the theoretical distance between the
transducer 𝑋𝑠0 and the transponder 𝑋𝑝 at the epoch of
signal transmission. 𝑓(𝑋𝑠, 𝑋𝑝) = the theoretical distance between the
transducer 𝑋𝑠1 and the transponder 𝑋𝑝 at the epoch of
signal receiving. ∆𝐶 = the sound speed bias ∆𝐶 = the sound speed bias 𝜏𝑖 = the travel time at epoch 𝑖 𝜀𝑖 = the random ranging error 𝜀𝑖 = the random ranging error Linearizing the observation equation: Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China 𝜎0
2 = 𝑉𝑇𝑃𝑉
𝑛−4
(10) As shown in Fig. 4, because of greater variation of 𝜏𝑛, the
performance of the coefficient matrix for the cross configuration
is better than circle configuration, but these two parameters are
still highly correlated. (10) where
𝑃 = the weight matrix of observations (an equal weight
assumption is used in this paper)
𝐷𝑋̂𝑋̂ = the covariance matrix of unknown parameters. 𝜎0
2 = the variance of unit weight
𝑉 = the observation residual vector. 𝑄𝑋̂𝑋̂ = the cofactor matrix of unknown parameters where Figure 4. Parameter correlation of cross configuration. 𝐷𝑋̂𝑋̂ = the covariance matrix of unknown parameters. 𝜎0
2 = the variance of unit weight Eq. (7) is based on the assumption that the parameters are linearly
independent, and thus cannot offer an adequate estimate when the
parameters are highly correlated with one another. In that case,
the determinant of 𝐴𝑇𝑃𝐴 approaches zero, making the solution
extremely unstable and leading to erroneous statistical results. Eq. (7) is based on the assumption that the parameters are linearly
independent, and thus cannot offer an adequate estimate when the
parameters are highly correlated with one another. In that case,
the determinant of 𝐴𝑇𝑃𝐴 approaches zero, making the solution
extremely unstable and leading to erroneous statistical results. The co-linear relationship of parameters is usually caused by
defects in the data or over-parameterization of the model, and the
performance indicators of the coefficient matrix may be used to
analyze the correlation of parameters. We studied the circle
configuration
and
crossover
configuration
for
seafloor
positioning, as shown in Fig. 2, for both configurations: The co-linear relationship of parameters is usually caused by
defects in the data or over-parameterization of the model, and the
performance indicators of the coefficient matrix may be used to
analyze the correlation of parameters. We studied the circle
configuration
and
crossover
configuration
for
seafloor
positioning, as shown in Fig. 2, for both configurations: Figure 4. Parameter correlation of cross configuration. Bayesian estimation, ridge estimation, and other methodologies
such as principal component analysis are extensively used for the
solution of ill-condition equations(Massy, 1965; Mandel, 1982;
Marin, Robert, 2007). These methods effectively address the
problem by providing external information or reducing
dimensionality. Linearizing the observation equation: Linearizing the observation equation: Some characteristics of ocean currents can be mirrored in the
spatial and temporal variation of sound velocity, particularly the
two-dimensional horizontal gradient. In general, the structure of
ocean sound velocity parallel to ocean current is more spatially
homogenous than that perpendicular to it(Yada et al., 2004). The
relationship between continuous horizontal gradients at different
depths and the real ocean field was investigated(Yokota,
Ishikawa, 2019). The findings showed that the gradient field
might respond to the Kuroshio flow (a warm current flowing
from the Philippine Sea east of Taiwan to Japan). This indicates
that GNSS-A technology may be utilized for more than only
seabed transponder positioning; the ocean gradient field derived
from acoustic data can also be used to determine the ocean
current route. This enables more research into the physical
properties of the water. 𝑉= 𝐴𝑑𝑋−𝐿
(3)
𝐹= 𝑓(𝑋𝑠0, 𝑋𝑝) + 𝑓(𝑋𝑠1, 𝑋𝑝)
(4)
𝐴=
[
𝜕𝐹1
𝜕𝑥
𝜕𝐹1
𝜕𝑦
𝜕𝐹1
𝜕𝑧
−𝜏1
⋮
⋮
⋮
𝜕𝐹𝑛
𝜕𝑥
𝜕𝐹𝑛
𝜕𝑦
𝜕𝐹𝑛
𝜕𝑧
−𝜏𝑛]
(5)
𝐿= [𝐶0𝜏1 −𝐹1 … 𝐶0𝜏𝑛−𝐹𝑛]𝑇
(6) (3)
(4) (5) (6) 𝑑𝑋 = the correction of parameters (3D coordinates and
sound velocity) 𝑑𝑋 = the correction of parameters (3D coordinates and
sound velocity) y
𝐴 = the coefficient matrix of observation equation
𝑉 = the observation residual vector
𝐶0 = Initial value of sound velocity
𝑛 = the number of observations In this paper, we estimate the spatial and temporal variation of
sound velocity using cubic B-spline curves based on a model
including the sound velocity parameter, and then we analyze
simulated data to demonstrate the model's effectiveness in
acoustic positioning. Finally, the coordinate of transponder and
sound velocity structure are estimated using acoustic data at a
depth over 3000m collected in July 2019. The acoustic ranging
equation is presented in section 2 and the sound velocity model
based on the cubic B-spline curve is proposed in section 3. To achieve optimal unbiased parameter estimation in linear
models, the Gauss-Markov theorem is commonly used to
estimate unknown parameters: 𝑑𝑋= (𝐴𝑇𝑃𝐴)−1𝐴𝑇𝑃𝐿
(7)
𝑋̂ = 𝑋0 + 𝑑𝑋
(8)
𝐷𝑋̂𝑋̂ = 𝜎0
2𝑄𝑋̂𝑋̂ = 𝜎0
2(𝐴𝑇𝑃𝐴)−1
(9) (7)
(8)
(9) This contribution has been peer-reviewed. rchives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License. 280 280 280 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. 3. SOUND VELOCITY PERTURBATION MODEL Because of the spatial and temporal variation of the sound speed,
the residuals of Eq. (3) will include periodic systematic errors
that directly represent the sound speed fluctuation. The time-
continuous property of the sound speed perturbation can be
satisfied by b-spline curves; however, if the temporal and spatial
variation of the sound speed is estimated simultaneously, the
smoothness of the curve must be controlled by hyperparameters
for the collinearity problem caused by the over-definition
model(Ikuta et al., 2008; Watanabe et al., 2020). On the other
hand, this method can also improve positioning precision as a
resilient way for modeling systematic errors(Yang, Qin, 2021). Figure 2. Geometric configuration. Figure 2. Geometric configuration. 𝐴= [
𝑐𝑜𝑠𝜃1 ∙𝑠𝑖𝑛𝜔1
𝑠𝑖𝑛𝜃1 ∙𝑠𝑖𝑛𝜔1
𝑐𝑜𝑠𝜔1
−𝜏1
⋮
𝑐𝑜𝑠𝜃𝑛∙𝑠𝑖𝑛𝜔𝑛
⋮
𝑠𝑖𝑛𝜃𝑛∙𝑠𝑖𝑛𝜔𝑛
⋮
𝑐𝑜𝑠𝜔𝑛
⋮
−𝜏𝑛
]
= [𝐴1
𝐴2
𝐴3
𝐴4]
(11) (11) where where
𝜃 = the azimuth angle of the line between transponder
and transducer onto the horizontal plane 𝜔 = the inclination angle between the zenith and the
line of sight from the transducer to the transponder
(shown in Fig. 1) Cubic B-spline curve model constructed on n+1 nodes may be
expressed as the following equation to derive the temporal
variation of the sound velocity and the two-dimensional spatial
gradient: For circle track, It is obvious that there exists 𝑘= 𝑐𝑜𝑠𝜔𝑛/𝜏𝑛
such that 𝐴3 + 𝑘∙𝐴4 = 0, implying that the coefficient matrix
is not full column rank, and the vertical coordinate parameter is
linearly related to the sound velocity parameter. The correlation
of all parameters is shown in Fig. 3. 𝑣𝑖= 𝐴𝑖𝑑𝑋̂𝑝+ 𝛥𝐶̂𝜏𝑖−(𝜌𝑖−𝜌𝑖
0)
(12)
𝑣𝑖= 𝜏𝑖(∑𝑎1𝑖𝐵𝑖,3(𝑡∗)
𝑛
𝑖=0
+ ∑𝑎2𝑖𝐵𝑖,3(𝑡∗) ∗𝑑𝑐𝑜𝑠𝜑
𝑛
𝑖=0
+ ∑𝑎3𝑖𝐵𝑖,3(𝑡∗) ∗
𝑛
𝑖=0
𝑑𝑠𝑖𝑛𝜑) (13)
𝑡∗=
𝑡−𝑡𝑖
𝑡𝑖+1 −𝑡𝑖
𝑡∈(𝑡𝑖, 𝑡𝑖+1)
(14) (12) Figure 3. Parameter correlation of circle configuration. (14) where
𝑣𝑖 = the residuals of Eq. (3) re
𝑣𝑖 = the residuals of Eq. (3)
𝑎1 = time-varying parameter of sound velocity
𝑎2, 𝑎3 = two-dimensional spatial gradient
𝐵𝑖,3(𝑡∗) = cubic B-spline basis function
𝜑 = the azimuth angle of the transducer relative to the
transponder. 𝑣𝑖 = the residuals of Eq. (3)
𝑎1 = time-varying parameter of sound velocity
𝑎2, 𝑎3 = two-dimensional spatial gradient
𝐵𝑖,3(𝑡∗) = cubic B-spline basis function
𝜑 = the azimuth angle of the transducer relative to the
transponder. Eq. (13) containing three kinds of parameters is to be solved by
least squares with constraints. Linearizing the observation equation: It should be noted that the linear combination of
strongly correlated parameters may be accurately estimated in the
collinearity problem, while collinearity has no effect on the
estimation of other uncorrelated parameters. This means that if
the variation range of sound velocity is controlled, the vertical
coordinates of the seafloor transponder will be quite stable, which
is advantageous for monitoring the vertical displacement of the
seafloor crust. Figure 2. Geometric configuration. This contribution has been peer-reviewed. This contribution has been peer-reviewed. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License.
281 This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License.
2 This contribution has been peer reviewed.
https://doi.org/10.5194/isprs-archives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License. 3. SOUND VELOCITY PERTURBATION MODEL To control the roughness of the Figure 3. Parameter correlation of circle configuration. This contribution has been peer-reviewed. https://doi org/10 5194/isprs archives XLVI 3 W1 2022 279 2022 | © Author(s) 2022 CC BY 4 0 License
281 281 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China Figure 6. Sound speed setting and estimation. curve, the integral of the second-order derivative of the B-spline
curve basis function is used as the a priori weight matrix of the
sound velocity perturbation parameters(Honsho, Kido, 2017;
Watanabe et al., 2020), which can be adjusted by the
hyperparameters. 𝑃𝑖,𝑗= 𝜇∫𝜕2𝐵𝑖,3(𝑡)
𝜕𝑡2
𝜕2𝐵𝑗,3(𝑡)
𝜕𝑡2
𝑑𝑡
(15)
𝑃0 = 𝑑𝑖𝑎𝑔( 𝑃𝑖,𝑗 102𝑃𝑖,𝑗 102𝑃𝑖,𝑗)
(16)
𝑎= (𝐸𝑇𝐸+ 𝑃0)−1(𝐸𝑇𝑣)
(17) (15) (17) where
𝑃0 = Prior weight matrix (set the weight matrices of 𝑎2
and 𝑎3 to be two orders of magnitude larger than the
weight matrix of 𝑎1, implying that the spatial variation
is smoother.)
𝐸 = the Jacobi matrix of Eq. (13) for all sound velocity
parameters. where 𝑃0 = Prior weight matrix (set the weight matrices of 𝑎2
and 𝑎3 to be two orders of magnitude larger than the
weight matrix of 𝑎1, implying that the spatial variation
is smoother.) where
𝑃0 = Prior weight matrix (set the weight matrices of 𝑎2
and 𝑎3 to be two orders of magnitude larger than the
weight matrix of 𝑎1, implying that the spatial variation
is smoother.) Figure 6. Sound speed setting and estimation. Data
Coordinates
Root mean squares (RMS)
E(m)
N(m)
U(m)
(cm)
(cm)
(cm)
Cross
Circle
-0.005
0.035
0.039
-0.016
-3000.333
-2329.809
0.5
3.5
3.9
1.6
33.3
67019.1
Circle + control
0.039
-0.013
-3000.214
3.9
1.3
21.4
Table 1. Positioning results of simulation data. Data
Coordinates
Root mean squares (RMS)
E(m)
N(m)
U(m)
(cm)
(cm)
(cm)
Cross
Circle
-0.005
0.035
0.039
-0.016
-3000.333
-2329.809
0.5
3.5
3.9
1.6
33.3
67019.1
Circle + control
0.039
-0.013
-3000.214
3.9
1.3
21.4
Table 1. Positioning results of simulation data. 𝐸 = the Jacobi matrix of Eq. (13) for all sound velocity
parameters. 3. SOUND VELOCITY PERTURBATION MODEL By this way, the temporal variation and horizontal spatial
gradient of oceanic sound speed can be derived, and the
horizontal spatial variation may have the capacity to reflect the
flow route of ocean currents. Table 1. Positioning results of simulation data. Table 1 shows the location results using simulated data. Given
the significant correlation of the circle configuration, the circle
data is separated into two instances for computation based on
whether the sound speed limitation is included or not, circle with
control is limited by introducing the average sound speed
calculated from the Cross data. The root mean square (RMS) of
the horizontal coordinates solved for all data is better than 5 cm. Due to the collinearity of the circle data without constraints,
positioning parameters have a large departure from the truth
value of its vertical coordinates, but the vertical location result of
the circle data with the constraint of sound velocity is similar to
cross, both of which are of the level of decimetres. It is also worth
noting that, in the circular walk configuration, the presence or
lack of vertical information does not affect the estimate of
horizontal coordinates, implying that collinearity has no effect on
the estimation of uncorrelated parameters. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License. This contribution has been peer-reviewed. This contribution has been peer-reviewed.
archives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License. 4.1 Analysis of simulation data Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China Figure 8. The change of coordinate with phase. Figure 9. The spatial position of observation data. Figure 8. The change of coordinate with phase. Figure 9. The spatial position of observation data. Figure 8. The change of coordinate with phase. Figure 9. The spatial position of observation data. Figure 8. The change of coordinate with phase. Section
Number of
segments
Time
Number of
observations
Data 1
1
15.81-15.88
514
Data 2
1
16.12-16.29
1119
Data 3
3
14.86-15.15
1889
15.28-15.71
2884
15.88-15.98
697
Table 2. Information of observation data. We also analyzed the influence of varying sound velocity's initial
phase on coordinates solved by the cross configuration. One
cycle of sound velocity fluctuation with different beginning
phases (0 to 24 hours) is established, as illustrated in Fig. 7. The
change of the root mean square of horizontal and vertical
coordinates with the initial phase is shown in Fig. 8. It can be
seen that the horizontal coordinate is greatly influenced, with a
maximum mutual difference of 1.6m for one cycle, but the
vertical coordinates are quite stable, with an overall fluctuation
of less than 3cm. Because sound velocity in the ocean has
obvious time-varying and weak spatially varying characteristics,
it is nearly impossible to guarantee the same sound velocity
structure at the symmetry points in the actual measurement,
which will almost certainly result in a horizontal coordinate
offset, and the magnitude of the offset is related to the initial
phase of the sound velocity change. The strong correlation
between the sound velocity parameters and the vertical
coordinates, on the other hand, provides us with a method of
solving the stable transponder vertical coordinates: it is just
essential to ensure that a complete period (typically roughly 24
hours) is observed, without focusing on the initial moment of
measurement. This is significant for monitoring the seafloor crust
since it is concerned with stability rather than the accuracy of the
vertical coordinates. Although the results shown in Table 1
indicates that the transponder vertical coordinates calculated
using cross data differ from the truth value by 33.3 cm, the
experiments in Fig. 4.1 Analysis of simulation data 8 imply that the technique can detect vertical
seabed displacements greater than 3 cm. Table 2. Information of observation data. The circular data (Data1/2) are processed in two ways: normally
(Table 3), and with the sound speed constraint calculated by
Data3 (Table 4). As shown in Table 3, the greatest differences in the E/N/U
components across different data sets are 0.478, 0.031, and 4.931
m. The collinearity of the model results in very poor repetition of
the U component, whereas the inferior repetition of the E
coordinate is thought to be owing to the same circumstance as in
Fig. 8. In other words, the sound velocity structure of Data3
results in a slower average sound velocity on the west side of the
transponder than on the east side, with no significant difference
between the north and south sides. After residual modeling, the square root of variance of all
coordinates was better than 10 cm. The 3D coordinates with the
best precision are those calculated from the Data3 with the
longest observation time. Meanwhile, the horizontal coordinate
precision of Data2 with a radius of 1.5 times the sea depth is
better than that of Data1, which is compatible with the results of
studies about geometric configuration(Zhao et al., 2016; Xue,
Yang, 2017; Chen et al., 2020). 4.1 Analysis of simulation data The measured sound velocity profile of 3000m and sound ray
tracking technique are used to simulate the acoustic signal's travel
time. The speed of ship is 3 knots per hour and the sample interval
is 12.4 seconds for simulating acoustic observation data. The
long and short periods of sound velocity change are set to 90
minutes and 24 hours, respectively. The effect of changing sound
velocity on measurement time is simulated based on the
following equation: ∆𝑉𝑖= 𝑐1 sin (2(𝑡−𝑡0)
𝑇𝑆
𝜋) + 𝑐2 sin (2(𝑡−𝑡0)
𝑇𝐿
𝜋)
(18)
𝜏𝑖=
𝑆𝑖
𝑉𝑖+ ∆𝑉𝑖
(19) (18) The point of Fig. 6 is to compare sound speed simulation to sound
speed estimation of cross data, with the red line representing the
simulated sound speed perturbation, the blue line showing sound
speed perturbation estimated by B-spline, and the black line
reflecting the difference between the two. It can be seen that the
two have a high degree of agreement, indicating that the
functional model's residual series completely reacts to the
fluctuation of the sound velocity and proving the superiority of
the B-spline curve for the estimation of continuous variation
regarding time series. (19) ere
𝑐1 = 0.13m/s
𝑐2 = 0.4m/s
𝑆𝑖 = Actual acoustic path length
𝑉𝑖 = sound velocity calculated by velocity profile where 𝑐2 = 0.4m/s 𝑆𝑖 = Actual acoustic path length 𝑆𝑖 = Actual acoustic path length 𝑉𝑖 = sound velocity calculated by velocity profile 𝑉𝑖 = sound velocity calculated by velocity profile Considering the influence of the ship track geometry
configuration on the positioning accuracy(Sato et al., 2013; Zhao
et al., 2016; Xue, Yang, 2017), one circular track with a radius of
about 1.5 times the water depth and cross track are used for
simulation experiments. The spatial location of the transponder
and sampled data are shown in Fig. 5. Figure 7. Phase simulator. Figure 7. Phase simulator. Figure 5. Spatial position. Figure 5. Spatial position. Figure 7. Phase simulator. Figure 5. Spatial position. Figure 5. Spatial position. 282 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License This contribution has been peer-reviewed. 4.2 Analysis of in-situ data It can
be seen that the vertical coordinates of Data1 and Data2 are both
closer to the vertical coordinate of Data3, but Data2 is still a long 283 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China Figure 12. 2D spatial gradient with constraint. way from the truth value. This phenomenon might be attributed
to two factors: (1) The speed of sound solved by Data3 differs
much from the real sound speed along Data2's sound path. (2) As
shown by the yellow line in Fig. 9, Data1 is not precise circle data,
with a tiny fraction of its track points near to the center of the
circle, making the vertical coordinate calculated by Data1 closer
to the true value. Figure 10. Time variation of the sound speed. Figure 12. 2D spatial gradient with constraint. ACKNOWLEDGEMENTS Figure 11. Horizontal distance from the transducer to the
transponder and spatial gradient. This research is financially supported by Wenhai Program of the
S&T Fund of Shandong Province for Pilot National Laboratory
for
Marine Science
and
Technology
(Qingdao)
(NO. 2021WHZZB1004), and the National Key Research &
Development Program of China (2020YFB0505804). 5. CONCLUSION The model with sound velocity parameter can be used to obtain
three-dimensional seafloor transponder coordinates, and the
cross track is beneficial for the vertical coordinate estimation. After the spatial and temporal variation of the sound speed was
estimated, the square root of variance of the coordinate was better
than 10 cm. Figure 10. Time variation of the sound speed. Fig. 10 shows the estimated sound velocity variation, which has
a clear trend of daily variation and short-period fluctuations
similar to internal waves, and the total temporal variation of
sound velocity is less than 1 m/s. The red line (Data2) is clearly
out of the black sine curve, which is related to the poorer vertical
coordinates obtained from the Data2 solution. As shown in Fig. 11, fixing the 2D spatial gradient directly with B splines is not
feasible because the local poor geometric design leads to local
mistakes of spatial gradient inversion. These scenarios occur
when the bottom transducer is close to the transponder for an
extended period (black dashed box), and the horizontal distance
approximating zero is insufficient to hold the sound velocity
information. Therefore, we tighten the constraint and force the
spatial gradient to remain constant during the observation. As
illustrated in Fig. 12, The east-west spatial gradient attained is
0.006 m/s/km, and the north-south spatial gradient is 0.028
m/s/km. The more active north-south sound velocity structure
during the observation might imply the presence of east-west
currents inside the ocean. For circle track, the sound velocity parameters are linearly
correlated with the vertical coordinate parameters, which will
lead to poor vertical coordinate estimation, but this does not
affect the horizontal coordinate estimation. For cross track, the
sound velocity parameters are strongly correlated with the
vertical coordinate parameters, which is beneficial for the vertical
displacement monitoring of the submarine crust. The simulation
experiment showed that the seafloor vertical displacement larger
than 3 cm could be detected as long as one cycle of observation
was guaranteed. The residuals reflect the fluctuation of the sound velocity. The
situ data showed that the variation of sound velocity in the
experimental sea area was less than 1m/s, and it had obvious daily
trends and short-period fluctuations. Meanwhile, the north-south
sound velocity gradient was more active than the east-west sound
velocity gradient, which might imply the existence of east-west
currents in the sea area during the observation period. 4.2 Analysis of in-situ data Data
set
Coordinates
Square root of variance
E(m)
N(m)
U(m)
𝑚𝐸(cm)
𝑚𝑁(cm)
𝑚𝑈(cm)
Data 1
0.504
0.445
0.254
7.7
7.7
2.6
Data 2
0.578
0.453
-4.195
5.3
5.3
5.5
Data 3
0.100
0.422
0.736
2.7
2.6
1.7
Table 3. Calculate normally. Data
set
Coordinates
Square root of variance
E(m)
N(m)
U(m)
𝑚𝐸(cm)
𝑚𝑁(cm)
𝑚𝑈(cm)
Data 1
0.483
0.432
0.617
7.7
7.7
2.6
Data 2
0.487
0.393
3.183
5.3
5.3
5.5
Data 3
0.100
0.422
0.736
2.7
2.6
1.7
Table 4. Calculate with sound speed constraint. Data
set
Coordinates
Square root of variance
E(m)
N(m)
U(m)
𝑚𝐸(cm)
𝑚𝑁(cm)
𝑚𝑈(cm)
Data 1
0.504
0.445
0.254
7.7
7.7
2.6
Data 2
0.578
0.453
-4.195
5.3
5.3
5.5
Data 3
0.100
0.422
0.736
2.7
2.6
1.7
Table 3. Calculate normally. From July 14 to 16, 2019, an acoustic localization experiment for
one seabed transponder at 3000 m water depth was carried out. The experiment involved three observation configurations. Fig. 9
displays the horizontal spatial link between the seabed
transponder and the observation data, where the yellow line
corresponds to data Data1, the red line to data Data2, and the blue
line to data Data3. Table 2 shows the complete information for
these three data sets, and it is noticeable that they are observations
from separate periods, allowing their results to be compared. Data3 is not a continuous data set, having gaps of 3 or 4 hours
between periods. Theoretically, the more B-spline time nodes
sampled in a continuous period, the better the modeling results,
but there may be a risk of overfitting. Time nodes are taken at
30min intervals in this article, and the number of period nodes is
proportionate to the duration of observation. Data
set
Coordinates
Square root of variance
E(m)
N(m)
U(m)
𝑚𝐸(cm)
𝑚𝑁(cm)
𝑚𝑈(cm)
Data 1
0.483
0.432
0.617
7.7
7.7
2.6
Data 2
0.487
0.393
3.183
5.3
5.3
5.5
Data 3
0.100
0.422
0.736
2.7
2.6
1.7
Table 4. Calculate with sound speed constraint. Table 4. Calculate with sound speed constraint. The horizontal coordinates and precision do not vary
considerably after introducing the sound velocity restriction, as
shown in Table 4, however, the vertical coordinates of the circle
data (Data1/2) differ dramatically from the previous ones. This contribution has been peer reviewed.
https://doi.org/10.5194/isprs-archives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License. This contribution has been peer-reviewed. REFERENCES Chen, G., Liu, Y., Liu, Y., Liu, J. 2020. Improving GNSS-
acoustic positioning by optimizing the ship's track lines and
observation combinations. Journal of Geodesy, 94 (6): 61. Chen, G., Liu, Y., Liu, Y., Liu, J. 2020. Improving GNSS-
acoustic positioning by optimizing the ship's track lines and
observation combinations. Journal of Geodesy, 94 (6): 61. Chen, G., Liu, Y., Liu, Y., Tian, Z., Li, M. 2019. Adjustment of
Transceiver Lever Arm Offset and Sound Speed Bias for GNSS-
Acoustic Positioning. Remote Sensing, 11 (13): 1606. Chen, H. H. 2014. Travel-time approximation of acoustic ranging
in GPS/Acoustic seafloor geodesy. Ocean Engineering, 84: 133-
144. Dosso, Stan, E. 1998. Array element localization for horizontal
arrays via Occam's inversion. Acoustical Society of America
Journal, 104 (2): 846-859. Figure 11. Horizontal distance from the transducer to the
transponder and spatial gradient. This contribution has been peer-reviewed. 284 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China Marin, J. M., Robert, C. P. 2007. Bayesian Core (Bayesian Core). Fujimoto, H. 2014. Seafloor Geodetic Approaches to Subduction
Thrust Earthquakes. monographs on environment earth &
planets meep. Massy, William F. 1965. Principal Components Regression in
Exploratory Statistical Research. Publications of the American
Statistical Association, 60 (309): 234-256. Fujita, Masayuki, Ishikawa, Tadashi, Mochizuki, Masashi, Sato,
Mariko, Toyama, Shin-Ichi, Katayama, Masato, Kawai, Koji, et
al. 2006. GPS/Acoustic seafloor geodetic observation: method of
data analysis and its application. Earth, Planets and Space, 58 (3):
265-275. Obana, K., Katao, H., Ando, M. 2000. Seafloor positioning
system
with
GPS-acoustic
link
for
crustal
dynamics
observation—a preliminary result from experiments in the sea—. Earth Planets & Space, 52 (6): 415-423. Honsho, C., Kido, M., Tomita, F., Uchida, N. 2019. Offshore
Postseismic Deformation of the 2011 Tohoku Earthquake
Revisited: Application of an Improved GPS ‐ Acoustic
Positioning Method Considering Horizontal Gradient of Sound
Speed Structure. Journal of Geophysical Research: Solid Earth,
124 (6). REFERENCES Osada, Y., Fujimoto, H., Miura, S., Sweeney, A., Kanazawa, T.,
Nakao, S., Sakai, S. I., Hildebrand, J. A., Chadwell, C. D. 2003. Estimation and correction for the effect of sound velocity
variation on GPS/Acoustic seafloor positioning: An experiment
off Hawaii Island. Earth,Planets&Space. Sato, M., Fujita, M., Matsumoto, Y., Saito, H., Ishikawa, T.,
Asakura, T. 2013. Improvement of GPS/acoustic seafloor
positioning precision through controlling the ship's track line. Journal of Geodesy, 87 (9): 825-842. Honsho, Chie, Kido, Motoyuki. 2017. Comprehensive Analysis
of
Traveltime
Data
Collected
Through
GPS-Acoustic
Observation of Seafloor Crustal Movements. Journal of
Geophysical Research: Solid Earth, 122 (10): 8583-8599. Spiess, F., N. 1980. Acoustic techniques for Marine Geodesy. Marine Geodesy, 4 (1): 13-27. Ikuta, Ryoya, Tadokoro, Keiichi, Ando, Masataka, Okuda,
Takashi, Sugimoto, Shingo, Takatani, Kazunori, Yada, Kazuyuki,
Besana, Glenda M. 2008. A new GPS-acoustic method for
measuring ocean floor crustal deformation: Application to the
Nankai Trough. Journal of Geophysical Research, 113 (B2). Spiess, F. N., Chadwell, C. D., Hildebrand, J. A., Young, L. E.,
Purcell, George H, Dragert, H. 1998. Precise GPS/Acoustic
positioning of seafloor reference points for tectonic studies. Physics of the Earth & Planetary Interiors, 108 (2): 101-112. Ishikawa, T., Yokota, Y., Watanabe, S. I., Nakamura, Y. 2020. History of On-Board Equipment Improvement for GNSS-A
Observation With Focus on Observation Frequency. Frontiers in
Earth Science, 8: 150. Tomita, F., Kido, M., Honsho, C., Matsui, R. 2019. Development
of a kinematic GNSS-Acoustic positioning method based on a
state-space model. Earth, Planets and Space, 71 (1). Kido, M., Osada, Y., Fujimoto, H. 2008a. Temporal variation of
sound speed in ocean: a comparison between GPS/acoustic and
in situ measurements. Earth Planets & Space, 60 (3): 229-234. Tomita, F., Kido, M., Ohta, Y., Iinuma, T., Hino, R. 2017. Along-
trench variation in seafloor displacements after the 2011 Tohoku
earthquake. Science Advances, 3 (7): e1700113. Kido, Motoyuki, Fujimoto, Hiromi, Miura, Satoshi, Osada,
Yukihito, Tsuka, Kentaro, Tabei, Takao. 2006. Seafloor
displacement at Kumano-nada caused by the 2004 off Kii
Peninsula earthquakes, detected through repeated GPS/Acoustic
surveys. Earth, Planets and Space, 58 (7): 911-915. Watanabe, S. I., Ishikawa, T., Yokota, Y., Nakamura, Y. 2020. GARPOS: analysis software for the GNSS-A seafloor
positioning with simultaneous estimation of sound speed
structure. Frontiers in Earth Science, 8 (2296-6463). Xu, P., Ando, M., Tadokoro, K. 2005. Precise, three-dimensional
seafloor geodetic deformation measurements using difference
techniques. Earth, Planets and Space, 57 (9): 795-808. Kido, Motoyuki, Osada, Yukihito, Fujimoto, Hiromi. Yang, Y., Qin, X. 2021. Resilient observation models for seafloor
geodetic positioning. Journal of Geodesy, 95 (7): 1-13. REFERENCES 2008b. Temporal variation of sound speed in ocean: a comparison
between GPS/acoustic and in situ measurements. Earth, Planets
and Space, 60 (3): 229-234. Xue, S, Yang, Y. 2017. Understanding GDOP minimization in
GNSS positioning: Infinite solutions, finite solutions and no
solution. Advances in Space Research, 59 (3): 775-785. Kinugasa, Natsuki, Tadokoro, Keiichi, Kato, Teruyuki, Terada,
Yukihiro. 2020. Estimation of temporal and spatial variation of
sound speed in ocean from GNSS-A measurements for
observation using moored buoy. Progress in Earth and Planetary
Science, 7 (1). Yada, K., Ikuta, R., Ando, M., Okuda, T., Takatani, K. 2004. Spatial variations in Acoustic velocity at Kuroshio region for the
accurate ocean-bottom positioning. AGU Fall Meeting Abstracts. Liu, J., Chen, G., Zhao, J., Gao, K., Liu, Y. 2019. Development
and Trends of Marine Space-Time Frame Network. Wuhan
Daxue Xuebao (Xinxi Kexue Ban)/Geomatics and Information
Science of Wuhan University, 44 (1): 17-37. Yan, J. 1999. Effects of earth curvature on two-dimensional ray
tracing in underwater acoustics. Applied Acoustics, 57 (2): 163-
177. Yang, F., Lu, X., Li, J., Han, L., Zheng, Z. 2011. Precise
Positioning of Underwater Static Objects without Sound Speed
Profile. Marine Geodesy, 34 (2): 138-151. Lu, X., Bian, S., Huang, M., Zhai, G. 2012. An Improved Method
for Calculating Average Sound Speed in Constant Gradient
Sound Ray Tracing Technology. Geomatics and Information
Science of Wuhan University, 37 (5): 590-593. Yang, Y., Qin, X. 2021. Resilient observation models for seafloor
geodetic positioning. Journal of Geodesy, 95 (7): 1-13. Mandel, J. 1982. Use of the Singular Value Decomposition in
Regression Analysis. American Statistician, 36 (1): 15-24. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License. 285 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China Yasuda, K., Tadokoro, K., Taniguchi, S., Kimura, H., Matsuhiro,
K. 2017. Interplate locking condition derived from seafloor
geodetic observation in the shallowest subduction segment at the
Central Nankai Trough, Japan. Geophysical Research Letters, 44
(8): 3572-3579. Yokota, Y., Ishikawa, T. 2019. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLVI-3-W1-2022-279-2022 | © Author(s) 2022. CC BY 4.0 License. Zielinski, Xueyi Geng Adam. 1999. Precise Multibeam Acoustic
Bathymetry. Marine Geodesy, 22 (3): 157-167. The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-3/W1-2022
7th Intl. Conference on Ubiquitous Positioning, Indoor Navigation and Location-Based Services (UPINLBS 2022), 18–19 March 2022, Wuhan, China REFERENCES Gradient field of undersea sound
speed structure extracted from the GNSS-A oceanography:
GNSS-A as a sensor for detecting sound speed gradient. SN
Applied Sciences, 1 (7). Yokota, Y., Ishikawa, T., Watanabe, S. I. 2018a. Gradient field
of undersea sound speed structure extracted from the GNSS-A
oceanography. Marine Geophysical Researches. Yokota, Yusuke, Ishikawa, Tadashi, Watanabe, Shun-Ichi. 2018b. Seafloor crustal deformation data along the subduction
zones around Japan obtained by GNSS-A observations. Scientific
Data, 5 (1): 180-182. Zhao, J., Zou, Y., Zhang, H., Wu, Y., Fang, S. 2016. A new
method for absolute datum transfer in seafloor control network
measurement. Journal of Marine Science & Technology, 21 (2):
216-226. Zielinski, Xueyi Geng Adam. 1999. Precise Multibeam Acoustic
Bathymetry. Marine Geodesy, 22 (3): 157-167. 286
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https://openalex.org/W2137937973
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https://acuresearchbank.acu.edu.au/download/df08d5816275cc8d1eb4ff2a1da4aac666e8256d979856b552a246adbd96c6bd/501463/OA_Storhaug_2013_Uric_acid_is_a_risk_factor.pdf
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English
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Uric acid is a risk factor for ischemic stroke and all-cause mortality in the general population: a gender specific analysis from The Tromsø Study
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BMC cardiovascular disorders
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cc-by
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Abstract Background: The role of serum uric acid as an independent predictor of cardiovascular disease and death is
uncertain in the general population. Adjustments for additional cardiovascular risk factors have not been consistent. We examined the association of serum uric acid with all-cause mortality, ischemic stroke and myocardial infarction
in a prospective population based study, with several traditional and non-traditional risk factors for cardiovascular
disease included in the model. Methods: A population-based prospective cohort study was performed among 2696 men and 3004 women. Endpoints were all-cause mortality after 15 years, and fatal or non-fatal myocardial infarction (MI) and ischemic
stroke after 12 years. Results: 1433 deaths, 659 MIs and 430 ischemic strokes occurred during follow-up. Fully adjusted Cox regression
analyses showed that per 1 SD (87 μmol/L) increase in serum uric acid level, the risk of all-cause mortality increased
in both genders (hazard ratios, HR men; 1.11, 95% CI 1.02-1.20, women; 1.16, 1.05-1.29). HRs and 95% CI for stroke
were 1.31, 1.14-1.50 in men, 1.13, 0.94-1.36 in women, and 1.22 (1.09, 1.35) in the overall population. No independent
associations were observed with MI. Results: 1433 deaths, 659 MIs and 430 ischemic strokes occurred during follow-up. Fully adjusted Cox regression
analyses showed that per 1 SD (87 μmol/L) increase in serum uric acid level, the risk of all-cause mortality increased
in both genders (hazard ratios, HR men; 1.11, 95% CI 1.02-1.20, women; 1.16, 1.05-1.29). HRs and 95% CI for stroke
were 1.31, 1.14-1.50 in men, 1.13, 0.94-1.36 in women, and 1.22 (1.09, 1.35) in the overall population. No independent
associations were observed with MI. Conclusion: Serum uric acid was associated with all-cause mortality in men and women, even after adjustment for
blood pressure, estimated GFR, urinary albumin/creatinine ratio, drug intake and traditional cardiovascular risk factors. After the same adjustments, serum uric acid was associated with 31% increased risk of stroke in men. Keywords: All-cause mortality, Gender, Myocardial infarction, Stroke, Uric acid Keywords: All-cause mortality, Gender, Myocardial infarction, Stroke, Uric acid to the 1970s, from less than 210 μmol/L to 360-390 μmol
[1]. Pre-menopausal women tend to have lower levels than
men, probably because of the uricosuric effect of estrogens
[2]. The relationship between serum uric acid and cardio-
vascular disease is not clear. Some epidemiologic studies
have reported a relationship between serum uric acid and
several cardiovascular conditions [3-10], whereas others
have not observed such links [11-15]. Storhaug et al. BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Storhaug et al. BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Open Access © 2013 Storhaug et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. * Correspondence: Ingrid.Toft@unn.no
2Department of Clinical Medicine, UiT The Arctic University of Norway,
Tromsø, Norway
3Section of Nephrology, University Hospital of North Norway, N-9038,
Tromsø, Norway
Full list of author information is available at the end of the article Uric acid is a risk factor for ischemic stroke and
all-cause mortality in the general population: a
gender specific analysis from The Tromsø Study Hilde M Storhaug1,2, Jon V Norvik3, Ingrid Toft2,3*, Bjorn O Eriksen2,3, Maja-Lisa Løchen4, Svetlana Zykova2,
Marit Solbu3, Sarah White5, Steve Chadban6 and Trond Jenssen2,7 © 2013 Storhaug et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Abstract The National Health
and Nutrition Examination Survey (NHANES I study) [3]
reported a significant risk for cardiovascular death with in-
creasing serum uric acid levels. The Framingham Heart
study, on the other hand, was not able to confirm these
findings when use of diuretics was adjusted for [11]. Two
large cohort studies from Japan and Korea, each with 9-10
years follow up could not confirm that serum uric acid was Outcomes The endpoints were death from any cause, first ever non-
fatal or fatal myocardial infarction or ischemic stroke. Ad-
judication of hospitalized and out-of-hospital events was
performed by an independent endpoint committee, who
thoroughly reviewed data from hospital and out-of-hospital
journals, autopsy records and death certificates. Event
ascertainment followed a detailed protocol according to
established diagnostic criteria. Each case was reviewed
separately. Stroke was defined according to the WHO
definition, only ischemic strokes were included [18]. Individuals who had died, moved, or emigrated from
Tromsø were identified through the population Registry
of Statistics Norway. The national 11-digit identification
number allowed a linkage to the population Registry of
Statistics Norway and ensured a complete follow-up status
for all-cause mortality until November 30th, 2010 (follow-
up time 15 years). The cardiovascular (CV) endpoint regis-
try was completed until December 31, 2007 (follow-up
time 12 years). Data were censored for date of registered
emigration, or deaths from causes other than myocardial
infarction and stroke. Study population The Tromsø Study is a series of population-based, pro-
spective surveys of inhabitants of the municipality of
Tromsø, Norway [16]. In 1994/95, 27.158 subjects were
screened (77% of eligible subjects). All participants at
the age of 55-75 years and 5-10% of the other age
groups ≥25 years were invited to a second visit including
a more comprehensive examination 4-6 weeks later. Of
the 9057 individuals who were invited 6862 participated
(attendance rate 75%). Persons with known previous MI
(n = 402), ischemic stroke (n = 190) or diabetes (n = 308),
defined as self-reported diabetes, use of antidiabetic medi-
cation, HbA1c > 6.5% or non-fasting plasma glucose ≥10.0
mmol/L, were excluded. Data on uric acid was available
in 5700 subjects (Figure 1). The Tromsø Study was
conducted by the University of Tromsø in cooperation
with The National Health Screening Service. The Regional
Committee for Medical Research Ethics approved the
study, and all participants gave their written consent. Measurements All measurements and information on risk factors were
obtained from baseline data of the 4th Tromsø Study in
1994 /95. Information about presence of diabetes, smoking
habits and physical activity was obtained from a self-
administered questionnaire. Blood pressure was recorded
in triplet (Dinamap) after 5-min seating, the mean of the
second and third measurement was used. Hypertension
was defined as systolic blood pressure (SBP) ≥140 mmHg
and/or diastolic pressure (DBP) ≥90 mmHg and/or current
use of antihypertensive medication. Physical activity was
classified as active (> 1 hour physical activity/week) or in-
active (all others). Smoking habits were classified as non-
smokers or current smokers. Serum HDL-cholesterol was
measured after precipitation of lower-density lipoprotein
with heparin and manganese chloride. Serum uric acid
was measured by photometry with COBAS® instruments
(Roche diagnostics, Switzerland) using an enzymatic col-
orimetric test, the uricase/ PAP method. Reference values Storhaug et al. BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Page 2 of 10 were 140-340 μmol/L (2.4-5.7 mg/100 mL) for females
and 200-415 μmol/L (3.4-7.0 mg/100 mL) for males. Cre-
atinine was analyzed by a modified Jaffe reaction, but since
creatinine-based estimation of GFR is better validated
for enzymatic creatinine measurements, 111 plasma
samples from the 1994/95 survey were thawed and rea-
nalysed with an enzymatic method (Modular P/Roche). Values were fitted to a linear regression model, and
recalibrated creatinine values were calculated for all
participants. Estimated GFR was calculated according
to the CKD-EPI formula [17]: eGFR = 141 × min(SCr/k,1)a ×
max(SCr/k,1)-1.209 × 0.993age × ([1.018 if female] and ×
[1.159 if black]) where SCr is serum creatinine (mg/dL),
k is 0.7 for females and -0.411 for males, min indicates
the minimum of SCr/k and max indicates the maxiumum
of SCr/k). Albuminuria was reported as albumin/creatinine
ratio (ACR, mg/mmol, urinary albumin and creatinine
analyses; kits from ABX Diagnostics, Montpellier, France). For each subject, ACR was measured in fresh samples of
each of 3 separate urine specimens and the mean of all 3
was used in the analyses. a risk factor for either cardiovascular disease or death
[12,13]. It has been argued that previous studies may not
have sufficiently accounted for differences in gender or for
risk factors being strongly related to serum uric acid levels,
e.g., use of diuretics or renal factors, such as glomerular fil-
tration rate (GFR) and renal dysfunction measured as urin-
ary albumin excretion. The purpose of the present study was to address uric
acid as putative cardiovascular risk factor in a Caucasian
population followed for more than 12 years. In order to
do so, we chose a stepwise approach with models in-
cluding various covariates that have and have not been
included in previous studies. Background Uric acid is the breakdown product of purines from DNA,
RNA, ATP and cAMP. In this process hypoxanthine is con-
verted by the enzyme xanthine oxidase to xanthine and fur-
ther to uric acid. Both steps induce the release of free
radicals. Uric acid may accumulate in the body due to in-
creased production (cell death, intake of alcohol or purine
rich diet) or decreased elimination (impaired renal function,
use of diuretics). Epidemiologic studies show that mean
uric acid levels in men increased gradually from the 1920s * Correspondence: Ingrid.Toft@unn.no
2Department of Clinical Medicine, UiT The Arctic University of Norway,
Tromsø, Norway
3Section of Nephrology, University Hospital of North Norway, N-9038,
Tromsø, Norway
Full list of author information is available at the end of the article Storhaug et al. BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Storhaug et al. BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Statistics Data are given as mean ± SD or median and interquar-
tile range. Uric acid was categorized into gender-specific
tertiles. Crude and age-adjusted incidence rates were cal-
culated as events per 1000 person years at risk. Age-
adjustment of incidence rates was performed on 10 year
age groups with the population of Tromsø in 1995 as the
standard population. Multiple linear regression analyses
were performed with uric acid as the dependent variable. Storhaug et al. BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Page 3 of 10 The Tromsø Study 1994/-95, Phase 2:
All participants aged 55-75 years and 5-10 %
of all other age groups 25 years invited
9057 invited, 6862 consented and attended
(75% of the invited)
The Tromsø Study 1994/-95, Phase 1:
All inhabitants of the municipality of
Tromsø > 25 years were invited
27.158 subjects attended
(77 % of the invited)
5700 individuals; 2696 men, 3004 women
Number of endpoints:
659 myocardial infarctions, 430 ischemic
strokes, and 1433 deaths from all causes
506 individuals excluded
due to previous myocardial
infarction (402) or stroke (190)
or both (86)
308 individuals excluded
due to the presence of diabetes*
348 individuals had missing
values for serum uric acid
Figure 1 Selection of the study population. * Self-reported diabetes mellitus and/or use of antidiabetic medication and/or HbA1c > 6.5%
and/or non-fasting glucose ≥10.0 mmol/L. The Tromsø Study 1994/-95, Phase 1:
All inhabitants of the municipality of
Tromsø > 25 years were invited Figure 1 Selection of the study population. * Self-reported diabetes mellitus and/or use of antidiabetic medication and/or HbA1c > 6.5%
and/or non-fasting glucose ≥10.0 mmol/L. Covariates were systolic blood pressure, body mass index
(BMI), high density lipoprotein cholesterol (HDL), total
cholesterol, smoking status and physical activity. Renal co-
variates (estimated GFR and ACR) and use of diuretics or
antihypertensive drugs were also added to the models in
the multivariable analyses. ACR was logarithmically trans-
formed. Cox proportional hazard models were used to in-
vestigate associations of uric acid with cardiovascular
outcomes and mortality, calculated per 1 SD (87 μmol/L)
change in uric acid, in unadjusted, age-adjusted and multi-
variable analyses. The proportional hazard assumption was
checked by visual inspection of the -log-log survival
curves. Tests for interactions and non-linearity (by quad-
ratic terms) were assessed in separate models. Non-linear effects were also explored in fractional polynomial regres-
sion models. P values < 0.05 were considered statistically
significant. Statistics Most analyses were run using SPSS software
version 15.0 (SPSS, INC, Chicago, Illinois), fractional poly-
nomial regression models were performed with STATA/
MP 12.1 (Stata Corp LP, College Station, Texas). Baseline characteristics Mean serum uric acid was 357 ± 84 μmol/L for men and
276 ± 70 μmol/L for women. Figure 2 shows serum uric
acid concentrations according to gender and age. In both
genders, increasing uric acid was associated with a poorer
risk profile in terms of elevated BMI, blood pressure, Storhaug et al. BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Page 4 of 10 Age Groups, Years
Serum Uric Acid, Women
Serum Uric Acid, Men
Serum
Uric Acid,
µmol/l
150
250
350
450
550
25 - 29
30 - 39
40 - 49
50 - 59
60 - 69
70
Figure 2 Serum uric acid levels by gender and age groups. Mean (SD). Serum Uric Acid, Women
Serum Uric Acid, Men Figure 2 Serum uric acid levels by gender and age groups. Mean (SD). tertiles of serum uric acid, are shown in Figure 3. Age-
adjusted incidence rate for myocardial infarction was sig-
nificantly higher in the upper serum uric acid tertile
among men, but not in women. Increasing crude and age-
adjusted incidence rates with higher levels of uric acid
were observed in both genders for ischemic stroke. ACR, and proportion of hypertensive persons. Physical ac-
tivity, renal function and HDL were lower with increasing
uric acid concentrations. Only a few subjects were using
diuretics and anti-hypertensive medication at baseline in
1994/95 (Table 1). Associations between serum uric acid and outcome
variables (Cox proportional hazard models) The correlation between uric acid and age differed in men
and women (men; r = -0.09, p < 0.001, women; r = 0.17, p <
0.001, Pearson’s correlation coefficients), and tested signifi-
cant for gender interaction (P < 0.001). Results of the mul-
tiple linear regression analyses are given in Table 2. In both
genders, standardized beta-coefficients were highest for
BMI, HDL-cholesterol, total cholesterol, and GFR. The
addition of renal factors (eGFR and ACR; model 5) into
the model including drug intake and traditional cardiovas-
cular risk factors, contributed significantly to variation of
serum uric acid; adjusted R2 increased from 0.19 to 0.23 in
men, from 0.20 to 0.27 in women, and from 0.20 to 0.40
in pooled analyses for men and women together. ACR was
independently associated with serum uric acid in women,
but not in men, and there was a significant interaction with
gender (P = 0.005). In both genders, P-values for linear, increasing trend
were <0.001 for all endpoints in multivariable, Cox pro-
portional hazard models where the lowest serum uric
acid tertile was reference (data not shown). In multivari-
able analyses calculated per 1 SD (87 μmol/L) increase
in uric acid (Table 3), uric acid was independently asso-
ciated with all-cause mortality in men (HR, 95% CI;
1.11, 95% CI 1.02-1.20), women (HR, 95% CI; 1.16, 1.05-
1.29), and in the total population (men and women; HR,
95% CI: 1.13, 1.06-1.21). In fully adjusted, multivariable
models, serum uric acid was not independently associated
with MI in any gender or in the mixed population. In both
genders, uric acid lost significance when lipids were intro-
duced as covariates. Serum uric acid was strongly associ-
ated with ischemic stroke in men after multivariable
adjustments (HR, 95% CI: 1.31, 1.14-1.50). In contrast, sig-
nificance was lost among women after adjustments for
blood pressure and BMI. Test for gender interaction was
not significant (P = 0.19). In multivariable analysis of the
mixed population, adjusted for sex, 87 μmol/L increase in
uric acid was associated with a 24% increase in risk of fu-
ture ischemic stroke (Table 3). Tests for non-linearity in
fractional polynomial models for associations of serum
uric acid with the three endpoints were not significant in Event rates Number of events during follow-up were 659 first ever
cases of fatal or non-fatal MI, 430 fatal or non-fatal is-
chemic strokes, and 1433 deaths from all causes. Median
observation time was 12.5 years for myocardial infarction
and ischemic stroke, and 15.7 years for all-cause mortality. Crude and age-adjusted incidence, for MI and ischemic
stroke, picturing absolute risk rates according to increasing Storhaug et al. Event rates BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Page 5 of 10 Page 5 of 10 Table 1 Baseline characteristics according to gender and serum uric acid tertiles Table 1 Baseline characteristics according to gender and serum uric acid tertiles Table 1 Baseline characteristics according to gender and serum uric acid tertiles
Men
SUA tertiles
I ≤317μmol/L
II 318-380 μmol/L
III 381-976 μmol/L
P value for linear trend
N
896
902
898
Age, years
60 ± 10
59 ± 10
59 ± 11
<0.001
Serum uric acid (SUA), μmol/L
276 ±33
348 ± 18
449 ± 70
-
BMI, kg/m2
24.7 ± 2.9
25.8 ± 3.0
27.3 ± 3.4
<0.001
SBP, mm Hg
144 ± 20
144 ± 20
146 ± 20
<0.001
DBP, mm Hg
84 ± 12
85 ± 12
86 ± 12
<0.001
Hypertension, n (%)
482 (54%)
546 (61%)
571 (64%)
<0.001
Total cholesterol, mmol/l
6.35 ± 1.19
6.40 ± 1.20
6.73 ± 1.20
<0.001
HDL cholesterol, mmol/l
1.51 ± 0.40
1.40 ± 0.39
1.26 ± 0.38
<0.001
GFR ckd-epi, ml/min/1.73m2
96.7 (90.9, 102.4)
95.5 (88.0, 102.5)
93.4 (84.0, 101.5)
<0.001
ACR mg/mmol
0.55 (0.38, 0.96)
0.51 (0.33, 1.01)
0.54 (0.34, 1.13)
0.021
Use of antihypertensive drugs, n (%)
74 (8%)
87 (10%)
155 (17%)
<0.001
Use of diuretics , n (%)
2 (0.2%)
5 (0.6%)
13 (1.4%)
<0.001
Current smoker, n (%)
351 (39%)
300 (33%)
294 (33%)
<0.001
Physically active, n (%)
301 (33%)
268 (30%)
244 (27%)
<0.001
Women
SUA tertiles
I ≤244 μmol/L
II 245-297 μmol/L
III 298-681 μmol/L
P value for linear trend
N
1002
1001
1001
Age, years
58 ± 11
60 ± 10
62 ± 9
<0.001
Serum uric acid (SUA), μmol/L
206 ± 28
269 ± 15
354 ± 54
-
BMI, kg/m2
24.0 ± 3.5
25.6 ± 3.8
27.7 ± 4,6
<0.001
SBP, mm Hg
138 ± 23
143 ± 23
151 ± 25
<0.001
DBP, mm Hg
78 ± 13
81 ± 12
85 ± 14
<0.001
Hypertension, n (%)
446 (45%)
534 (53%)
679 (68%)
<0.001
Total cholesterol, mmol/l
6.63 ± 1.39
6.82 ± 1.29
7.14 ± 1.32
<0.001
HDL cholesterol, mmol/l
1.76 ± 0.43
1.69 ± 0.43
1.54 ± 0.42
<0.001
GFR ckd-epi, ml/min/1.73m2
96.9 (90.5,103.0)
93.6 (86.0, 99.4)
89.7 (79.4, 97.1)
<0.001
ACR, mg/mmol
0.62 (0.41, 0.96)
0.58 (0.41, 0.93)
0.64 (0.42, 1.14)
<0.001
Use of diuretics, n (%)
3 (0.3%)
7 (0.7%)
39 (3.9%)
<0.001
Use of antihypertensive drugs, n (%)
65 (6.5%)
107 (11%)
148 (15%)
<0.001
Current smoker, n (%)
339 (34%)
315 (32%)
278 (28%)
0.001
Physically active, n (%)
162 (16%)
177 (18%)
118 (12%)
<0.002
The Tromsø Study 1994/95. Event rates BMI: Body mass index. SBP: Systolic blood pressure. DBP: Diastolic blood pressure Hypertension: SBP ≥140 mmHg and/or DBP ≥90 mmHg and/or self-reported use
of antihypertensive medication. Physical active: Self-reported hard physical activity ≥1 hr per week (yes / no). GFR: Glomerular Filtration Rate according to the CKD EPI formula [17]. ACR: Urinary albumin/creatinine ratio (mean of 3 urine samples). The Tromsø Study 1994/95. BMI: Body mass index. SBP: Systolic blood pressure. DBP: Diastolic blood pressure Hypertension: SBP ≥140 mmHg and/or DBP ≥90 mmHg and/or self-reported use
of antihypertensive medication. Physical active: Self-reported hard physical activity ≥1 hr per week (yes / no). GFR: Glomerular Filtration Rate according to the CKD EPI formula [17]. ACR: Urinary albumin/creatinine ratio (mean of 3 urine samples). higher uric acid levels was not significantly associated with
myocardial infarction in men (P = 0.26) or women (P =
0.27) after multivariable adjustments. any gender. Cox regression analyses were also performed
without exclusion of persons with diabetes and previous
cardiovascular disease. Risk estimates for uric acid and
myocardial infarction in fully adjusted analyses did not
change substantially (HR and 95% CI for men were 1.06,
0.99-1.14, p = 0.06 and for women 1.04, 0.92-1.14, p = 0.5). W
l
t tifi d th
t d
l ti
di
t any gender. Cox regression analyses were also performed
without exclusion of persons with diabetes and previous
cardiovascular disease. Risk estimates for uric acid and
myocardial infarction in fully adjusted analyses did not
change substantially (HR and 95% CI for men were 1.06,
0.99-1.14, p = 0.06 and for women 1.04, 0.92-1.14, p = 0.5). We also stratified the study population according to serum
uric acid levels higher or lower than the hospital labora-
tory’s upper reference range. Belonging to the group with Discussion In this 12-15 year prospective study of 5700 men and
women from the general population where persons with
known diabetes or cardiovascular disease were excluded,
a 1 SD (87 μmol/L) increase in serum uric acid was We also stratified the study population according to serum
uric acid levels higher or lower than the hospital labora-
tory’s upper reference range. Belonging to the group with Table 2 Multiple regression analysis with cardiovascular and renal covariants, and serum uric acid as dependent variable
Men n = 2696
Women n = 3004
Men and women n = 5700
β (95% CI*)
Std. β-coeffcent.ϯ
P-value
β (95% CI*)
Std. β-coeffcent.ϯ
P-value
β (95% CI*)
Std. β-coeffcent.ϯ
P-value
Model 1
Intercept
175 (143, 208)
-
<0.001
71 (52, 90)
-
<0.001
128 (108, 148)
-
<0.001
Age
−0.70 (-0.10, -0.39)
−0.09
<0.001
0.55 (0.30, 0.80)
0.08
<0.001
−0.39 (-0.61, -0.17)
−0.05
0.001
BMI
8.29 (7.38, 9.21)
0.33
<0.001
5.31 (4.73, 5.88)
0.32
<0.001
6.79 (6.21, 7.37)
0.30
<0.001
SBP
0.06 (-0.10, 0.21)
0.01
0.49
0.24 (0.13, 0.35)
0.08
< 0.001
0.24 (0.13, 0.35)
0.06
<0.001
Adjusted R2
0.12
0.15
0.10
Model 2
HDL-cholesterol
−44.42 (-52.09, -36.76)
−0.22
<0.001
-27.68 (-33.12,-22.23)
−0.17
<0.001
−61.58 (-66.42, -56.74)
−0.32
<0.001
Cholesterol
10.12 (7.70, 12.54)
0.15
<0.001
6.38 (4.50, 8.26)
0.12
<0.001
6.33 (4.67, 7.99)
0.10
<0.001
Adjusted R2
0.17
0.18
0.19
Model 3
Antihypertensive
Drugs
26.10 (16.76, 35.44)
0.10
<0.001
14.19 (6.55, 21.83)
0.06
0.001
20.37 (13.66, 27.08)
0.07
<0.001
Diuretics
66.90 (33.13, 99.98)
0.07
<0.001
69.14 (51.01, 87.28)
0.13
<0.001
54.52 (35.75, 73.29)
0.07
<0.001
Adjusted R2
0.18
0.20
0.20
Model 4
Current smoker
−7.73 (-13.81, -1.63)
−0.04
0.01
2.07 (-3.02, 7.16)
0.01
0.4
−0.80 (-5.23, 3.62)
−0.004
0.7
Physical activity
−7.29 (-13.56, -1.02)
−0.04
0.02
1.17 (-5.27, 7.61)
0.01
0.7
11.28 (6.35, 16.20)
0.05
<0.001
Adjusted R2
0.19
0.20
0.20
Model 5
GFR ckd-epi
−1.76 (-2.05, -1.48)
−0.28
<0.001
−1.73 (-1.94, -1.51)
−0.32
<0.001
−1.74 (-1.91,-1.56)
−0.27
<0.001
logACR
4.09 (-2.58, 10.76)
0.02
0.23
15.41 (9.28, 21.55)
0.08
< 0.001
8.09 (3.58, 12.61)
0.04
<0.001
Gender
- -
-
-
- -
-
-
76.87 (73.08, 80.66)
0.44
<0.001
Adjusted R2
0.23
0.27
0.40
*Confidence interval. ϯStandarized β-coeffcient. Gender: 1 = male, 0 = female, GFR: Glomerular filtration rate according to the CKD EPI formula [17]. ACR: urinary albumin/creatinine ratio (mean of 3 urine samples) logACR: logarithmic transformation of ACR. Model 1: Age, body mass index. mic transformation of ACR. Model 1: Age, body mass index.
al cholesterol. Model 3: Model 2 + use of diuretics and other anti-hypertensive medication. Model 4: Model 3 + current smoking and
o = 0), current smoking (yes = 1 / no = 0). Model 5: Model 4 + renal factors added (GFR the CKD-EPI and logACR). *Confidence interval. ϯStandarized β-coeffcient. Gender: 1 = male, 0 = female, GFR: Glomerular filtration rate according to the CKD EPI formula [17].
ACR: urinary albumin/creatinine ratio (mean of 3 urine samples) logACR: logarithmic transformation of ACR. Model 1: Age, body mass index.
(BMI), systolic blood pressure (SBP). Model 2: Model 1 + HDL-cholesterol and total cholesterol. Model 3: Model 2 + use of diuretics and other anti-hypertensive medication. Model 4: Model 3 + current smoking and
physical activity (self-reported hard physical activity ≥1 hr per week; yes = 1 / no = 0), current smoking (yes = 1 / no = 0). Model 5: Model 4 + renal factors added (GFR the CKD-EPI and logACR). ϯStandarized β-coeffcient. Gender: 1 = male, 0 = female, GFR: Glomerular filtration rate according to the CKD EPI formula [17].
creatinine ratio (mean of 3 urine samples) logACR: logarithmic transformation of ACR Model 1: Age body mass index Discussion (BMI), systolic blood pressure (SBP). Model 2: Model 1 + HDL-cholesterol and total cholesterol. Model 3: Model 2 + use of diuretics and other anti-hypertensive medication. Model 4: Model 3 + current smoking and
physical activity (self-reported hard physical activity ≥1 hr per week; yes = 1 / no = 0), current smoking (yes = 1 / no = 0). Model 5: Model 4 + renal factors added (GFR the CKD-EPI and logACR). Table 2 Multiple regression analysis with cardiovascular and renal covariants, and serum uric acid as dependent variable Table 2 Multiple regression analysis with cardiovascular and renal covariants, and serum uric acid as dependent variable
Men n = 2696
Women n = 3004
Men and women n = 5700 n analysis with cardiovascular and renal covariants, and serum uric acid as dependent variable Table 2 Multiple regression analysis with cardiovascular and renal covariants, and serum uric acid Storhaug et al. BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Storhaug et al. BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Page 7 of 10 Page 7 of 10 First ever fatal or non-fatal
myocardial infarction
5
10
15
20
25
‡
‡
‡
‡
Serum Uric Acid
First ever fatal or non-fatal
ischemic stroke
Serum Uric Acid
5
10
15
20
25
‡
†
†
†
Age-adjusted incident rates
Crude incident rates
Incidence Rates (per 1000 person-years at risk)
MEN
MEN
MEN
WOMEN WOMEN WOMEN
Tertile I
Tertile II
Tertile III
Tertile I
Tertile II
Tertile III
317
318-380
381
244
245-297
298
µmol/L
µmol/L
µmol/L
µmol/L
µmol/L
µmol/L
MEN
MEN
MEN
WOMEN
WOMEN
WOMEN
Tertile I
Tertile II
Tertile III
Tertile I
Tertile II
Tertile III
317
318-380
381
244
245-297
298
µmol/L
µmol/L
µmol/L
µmol/L
µmol/L
µmol/L
Figure 3 Crude and age-adjusted incidence rates by serum uric acid tertiles. † : P < 0.05. ‡ : P < 0.001 (differences between tertiles were
tested for using a normal test with continuity correction, tertile 1 was reference). BMI were included as covariates. However, no gender inter-
action was observed. Differences in risk estimates for stroke
between genders, may relate to gender-specific differences
in vascular biology such as adaption to pro-inflammatory
stress. Vlachopoulos et al. [25] reported that in newly diag-
nosed hypertensive persons, uric acid was associated with
increased aortic stiffness in both genders, however a nega-
tive association with arterial wave reflection was observed
only in women. Discussion Such differences like this in vascular func-
tion could influence the tendency of developing stroke. The
Framingham study [11] also reported lack of independent
association of uric acid with stroke, but this is in contrast
to the gender specific analyses of the AMORIS-study [5]. However, BMI and use of antihypertensive medication was
not accounted for in the AMORIS study [5]. significantly associated with 31% increased risk for ische-
mic stroke in men, and all-cause mortality risk was in-
creased in both genders, with 11% in men, and 16% in
women, after multivariable adjustments. Gender-adjusted,
multivariable analyses of pooled data from both men and
women showed, for each 1 SD increase in serum uric acid,
a 22% increased risk for ischemic stroke, and 13% in-
creased risk for all-cause mortality. The association between serum uric acid and ischemic
stroke is in accordance with previous studies [5,19,20]. It
has been suggested that serum uric acid may have harm-
ful effects on platelet function [21] and cause endothelial
dysfunction [22]. Vannorsdall et al. [23] reported that even
a mild elevation of serum uric acid was associated with
cerebral ischemia among community-dwelling adults. It
was suggested that impaired vascular tone and endothelial
dysfunction could contribute to ischemic changes, because
they permit cerebrospinal fluid to cross the blood-brain
barrier and cause areas of edema [23]. We observed a significant association of serum uric
acid with all-cause mortality, with a modest increase in
mortality risk in both genders. In the Framingham study
[11], no association was observed with all-cause mortal-
ity among the genders after adjustments for age, blood
pressure, smoking, BMI, total cholesterol, intake of alcohol
and medication. On the other hand, the NHANES study
[3] reported a 13% increased mortality risk in women in
fully adjusted analyses, but only non-significant associations
in men. The fact that serum uric acid levels in women tend
to increase during the fifth to the seventh decade due to
postmenopausal reduction in uric acid excretion [2,26],
whereas a flat (Figure 2) or slightly declining curve with
ageing is seen in men [11], may influence the association
with endpoints among the genders. Quite opposite to this, it has been claimed that treat-
ment with uric acid in combination with thrombolysis
would be of benefit to patients suffering from acute
stroke [24]. Discussion Uric acid is one of the most important en-
dogenous antioxidants in the human brain, and high circu-
lating uric acid could play a role against the deleterious
effect of free radicals produced upstream in the synthesis of
uric acid [24]. Accordingly, a J-curve relationship has been
observed between serum uric acid and occurrence of ische-
mic stroke [19]. Our study did, however, not reveal any
non-linear associations between serum uric acid and any of
the endpoints. In women, serum uric acid lost significance
as predictor for ischemic stroke when blood pressure and No independent association between increment in uric
acid and MI was observed in the present study. However, Storhaug et al. Discussion BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Page 8 of 10 Page 8 of 10 Table 3 Associations of serum uric acid with adverse cardiovascular events and all-cause mortality
Hazard ratio (95% CI) per 1 SD increase in serum uric acid (87 μmol/L)
Men (n = 2696)
P-value
Women (n = 3004)
P-value
Men and women ( n = 5700)
P-value
All-cause mortality
Unadjusted
1.01 (0.94, 1.09)
0.6
1.24 (1.14, 1.35)
<0.001
1.10 (1.04, 1.17)
<0.001
Age-adjusted
1.11 (1.03, 1.19)
0.005
1.12 (1.03, 1.22)
0.008
1.11 (1,05, 1.18)
<0.001
Multivariable adjustment
Model 1
1.13 (1.04, 1.22)
0.003
1.22 (1.12, 1.33)
<0.001
1.19 (1.14, 1.25)
<0.001
Model 2
1.13 (1.05, 1.23)
0.003
1.20 (1.10, 1.31)
0.001
1.17 (1.01, 1.24)
<0.001
Model 3
1.13 (1.04, 1.22)
0.005
1.20 (1.09, 1.32)
<0.001
1.16 (1,09, 1.23)
<0.001
Model 4
1.12 (1.04, 1.22)
0.005
1.20 (1.09, 1.31)
<0.001
1.16 (1.09, 1.23)
<0.001
Model 5
1.11 (1.02, 1.20)
0.02
1.16 (1.05, 1.29)
0.004
1.13 (1.06, 1.21)
<0.001
Myocardial infarction
Unadjusted
1.15 (1.05, 1.26)
0.003
1.38 (1.20, 1.59)
<0.001
1.21 (1.12, 1.31)
<0.001
Age-adjusted
1.22 (1.11, 1.34)
<0.001
1.21 (1.06, 1.40)
0.006
1.23 (1.14, 1.33)
<0.001
Multivariable adjustment
Model 1
1.16 (1.05, 1.28)
0.004
1.16 (1.01, 1.35)
0.049
1.16 (1.08, 1.27)
<0.001
Model 2
1.07 (0.95, 1.19)
0.25
1.09 (0.93, 1.27)
0.26
1.09 (0.99, 1.18)
0.09
Model 3
1.05 (0.94, 1.17)
0.3
1.10 (0.94, 1.29)
0.22
1.09 (0.10, 1.18)
0.06
Model 4
1.05 (0.95, 1.17)
0.3
1.09 (0.93, 1.27)
0.3
1.08 (0.99, 1.18)
0.09
Model 5
1.05 (0.94, 1.17)
0.4
1.06 (0.90, 1.25)
0.5
1.06 (0.97, 1.16)
0.19
Ischemic stroke
Unadjusted
1.13 (1.01, 1.28)
0.049
1.37 (1.17, 1.61)
<0.001
1.21 (1.10, 1.33)
<0.001
Age-adjusted
1.23 (1.08, 1.40)
0.001
1.20 (1.03, 1.40)
0.02
1.22 (1.11, 1.35)
<0.001
Multivariable adjustment
Model 1
1.25 (1.10, 1.42)
0.001
1.13 (0.95, 1.33)
0.17
1.19 (1.07, 1.31)
0.001
Model 2
1.29 (1.13, 1.47)
<0.001
1.13 (0.95, 1.34)
0.17
1.22 (1.09, 1.35)
<0.001
Model 3
1.28 (1.11, 1.46)
<0.001
1.13 (0.95, 1.35)
0.18
1.21 (1.09, 1.35)
<0.001
Model 4
1.28 (1.12, 1.47)
<0.001
1.12 (0.94, 1.34)
0.20
1.22 (1.09, 1.35)
<0.001
Model 5
1.31 (1.14, 1.50)
<0.001
1.13 (0.94, 1.36)
0.21
1.24 (1.11, 1.38)
<0.001
The Tromsø Study. Discussion Model 1 Age body mass index (BMI) systolic blood pressure (SBP) and diastolic blood pressure (DBP) f serum uric acid with adverse cardiovascular events and all-cause mortality
Hazard ratio (95% CI) per 1 SD increase in serum uric acid (87 μmol/L verse cardiovascular events and all-cause mortalit significance did not depend on renal function or ACR as
expected, but on lipids. Total- and HDL-cholesterol abol-
ished the effect of uric acid on future MI. This is in con-
sistence with a recent study [27] on the predictive value
of uric acid on cardiovascular mortality among persons
with type 2 diabetes in the general population. The in-
clusion of HDL-cholesterol, LDL-cholesterol, triglycerides,
BMI, HbA1c and blood pressure as covariates abolished the
significance of a 40% risk increment found in subjects with
uric acid levels > 375 μmol/L in sex and age-adjusted ana-
lyses [27]. Subgroups with known diabetes or cardiovascular disease were excluded in the present study, as we aimed to
examine the role of uric acid in a low-risk population. Suba-
nalyses where persons with diabetes or preexisting cardio-
vascular disease were included, did not change our results. Neither did stratification according to uric acid values
above or less than the upper reference range. This is in con-
trast to the NHANES study [3], where an increased risk of
ischemic heart disease mortality was observed. Hazard ra-
tios for each 59 μmol/l increase in uric acid level were re-
ported to be 1.17 (95% CI 1.06-1.28) in men and 1.30 (95%
CI 1.17-1.45) in women [3]. However, in that study, BMI Storhaug et al. BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Storhaug et al. BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Page 9 of 10 risk factors and the use of antihypertensive medication
and diuretics. was higher than in the present study, and covariates such as
HDL-cholesterol and drug intake were not accounted for. The Framingham Study [11] is one of the largest stud-
ies on the association of uric acid with CVD in the gen-
eral population. Our study differs from the Framingham
Study [11] in many ways. Mean age was 47 years in the
Framingham study compared with 60 years in the present
study, and thus mortality rate was lower in the Framingham
study (12.4 per 1000 person years compared to 18.9 per
1000 person years). Discussion In general, studies that have failed to
discern associations between uric acid and cardiovascular
disease have had shorter observation time, included youn-
ger persons, and had low number of events per-person-
years. As such they may have lacked the power to identify
the contribution of hyperuricemia to cardiovascular out-
comes [28]. The observation time in our study was longer
than in most previous studies, and this may explain why we
were able to detect associations in a study population where
high-risk subjects had been excluded (e.g., subjects with
diabetes and known CVD). It has been claimed that uric
acid is a more important risk factor among Afro Americans
than among Caucasians [3,11]. Our study shows that uric
acid is a risk factor in the Caucasian population as well. Authors’ contributions
d d Study design: HMS, IT, TJ. Data collection: MLL. Data analyses: HMS, IT, TJ,
BOE. Writing the first draft: HMS, IT, TJ. Data interpretation, discussion and
preparation of the final manuscript: HMS, IT, JVN, BOE, MLL, MDS, SNZ, SW,
SC, TJ. All authors read and approved the final manuscript. Competing interests
Th
h
h Competing interests
The authors have no conflict of interest to disclose related to the present study. Acknowledgements
Th
k This work was supported by grants from the local Health Authorities (Helse Nord). Several mechanisms could cause the uric acid metabolic
pathway to be a CV risk factor. Uric acid may stimulate
vascular smooth cell proliferation, and reduce vascular ni-
tric oxide production. For a review, see [29]. The action of
xanthine oxidase leads to generation of superoxide anions
[30]. This could mean that xantine oxidase activity is the
key risk factor, with uric acid just an epiphenomenon. Uric
acid per se has been described as a scavenger with antioxi-
dant effects [29-32]. It is noteworthy that some prelimin-
ary intervention studies have shown that the xanthine
oxidase inhibitor Allopurinol lowered blood pressure in
hypertensive adolescents [33], and had anti-ischemic ef-
fects in patients with angina pectoris [34]. Allopurinol also
reduced cardiovascular and hospitalization risk in a small
study of patients with renal failure [35]. A recent study
found that hyperuricemia was significantly associated with
poor outcomes in heart failure patients without chronic
kidney disease, but not in hyperuricemic persons with
renal failure [36]. The latter could suggest that hyperurice-
mia may predict poor outcomes primarily as a marker of
xanthine oxidase activity, and not due to impaired renal
excretion of uric acid. Future intervention studies will
hopefully explore this important issue further. Author details
1 1Section of Haematology, University Hospital of North Norway, Tromsø,
Norway. 2Department of Clinical Medicine, UiT The Arctic University of
Norway, Tromsø, Norway. 3Section of Nephrology, University Hospital of
North Norway, N-9038, Tromsø, Norway. 4Department of Community
Medicine, UiT The Arctic University of Norway, Tromsø, Norway. 5Renal
Division, The George Institute for International Health, University of Sydney,
Sydney, Australia. 6Renal Medicine, Royal Prince Alfred Hospital, Camperdown, Sydney, Australia. 7Department of Nephrology, Oslo University
Hospital Rikshospitalet, Oslo, Norway. Conclusion After multivariable adjustment, serum uric acid was sig-
nificantly associated with increased risk of future ische-
mic stroke in men and with all-cause mortality in both
genders. Associations of uric acid with myocardial in-
farction lost significance after adjustments for lipids. We
conclude that serum uric acid is an independent marker
of ischemic stroke in men, and all-cause mortality in both
genders in a Caucasian, general population. Gender-specific
analyses should be given priority in future studies. 1.
Feig DI, Kang DH, Johnson RJ: Uric acid and cardiovascular risk.
N Engl J Med 2008, 359:1811–1821. 1. Feig DI, Kang DH, Johnson RJ: Uric acid and cardiovascular risk. N Engl J Med 2008, 359:1811–1821. 1. Feig DI, Kang DH, Johnson RJ: Uric acid and cardiovascular risk. N Engl J Med 2008, 359:1811–1821. 2. Wingrove CS, Walton C, Stevenson JC: The effect of menopause on serum
uric acid levels in non-obese healthy women. Metabolism 1998, 47:435–438 3. Fang J, Alderman M: Serum uric acid and cardiovascular mortality. JAMA
2000, 283:2404–2410. 4. Madero M, Sarnak MJ, Wang X, Greene T, Beck GJ, Kusec JW, Collins AJ,
Levey AS, Menin V: Uric acid and long-term outcomes in CKD. Am J Kidney Dis 2009, 53:796–803. 4. Madero M, Sarnak MJ, Wang X, Greene T, Beck GJ, Kusec JW, Collins AJ,
Levey AS, Menin V: Uric acid and long-term outcomes in CKD. Am J Kidney Dis 2009, 53:796–803. 5. Holme I, Aastveit AH, Hammar N, Jungner I, Walldius G: Uric acid and risk
of myocardial infarction, stroke and congestive heart failure in 417734
men and women in the Apolipoprotein Mortality RISK study (AMORIS). J Intern Med 2009, 266:558–570. 6. Strasak AM, Kelleher CC, Brant LJ, Rapp K, Ruttmann E, Concin H, Diem G,
Pfeiffer KP, Ulmer H: Serum uric acid is an independent predictor for all
major forms of cardiovascular death in 28,613 elderly women: a
prospective 21-year follow-up study. Int J Cardiol 2008, 125:232–239. A major shortcoming of our study is that serum uric
acid was done as a single measurement. Furthermore,
assessment was done in a population of Caucasians only,
so our results may not pertain to other ethnical groups. The study is strengthened by the solid attendance rate,
a long follow-up time, the thorough validation of end-
points, and the ability to correct for confounding risk fac-
tors such as renal function, ACR, traditional cardiovascular 7. Wu YQ, Li J, Xu YX, Wang YL, Luo YY, Hu DY, Liu WJ, Yang M, Pi L, Wang MS,
Wang JY, Zhao SM, LI MJ: Predictive value of serum uric acid on
cardiovascular disease and all-cause mortality in urban Chinese patients. Chin Med J (Engl) 2010, 123:1387–1391. 7. Wu YQ, Li J, Xu YX, Wang YL, Luo YY, Hu DY, Liu WJ, Yang M, Pi L, Wang MS,
Wang JY, Zhao SM, LI MJ: Predictive value of serum uric acid on
cardiovascular disease and all-cause mortality in urban Chinese patients. Chin Med J (Engl) 2010, 123:1387–1391. 8. Storhaug et al. BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Moriarity JT, Folsom AR, Iribarren C, Nieto FJ, Rosamond WD: Serum uric
acid and risk of coronary heart disease: Atherosclerosis Risk in
Communities (ARIC) Study. Ann Epidemiol 2000, 10:136–143. 15. Wannamethee SG, Shaper AG, Whincup PH: Serum urate and the risk of
major coronary heart disease events. Heart 1997, 78:147–153. doi:10.1186/1471-2261-13-115
Cite this article as: Storhaug et al.: Uric acid is a risk factor for ischemic
stroke and all-cause mortality in the general population: a gender spe-
cific analysis from The Tromsø Study. BMC Cardiovascular Disorders
2013 13:115. 16. Jacobsen BK, Eggen AE, Mathiesen EB, Wilsgaard T, Njølstad I:
Cohort profile: The Tromsø Study. Int J Epidemiol 2012, 41:961–967. 17. Levey AS, Stevens LA, Schmid CH, Zhang YL, Castro AF, Feldman HI,
Kusek JW, Egges P, Van Lente F, Greene T, Coresh J: A new equation to
estimate glomerular filtration rate. Ann Intern Med 2009, 150:604–612. 18. WHO MONICA Project Principal Investigators: The World Health
Organization MONICA Project (monitoring trends and determinants in
cardiovascular disease): a major international collaboration. J Clin Epidemiol 1988, 41:105–114. 19. Lehto S, Niskanen L, Ronnemaa T, Laakso M: Serum uric acid is a strong
predictor of stroke in patients with non-insulin-dependent diabetes
mellitus. Stroke 1998, 29:635–639. 20. Kim SY, Guevara JP, Kim KM, Choi HK, Heitjan DF, Albert DA: Hyperuricemia
and risk of stroke: a systematic review and meta-analysis. Arthritis Rheum
2009, 61:885–892. 21. Mustard JF, Murphy EA, Ogryzlo MA, Smythe HA: Blood coagulation and
platelet economy in subjects with primary gout. Can Med Assoc J 1963,
89:1207–1211. 22. Kang DH, Park SK, Lee IK, Johnson RJ: Uric acid-induced C-reactive protein
expression: implication on cell proliferation and nitric oxide production
of human vascular cells. J Am Soc Nephrol 2005, 16:3553–3562. 23. Vannorsdall TD, Jinnah HA, Gordon B, Kraut M, Schretlen DJ: Cerebral ischemia
mediates the effect of serum uric acid on cognitive function. Stroke 2008,
39:3418–3420. 24. Amaro S, Urra X, Gomez-Choco M, Obach V, Cervera A, Vargas M, Torres F,
Rios J, Planas AM, Chamorro A: Uric acid levels are relevant in patients
with stroke treated with thrombolysis. Stroke 2011, 42:S28–S32. 25. Vlachopoulos C, Xaplanteris P, Vyssoulis G, Bratsas A, Baou K, Tzamou V,
Aznaouridis K, Dima I, Lazaros G, Stefanadis C: Association of serum uric
acid level with aortic stiffness and arterial wave reflections in newly
diagnosed, never-treated hypertension. Am J Hypertens 2011, 24:33–39. 26. Storhaug et al. BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 9. Liese AD, Hense HW, Lowel H, Doring A, Tietze M, Keil U: Association of
serum uric acid with all-cause and cardiovascular disease mortality and
incident myocardial infarction in the MONICA Augsburg cohort. WHO Monit Trends Determinants Cardiovasc Dis. Epidemiol 1999, 10:391–397. 31. Higgins P, Dawson J, Walters M: The potential for xanthine oxidase
inhibition in the prevention and treatment of cardiovascular and
cerebrovascular disease. Cardiovasc Psychiatry Neurol 2009. http://www. hindawi.com/journals/cpn/2009/282059. y
g
g
WHO Monit Trends Determinants Cardiovasc Dis. Epidemiol 1999, 10:391–397. 10. Bengtsson C, Lapidus L, Stendahl C, Waldenstrom J: Hyperuricaemia and
risk of cardiovascular disease and overall death. A 12-year follow-up of
participants in the population study of women in Gothenburg, Sweden. Acta Med Scand 1988, 224:549–555. 32. Nakagawa T, Kang DH, Feig D, Sanchez-Lozada LG, Srinivas TR, Sautin Y,
Ejaz AA, Segal M, Johnson RJ: Unearthing uric acid: an ancient factor with
recently found significance in renal and cardiovascular disease. Kidney Int
2006, 69:1722–1725. 33. Feig DI, Soletsky B, Johnson RJ: Effect of allopurinol on blood pressure of
adolescents with newly diagnosed essential hypertension: a randomized
trial. JAMA 2008, 300:924–932. 11. Culleton BF, Larson MG, Kannel WB, Levy D: Serum uric acid and risk for
cardiovascular disease and death: the Framingham Heart Study. Ann Intern Med 1999, 131:7–13. 34. Noman A, Ang DS, Ogston S, Lang CC, Struthers AD: Effect of high-dose
allopurinol on exercise in patients with chronic stable angina: a rando-
mised, placebo controlled crossover trial. Lancet 2010, 375:2161–2167. 12. Sakata K, Hashimoto T, Ueshima H, Okayama A: Absence of an association
between serum uric acid and mortality from cardiovascular disease:
NIPPON DATA 80, 1980-1994. National integrated projects for prospective
observation of non-communicable diseases and its trend in the aged. Eur J Epidemiol 2001, 17:461–468. 35. Goicoechea M, de Vinuesa SG, Verdalles U, Ruiz-Caro C, Amphuero J,
Rinchon A, Arroyo D, Luro J: Effect of allopurinol in chronic kidney
disease progression and cardiovascular risk. Clin J Am Soc Nephrol 2010,
5:1388–1393. 13. Jee SH, Lee SY, Kim MT: Serum uric acid and risk of death from cancer,
cardiovascular disease or all causes in men. Eur J Cardiovasc Prev Rehabil
2004, 11:185–191. 36. Filippatos GS, Ahmed MI, Gladden JD, Mujib M, Aban IB, Love TE, Sanders PW,
Pitt B, Anker SD, Ahmed A: Hyperuricaemia, chronic kidney disease, and
outcomes in heart failure: potential mechanistic insights from
epidemiological data. Eur Heart J 2011, 32:712–720. 14. 1.
Feig DI, Kang DH, Johnson RJ: Uric acid and cardiovascular risk.
N Engl J Med 2008, 359:1811–1821. Niskanen LK, Laaksonen DE, Nyyssonen K, Alfthan G, Lakka HM, Lakka TA,
Salonen JT: Uric acid level as a risk factor for cardiovascular and all-cause
mortality in middle-aged men: a prospective cohort study. Arch Intern Med 2004, 164:1546–1551. 8. Niskanen LK, Laaksonen DE, Nyyssonen K, Alfthan G, Lakka HM, Lakka TA,
Salonen JT: Uric acid level as a risk factor for cardiovascular and all-cause
mortality in middle-aged men: a prospective cohort study. Arch Intern Med 2004, 164:1546–1551. Page 10 of 10 Page 10 of 10 Storhaug et al. BMC Cardiovascular Disorders 2013, 13:115
http://www.biomedcentral.com/1471-2261/13/115 Levine W, Dyer AR, Shekelle RB, Schoenberger JA, Stamler J: Serum uric
acid and 11.5-year mortality of middle-aged women: findings of the
Chicago Heart Association Detection Project in Industry. J Clin Epidemiol
1989, 42:257–267. Submit your next manuscript to BioMed Central
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and take full advantage of: 27. Panero F, Gruden G, Perotto M, Fornengo P, Barutta F, Runzo C, Ghezzo G,
Cavallo-Perin P, Bruno G: Uric acid is not an independent predictor of
cardiovascular mortality in a population-based study. Atherosclerosis 2012,
221:183–188. • Convenient online submission 28. Krishnan E, Sokolove J: Uric acid in heart disease: a new C-reactive
protein? Curr Opin Rheumatol 2011, 23:174–177. 29. Dawson J, Quinn T, Walters M: Uric acid reduction: a new paradigm in the
management of cardiovascular risk? Curr Med Chem 2007, 14:1879–1886. 30. Berry C, Hamilton CA, Brosnan MJ, Magill FG, Berg GA, McMurray JJ,
Dominiczak AF: Investigation into the sources of superoxide in human
blood vessels: Angiotensin II increases superoxide production in human
internal mammary arteries. Circulation 2000, 101:2206–2212.
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https://openalex.org/W3205783061
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https://www.frontiersin.org/articles/10.3389/fmed.2021.749318/pdf
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English
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Liver Histopathological Analysis of 24 Postmortem Findings of Patients With COVID-19 in China
|
Frontiers in medicine
| 2,021
|
cc-by
| 6,706
|
Liver Histopathological Analysis of 24
Postmortem Findings of Patients
With COVID-19 in China
Huikuan Chu 1†, Li Peng 2†, Lilin Hu 1, Yixin Zhu 3, Jinfang Zhao 4, Hua Su 5, Lin Yao 1,
Qingjing Zhu 6, Xiu Nie 2*, Ling Yang 1* and Xiaohua Hou 1* SARS-CoV-2 induced liver injury
might be independent of pre-existing Schistosoma infection or obstructive cholestasis. Patients combined with respiratory failure had more severe hepatocellular necrosis and
male patients were more susceptible to liver injury. Although coronavirus particles or viral
inclusions were not detected in the liver tissues for all cases, vacuolar degenerations in
hepatocytes, edematous of mitochondria with the disruption of cristae, and expansions
of the endoplasmic reticulum were observed. In conclusion, pathologic changes of liver
tissues provide us a further understanding of liver injury in COVID-19 patients. Changes
in the liver seem to be related to the underlying diseases/conditions. Keywords: liver histopathological analysis, COVID-19 patients, transmission electron microscopy, edematous of
mitochondria, expansions of endoplasmic reticulum Edited by:
Jinhang Gao,
Sichuan University, China
Reviewed by:
Yang Tai,
Sichuan University, China
Yongyin Li,
Southern Medical University, China
*Correspondence:
Xiu Nie
niexiuyishi@126.com
Ling Yang
hepayang@163.com
Xiaohua Hou
houxh@hust.edu.cn
†These authors have contributed Edited by:
Jinhang Gao,
Sichuan University, China Reviewed by:
Yang Tai,
Sichuan University, China
Yongyin Li,
Southern Medical University, China †These authors have contributed
equally to this work †These authors have contributed
equally to this work Specialty section:
This article was submitted to
Gastroenterology,
a section of the journal
Frontiers in Medicine
Received: 29 July 2021
Accepted: 13 September 2021
Published: 11 October 2021 Specialty section:
This article was submitted to
Gastroenterology,
a section of the journal
Frontiers in Medicine INTRODUCTION The Corona Virus Disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome
coronavirus 2 (SARS-Cov-2), has been considered as a public health emergency of international
concern by the World Health Organization (WHO). Currently, SARS-CoV-2 has spread to over
200 countries and areas with 21, 294, 845 confirmed cases, including 761, 779 deaths globally until
August 16, 2020 (1). COVID-19 mainly affects the lower respiratory tract. Besides, it also induces
acute kidney injury and liver dysfunction in some patients (2–5). Received: 29 July 2021
Accepted: 13 September 2021
Published: 11 October 2021 Liver Histopathological Analysis of 24
Postmortem Findings of Patients
With COVID-19 in China
Huikuan Chu 1†, Li Peng 2†, Lilin Hu 1, Yixin Zhu 3, Jinfang Zhao 4, Hua Su 5, Lin Yao 1,
Qingjing Zhu 6, Xiu Nie 2*, Ling Yang 1* and Xiaohua Hou 1* Huikuan Chu 1†, Li Peng 2†, Lilin Hu 1, Yixin Zhu 3, Jinfang Zhao 4, Hua Su 5, Lin Yao 1,
Qingjing Zhu 6, Xiu Nie 2*, Ling Yang 1* and Xiaohua Hou 1* 1 Division of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan, China, 2 Department of Pathology, Union Hospital, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan, China, 3 Department of Medicine, University of California San Diego, La Jolla, CA, United States,
4 Center for Life Sciences, Tsinghua University, Beijing, China, 5 Department of Nephrology, Union Hospital, Tongji Medical
College, Huazhong University of Science and Technology, Wuhan, China, 6 Liver and Infectious Diseases Department, Wuhan
Jinyintan Hospital, Wuhan, China Although the pathologic investigation of liver injury was observed in a couple of cases
in China, the detailed description of liver histopathologic and ultrastructural changes in
a relatively larger series of liver tissues from COVID-19 patients is lacking. Samples from
the liver were obtained from 24 COVID-19 cases from February 1 to April 1, 2020. Light
microscopy showed that all liver sections had different degrees of liver injury manifested
as swelling of the hepatocytes, hepatocellular necrosis, steatosis, lobular inflammation,
portal inflammation, dilatation of sinusoids, and so on. SARS-CoV-2 induced liver injury
might be independent of pre-existing Schistosoma infection or obstructive cholestasis. Patients combined with respiratory failure had more severe hepatocellular necrosis and
male patients were more susceptible to liver injury. Although coronavirus particles or viral
inclusions were not detected in the liver tissues for all cases, vacuolar degenerations in
hepatocytes, edematous of mitochondria with the disruption of cristae, and expansions
of the endoplasmic reticulum were observed. In conclusion, pathologic changes of liver
tissues provide us a further understanding of liver injury in COVID-19 patients. Changes
in the liver seem to be related to the underlying diseases/conditions. Although the pathologic investigation of liver injury was observed in a couple of cases
in China, the detailed description of liver histopathologic and ultrastructural changes in
a relatively larger series of liver tissues from COVID-19 patients is lacking. Samples from
the liver were obtained from 24 COVID-19 cases from February 1 to April 1, 2020. Light
microscopy showed that all liver sections had different degrees of liver injury manifested
as swelling of the hepatocytes, hepatocellular necrosis, steatosis, lobular inflammation,
portal inflammation, dilatation of sinusoids, and so on. ORIGINAL RESEARCH
published: 11 October 2021
doi: 10.3389/fmed.2021.749318 Clinical Information of the Patients Clinical Information of the Patients
The 24 patients with COVID-19 included 16(67%) males and
8(33%) females, with an average age of 68.3 years (range,
42–91 years). All cases had positive results for SARS-CoV-2
by nucleic acid testing and characteristic radiologic alterations
in lungs. Ten (41.7%) patients had a history of hypertension
or coronary heart diseases or both. One patient had both
coronary heart diseases and diabetes. Six (25%) patients had Statistical Analysis Categorical
variables
were
presented
as
numbers
and
percentages. Continuous variables were expressed as median
(25th−75th
percentile). The
relationships
of
pathologic
changes and clinical liver function and inflammatory data
were
examined
using
Spearman
Rho’s
(ρ)
correlation. Pathologic
features
among
those
with
and
without
macrovesicular steatosis/liver fibrosis and among those with
and without antiviral intervention/combination therapeutic
intervention/combined diseases were compared using Fisher’s
exact test. Pathologic features among those with different degrees
of liver injury and among those with and without antiviral
intervention/combination therapeutic intervention/combined
diseases were compared using the Mann–Whitney U test. All statistical analyses were performed using SPSS (Statistical
Package for the Social Sciences) (version 25.0, IBM Corp,
Armonk, NY, USA). A significance level of P ≤0.05 was used for
all models (two-sided). Tissue Sampling and Processing Samples from the liver were obtained with ultrasound guidance
and percutaneous multiple punctures of the liver from 24
COVID-19 cases with a postmortem interval of 2 h from
February 1 to April 1, 2020. Tissue specimens were fixed in 3.7%
formaldehyde for 48–72 h before the following procedures. Four
micrometer sections were cut and stained with hematoxylin-
eosin. Slides were examined and analyzed by two pathologists
blinded to the study. For EM examination, tissue specimens were fixed in 2.5%
glutaraldehyde for 24–48 h. After 1% osmic acid post-fixation and
gradient dehydration, Epon-embedded, toluidine blue-stained
“semi-thin” sections were examined, and selected areas were
chosen for thin sections. Thin sections were then cut and stained
with uranyl acetate and lead citrate. EM grids were then viewed
with a transmission electron microscope (HT-7800; Hitachi,
Tokyo, Japan). Patients This study was conducted in accordance with the principles of
the Declaration of Helsinki and approved by the Institutional
Ethics Board of Union Hospital, Tongji Medical College,
Huazhong University of Science and Technology (2020. 0043-
1). Written informed consent was obtained from the next of
kin of each enrolled case. According to the World Health
Organization Interim Guidance, the diagnosis of COVID-19 was
confirmed based on real-time reverse-transcriptase polymerase-
chain-reaction (RT-PCR) assay for nasal and pharyngeal swab
specimens (9). Their electronic medical records, the results of
the laboratory, and imaging examinations were all obtained and
reviewed. The degree of severity of liver pathological changes was
defined using pathologic diagnosis and the grading guideline of
common pathological changes after liver transplantation (II) (17)
and Scheure scoring system (18, 19). Citation: Chu H, Peng L, Hu L, Zhu Y, Zhao J,
Su H, Yao L, Zhu Q, Nie X, Yang L and
Hou X (2021) Liver Histopathological
Analysis of 24 Postmortem Findings of
Patients With COVID-19 in China. Front. Med. 8:749318. doi: 10.3389/fmed.2021.749318 On
the
basis
of
previous
reports
from
China,
a
subset
of
COVID-19
patients
had liver impairment with abnormal levels of alanine aminotransferase (ALT) and/or
aspartate aminotransferase (AST) accompanied by slightly elevated bilirubin levels during
disease
progression
(6–8). The
incidence
of
liver
injury
in
COVID-19
patients
was October 2021 | Volume 8 | Article 749318 Frontiers in Medicine | www.frontiersin.org Liver Analysis of COVID-19 Patients Chu et al. Biomedical Technology Co., Ltd, Xiamen, China) and a real-
time reverse transcriptase polymerase chain reaction (real-time
RT-PCR) assay was run on the Mx3000P qPCR system with a
2019-nCoV nucleic acid detection kit (Catalog Z-RR-0479-02-
25, from Shanghai Zhijiang Biotechnology Co., Ltd, Shanghai,
China) according to the manufacturer’s protocol. common, especially in some severe and deceased cases (8–
10). The pathologic investigation of liver injury was observed
in several studies worldwide (11–15). They centered on the
pathologic findings using light microscopy and only one paper
described the change of organelles using electron microscopy
(15). Besides, pathologic investigation of liver injury was
observed only in a couple of cases in China (16), the detailed
description of pathologic findings using both light microscopy
and transmission electron microscopy observations in a relatively
larger series of liver tissues from COVID-19 patients in China
is lacking. In this study, we report on our experience of liver
findings at autopsy in 24 deceased patients with COVID-19. g
Two target genes, the RdRp, nucleocapsid protein (N) and
E genes, were simultaneously amplified and monitored during
the real-time RT-PCR assay. The primers for target 1 (RdRp)
were RdRp_SARSr-F 5′-GTGARATGGTCATGTGTGGCGG-
3′, RdRp_SARSr-R 5′-CARATGTTAAASACACTATTAGCATA-
3′, RdRP_SARSr-P1 5′-CCAGGTGGWACRTCATCMGGTGAT
GC-3′, RdRp_SARSr-P2 5′-CAGGTGGAACCTCATCAGGAG
ATGC-3′ and E_Sarbeco_F 5′-ACAGGTACGTTAATAGTTAAT
AGCGT-3′. The primers for target 2 (N) were N_Sarbeco_R
5′-GAGGAACGAGAAGAGGCTTG-3′ and N_Sarbeco_P 5′-
ACTTCCTCAAGGAACAACATTGCCA-3′
and the primers
for target 3 (E) were N_Sarbeco_F 5′-CACATTGGCACC
CGCAATC-3′, E_Sarbeco_R 5′-ATATTGCAGCAGTACGCA
CACA-3′ and E_Sarbeco_P1 5′-ACACTAGCCATCCTTACTG
CGCTTCG-3′. A cycle threshold (Ct) value of 43 or less
was defined as a positive, and a Ct value of more than
43 was defined as a negative. These testing criteria were
based on recommendations by the National Institute for
Viral Disease Control and Prevention (China) (http://ivdc. chinacdc.cn/kyjz/202001/t20200121_211337. html). Positive and
negative controls were included. Light Microscopy Findings Initial value
Peak value
ALT (U/L) (N = 24)
30 (23.5, 34.75)
124 (46, 629.5)
AST (U/L) (N = 24)
42 (27.3, 58.5)
96 (60, 817)
LDH (U/L) (N = 24)
465 (246.8, 591)
614.5 (452.8, 2,072)
TBIL (umol/l) (N = 24)
12.2 (9.2, 21.3)
38.8 (20.3, 49.1)
ALP (U/L) (N = 24)
71.5 (60.8, 90.5)
111.5 (77, 178.8)
GGT (U/L) (N = 24)
40.5 (20.3, 65.8)
75.5 (38.8, 165.5)
Albumin (g/l) (N = 24)
27.8 (25.6, 31.8)
21.9 (17.3, 24.1)
hs-TnI (ng/l) (N = 18)
19.55 (7.7, 61.1)
227.2 (49.2, 1,876)
CK-MB (U/L) (N = 16)
14 (12, 22)
47 (26, 101.8)
WBC (G/L) (N = 24)
8.735 (5.5, 12.4)
21.5 (15.3, 27.8)
LY (G/L) (N = 24)
0.49 (0.33, 0.72)
0.24 (0.19, 0.31)
PLT (G/L) (N = 23)
146 (108, 233)
33 (22, 88)
Hb (g/l) (N = 23)
121 (105, 144)
70 (61, 87)
Cr (umol/l) (N = 24)
68.5 (61, 98.5)
122.5 (92.1, 208.4)
BUN (mmol/l) (N = 24)
7.2 (4.9, 11.2)
19.6 (15.8, 32.9)
D-dimer (ug/ml) (N = 24)
1.3 (0.7, 7.8)
8 (6.5, 8)
PCT (ng/ml) (N = 22)
0.14 (0.11, 0.37)
5.7 (2.3, 25.4)
CRP (mg/l) (N = 24)
82.8 (45.1, 118)
153.4 (118.4, 173.9)
All statistics are presented as median (interquartile range). ALT,
alanine
aminotransferase;
ALP,
Alkaline
phosphatase;
AST,
aspartate
aminotransferase; BUN, blood urea nitrogen; CK, creatine kinase; Cr, creatinine;
CRP, C-reactive protein; DM, diabetes; GGT, γ-glutamyl transpeptidase; Hb, hemoglobin;
hs-TnI, high sensitive troponin I; LDH, lactate dehydrogenase; LY, lymphocytes; PCT,
procalcitonin; PLT, platelet; TBIL, total bilirubin; WBC, white blood cell. TABLE 2 | Summary of laboratory results. Previous study found that 54–75% COVID-19 patients
presented
steatosis
(11,
12,
14,
15). Consistently,
we
found 20 from 24 cases (83.3%) demonstrated steatosis,
15
cases
presented
microvesicular
steatosis
(Figure 1C)
and the other 5 presented both microvesicular steatosis
and
Macrovesicular
steatosis. There
was
no
relationship
between liver function test at both initial and peak time
points and steatosis. Microvesicular steatosis was correlated
with antiviral therapy combined with invasive mechanical
ventilation
intervention,
antiviral
therapy
combined
with
ECMO
intervention
and
invasive
mechanical
ventilation
(Supplementary Tables 1–3). 28.6–47% of COVID-19 patients were reported to have
hepatic necrosis (13, 15), while all cases with different
degrees of hepatic necrosis were presented in our study,
moderate necrosis was observed in 5 cases and severe
hepatocellular necrosis was observed in 10 cases (Figure 1D). Light Microscopy Findings g
py
g
Light microscopy showed that all liver sections had different
degrees of liver injury manifested as swelling of the hepatocytes,
hepatocellular
necrosis,
steatosis,
lobular
inflammation,
portal inflammation, dilatation of sinusoids, cholestasis, and
fibrosis (Table 3). A detailed assessment of each feature is
provided below. All cases presented with slight to moderate swelling of the
hepatocytes (Figure 1A): 13 mild and 11 moderate. The degree
of hepatocyte swelling was positively correlated with levels of
LDH at both initial and peak time points, while there was no
relationship between the swelling degree of the hepatocytes and
therapeutic interventions (Supplementary Tables 1–3). A previous study reported that about 38% of the patients
dying of COVID-19 had cholestasis (14). Similarly, we found
a total of eight from 24 patients (33.3%) had cholestasis
(Figure 1B): 7 cases had mild cholestasis and one case
had
moderate
cholestasis. There
was
no
relationship
between
liver
function
tested
at
the
initial
time
point
and
cholestasis. However,
the
levels
of
ALT,
AST,
and
LDH tested at peak time point were positively correlated
with cholestasis, and cholestasis was also correlated with
antiviral
therapy
combined
with
ECMO
intervention
(Supplementary Tables 1–3). TABLE 2 | Summary of laboratory results. Real-Time Reverse Transcription
Polymerase Chain Reaction Assay for
SARS-COV-2 in Tissue Formalin-fixed, paraffin-embedded (FFPE) tissue blocks were
used for RNA extraction. Total RNA was extracted using a
sample RNA isolation kit (Catalog No. ADx-FF04, fromIde October 2021 | Volume 8 | Article 749318 Frontiers in Medicine | www.frontiersin.org 2 Liver Analysis of COVID-19 Patients Chu et al. TABLE 1 | Clinical information of patients with COVID-19 (N = 24). Demographics and combined diseases
Characters
Age (years)
66 (60,78)
Sex (n, %)
Female
8 (33%)
Male
16 (67%)
Chronic liver diseases (n, %)
With
4 (16.7%)
Without
20 (83.3%)
Diabetes (n, %)
With
1 (4.2%)
Without
23 (95.8%)
Hypertension (n, %)
With
8 (33.3%)
Without
16 (66.7%)
Coronary heart disease (n, %)
With
5 (20.8%)
Without
19 (79.2%)
Chronic kidney disease (n, %)
With
1 (4.2%)
Without
23 (95.8%)
Cancer (n, %)
With
6 (25.0%)
Without
18 (75.0%)
All statistics are presented as median (interquartile range) or N (%). Light Microscopy Findings All statistics are presented as median (interquartile range). All statistics are presented as median (interquartile range). Light Microscopy Findings The level of total bilirubin at the initial time point was
positively related to hepatic necrosis. The level of albumin
at the peak time point was negatively related to hepatic
necrosis. The levels of ALT, AST, and LDH at the peak
time point were positively correlated with hepatic necrosis. Hepatic necrosis was also correlated with antiviral therapy
combined with invasive mechanical ventilation intervention
(Supplementary Tables 1–3). ALT,
alanine
aminotransferase;
ALP,
Alkaline
phosphatase;
AST,
aspartate
aminotransferase; BUN, blood urea nitrogen; CK, creatine kinase; Cr, creatinine;
CRP, C-reactive protein; DM, diabetes; GGT, γ-glutamyl transpeptidase; Hb, hemoglobin;
hs-TnI, high sensitive troponin I; LDH, lactate dehydrogenase; LY, lymphocytes; PCT,
procalcitonin; PLT, platelet; TBIL, total bilirubin; WBC, white blood cell. Lobular inflammation was observed in all cases and most of
them were moderate to severe (Figure 1E). The levels of ALT,
AST, LDH, and PCT at the peak time point were positively
correlated with lobular inflammation. Lobular inflammation was
also correlated with antiviral therapy combined with ECMO
intervention (Supplementary Tables 1–3). a history of tumor. Three patients had Schistosoma infection
and one patient had obstructive cholestasis. One patient had a
history of hepatocellular carcinoma. The clinical information and
laboratory results are summarized in Tables 1, 2. Mild to moderate portal inflammation was observed in 23
cases (Figure 1F). The levels of ALT, AST, and LDH at the initial October 2021 | Volume 8 | Article 749318 Frontiers in Medicine | www.frontiersin.org 3 Liver Analysis of COVID-19 Patients Chu et al. TABLE 3 | The pathologic abnormalities of liver using light microscopy observations in 24 cases of deceased patients with COVID-19. Light Microscopy Findings ID
Swelling
of the
hepatocytes
Cholestasis Hepatocellular
necrosis
Steatosis
Lobular
inflammation
Portal
inflammation
Fibrosis
GS
Dilatation
of
sinusoids
Microvesicular
steatosis
(Relative
area)
Macrovesicular
steatosis
SJ1
Moderate
Mild
Mild
60%
0
2
1
1
G2S1
Moderate
SJ2
Moderate
N
Mild
70%
0
2
0–1
0
G2S0
Mild
SJ3
Moderate
Moderate
Mild
0
0
2
2
1
G2S1
Mild
SJ4
Mild
N
Mild
0
0
2–3
0
1
G2-
3S1
Moderate
SJ5
Mild
Mild
Severe
5%
0
2–3
1
1
G2-
3S1
N
SJ6
Mild
N
Mild
50%
0
1
1
0
G1S0
Mild
SJ7
Mild
N
Mild
10%
0
1
1
0
G1S0
N
SJ8
Moderate
Mild
Severe
50%
0
3
2
0
G3S0
Mild
SJ9
Moderate
N
Mild
50%
0
2
1
0
G2S0
Mild
SJ10
Mild
Mild
Severe
20%
0
3
1
0
G3S0
Moderate
SJ11
Mild
N
Moderate
40%
0
2
1
0
G2S0
Moderate
SJ12
Moderate
N
Moderate
10%
0
2
1
0
G3S0
Moderate
SJ13
Mild
N
Severe
50%
0
2
1
0
G2S0
Moderate
SJ14
Mild
N
Mild
80%
10%
2
1
0
G2S0
N
SJ15
Mild
N
Moderate
30%
50%
2–3
2
3
G2-
3S3
Moderate
SJ16
Moderate
Mild
Severe
60%
0
3
1
0
G3S0
Mild
SJ17
Mild
N
Mild
70–80%
20%
2
2
1
G2S1
N
SJ18
Mild
N
Severe
80%
0
3
1
0
G3S0
Mild
SJ19
Mild
N
Severe
20%
0
3
1
0
G3S0
Mild
SJ20
Moderate
N
Moderate
70%
0
3
1
0
G3S0
Mild
SJ21
Moderate
Mild
Severe
0
0
4
2
0
G4S0
Mild
SJ22
Moderate
Mild
Severe
0
0
3
2
1
G3S1
N
SJ23
Moderate
N
Severe
10%
60%
2–3
1–2
0
G2-
3S0
N
SJ24
Mild
N
Moderate
80%
10%
2
1–2
1
G2S1
N
ID, identification number; N, no. 3 | The pathologic abnormalities of liver using light microscopy observations in 24 cases of deceased patients with COVID-19. Liver Injury Is Not Associated With
SARS-COV-2 Infection in Liver Tissue necrosis was observed in one patient among the other
four patients without respiratory failure. Severe hepatocellular
necrosis and lobular inflammation were observed in two
patients who were presented with severe respiratory failure
and performed ECMO treatment. Moreover, microvesicular
steatosis was correlated with invasive mechanical ventilation
which is a therapeutic method for severe respiratory failure
(Supplementary Table 3). These indicated that hypoxia may
contribute to liver injury. Previous studies speculated that the virus may directly cause
liver damage. In order to further confirm this hypothesis, we
performed RT-PCR to detect SARS-COV-2 in liver tissues. Surprisingly, tests on the liver tissue from all cases were negative,
which is consistent with the result from our transmission electron
microscopy observations. This result indicated that liver injury in
these cases is not directly caused by SARS-COV-2. Transmission Electron Microscopy
Observations (A) vacuolar degenerations in hepatocytes (arrows),
(B) edematous of mitochondria and the disruption of cristae (arrows), (C) expansion of endoplasmic reticulum (arrows). Bars = 5 µm (A), 500 nm (B), 2 µm (C). Transmission Electron Microscopy
Observations time point were positively correlated with portal inflammation
while there was no relationship between portal inflammation and
therapeutic intervention (Supplementary Tables 1–3). Coronavirus particles were also identified in the liver of deceased
SARS-CoV-2 patients (16). In order to check whether there
were viral particles or inclusions in our patients, we observed
the liver tissues using transmission electron microscopy. Inconsistent with previous reports, coronavirus particles or viral
inclusions were not detected in the liver tissues for any cases. Vacuolar degenerations in hepatocytes were noted (Figure 2A). Hepatocytes also exhibited marked edematous of mitochondria
with the disruption of cristae (Figure 2B). The expansions of
the endoplasmic reticulum were also observed (Figure 2C). These indicated that liver injury may be associated with other
underlying conditions instead of the direct damage induced
by Coronavirus. Eight
cases
presented
with
hepatic
fibrosis
(Figure 1G). There
was
no
relationship
between
liver
function
test
at
both
initial
and
peak
time
points
and
hepatic
fibrosis. There
was
also
no
relationship
between
hepatic
fibrosis
and
therapeutic
intervention
(Supplementary Tables 1–3). Mild to moderate dilatation of sinusoids were observed
in
17
cases
(Figure 1H). There
was
no
relationship
between
liver
function
test
at
both
initial
and
peak
time
points
and
dilatation
of
sinusoids. Dilatation
of
sinusoids
was
correlated
with
Umifenovir
intervention
(Supplementary Tables 1–3). October 2021 | Volume 8 | Article 749318 Frontiers in Medicine | www.frontiersin.org Liver Analysis of COVID-19 Patients Chu et al. FIGURE 1 | Spectrum of pathologic abnormalities of liver tissues from postmortem of patients with COVID-19. (A) swelling of the hepatocytes, (B) cholestasis, (C)
steatosis, (D) hepatocellular necrosis. (E) lobular inflammation, (F) portal inflammation, (G) fibrosis, (H) dilatation of sinusoids, Bars = 50 µm (A–D). FIGURE 1 | Spectrum of pathologic abnormalities of liver tissues from postmortem of patients with COVID-19. (A) swelling of the hepatocytes, (B) cholestasis, (C)
steatosis, (D) hepatocellular necrosis. (E) lobular inflammation, (F) portal inflammation, (G) fibrosis, (H) dilatation of sinusoids, Bars = 50 µm (A–D). FIGURE 2 | Spectrum of ultrastructural abnormalities of liver tissues from postmortem of patients with COVID-19. (A) vacuolar degenerations in hepatocytes (arrows),
(B) edematous of mitochondria and the disruption of cristae (arrows), (C) expansion of endoplasmic reticulum (arrows). Bars = 5 µm (A), 500 nm (B), 2 µm (C). FIGURE 2 | Spectrum of ultrastructural abnormalities of liver tissues from postmortem of patients with COVID-19. The Degree of Liver Injury Is Not
Associated With Combined Diseases in
COVID-19 The Degree of Liver Injury Is Associated
With Severity of Hypoxemia in COVID-19
Twenty cases combined with respiratory failure. Among them,
nine cases had severe hepatocellular necrosis and five cases had
moderate hepatocellular necrosis, while severe hepatocellular COVID-19 Twenty cases combined with respiratory failure. Among them,
nine cases had severe hepatocellular necrosis and five cases had
moderate hepatocellular necrosis, while severe hepatocellular Three cases combined with Schistosoma infection presented
with slight to moderate swelling of the hepatocytes, no or October 2021 | Volume 8 | Article 749318 Frontiers in Medicine | www.frontiersin.org 5 Liver Analysis of COVID-19 Patients Chu et al. TABLE 4 | Exposure to hepatotoxic factors and treatment history. ID
Exposure
to
hepatotoxic
factors
Antivirals
Antifungal drugs
Steroid
Invasive
mechanical
ventilation
CRRT
ECMO
SJ1
Schistosoma
infection
Umifenovir
Y
Y
Y
N
N
SJ2
N
Umifenovir, Ribavirin
N
Y
Y
Y
N
SJ3
Obstructive
cholestasis
Umifenovir, Ribavirin
N
Y
N
N
N
SJ4
Schistosoma
infection
Umifenovir, Ribavirin
N
N
N
N
N
SJ5
N
Umifenovir
Y
Y
Y
N
N
SJ6
N
Umifenovir, Ribavirin
N
Y
Y
Y
N
SJ7
N
Umifenovir
N
N
N
N
N
SJ8
N
Umifenovir, Ribavirin
Y
Y
Y
N
N
SJ9
N
Umifenovir
Y
Y
Y
Y
N
SJ10
N
N
N
Y
N
N
N
SJ11
N
Ganciclovir, Oseltamivir,
Umifenovir
N
Y
Y
N
N
SJ12
N
Umifenovir, Interferon α2β
Y
Y
Y
N
N
SJ13
N
Oseltamivir, Umifenovir,
Ribavirin, Interferon α2β,
Lopinavir and Ritonavir
Y
Y
Y
N
N
SJ14
N
Interferon α2β, Ganciclovir,
Ribavirin
N
Y
Y
N
N
SJ15
N
Umifenovir, Ribavirin
Y
Y
Y
N
N
SJ16
N
Umifenovir, Ribavirin
Y
Y
Y
N
N
SJ17
N
Umifenovir
Y
Y
Y
Y
N
SJ18
N
Umifenovir, Ribavirin
Y
Y
Y
Y
N
SJ19
N
Umifenovir, Ribavirin
Y
Y
Y
N
N
SJ20
N
Umifenovir
Y
N
Y
Y
N
SJ21
N
Umifenovir, Ribavirin,
Interferon α2β
Y
Y
Y
Y
Y
SJ22
N
Ribavirin
Y
Y
Y
Y
Y
SJ23
N
Interferon α2β, Umifenovir,
Ribavirin
Y
Y
Y
N
N
SJ24
Schistosoma
infection
Ribavirin, Ganciclovir
N
Y
Y
Y
N
CRRT, continuous renal replacement therapy; ECMO, Extracorporeal Membrane Oxygenation; ID, identification number; N, no; Y, yes. TABLE 4 | Exposure to hepatotoxic factors and treatment history. CRRT, continuous renal replacement therapy; ECMO, Extracorporeal Membrane Oxygenation; ID, identification number; N, no; Y, yes. COVID-19. This is the first detailed report of liver pathologic
presentations using both light microscopy and transmission
electron microscopy observations from a large series of liver
tissues in patients with SARS-CoV-2 infection in China. COVID-19 Our
biopsy study demonstrates the range of abnormalities present
and the specific hepatocellular necrosis that might be induced by
hypoxia in COVID-19 patients, and thus may provide important
information for future clinicopathologic studies in severely
ill patients with COVID-19 infection and liver injury. We
observed mild to moderate cholestasis. This is in accordance with
mechanisms known for SARS-CoV-2 infection via angiotensin-
converting enzyme II (ACE2) on bile duct epithelial cells in
the liver (20). We also show findings that suggest distinct
mechanisms of this novel coronavirus infection, significant
hepatocellular necrosis. Thus, these pathologic observations mild cholestasis, and mild to moderate hepatocellular necrosis. One case combined with obstructive cholestasis presented with
moderate swelling of the hepatocytes, moderate cholestasis, and
mild hepatocellular necrosis (Table 4). These results indicated
that SARS-CoV-2 induced liver injury might be independent
of pre-existing Schistosoma infection or obstructive cholestasis. Moreover, we analyzed the relationships between combined
diseases and liver injury and found that there was no relationship
between them (Supplementary Table 4). DISCUSSION About 80% of the patients were non-
severe patients in previous studies (24, 25), while all the
patients
in
our
study
were
severe
cases
and
deceased
in
the
end. Besides,
the
mechanisms
of
liver
injury
might be different with different degrees of severity of
COVID-19 infection. In this study, we did not detect viral RNA, viral particles,
or inclusions from liver tissues for all patients, which is
inconsistent with previous studies (11, 14–16). It is probable
that there was no virus in the liver tissue for these patients. It is also possible that the virus was cleared already, and the
clearance of the virus may be related to different stages of
disease progression. Vascular thrombosis was described in several articles (11, 12,
15), while we did not find vascular thrombosis in our paper. This may be related with a lower level of platelets and D-dimer
value compared to previous study. Only five patients had a low
level of platelets (<100 G/L) in the study of Sonzogni et al. (11), while a total of 19/23 (82.6%) patients had a low level of
platelets (<100 G/L) with an average of 33 G/L at peak time
point in our paper. The average value of D-dimer was 8 ug/ml
(80 ng/dl) in our paper, which is lower than previous report with
96% patients having a very high value of D-dimer values ≥500
ng/dL (11). p
g
In summary, we observed pathologic changes of liver injury
using light microscopy and transmission electron microscopy
in some deceased patients with COVID-19. We described the
liver injury patterns and the severity of liver injury and found
that 41.7% of the patients had severe hepatocyte necrosis. Liver injury may be related with systemic inflammation and
hypoxia induced by COVID-19 infection, hepatotoxic effects
from antifungal agents and antiviral therapy, and organ failure
that needs invasive mechanical ventilation and/or ECMO
intervention. However, there are still some limitations for our
study, such as a relatively small number of cases. Further
studies with a larger number of cases are needed for more
comprehensive understandings of liver injury in COVID-
19 patients. In this study, we did not detect viral RNA or viral particles
from liver tissues for all patients. Lagana et al. identified viral
RNA in some liver tissue samples, but there was no significant
correlations between PCR positivity and any histologic finding
(14). DISCUSSION In the present study, we report the liver histopathologic,
ultrastructural, and RT-PCR findings from biopsies of 24 patients
who died from respiratory failure or circulatory failure due to October 2021 | Volume 8 | Article 749318 Frontiers in Medicine | www.frontiersin.org 6 Liver Analysis of COVID-19 Patients Chu et al. may provide a basis for further understanding of COVID-
19. positively correlated with lobular inflammation. Thus, systemic
inflammation induced by virus and hypoxia which is induced
by respiratory failure might contribute to liver injury. Moreover,
organ failure may be also related with liver injury, as invasive
mechanical ventilation and/or ECMO intervention which were
therapeutic methods for organ failure were related with liver
injury. Besides, the adverse effects of some drugs may be another
factor to cause liver injury (22, 23). In our study, antiviral
drugs were widely used for our patients, and patients treated
with two or more kinds of antiviral drugs seem to have more
severe hepatocyte necrosis and liver injury (Table 3). In addition,
patients who had a history of antifungal drugs usage seem to have
more severe hepatocyte necrosis and liver injury (Table 3). The detailed description of pathologic findings has been
described by several studies. Hepatic necrosis, portal tract
inflammation,
lobular
inflammation,
steatosis,
increased
number of histiocytes and megakaryocytes and platelet-fibrin
microthrombi were described by Zhao et al. (15). Bradley et al. demonstrated the presence of acute congestion, centrilobular
necrosis and mild periportal lymphocytic inflammation (13). Sonzogni et al. reported fibrosis, mild to moderate lobular
inflammation, mild portal inflammation, parenchymal confluent
necrosis, steatosis, vascular thrombosis, vascular alterations
including portal vein parietal fibrosis, herniated portal vein in
periportal parenchyma and periportal abnormal vessels (11). Lagana et al. found lobular necroinflammation and portal
inflammation, lobular apoptosis, steatosis, and cholestasis
(14). Diaz et al. wrote a meta-analysis to summarize the
presentations including hepatic steatosis, congestion of hepatic
sinuses and necrosis, vascular thrombosis and other vascular
alterations, fibrosis, Kupffer cell proliferation or hyperplasia,
portal and lobular inflammation (12). We found similar
presentations including hepatocellular necrosis, cholestasis,
steatosis,
lobular
inflammation,
portal
inflammation
and
fibrosis. Moreover, we found swelling of the hepatocytes, and
dilatation of sinusoids. Our data confirmed that 41.7% of the patients with
liver
injury
presented
as
severe
hepatocellular
necrosis,
which was higher than those found in previous studies
(24, 25). This might be related to the severity of the
COVID-19 patients. DISCUSSION Thus, we conclude that liver damage was not caused by virus
directly. We observed significant hepatocellular necrosis, and the
change of ultrastructural features of hepatocytes, such as vacuolar
degenerations, edematous of mitochondria with the disruption
of cristae and the expansions of endoplasmic reticulum in
cases without viral particles or inclusions detected in their
liver tissues. These indicate that hepatocellular injury may be
induced by other mechanisms instead of SARS-CoV-2 infection
directly. Hypoxia, presenting as one of the common symptoms
in COVID-19 patients, may be related to liver injury (21). Our
data confirmed that more patients with severe hepatocellular
necrosis had respiratory failure compared to patients with mild
or moderate hepatocellular necrosis. Moreover, microvesicular
steatosis was correlated with invasive mechanical ventilation
which is a therapeutic method for severe respiratory failure. We
also found that the level of PCT at the peak time point was 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 author/s. Frontiers in Medicine | www.frontiersin.org REFERENCES 11. Sonzogni G, Previtali M, Seghezzi M, Grazia Alessio A, Gianatti L, Licini D, et
al. Liver histopathology in severe COVID 19 respiratory failure is suggestive
of vascular alterations. Liver Int. (2020) 40:2110–6. doi: 10.1111/liv.14601 1. World Health Organization. Weekly Operational Update on COVID-19
25 September 2020. (2000). Available online at: https://www.who.int/docs/
default-source/coronaviruse/weekly-updates/wou-25-september-2020-for-
cleared.pdf (accessed September 27, 2020). 1. World Health Organization. Weekly Operational Update on COVID-19
25 September 2020. (2000). Available online at: https://www.who.int/docs/
default-source/coronaviruse/weekly-updates/wou-25-september-2020-for-
cleared.pdf (accessed September 27, 2020). 12. Diaz LA, Idalsoaga F, Cannistra M, Candia R, Cabrera D, Barrera F, et al. High prevalence of hepatic steatosis and vascular thrombosis in COVID-19:
a systematic review and meta-analysis of autopsy data. World J Gastroenterol. (2020) 26:7693–706. doi: 10.3748/wjg.v26.i48.7693 2. Wang D, Hu B, Hu C, Zhu F, Liu X, Zhang J, et al. Clinical characteristics of
138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in
Wuhan, China. JAMA. (2020) 323:1061–9. doi: 10.1001/jama.2020.1585 2. Wang D, Hu B, Hu C, Zhu F, Liu X, Zhang J, et al. Clinical characteristics of
138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in
Wuhan, China. JAMA. (2020) 323:1061–9. doi: 10.1001/jama.2020.1585 13. Bradley BT, Maioli H, Johnston R, Chaudhry I, Fink SL, Xu H,
et al. Histopathology and ultrastructural findings of fatal COVID-19
infections in Washington State: a case series. Lancet. (2020) 396:320–32. doi: 10.1016/S0140-6736(20)31305-2 3. Yang X, Yu Y, Xu J, Shu H, Xia J, Liu H, et al. Clinical course and outcomes
of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a
single-centered, retrospective, observational study. Lancet Respir Med. (2020)
8:475–81. doi: 10.1016/S2213-2600(20)30079-5 3. Yang X, Yu Y, Xu J, Shu H, Xia J, Liu H, et al. Clinical course and outcomes
of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a
single-centered, retrospective, observational study. Lancet Respir Med. (2020)
8:475–81. doi: 10.1016/S2213-2600(20)30079-5 14. Lagana SM, Kudose S, Iuga AC, Lee MJ, Fazlollahi L, Remotti HE, et al. Hepatic pathology in patients dying of COVID-19: a series of 40 cases
including clinical, histologic, and virologic data. Mod Pathol. (2020) 33:2147–
55. doi: 10.1038/s41379-020-00649-x 4. Cui Y, Tian M, Huang D, Wang X, Huang Y, Fan L, et al. A 55-day-old
female infant infected with 2019 novel coronavirus disease: presenting with
pneumonia, liver injury, and heart damage. J Infect Dis. (2020) 221:1775–81. doi: 10.1093/infdis/jiaa113 15. Zhao CL, Rapkiewicz A, Maghsoodi-Deerwester M, Gupta M, Cao W,
Palaia T, et al. REFERENCES Pathological findings in the postmortem liver of patients
with coronavirus disease 2019 (COVID-19). Hum Pathol. (2021) 109:59–68. doi: 10.1016/j.humpath.2020.11.015 5. Special Expert Group for Control of the Epidemic of Novel Coronavirus
Pneumonia of the Chinese Preventive Medicine Association. An update
on the epidemiological characteristics of novel coronavirus pneumonia
COVID-19. Zhonghua Liu Xing Bing Xue Za Zhi. (2020) 41:139–44. doi: 10.3760/cma.j.issn.0254-6450.2020.02.002 16. Wang Y, Liu S, Liu H, Li W, Lin F, Jiang L, et al. SARS-CoV-2 infection of the
liver directly contributes to hepatic impairment in patients with COVID-19. J
Hepatol. (2020) 73:807–16. doi: 10.1016/j.jhep.2020.05.002 17. National hepatobiliary tumor and Transplantation Pathology Collaboration
Group. Pathologic diagnosis and grading guideline of common pathological
changes after liver transplantation (II). Prac J Organ Transplant. (2015) 3:1–7. 6. Xu XW, Wu XX, Jiang XG, Xu KJ, Ying LJ, Ma CL, et al. Clinical findings
in a group of patients infected with the 2019 novel coronavirus (SARS-Cov-
2) outside of Wuhan, China: retrospective case series. BMJ. (2020) 368:m606. doi: 10.1136/bmj.m606 18. Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Scheuer PJ. Classification of
chronic hepatitis: diagnosis, grading and staging. Hepatology. (1994) 19:1513–
20. doi: 10.1002/hep.1840190629 7. Shi H, Han X, Jiang N, Cao Y, Alwalid O, Gu J, et al. Radiological findings
from 81 patients with COVID-19 pneumonia in Wuhan, China: a descriptive
study. Lancet Infect Dis. (2020) 20:425–34. doi: 10.1016/S1473-3099(20)
30086-4 19. Scheuer PJ. Classification of chronic viral hepatitis: a need for reassessment. J
Hepatol. (1991) 13:372–4. doi: 10.1016/0168-8278(91)90084-O 8. Guan WJ, Ni ZY, Hu Y, Liang WH, Ou CQ, He JX, et al. Clinical characteristics
of Coronavirus Disease 2019 in China. N Engl J Med. (2020) 382:1708–20. doi: 10.1056/NEJMoa2002032 20. Chai X, Hu L, Zhang Y, Han W, Lu Z, Ke A, et al. Specific ACE2 expression
in cholangiocytes may cause liver damage after 2019-nCoV infection. bioRxiv. (2020) doi: 10.1101/2020.02.03.931766 9. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of
patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. (2020) 395:497–506. doi: 10.1016/S0140-6736(20)30183-5 21. Yang L, Wang W, Wang X, Zhao J, Xiao L, Gui W, et al. Creg in hepatocytes
ameliorates liver ischemia/reperfusion injury in a TAK1-dependent manner
in mice. Hepatology. (2019) 69:294–313. doi: 10.1002/hep.30203 10. Zhang B, Zhou X, Qiu Y, Song Y, Feng F, Feng J, et al. Clinical characteristics
of 82 cases of death from COVID-19. PLoS ONE. (2020) 15:e0235458. FUNDING Hospital, Tongji Medical College, Huazhong University of
Science and Technology. The next of kin of each enrolled
patient/participant provided their written informed consent
to
participate
in
this
study. Written
informed
consent
was obtained from the individual(s) for the publication
of any potentially identifiable images or data included in
this article. The study was supported by Urgent projects of scientific
and technological research on COVID-19 funded by Hubei
province (No. 2020FCA014 to XH), Key Special Projects of the
Hubei Provincial Department of Science and Technology (No. 2019ACA133 to LY), National Natural Science Foundation of
China (No. 82072333 to XN), and the China Postdoctoral Science
Foundation (No. 2020M670289 and 2020T130065ZX to JZ). SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fmed. 2021.749318/full#supplementary-material ACKNOWLEDGMENTS HC: conceptualization, methodology, validation, formal analysis,
investigation, and writing—original draft. LP: methodology,
validation, formal analysis, investigation, and visualization. LH
and JZ: methodology, validation, and formal analysis. YZ:
methodology, formal analysis, and language correction. HS:
formal analysis, investigation, and visualization. LYao and QZ:
methodology, validation, formal analysis, and investigation. XN, LYan, and XH: conceptualization, investigation, resources,
writing—review and editing, and supervision. All authors
contributed to the article and approved the submitted version. The authors appreciate the language correction and technical
support from Dr. Brant Wang from Department of Pathology,
Inova Fairfax Hospital, 3300 Gallows Road, Falls Church,
VA, USA. ETHICS STATEMENT The studies involving human participants were reviewed
and approved by the Institutional Ethics Board of Union October 2021 | Volume 8 | Article 749318 7 Liver Analysis of COVID-19 Patients Chu et al. Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. 25. Ji D, Qin E, Xu J, Zhang D, Cheng G, Wang Y, et al. Non-alcoholic fatty liver
diseases in patients with COVID-19: a retrospective study. J Hepatol. (2020)
73:451–3. doi: 10.1016/j.jhep.2020.03.044 24. Cai Q, Huang D, Yu H, Zhu Z, Xia Z, Su Y, et al. COVID-19: abnormal liver
function tests. J Hepatol. (2020) 73:566–74. doi: 10.1016/j.jhep.2020.04.006 23. Marty FM, Vidal-Puigserver J, Clark C, Gupta SK, Merino E, Garot
D, et al. Intravenous zanamivir or oral oseltamivir for hospitalised
patients
with
influenza:
an
international,
randomised,
double-blind,
double-dummy, phase 3 trial. Lancet Respir Med. (2017) 5:135–46.
doi: 10.1016/S2213-2600(16)30435-0 Frontiers in Medicine | www.frontiersin.org REFERENCES doi: 10.1371/journal.pone.0235458 22. Yancheva N, Tzonev R. A case of late presentation of darunavir-
related
cholestatic
hepatitis. Int
J
STD
AIDS. (2019)
30:620–2. doi: 10.1177/0956462419826723 October 2021 | Volume 8 | Article 749318 Frontiers in Medicine | www.frontiersin.org 8 Liver Analysis of COVID-19 Patients Chu et al. Publisher’s Note: All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations, or those of
the publisher, the editors and the reviewers. Any product that may be evaluated in
this article, or claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher. 23. Marty FM, Vidal-Puigserver J, Clark C, Gupta SK, Merino E, Garot
D, et al. Intravenous zanamivir or oral oseltamivir for hospitalised
patients
with
influenza:
an
international,
randomised,
double-blind,
double-dummy, phase 3 trial. Lancet Respir Med. (2017) 5:135–46. doi: 10.1016/S2213-2600(16)30435-0 24. Cai Q, Huang D, Yu H, Zhu Z, Xia Z, Su Y, et al. COVID-19: abnormal liver
function tests. J Hepatol. (2020) 73:566–74. doi: 10.1016/j.jhep.2020.04.006 Copyright © 2021 Chu, Peng, Hu, Zhu, Zhao, Su, Yao, Zhu, Nie, Yang and Hou. This is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) and the copyright owner(s) are credited
and that the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted which does not
comply with these terms. 25. Ji D, Qin E, Xu J, Zhang D, Cheng G, Wang Y, et al. Non-alcoholic fatty liver
diseases in patients with COVID-19: a retrospective study. J Hepatol. (2020)
73:451–3. doi: 10.1016/j.jhep.2020.03.044 Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. October 2021 | Volume 8 | Article 749318 Frontiers in Medicine | www.frontiersin.org 9
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Evolving evidence in the treatment of primary and recurrent posterior cruciate ligament injuries, part 2: surgical techniques, outcomes and rehabilitation
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Knee surgery, sports traumatology, arthroscopy
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Knee Surgery, Sports Traumatology, Arthroscopy (2021) 29:682–693
https://doi.org/10.1007/s00167-020-06337-2 Knee Surgery, Sports Traumatology, Arthroscopy (2021) 29:682–693
https://doi.org/10.1007/s00167-020-06337-2 KNEE KNEE Philipp W. Winkler1,2 · Bálint Zsidai3 · Nyaluma N. Wagala2 · Jonathan D. Hughes2 · Alexandra Horvath4 ·
Eric Hamrin Senorski5 · Kristian Samuelsson3,6 · Volker Musahl2 Received: 16 September 2020 / Accepted: 13 October 2020 / Published online: 30 October 2020
© The Author(s) 2020 Evolving evidence in the treatment of primary and recurrent posterior
cruciate ligament injuries, part 2: surgical techniques, outcomes
and rehabilitation Philipp W. Winkler1,2 · Bálint Zsidai3 · Nyaluma N. Wagala2 · Jonathan D. Hughes2 · Alexandra Horvath4 ·
Eric Hamrin Senorski5 · Kristian Samuelsson3,6 · Volker Musahl2 Philipp W. Winkler1,2 · Bálint Zsidai3 · Nyaluma N. Wagala2 · Jonathan D. Hughes2 · Alexandra Horvath4 ·
Eric Hamrin Senorski5 · Kristian Samuelsson3,6 · Volker Musahl2 Introduction displayed a shift in favor of operative treatment [17, 61,
79]. Indications for operative treatment may be based on
increased translational and rotational tibial movement
compared to contralateral PCL intact knees (as measured
on in vivo kinematic analyses) [43]. An increasing number
of intra-articular injuries (meniscus, cartilage), pathologi-
cal changes of the anterior cruciate ligament (ACL), and
an increased joint contact pressure (tibiofemoral and patel-
lofemoral) have been observed in PCL deficient patients
[20]. Posterior cruciate ligament reconstruction (PCL-R) tech-
niques have been studied and evolved over the past dec-
ades, providing a solid and evidence-based foundation for
the operative management of posterior cruciate ligament
(PCL) injuries [1, 39, 41, 45, 77, 88]. Single-bundle (SB)
and double-bundle (DB) PCL-R based on the transtibial or
tibial inlay technique and implemented by an open, arthro-
scopically assisted, or all-arthroscopic approach, all offer
certain advantages and disadvantages [1, 8, 15, 16, 34, 40,
45, 75, 77, 87, 89]. On the other hand, the PCL is charac-
terized by a strong intrinsic healing capability, making the
non-operative treatment approach a viable option, espe-
cially for partial PCL tears and tibial avulsion injuries of
the PCL [2, 3, 26, 71, 72]. Good subjective and objective
long-term outcomes after non-operative treatment with a
fairly low prevalence of 11% of moderate to severe osteo-
arthritis (OA) after more than 14 years follow-up, keep the
debate about the optimal treatment approach, operative
vs. non-operative, in PCL injured patients ongoing [71]. As a result, a wide range of viable treatment options is
available, enabling an individualized treatment approach
based on the injury pattern and the patient’s compliance
and demands. A failure rate of 1–25% after primary PCL-R is reported,
mounting to 45% if unfavorable patient-reported outcomes
(Knee Injury and Osteoarthritis Outcome Score < 40 points)
are considered as subjective failure [4, 36, 41, 46, 81, 88]. However, inadequate reporting and varying definitions of
failures require caution in the interpretation of failure rates. Debilitating pain and functional impairment during daily
activities combined with a PTT of ≥ 10 mm or confirmed
graft failure based on magnetic resonance imaging (MRI)
scans are accepted indications for revision PCL-R [58, 59]. The aim of revision PCL-R is to address the cause of initial
failure and eliminate concurrent pathological changes caus-
ing pain and instability [41, 59]. Introduction Therefore, in case of revi-
sion PCL-R, extended diagnostic work-up including MRI,
hip-knee-ankle radiographs, and computed tomography (CT)
scans are required to assess concomitant injuries, lower limb
alignment, and prior bone tunnel placement. The require-
ment of bone grafting due to prior semi-anatomic bone
tunnels or the need for corrective osteotomies sometimes
require staged revision PCL-R. In this review, we present the indications, techniques,
and outcomes of operative and non-operative treatment of
primary and recurrent PCL injuries. Moreover, risk fac-
tors associated with recurrent PCL deficiency and future
perspectives are outlined. Abstract Isolated and combined posterior cruciate ligament (PCL) injuries are associated with severe limitations in daily, professional,
and sports activities as well as with devastating long-term effects for the knee joint. As the number of primary and recurrent
PCL injuries increases, so does the body of literature, with high-quality evidence evolving in recent years. However, the
debate about the ideal treatment approach such as; operative vs. non-operative; single-bundle vs. double-bundle reconstruc-
tion; transtibial vs. tibial inlay technique, continues. Ultimately, the goal in the treatment of PCL injuries is restoring native
knee kinematics and preventing residual posterior and combined rotatory knee laxity through an individualized approach. Certain demographic, anatomical, and surgical risk factors for failures in operative treatment have been identified. Failures
after PCL reconstruction are increasing, confronting the treating surgeon with challenges including the need for revision PCL
reconstruction. Part 2 of the evidence-based update on the management of primary and recurrent PCL injuries will summarize
the outcomes of operative and non-operative treatment including indications, surgical techniques, complications, and risk
factors for recurrent PCL deficiency. This paper aims to support surgeons in decision-making for the treatment of PCL injuries
by systematically evaluating underlying risk factors, thus preventing postoperative complications and recurrent knee laxity. Level of evidence V. y
Transtibial · Tibial inlay Abbreviations
ACL
Anterior cruciate ligament
ALB
Anterolateral bundle
AP
Anterior–posterior
DB
Double-bundle
IKDC
International Knee Documentation Committee
MCL
Medial collateral ligament
OA
Osteoarthritis
PCL
Posterior cruciate ligament
PCL-R
Posterior cruciate ligament reconstruction
PMB
Posteromedial bundle
PMC
Posteromedial corner
PTS
Posterior tibial slope
PTT
Posterior tibial translation
ROM
Range of motion
RTS
Return-to-sports
SB
Single-bundle
* Philipp W. Winkler
ph.winkler@tum.de
1
Department for Orthopaedic Sports Medicine, Klinikum
Rechts der Isar, Technical University of Munich, Ismaninger
Str. 22, 81675 Munich, Germany
2
Department of Orthopaedic Surgery, UPMC Freddie Fu
Sports Medicine Center, University of Pittsburgh, 3200
S. Water St., Pittsburgh, PA 15203, USA
3
Department of Orthopaedics, Sahlgrenska University
Hospital, Mölndal, Sweden
4
Department of Internal Medicine and Clinical Nutrition,
Institute of Medicine, Sahlgrenska Academy, University
of Gothenburg, Gothenburg, Sweden
5
Department of Health and Rehabilitation, Institute
of Neuroscience and Physiology, Sahlgrenska Academy,
University of Gothenburg, Gothenburg, Sweden
6
Department of Orthopaedics, Institute of Clinical Sciences,
Sahlgrenska Academy, University of Gothenburg,
Gothenburg, Sweden * Philipp W. Winkler
ph.winkler@tum.de Vol:1 .(123456789
3 Knee Surgery, Sports Traumatology, Arthroscopy (2021) 29:682–693 683 Indications for PCL‑R Owing to the anatomy of the PCL and the complex nature of
PCL injuries, there is no consensus for a specific operative
technique when considering PCL-R. Many biomechanical
and clinical research efforts have enabled the development
of techniques focusing on anatomic restoration of native
knee kinematics [28, 66, 85]. Variations between the dif-
ferent reconstructive techniques depend primarily on tun-
nel placement, graft choice, graft positioning and fixation,
and the choice of an arthroscopic or open surgical approach
(Fig. 1). While studies have reported that both SB and DB
PCL-R improve knee kinematics, biomechanical results have
recently shown DB PCL-R to closer approximate the native
state of the ligament [15, 23, 85]. Operative treatment of PCL injuries is indicated for
patients with symptomatic grade III (complete) tears dis-
playing inadequate functional improvement in response to
non-operative treatment. Furthermore, patients with PCL
injuries with high-grade knee laxity, or combined with
intraarticular or capsuloligamentous injuries should be
considered for operative treatment [55, 79]. A side-to-side
difference in posterior tibial translation (PTT) greater than
8 mm revealed by stress-radiography indicates a complete
PCL tear and presents an indication for operative treat-
ment for symptomatic patients [29, 68, 69]. Additionally,
the patient’s demands are essential in treatment decision-
making, leading to the recommendation of PCL-R in an
athletic population [39]. There is currently insufficient
evidence in the literature to support a definitive treatment
protocol; however, the historical preference towards non-
operative treatment of isolated PCL injuries has recently The focus of anatomic SB PCL-R is to restore knee
kinematics by aligning the path of the PCL graft with the
native course of the stronger and more prominent anterolat-
eral bundle (ALB). On the other hand, DB PCL-R aims to
provide an alternative method by restoring the synergistic 1 3 Knee Surgery, Sports Traumatology, Arthroscopy (2021) 29:682–693 684 onality of the ALB and posteromedial bundle (PMB)
ser approximate the native anatomy [5, 79]. bial graft fixation during anatomic SB and DB PCL-R
e performed using the transtibial or the tibial inlay
ique by an all-arthroscopic, arthroscopic assisted,
or an open approach [8, 42, 62, 76]. Single‑bundle (SB) vs. double‑bundle (DB) Biomechanical studies have demonstrated that only anatomic
DB PCL-R is able to restore native knee laxity across the
entire range-of-motion and that SB PCL-R leads to residual
posterior laxity [31, 32, 66]. Consequently, several clinical
studies have advocated DB PCL-R and have demonstrated
improved clinical and functional outcomes compared to SB
PCL-R [15, 36, 45]. A randomized controlled trial compar-
ing isolated SB (n = 22) and DB (n = 24) allograft PCL-R
reported significant improvements in patient-reported
outcomes (International Knee Documentation Committee
(IKDC) Subjective Knee Form, Lysholm Score, Tegner
Activity Scale) and reduced knee laxity (KT-1000) for both
techniques with a minimum of two-years of follow-up [45]. The DB group showed superior results in knee laxity as well
as objective and subjective IKDC compared to the SB group
[45]. Another randomized controlled trial showed similar
results with significantly less residual PTT measured on
posterior stress radiographs in patients undergoing isolated
DB (n = 28) PCL-R compared to SB (n = 25) PCL-R [88]. However, the authors questioned the clinical relevance of
the statistically significant difference of 1.4 mm between
the groups [88]. Accordingly, no difference in clinical and
radiological outcomes, failure and survival rates between
SB (n = 28) and DB (n = 36) PCL-R could be observed in
long-term follow-up (minimum 10 years) [89]. The tibial inlay techniques have been developed to
facilitate graft fixation by employing bone troughs for tib-
ial graft insertion and thereby restoring the original PCL
anatomy while diminishing PCL graft stress by avoiding the
killer-turn effect [8]. The open or arthroscopically assisted
approach to the tibial inlay technique requires the patient to
be positioned in the lateral decubitus or prone position, or
in supine position if the hip and knee are freely movable. After careful dissection, the bone block of the graft can be
placed in the created trough and a screw is used for tibial
graft fixation [8, 42].i i
Posterior cruciate ligament graft fixation is usually per-
formed using interference screws, or suspensory fixation
techniques [14, 21, 60, 79]. To optimize graft loads and to
restore native knee kinematics, the ideal knee flexion angle
during graft fixation is important [24, 31, 32]. Consequently,
it has been demonstrated that graft fixation angles ranging
from 75° to 105° of knee flexion restore knee kinematics to
the same extent after SB PCL-R, albeit failing to restore lax-
ity compared to the intact state [31]. Indications for PCL‑R Positioning of a PCL
drill guide medial to the tibial tubercle just proximal to the
pes anserinus, aiming for a sagittal angle of 45°, allows
subsequent insertion of a guidewire to reach the anatomic
tibial PCL insertion zone when performing the transtibial
Schematic illustration of
nstibial and tibial inlay
or cruciate ligament
truction techniques. -bundle (a) and double-
(b) transtibial technique
knee, anterior view). -bundle (c) and double-
(d) tibial inlay technique
knee, posterior view) Fig. 1 Schematic illustration of
the transtibial and tibial inlay
posterior cruciate ligament
reconstruction techniques. Single-bundle (a) and double-
bundle (b) transtibial technique
(right knee, anterior view). Single-bundle (c) and double-
bundle (d) tibial inlay technique
(right knee, posterior view) Fig. 1 Schematic illustration of
the transtibial and tibial inlay
posterior cruciate ligament
reconstruction techniques. Single-bundle (a) and double-
bundle (b) transtibial technique
(right knee, anterior view). Single-bundle (c) and double-
bundle (d) tibial inlay technique
(right knee, posterior view) functionality of the ALB and posteromedial bundle (PMB)
to closer approximate the native anatomy [5, 79].i or an open approach [8, 42, 62, 76]. Positioning of a PCL
drill guide medial to the tibial tubercle just proximal to the
pes anserinus, aiming for a sagittal angle of 45°, allows
subsequent insertion of a guidewire to reach the anatomic
tibial PCL insertion zone when performing the transtibial functionality of the ALB and posteromedial bundle (PMB)
to closer approximate the native anatomy [5, 79].i Tibial graft fixation during anatomic SB and DB PCL-R
can be performed using the transtibial or the tibial inlay
technique by an all-arthroscopic, arthroscopic assisted, Tibial graft fixation during anatomic SB and DB PCL-R
can be performed using the transtibial or the tibial inlay
technique by an all-arthroscopic, arthroscopic assisted, 1 3 1 3 Knee Surgery, Sports Traumatology, Arthroscopy (2021) 29:682–693 685 than 1.5 mm has to be questioned [6, 7, 44, 80]. The use
of allografts was associated with a shorter operation time,
while autografts were associated with donor-site morbidity. With respect to patient-reported outcomes and graft failure
rates, no difference between allografts and autografts for
PCL-R could be demonstrated [6, 7]. Therefore, no appreci-
able difference seems to exist between the usage of allog-
enous or autologous tissue for PCL-R [25, 46, 82]. Indications for PCL‑R technique (a more detailed description of radiographic land-
marks for the native femoral and tibial insertion zones of
the PCL is given in Part 1 of the evidence-based update
on the management of primary and recurrent PCL injuries)
[18, 74]. A frequently reported drawback of the transtibial
technique is the formation of an acute angle by the PCL graft
exiting the tibial tunnel, also known as the “killer turn” [53]. Graft degeneration, abrasion, and delayed or incomplete
graft maturation are attributed to the killer-turn effect, which
is believed to cause residual posterior laxity and increase the
failure rate after transtibial PCL-R [8, 39, 84]. Protection of
the posterior neurovascular bundle is of utmost importance. One study has shown that the mean distance between the
popliteal artery and the posterior tibial cortex 5 mm distal
to the joint line was significantly greater at 90° compared
to 0° of knee flexion (7.7 ± 3.8 mm vs. 1.6 ± 1.3 mm) [33]. Accordingly, tibial tunnel drilling with fluoroscopic guid-
ance is recommended at 90° knee flexion to prevent injury
of the neurovascular structures. Tibial inlay vs. transtibial Failures after primary PCL-R are reported to cause severe
impairments in daily living and are a burdening condition
for most patients [41, 58, 59, 81]. In such cases, a revision
PCL-R is required and is sometimes considered as a salvage
procedure. The rate of revision surgery after isolated PCL-R
is reported to be 3% and slightly higher (3.4%) after com-
bined PCL-R [46]. However, only a few studies and case
reports have reported on the outcomes of revision PCL-R
[16, 41, 42, 47, 59, 81]. In one study, revision PCL-R using
quadriceps tendon-bone autografts in a DB technique has
significantly improved patient-reported outcomes, activities
of daily living, sports activity level, occupational rate, and
PTT based on posterior stress radiographs in 15 patients
after a mean follow-up of 44 months [59]. Two of the sub-
jects underwent revision PCL-Rs that failed a second time
[59]. A recently published study has reported similar results
after 22 revision PCL-Rs using Achilles tendon allografts
with a DB tibial inlay technique [41]. Patient-reported out-
comes and objective evaluation increased significantly while
PTT, based on posterior stress radiographs as well as knee
laxity (measured by KT-1000 arthrometry), decreased sig-
nificantly after a mean follow-up of 40 months. Addition-
ally, based on the Tegner Activity Scale, 77% of patients
undergoing revision PCL-R were able to return to normal
activities of daily living [41]. However, research has shown
that 46% of failed PCL operative procedures are not amena-
ble to revision PCL-R mostly due to advanced degenerative
changes and OA [58]. Given that revision PCL-R is inher-
ently related to an alteration of the native anatomy and the
bony landmarks due to the primary PCL-R, revision surger-
ies represent more challenging procedures [59]. Therefore,
it is recommended to perform a comprehensive diagnostic
work-up including patient history, thorough clinical exami-
nation, gait analysis, AP, lateral, and weight-bearing hip-
knee-ankle radiographs as well as MRI and CT scans prior
to considering revision PCL-R. Consequently, risk factors
related to failures in the operative treatment of PCL injuries
can be assessed to facilitate treatment decision-making [58,
59] In the early 1990s, a new technique for tibial graft fixation in
PCL-R, termed the tibial inlay technique, was introduced [8,
27]. The tibial inlay technique was subsequently advocated
to prevent increased graft stress, degeneration, and abrasion
caused by the so-called ‘’killer turn’’ at the proximal tibial
tunnel aperture in transtibial PCL-R [8]. Single‑bundle (SB) vs. double‑bundle (DB) In the case of DB PCL-
R, PMB graft fixation at 0° and ALB graft fixation at 90°
or 105° of knee flexion best restores native knee kinematics
while avoiding excessive restriction of the tibiofemoral joint
[32]. Recently, remnant preservation in SB and DB transti-
bial PCL-R has gained increasing interest. Preservation of
the remnant PCL fibers and meniscofemoral ligaments is
believed to provide graft protection by stabilizing the graft
and additionally reducing the killer-turn effect by cushioning
the graft at the proximal aperture of the tibial tunnel [38, 39,
78, 88]. Therefore, improved graft healing and maturation
is assumed by enhanced revascularization and regenera-
tion of mechanoreceptors [38, 39]. In one study, improved
patient-reported outcomes, extensor and flexor muscle peak
torque, functional performance, and decreased PTT, based
on posterior sagittal stress radiographs, have been reported
in 52 isolated SB transtibial PCL-R with remnant preserva-
tion after a mean follow-up duration of 30 months [39]. In Graft choice is crucial in PCL-R and includes allografts
and autografts as well as soft tissue-only, bone-tendon, and
bone-tendon-bone grafts. While soft tissue-only grafts are
often preferred for the transtibial technique, tendon-bone
grafts are usually used when performing the tibial inlay tech-
nique [8, 42, 67]. Less residual PTT has been demonstrated
when using autografts compared to allografts for PCL-R [6,
7]. Although a statistically significant difference between
autograft and allograft use was demonstrated, the clinical
relevance of a mean side-to-side difference in PTT of less 1 Knee Surgery, Sports Traumatology, Arthroscopy (2021) 29:682–693 686 this study cohort, acute PCL-R (mean time from injury to
surgery, 2.4 months) enabled remnant preservation, which
resulted in normal graft appearance (signal intensity, gross
appearance) in almost 79% of patients as assessed by postop-
erative MRI (mean time from surgery to MRI, 15.8 months)
[39]. Similar results have also been shown for combined
PCL and posterolateral corner reconstruction [38]. for remnant preservation [40, 70]. However, the tibial inlay
technique demonstrates advantages for the treatment of chronic
PCL injuries and for the increasing number of revision PCL-
Rs [8, 41, 42, 59]. Consequently, future research should focus
on identifying specific indications for each technique and thus
facilitate surgical decision-making. Tibial inlay vs. transtibial However, a recently
published study reported that remnant preservation in acute
transtibial PCL-R enables to avoid the negative influence of
the killer turn by a cushioning effect of the remnant PCL fib-
ers [39]. Unfortunately, remnant preservation is not possible
in chronic PCL deficiency and revision PCL-R, and, there-
fore, the tibial inlay technique has been suggested as a viable
treatment alternative for such cases [41, 42, 59]. In spite of
a biomechanically confirmed superiority of the tibial inlay
technique compared to the transtibial technique in terms of
residual posterior tibial laxity and graft degeneration [9], this
is not translated into clinical outcomes [40, 50, 70, 75]. Since
research has shown that there is no significant correlation
between residual posterior tibial laxity and patient-reported
outcomes, the biomechanically suggested superiority of the
tibial inlay compared to the transtibial technique needs to be
questioned [64, 73]. In one study, the tibial inlay technique
was compared to the transtibial technique in 66 isolated PCL-
Rs at a mean follow-up of 148 months [77]. Patients under-
going isolated tibial inlay PCL-R (n = 30) with bone-patellar
tendon-bone autograft showed no difference in postoperative
patient-reported outcomes (Lysholm Score and Tegner Activ-
ity Scale), manual laxity testing (posterior drawer test at 90°
knee flexion), instrumented posterior laxity testing (stress
radiographs at 90° knee flexion), and progression of OA com-
pared to patients undergoing transtibial PCL-R (n = 36) with
hamstring tendon autograft [77]. Consistently, most studies
report statistically significant improvement in clinical and
functional outcomes postoperatively compared to preopera-
tively [50, 70, 77]. However, regardless of the performed tibial
fixation technique, considerable rates of residual posterior
tibial laxity are reported. One comparative study has shown
that 46% and 57% of patients undergoing transtibial and tibial
inlay PCL-R, respectively, reported residual episodes of sub-
jective instability [50]. On the other hand, the all-arthroscopic
transtibial technique is surgically less demanding, avoids an
invasive surgical approach, has a reduced operation time, has
a lower risk of complications, and allows for the possibility 1 3 3 3 687 Knee Surgery, Sports Traumatology, Arthroscopy (2021) 29:682–693 Failure analysis and survivorship in PCL‑R replacement, and PCL thermoplasty) reported that 71% of
patients complained of moderate to severe pain during daily
activities, 49% described their own knee condition as poor,
and 75% have completely quit sports activities after a mean
time of 42 months after the failed operative PCL treatment
[58]. However, synthetic graft replacement and thermoplasty
as a treatment for PCL injuries have been abandoned, which
needs to be considered when interpreting the reported poor
results. To avoid the detrimental effects of failed PCL-R, it
is essential to be aware of the risk factors and understand Unlike in ACL reconstructive procedures, less is known
about risk factors and causes of failure in the treatment of
PCL injuries [16, 41, 58, 81]. Failed operative treatment of
isolated and combined PCL injuries is associated with severe
limitations in daily, professional, and sports activities [58,
59, 81]. One study investigating 52 failed operative PCL pro-
cedures (including SB PCL-R, PCL repair, synthetic graft Fig. 2 Posterior cruciate ligament graft failure. Patient with atrau-
matic PCL graft failure of the right knee. T2-weighted sagittal (a) and
axial (b) MR images showing PCL graft failure and misplaced tibial
tunnel (too anterior and too proximal). Note scarring of remnant PCL
fibers imitating PCL continuity (white arrows). Arthroscopic images
demonstrating graft failure (c), misplaced tibial tunnel (d), and revi-
sion PCL graft (e). Postoperative anterior–posterior (f) and lateral
(g) radiographs demonstrating new anatomic tibial tunnel. wh
black dashed lines, misplaced tibial tunnel; yellow dashed lines, n
anatomic tibial tunnel; black arrow, new anatomic tibial tunnel d
ing revision PCL reconstruction; *deficient PCL graft, ACL ante
cruciate ligament, MFC medial femoral condyle, MR magnetic re
nance, PCL posterior cruciate ligament (g) radiographs demonstrating new anatomic tibial tunnel. white/
black dashed lines, misplaced tibial tunnel; yellow dashed lines, new
anatomic tibial tunnel; black arrow, new anatomic tibial tunnel dur-
ing revision PCL reconstruction; *deficient PCL graft, ACL anterior
cruciate ligament, MFC medial femoral condyle, MR magnetic reso-
nance, PCL posterior cruciate ligament Fig. 2 Posterior cruciate ligament graft failure. Patient with atrau-
matic PCL graft failure of the right knee. T2-weighted sagittal (a) and
axial (b) MR images showing PCL graft failure and misplaced tibial
tunnel (too anterior and too proximal). Note scarring of remnant PCL
fibers imitating PCL continuity (white arrows). Arthroscopic images
demonstrating graft failure (c), misplaced tibial tunnel (d), and revi-
sion PCL graft (e). Failure analysis and survivorship in PCL‑R Postoperative anterior–posterior (f) and lateral 1 3 Knee Surgery, Sports Traumatology, Arthroscopy (2021) 29:682–693 688 [15, 16, 36, 59, 67, 70, 77, 81, 87, 90]. Iatrogenic injuries
to the neurovascular structures of the posterior part of the
knee represent the most dreaded complications in PCL-R. The occurrence is rare, yet several case reports about pop-
liteal artery lacerations, occlusions, and also popliteal vein
injuries have been published [51, 57, 86]. Precise anatomic
knowledge and fluoroscopically guided tibial tunnel drilling
in 90° knee flexion may help to prevent iatrogenic neurovas-
cular injuries [33, 54]. In the setting of revision PCL-R, scar
formation has to be considered, which may alter the natural
course of the neurovascular structures. the underlying causes of failure. In more than 50% of failed
PCL-R, multiple factors for failure have been identified [16,
41, 58]. Posterolateral corner deficiency and femoral or tibial
tunnel misplacement have been shown to be the most com-
mon causes of failure, accounting for 40–77% and 33–41%,
respectively (Fig. 2) [41, 58]. An incorrect tunnel placement
is characterized by a too proximal (deep) and posterior (low)
position on the femoral site or a too anterior and proximal
position on the tibial site, resulting in a vertical position of
the graft [56, 58]. Further risk factors are varus malalign-
ment, primary PCL suture repair, biological and technical
failures, wrong surgical decision-making, a too early or too
progressive rehabilitation protocol, and a low annual volume
of PCL-Rs performed by the operating surgeon [16, 58, 81]. the underlying causes of failure. In more than 50% of failed
PCL-R, multiple factors for failure have been identified [16,
41, 58]. Posterolateral corner deficiency and femoral or tibial
tunnel misplacement have been shown to be the most com-
mon causes of failure, accounting for 40–77% and 33–41%,
respectively (Fig. 2) [41, 58]. An incorrect tunnel placement
is characterized by a too proximal (deep) and posterior (low)
position on the femoral site or a too anterior and proximal
position on the tibial site, resulting in a vertical position of
the graft [56, 58]. Further risk factors are varus malalign-
ment, primary PCL suture repair, biological and technical
failures, wrong surgical decision-making, a too early or too
progressive rehabilitation protocol, and a low annual volume
of PCL-Rs performed by the operating surgeon [16, 58, 81]. Failure analysis and survivorship in PCL‑R A meta-analysis reported that the incidence of periop-
erative complications is 1.7 times higher for the tibial inlay
compared to the transtibial technique. However, no statisti-
cally significant difference was noted [40]. The open tibial
inlay technique requires careful dissection of the popliteal
fossa and poses the risk of neurovascular injuries [8, 42,
81]. Consequently, all-arthroscopic tibial inlay techniques
have evolved and have been shown to be biomechanically
comparable to an open tibial inlay technique [91]. Additional
reported complications related to the tibial inlay technique
include fractures of the tibial bone plug during graft fixation
[34]. Double-bundle PCL-R is surgically challenging and
requires precise knowledge of anatomy and bony landmarks
for accurate tunnel placement [5, 56]. One study reported
a fracture of the separating bony bridge between the ALB
and PMB femoral tunnel in DB PCL-R [87]. Furthermore,
the tibial inlay and DB techniques are associated with a sig-
nificantly longer operation time compared to the transtibial
and SB techniques, increasing the overall risk of peri- and
postoperative complications [45, 70]. p
y
p
g
g
Recently, a biomechanical study has shown an inverse
correlation between the graft force after PCL-R and the pos-
terior tibial slope (PTS). Irrespective of the loading condi-
tion and the knee flexion angle, a flatter (reduced) PTS leads
to increasing PCL graft forces [10]. Clinically, a significant
negative correlation between PTS and residual PTT meas-
ured on posterior stress radiographs and a significant posi-
tive correlation between PTS and the reduction of PTT from
pre- to postoperative has been shown for SB PCL-R [22]. However, this observation has not been demonstrated for
DB PCL-R [11]. Additionally, a significantly lower PTS has
been observed in patients undergoing primary PCL-R com-
pared to sex- and age-matched controls without ligamentous
injury (6° vs. 9°) [12]. Analysis of the injury mechanism
revealed that the PTS was significantly lower in non-contact
PCL injuries compared to contact injuries (5° vs. 6°) [12],
highlighting the impact of the PTS on AP laxity. At 15 years follow-up with graft failure as the endpoint
(need for revision PCL-R, high tibial osteotomy, arthro-
plasty, complete graft tear based on MRI, or > 10 mm
side-to-side difference in PTT based on posterior stress
radiographs), the survival rates have been reported to be
approximately 82% and 84% for SB (n = 28) and DB (n = 36)
Achilles tendon allograft PCL-R, respectively [89]. Non‑operative treatment, rehabilitation,
and return‑to‑sports (RTS) Within the past 10 years (2010s), studies have consistently
supported non-operative treatment for isolated grade I, grade
II, and nondisplaced tibial avulsion PCL injuries [52, 63,
65, 71, 72]. There continues to be a debate regarding the
management of isolated grade III injuries as there is limited
data on the outcomes following non-operative treatment. A
prospective cohort study in high-level athletes with grade II
(n = 25) and grade III (n = 21) isolated acute PCL injuries
showed that approximately 83% of athletes were able to par-
ticipate at a competitive sports level (mean Tegner Activity
Scale, 9) after non-operative treatment at an average follow-
up period of 5 years [2]. In addition, an epidemiological
study demonstrated a median lay-off time of 31 days after
PCL injury for professional male soccer players. However,
these prospectively collected data in men’s professional soc-
cer included all grades of PCL injuries as well as opera-
tively and non-operatively treated athletes [48]. Accordingly, Complications in PCL‑R However, it has been shown that non-
operative treatment leads to significantly more subsequent
meniscal injuries as well as a higher rate of OA and a higher
conversion rate to total knee arthroplasty compared to opera-
tive treatment [83]. exercises are performed in the prone position to minimize
hamstring activity and to counteract the gravity-induced pos-
terior tibial sag [2, 35, 65]. The following weeks are accom-
panied by advancement to full weight-bearing with strong
emphasis on quadriceps strengthening. Jogging and sport-
specific exercises are often initiated in the sixth postopera-
tive month. Full ROM, quadriceps strength, and a firm end-
point in the posterior drawer test are required before return
to cutting and pivoting sports [35, 65]. This can take up to
12 months, however, quicker recovery with return to sports
at 16 weeks has been reported in high-level athletes [2]. Following PCL-R, weight-bearing as tolerated with a
knee brace providing posterior tibial support and locked in
full extension is recommended for the first 3–6 weeks, fol-
lowed by functional bracing for up to 6 months, to promote
healing and prevent a fixed posterior tibial subluxation [19,
35, 65]. The authors’ recommendation for non-operative
treatment and postoperative rehabilitation is illustrated in
Fig. 3. Future research It is unknown which demographic, surgical, or patient-
related factors reliably predict clinical and functional out-
comes for both operative and non-operative treatment. Since
non-operative treatment may yield satisfactory outcomes,
an initial non-operative treatment of PCL injuries with an
optional delayed PCL-R is often recommended. However,
the optimal timing of operative PCL-R (early vs. delayed
surgery) as well as the most appropriate timing of post-
injury/postoperative rehabilitation (early vs. delayed) is not
sufficiently supported by high-quality evidence, which is
even more pronounced in combined PCL injuries. Therefore,
a prospective randomized multi-center clinical trial—Sur-
gical Timing and Rehabilitation (STaR) Trial for Multiple
Ligament Knee Injuries—is currently ongoing to provide
evidence for the optimal timing of operative treatment and
non-operative/postoperative rehabilitation [49]. Rehabilitation protocols whether for non-operative treat-
ment or postoperative care, are inconsistently reported in
the literature [65]. Agreement exists in the combination of
temporary immobilization/bracing and exercise therapy. Accordingly, appropriate stabilization by initial static and
later functional bracing accompanied by progressive exer-
cise therapy is important, whether post-injury or postopera-
tively, to support the healing process of the PCL [3, 26, 30]. A dynamic anterior drawer brace facilitates end-to-end con-
tact between the torn PCL fibers by applying an anteriorly
directed force along the proximal tibia [37]. Studies dem-
onstrated a reduction of PTT based on instrumented laxity
measurement following non-operative treatment using static
and dynamic braces with posterior tibial support [26, 30]. Initially, partial weight-bearing is recommended and ROM Complications in PCL‑R Complications in isolated and combined PCL-R have been
reported to occur in up to 53% of surgeries and can be
divided into complications generally associated with opera-
tive procedures and in complications which are inherently
related to the different techniques in PCL-R [15, 34, 36, 38,
40, 45, 59, 67, 70, 77, 81, 88, 90]. Well-known complica-
tions in knee surgery and thus also described in PCL-R are
postoperative hematoma, surgical site infections, arthrofibro-
sis, reflex sympathetic dystrophy syndrome, anterior knee
pain, paresthesia, neurovascular injuries, deep vein throm-
bosis, and graft failure with recurrent pain and instability 1 3 3 Knee Surgery, Sports Traumatology, Arthroscopy (2021) 29:682–693 689 initial non-operative management based on functional brac-
ing and rehabilitation with optional delayed PCL-R seems to
be reasonable for isolated acute PCL injuries, even for high-
level athletes with grade III PCL injuries [2]. Although the
PCL has a strong intrinsic healing capability, residual pos-
terior laxity is a serious and frequently observed disadvan-
tage of non-operative management [52, 71, 72]. However,
the subjective and objective outcomes after non-operative
treatment are promising [3, 26, 30, 63, 71, 72]. One pro-
spective study demonstrated increased knee laxity based on
manual testing in 9% of patients following non-operative
treatment after a mean follow-up of 14 years. Additionally,
instrumented laxity testing (KT-1000) revealed a mean side-
to-side difference of 3 mm [71]. Nevertheless, the majority
of patients were able to regain functional range-of-motion
(ROM) and sufficient quadriceps strength to return to activi-
ties of daily living, with 45% participating in jumping and
pivoting activities [71]. Furthermore, no correlation between
functional outcomes and grade of laxity could be observed
[71]. While non-operative management remains an integral
part of the management of isolated PCL injures, it is impor-
tant to acknowledge that unsatisfactory outcomes may occur. One study showed that patients undergoing non-operative
treatment of isolated PCL injuries occasionally experienced
pain and swelling in 81% and 56% of patients, respectively
[13]. Additionally, a considerable number of PCL deficient
patients developed subsequent meniscal injuries requiring
subsequent surgery as well as a deterioration of the articu-
lar cartilage on average 13 years after the injury, indicating
residual knee laxity [13]. This is also supported by the devel-
opment of moderate to severe OA in approximately 11% of
patients at long-term follow-up [71]. There is a paucity of
studies comparing operative and non-operative treatment in
PCL deficient patients. Fig. 3 Non-operative and
postoperative treatment protocol
for posterior cruciate ligament
injuries. PCL posterior cruciate
ligament, PT physical therapy,
PTS posterior tibial support,
ROM (ex/flex) range of motion
(extension to flexion), w week Conclusion As demonstrated in this two-part review PCL injuries are
complex and commonly associated with neurovascular
compromise and multiple ligament knee injuries involving
the PLC and less commonly MCL. Clinicians must have a
thorough understanding of anatomy and biomechanics to
aid in decision-making, analysis of concomitant injuries,
and operative treatment. Treatment algorithms following
history and physical examination involve advanced imag-
ing including stress radiographs and MRI. Exact treatment
decisions are finalized and often revised after examination 1 3 690 Knee Surgery, Sports Traumatology, Arthroscopy (2021) 29:682–693 Fig. 3 Non-operative and
postoperative treatment protocol
for posterior cruciate ligament
injuries. PCL posterior cruciate
ligament, PT physical therapy,
PTS posterior tibial support,
ROM (ex/flex) range of motion
(extension to flexion), w week References 1. Aglietti P, Giron F, Losco M, Cuomo P, Ciardullo A, Mondanelli
N (2010) Comparison between single-and double-bundle ante-
rior cruciate ligament reconstruction: a prospective, randomized,
single-blinded clinical trial. Am J Sports Med 38:25–34 p
18. Fanelli GC (2020) PCL transtibial tunnel reconstruction. Sports
Med Arthrosc Rev 28:8–13 19. Fanelli GC (2008) Posterior cruciate ligament rehabilitation: how
slow should we go? Arthroscopy 24:234–235 2. Agolley D, Gabr A, Benjamin-Laing H, Haddad FS (2017) Suc-
cessful return to sports in athletes following non-operative man-
agement of acute isolated posterior cruciate ligament injuries:
medium-term follow-up. Bone Jt J 99:774–778 20. Grassmayr MJ, Parker DA, Coolican MR, Vanwanseele B (2008)
Posterior cruciate ligament deficiency: biomechanical and biologi-
cal consequences and the outcomes of conservative treatment. A
systematic review. J Sci Med Sport 11:433–443f 3. Ahn JH, Lee SH, Choi SH, Wang JH, Jang SW (2011) Evaluation
of clinical and magnetic resonance imaging results after treatment
with casting and bracing for the acutely injured posterior cruciate
ligament. Arthroscopy 27:1679–1687 21. Gupta A, Lattermann C, Busam M, Riff A, Bach BR Jr, Wang VM
(2009) Biomechanical evaluation of bioabsorbable versus metal-
lic screws for posterior cruciate ligament inlay graft fixation: a
comparative study. Am J Sports Med 37:748–753 4. Ahrend M, Ateschrang A, Döbele S, Stöckle U, Grünwald L,
Schröter S et al (2016) Return to sport after surgical treatment of
a posterior cruciate ligament injury: a retrospective study of 60
patients. Orthopade 45:1027–1038 22. Gwinner C, Weiler A, Roider M, Schaefer FM, Jung TM (2017)
Tibial slope strongly influences knee stability after posterior cruci-
ate ligament reconstruction: a prospective 5- to 15-year follow-up. Am J Sports Med 45:355–361 5. Anderson CJ, Ziegler CG, Wijdicks CA, Engebretsen L, LaPrade
RF (2012) Arthroscopically pertinent anatomy of the anterolateral
and posteromedial bundles of the posterior cruciate ligament. J
Bone Jt Surg Am 94:1936–1945 23. Harner CD, Janaushek MA, Kanamori A, Yagi M, Vogrin TM,
Woo SL (2000) Biomechanical analysis of a double-bundle
posterior cruciate ligament reconstruction. Am J Sports Med
28:144–151 6. Ansari AS, Dennis BB, Horner NS, Zhu M, Brookes C, Khan M
et al (2019) Influence of graft source on postoperative activity
and joint laxity in posterior cruciate ligament reconstruction: a
systematic review. Arthroscopy 35:262-274.e266 24. References Harner CD, Janaushek MA, Ma CB, Kanamori A, Vogrin TM,
Woo SL (2000) The effect of knee flexion angle and application
of an anterior tibial load at the time of graft fixation on the biome-
chanics of a posterior cruciate ligament-reconstructed knee. Am J
Sports Med 28:460–465 7. Belk JW, Kraeutler MJ, Purcell JM, McCarty EC (2018) Autograft
versus allograft for posterior cruciate ligament reconstruction: an
updated systematic review and meta-analysis. Am J Sports Med
46:1752–1757 25. Hudgens JL, Gillette BP, Krych AJ, Stuart MJ, May JH, Levy BA
(2013) Allograft versus autograft in posterior cruciate ligament
reconstruction: an evidence-based systematic review. J Knee Surg
26:109–115 8. Berg EE (1995) Posterior cruciate ligament tibial inlay reconstruc-
tion. Arthroscopy 11:69–76 9. Bergfeld JA, McAllister DR, Parker RD, Valdevit AD, Kambic
HE (2001) A biomechanical comparison of posterior cruciate liga-
ment reconstruction techniques. Am J Sports Med 29:129–136 26. Jacobi M, Reischl N, Wahl P, Gautier E, Jakob RP (2010) Acute
isolated injury of the posterior cruciate ligament treated by a
dynamic anterior drawer brace: a preliminary report. J Bone Jt
Surg Br 92:1381–1384 10. Bernhardson AS, Aman ZS, DePhillipo NN, Dornan GJ, Storaci
HW, Brady AW et al (2019) Tibial slope and its effect on graft
force in posterior cruciate ligament reconstructions. Am J Sports
Med 47:1168–1174 27. Jakob RP, Rüegsegger M (1993) Therapy of posterior and poste-
rolateral knee instability. Orthopade 22:405–413 28. Johannsen AM, Anderson CJ, Wijdicks CA, Engebretsen L,
LaPrade RF (2013) Radiographic landmarks for tunnel position-
ing in posterior cruciate ligament reconstructions. Am J Sports
Med 41:35–42fl 11. Bernhardson AS, DePhillipo NN, Aman ZS, Kennedy MI, Dor-
nan GJ, LaPrade RF (2019) Decreased posterior tibial slope does
not affect postoperative posterior knee laxity after double-bundle
posterior cruciate ligament reconstruction. Am J Sports Med
47:318–323 29. Jung TM, Reinhardt C, Scheffler SU, Weiler A (2006) Stress radi-
ography to measure posterior cruciate ligament insufficiency: a
comparison of five different techniques. Knee Surg Sports Trau-
matol Arthrosc 14:1116–1121 12. Bernhardson AS, DePhillipo NN, Daney BT, Kennedy MI, Aman
ZS, LaPrade RF (2019) Posterior tibial slope and risk of posterior
cruciate ligament injury. Am J Sports Med 47:312–317 cruciate ligament injury. Am J Sports Med 47:312–317 30. Jung YB, Tae SK, Lee YS, Jung HJ, Nam CH, Park SJ (2008)
Active non-operative treatment of acute isolated posterior cruci-
ate ligament injury with cylinder cast immobilization. Compliance with ethical standards under anesthesia and afford availability of multiple surgi-
cal tools and graft choices as well as the flexibility of the
experienced surgeon. Good to excellent outcomes with high
patient satisfaction can be achieved. High-quality and large-
scale studies are needed to provide further evidence for an
individualized treatment approach pursuing the ultimate goal
of restoring native knee kinematics and facilitating a return
to daily, professional, and sports activities. Conflict of interest VM reports educational grants, consulting fees,
and speaking fees from Smith & Nephew plc, educational grants from
Arthrex, is a board member of the International Society of Arthros-
copy, Knee Surgery and Orthopaedic Sports Medicine (ISAKOS),
and deputy editor-in-chief of Knee Surgery, Sports Traumatology, Ar-
throscopy (KSSTA). In addition, VM has a patent Quantified injury
diagnostics-U.S. Patent No. 9,949,684, Issued on April 24, 2018 issued
to University of Pittsburgh. Ethical approval Not applicable. Author contributions All listed authors have contributed substantially
to this work: PWW, BZ, NNW, AH and JDH performed the literature
review and primary manuscript preparation. VM, EHS and KS assisted
with literature review, initial drafting of the manuscript, as well as
editing and final draft preparation. All authors read and approved the
final manuscript. Informed consent Not applicable. 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 Funding Open Access funding enabled and organized by Projekt
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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/. 15. Chahla J, Moatshe G, Cinque ME, Dornan GJ, Mitchell JJ, Ridley
TJ et al (2017) Single-bundle and double-bundle posterior cruciate
ligament reconstructions: a systematic review and meta-analysis
of 441 patients at a minimum 2 years’ follow-up. Arthroscopy
33:2066–2080 16. Compliance with ethical standards Cooper DE, Stewart D (2004) Posterior cruciate ligament recon-
struction using single-bundle patella tendon graft with tibial inlay
fixation: 2- to 10-year follow-up. Am J Sports Med 32:346–360 ation: 2- to 10-year follow-up. Am J Sports Med 32:346–3 i
17. Devitt BM, Dissanayake R, Clair J, Napier RJ, Porter TJ, Feller
JA et al (2018) Isolated posterior cruciate reconstruction results in
improved functional outcome but low rates of return to preinjury
level of sport: a systematic review and meta-analysis. Orthop J
Sports Med 6:2325967118804478 References Markolf KL, Zemanovic JR, McAllister DR (2002) Cyclic loading
of posterior cruciate ligament replacements fixed with tibial tunnel
and tibial inlay methods. J Bone Jt Surg Am 84:518–524 p
37. LaPrade RF, Smith SD, Wilson KJ, Wijdicks CA (2015) Quanti-
fication of functional brace forces for posterior cruciate ligament
injuries on the knee joint: an in vivo investigation. Knee Surg
Sports Traumatol Arthrosc 23:3070–3076 54. Marom N, Ruzbarsky JJ, Boyle C, Marx RG (2020) Complications
in posterior cruciate ligament injuries and related surgery. Sports
Med Arthrosc Rev 28:30–33 55. Montgomery SR, Johnson JS, McAllister DR, Petrigliano FA
(2013) Surgical management of PCL injuries: indications, tech-
niques, and outcomes. Curr Rev Musculoskelet Med 6:115–123 p
38. Lee DW, Jang HW, Lee YS, Oh SJ, Kim JY, Song HE et al (2014)
Clinical, functional, and morphological evaluations of posterior
cruciate ligament reconstruction with remnant preservation: mini-
mum 2-year follow-up. Am J Sports Med 42:1822–1831 56. Narvy SJ, Pearl M, Vrla M, Yi A, Hatch GF 3rd (2015) Anatomy
of the femoral footprint of the posterior cruciate ligament: a
systematic review. Arthroscopy 31:345–354 39. Lee DW, Kim JG, Yang SJ, Cho SI (2019) Return to sports and
clinical outcomes after arthroscopic anatomic posterior cruciate
ligament reconstruction with remnant preservation. Arthroscopy
35:2658-2668.e2651 57. Nemani VM, Frank RM, Reinhardt KR, Pascual-Garrido C,
Yanke AB, Drakos M et al (2012) Popliteal venotomy during
posterior cruciate ligament reconstruction in the setting of a
popliteal artery bypass graft. Arthroscopy 28:294–299 40. Lee DY, Kim DH, Kim HJ, Ahn HS, Lee TH, Hwang SC (2018)
Posterior cruciate ligament reconstruction with transtibial or tibial
inlay techniques: a meta-analysis of biomechanical and clinical
outcomes. Am J Sports Med 46:2789–2797 58. Noyes FR, Barber-Westin SD (2005a) Posterior cruciate liga-
ment revision reconstruction, part 1: causes of surgical failure
in 52 consecutive operations. Am J Sports Med 33:646–654 p
41. Lee SH, Jung YB, Lee HJ, Jung HJ, Kim SH (2012) Revision
posterior cruciate ligament reconstruction using a modified tibial-
inlay double-bundle technique. J Bone Jt Surg Am 94:516–522 59. Noyes FR, Barber-Westin SD (2005b) Posterior cruciate liga-
ment revision reconstruction, part 2: results of revision using a
2-strand quadriceps tendon-patellar bone autograft. Am J Sports
Med 33:655–665f 42. Lee SH, Jung YB, Rhee SM, Lee HJ, Jung HJ (2014) Revision
posterior cruciate ligament reconstruction with a modified tibial-
inlay double-bundle technique. JBJS Essent Surg Tech 4:e1 60. References Nuelle CW, Milles JL, Pfeiffer FM, Stannard JP, Smith PA,
Kfuri M Jr et al (2017) Biomechanical comparison of five pos-
terior cruciate ligament reconstruction techniques. J Knee Surg
30:523–531 43. Li G, Papannagari R, Li M, Bingham J, Nha KW, Allred D et al
(2008) Effect of posterior cruciate ligament deficiency on in vivo
translation and rotation of the knee during weightbearing flexion. Am J Sports Med 36:474–479 61. Owesen C, Sandven-Thrane S, Lind M, Forssblad M, Granan LP,
Årøen A (2017) Epidemiology of surgically treated posterior cru-
ciate ligament injuries in Scandinavia. Knee Surg Sports Trauma-
tol Arthrosc 25:2384–2391 44. Li J, Kong F, Gao X, Shen Y, Gao S (2016) Prospective rand-
omized comparison of knee stability and proprioception for poste-
rior cruciate ligament reconstruction with autograft, hybrid graft,
and γ-irradiated allograft. Arthroscopy 32:2548–2555 62. Panchal HB, Sekiya JK (2011) Open tibial inlay versus arthro-
scopic transtibial posterior cruciate ligament reconstructions. Arthroscopy 27:1289–1295 45. Li Y, Li J, Wang J, Gao S, Zhang Y (2014) Comparison of single-
bundle and double-bundle isolated posterior cruciate ligament
reconstruction with allograft: a prospective, randomized study. Arthroscopy 30:695–700 63. Parolie JM, Bergfeld JA (1986) Long-term results of nonoperative
treatment of isolated posterior cruciate ligament injuries in the
athlete. Am J Sports Med 14:35–38 46. Lind M, Nielsen TG, Behrndtz K (2018) Both isolated and
multi-ligament posterior cruciate ligament reconstruction results
in improved subjective outcome: results from the Danish Knee
Ligament Reconstruction Registry. Knee Surg Sports Traumatol
Arthrosc 26:1190–1196 64. Patel DV, Allen AA, Warren RF, Wickiewicz TL, Simonian PT
(2007) The nonoperative treatment of acute, isolated (partial or
complete) posterior cruciate ligament-deficient knees: an inter-
mediate-term follow-up study. HSS J 3:137–146fi 65. Pierce CM, O’Brien L, Griffin LW, Laprade RF (2013) Posterior
cruciate ligament tears: functional and postoperative rehabilita-
tion. Knee Surg Sports Traumatol Arthrosc 21:1071–1084 47. Lubis AMT, Kuncoro MW (2019) Revision of failed-posterior
cruciate ligament (PCL) reconstruction due to tibial tunnel mis-
placement: a case report. Ann Med Surg (Lond) 48:105–108 66. Race A, Amis AA (1998) PCL reconstruction. In vitro biomechan-
ical comparison of “isometric” versus single and double-bundled
“anatomic” grafts. J Bone Jt Surg Br 80:173–179 48. Lundblad M, Hägglund M, Thomeé C, Hamrin Senorski E,
Ekstrand J, Karlsson J et al (2020) Epidemiological data on LCL
and PCL injuries over 17 seasons in men’s professional soccer:
the UEFA elite club injury study. References Knee Surg
Sports Traumatol Arthrosc 16:729–733 13. Boynton MD, Tietjens BR (1996) Long-term followup of the
untreated isolated posterior cruciate ligament-deficient knee. Am
J Sports Med 24:306–310 14. Campbell RB, Torrie A, Hecker A, Sekiya JK (2007) Comparison
of tibial graft fixation between simulated arthroscopic and open
inlay techniques for posterior cruciate ligament reconstruction. Am J Sports Med 35:1731–1738 31. Kennedy NI, LaPrade RF, Goldsmith MT, Faucett SC, Rasmussen
MT, Coatney GA et al (2014a) Posterior cruciate ligament graft
fixation angles, part 1: biomechanical evaluation for anatomic
single-bundle reconstruction. Am J Sports Med 42:2338–2345 1 3 692 Knee Surgery, Sports Traumatology, Arthroscopy (2021) 29:682–693 49. Lynch AD, Chmielewski T, Bailey L, Stuart M, Cooper J, Coady
C et al (2017) Current concepts and controversies in rehabilitation
after surgery for multiple ligament knee injury. Curr Rev Muscu-
loskelet Med 10:328–345 32. Kennedy NI, LaPrade RF, Goldsmith MT, Faucett SC, Rasmussen
MT, Coatney GA et al (2014b) Posterior cruciate ligament graft
fixation angles, part 2: biomechanical evaluation for anatomic
double-bundle reconstruction. Am J Sports Med 42:2346–2355 33. Kim J, Allaire R, Harner CD (2010) Vascular safety during high
tibial osteotomy: a cadaveric angiographic study. Am J Sports
Med 38:810–815 50. MacGillivray JD, Stein BE, Park M, Allen AA, Wickiewicz TL,
Warren RF (2006) Comparison of tibial inlay versus transtibial
techniques for isolated posterior cruciate ligament reconstruction:
minimum 2-year follow-up. Arthroscopy 22:320–328 34. Kim SJ, Kim TE, Jo SB, Kung YP (2009) Comparison of the
clinical results of three posterior cruciate ligament reconstruction
techniques. J Bone Jt Surg Am 91:2543–2549f minimum 2-year follow-up. Arthroscopy 22:320–328 51. Makino A, Costa-Paz M, Aponte-Tinao L, Ayerza MA, Muscolo
DL (2005) Popliteal artery laceration during arthroscopic poste-
rior cruciate ligament reconstruction. Arthroscopy 21:1396 q
g
35. Klose M, Forkel P, Imhoff A (2018) Rehabilitation after recon-
struction of the posterior cruciate ligament. MMW Fortschr Med
160:69–71 52. Mariani PP, Margheritini F, Christel P, Bellelli A (2005) Evalu-
ation of posterior cruciate ligament healing: a study using mag-
netic resonance imaging and stress radiography. Arthroscopy
21:1354–1361 36. LaPrade RF, Cinque ME, Dornan GJ, DePhillipo NN, Geeslin AG,
Moatshe G et al (2018) Double-bundle posterior cruciate ligament
reconstruction in 100 patients at a mean 3 years’ follow-up: out-
comes were comparable to anterior cruciate ligament reconstruc-
tions. Am J Sports Med 46:1809–1818 53. References Open Access J Sports Med
11:105–112 67. Rhatomy S, Abadi MBT, Setyawan R, Asikin AIZ, Soekarno NR,
Imelda LG et al (2020) Posterior cruciate ligament reconstruction
with peroneus longus tendon versus hamstring tendon: a com-
parison of functional outcome and donor site morbidity. Knee 1 3 693 Knee Surgery, Sports Traumatology, Arthroscopy (2021) 29:682–693 Surg Sports Traumatol Arthrosc. https://doi.org/10.1007/s0016
7-020-06077-3 combined posterior cruciate ligament reconstruction in a military
population. Orthop J Sports Med 7:2325967119875139 combined posterior cruciate ligament reconstruction in a military
population. Orthop J Sports Med 7:2325967119875139 68. Schulz MS, Steenlage ES, Russe K, Strobel MJ (2007) Distribu-
tion of posterior tibial displacement in knees with posterior cruci-
ate ligament tears. J Bone Jt Surg Am 89:332–338 82. Wang CJ, Chan YS, Weng LH, Yuan LJ, Chen HS (2004) Com-
parison of autogenous and allogenous posterior cruciate ligament
reconstructions of the knee. Injury 35:1279–1285 g
g
69. Sekiya JK, Whiddon DR, Zehms CT, Miller MD (2008) A clini-
cally relevant assessment of posterior cruciate ligament and pos-
terolateral corner injuries. Evaluation of isolated and combined
deficiency. J Bone Jt Surg Am 90:1621–1627 83. Wang SH, Chien WC, Chung CH, Wang YC, Lin LC, Pan RY
(2018) Long-term results of posterior cruciate ligament tear with
or without reconstruction: a nationwide, population-based cohort
study. PLoS ONE 13:e0205118 i
y
g
70. Seon JK, Song EK (2006) Reconstruction of isolated posterior
cruciate ligament injuries: a clinical comparison of the transtibial
and tibial inlay techniques. Arthroscopy 22:27–32 84. Weimann A, Wolfert A, Zantop T, Eggers AK, Raschke M,
Petersen W (2007) Reducing the “killer turn” in posterior cruci-
ate ligament reconstruction by fixation level and smoothing the
tibial aperture. Arthroscopy 23:1104–1111 71. Shelbourne KD, Clark M, Gray T (2013) Minimum 10-year
follow-up of patients after an acute, isolated posterior cruci-
ate ligament injury treated nonoperatively. Am J Sports Med
41:1526–1533 85. Wijdicks CA, Kennedy NI, Goldsmith MT, Devitt BM, Michalski
MP, Årøen A et al (2013) Kinematic analysis of the posterior
cruciate ligament, part 2: a comparison of anatomic single- versus
double-bundle reconstruction. Am J Sports Med 41:2839–2848 72. Shelbourne KD, Davis TJ, Patel DV (1999) The natural history of
acute, isolated, nonoperatively treated posterior cruciate ligament
injuries. A prospective study. Am J Sports Med 27:276–283 86. Wu RW, Hsu CC, Wang CJ (2003) Acute popliteal artery occlu-
sion after arthroscopic posterior cruciate ligament reconstruction. Arthroscopy 19:889–893 73. References Shelbourne KD, Muthukaruppan Y (2005) Subjective results of
nonoperatively treated, acute, isolated posterior cruciate ligament
injuries. Arthroscopy 21:457–461 87. Xu M, Zhang Q, Dai S, Teng X, Liu Y, Ma Z (2019) Double
bundle versus single bundle reconstruction in the treatment of pos-
terior cruciate ligament injury: a prospective comparative study. Indian J Orthop 53:297–303 j
py
74. Shin J, Maak TG (2018) Arthroscopic transtibial PCL reconstruc-
tion: surgical technique and clinical outcomes. Curr Rev Muscu-
loskelet Med 11:307–315f 88. Yoon KH, Bae DK, Song SJ, Cho HJ, Lee JH (2011) A prospec-
tive randomized study comparing arthroscopic single-bundle and
double-bundle posterior cruciate ligament reconstructions pre-
serving remnant fibers. Am J Sports Med 39:474–480 75. Shin YS, Kim HJ, Lee DH (2017) No clinically important differ-
ence in knee scores or instability between transtibial and inlay
techniques for PCL reconstruction: a systematic review. Clin
Orthop Relat Res 475:1239–1248 i
89. Yoon KH, Kim EJ, Kwon YB, Kim SG (2019) Minimum 10-year
results of single- versus double-bundle posterior cruciate ligament
reconstruction: clinical, radiologic, and survivorship outcomes. Am J Sports Med 47:822–827 76. Shon OJ, Lee DC, Park CH, Kim WH, Jung KA (2010) A compar-
ison of arthroscopically assisted single and double bundle tibial
inlay reconstruction for isolated posterior cruciate ligament injury. Clin Orthop Surg 2:76–84 90. Zayni R, Hager JP, Archbold P, Fournier Y, Quelard B, Cham-
bat P et al (2011) Activity level recovery after arthroscopic PCL
reconstruction: a series of 21 patients with a mean follow-up of
29 months. Knee 18:392–395 77. Song EK, Park HW, Ahn YS, Seon JK (2014) Transtibial versus
tibial inlay techniques for posterior cruciate ligament reconstruc-
tion: long-term follow-up study. Am J Sports Med 42:2964–2971 91. Zehms CT, Whiddon DR, Miller MD, Quinby JS, Montgomery
SL, Campbell RB et al (2008) Comparison of a double bundle
arthroscopic inlay and open inlay posterior cruciate ligament
reconstruction using clinically relevant tools: a cadaveric study. Arthroscopy 24:472–480 78. Song JG, Kim HJ, Han JH, Bhandare NN, Shetty GM, Kang SB
et al (2015) Clinical outcome of posterior cruciate ligament recon-
struction with and without remnant preservation. Arthroscopy
31:1796–1806 79. Spiridonov SI, Slinkard NJ, LaPrade RF (2011) Isolated and
combined grade-III posterior cruciate ligament tears treated with
double-bundle reconstruction with use of endoscopically placed
femoral tunnels and grafts: operative technique and clinical out-
comes. References J Bone Jt Surg Am 93:1773–1780 Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. 80. Sun X, Zhang J, Qu X, Zheng Y (2015) Arthroscopic posterior
cruciate ligament reconstruction with allograft versus autograft. Arch Med Sci 11:395–401 81. Tucker CJ, Cotter EJ, Waterman BR, Kilcoyne KG, Cameron
KL, Owens BD (2019) Functional outcomes after isolated and 1 3 1 3
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Copyright 2006 by Revista Estudos Feministas.
1 Projeto Universal CNPq para o período 2005/2007. O projeto prevê a organização da rede, pesquisas
comparativas, encontros e publicações sobre a temática de conjugalidades de pessoas do mesmo sexo e
parentalidades de pais não heterossexuais.
2 BUTLER, 2003, FASSIN, 1999 e 2000; SCOTT, 2005; e ALMEIDA, 2005.
3 GIDDENS, 1993; ROUDINESCO, 2003; DELAISY, 2000; CASTELLS, 2001; GODELIER, 2004; BOURDIEU, 1999; e
BAUMAN, 2004. Dossiê
Dossiê
Dossiê
Dossiê
Dossiê Dossiê
Dossiê
Dossiê
Dossiê
Dossiê ANA PAULA UZIEL, LUIZ MELLO E MIRIAM PILAR GROSSI ANA PAULA UZIEL, LUIZ MELLO E MIRIAM PILAR GROSSI LGBT – baseiam-se em argumentos muito semelhantes.4 A partir desse intenso debate,
inúmeras pesquisas têm sido publicadas, o que demonstra o crescente interesse na
sociedade francesa sobre essas temáticas.5 No Brasil, os estudos sobre homossexualidade
têm como marco os trabalhos de Carmen Dora Guimarães, Edward MacRae, Luiz Mott,
Nestor Perlonguer e Peter Fry, mas é a partir dos anos 1990 que as pesquisas passaram a
contemplar de maneira mais destacada a questão da conjugalidade entre pessoas do
mesmo sexo, sendo referências primeiras os trabalhos de Jurandir Freire Costa e Maria
Luiza Heilborn.6 LGBT – baseiam-se em argumentos muito semelhantes.4 A partir desse intenso debate,
inúmeras pesquisas têm sido publicadas, o que demonstra o crescente interesse na
sociedade francesa sobre essas temáticas.5 No Brasil, os estudos sobre homossexualidade
têm como marco os trabalhos de Carmen Dora Guimarães, Edward MacRae, Luiz Mott,
Nestor Perlonguer e Peter Fry, mas é a partir dos anos 1990 que as pesquisas passaram a
contemplar de maneira mais destacada a questão da conjugalidade entre pessoas do
mesmo sexo, sendo referências primeiras os trabalhos de Jurandir Freire Costa e Maria
Luiza Heilborn.6 Com a apresentação do Projeto de Lei nº 1.151/95, que institui a união civil entre
pessoas do mesmo sexo, de autoria da então deputada Marta Suplicy, os debates sobre
conjugalidade homossexual tomaram uma proporção antes inimaginável, tornando-se
objeto de programas de televisão, matérias de jornais e revistas, pronunciamentos de
políticos, discursos de líderes religiosos e ativistas LGBT, abaixo-assinados pró e contra,
novelas, conversas em família, discussões em mesa de bar e debates acadêmicos. Desde
então, não só os homossexuais estão cada vez mais visíveis na sociedade brasileira, mas
também a idéia de uma “família homossexual” começa a disputar espaço com outras
nas lutas de poder em torno das definições socialmente legítimas de conjugalidade e
parentalidade. 4 No debate francês destacamos os trabalhos de Sylviane AGACINSKI, 1998; Daniel BORRILLO, 2005; BORRILLO,
FASSIN e Marcela IACUB, 2001; Anne CADORET, 2002; Eric DUBREUIL, 1998; Clarisse FABRE e Eric FASSIN, 2003.
5 Entre as inúmeras publicações destacamos DUBREUIL, 1998; Stéphane NADAUD, 2002; Fione TASKER e Susan
GOLOMBOK, 2002; e Martine GROSS e Mathieu PEYCERÉ, 2005.
6 GUIMARÃES, 2004; MACRAE, 1990; MOTT, 1989; PERLONGHER, 1987; FRY, 1982; COSTA, 1992; e HEILBORN, 2004.
7 Miriam GROSSI, 2004a e 2004b; Moisés LOPES, 2005; Camila MEDEIROS, 2004; Luiz MELLO, 2005a e 2005b;
Luiz António RALA, 1999; Flávio TARNOVSKI, 2002 e 2004; e Anna Paula UZIEL, 2002 e 2004.
8 Ver os estudos de Maria Berenice DIAS, 2001 e 2003; Roger RIOS, Célio GOLIN e Fernando POCAHY, 2003; e
Fabiana SPENGLER, 2003. CONJUGALIDADES E PARENTALIDADES
DE GAYS, LÉSBICAS E TRANSGÊNEROS
NO BRASIL Este Dossiê reúne artigos de pesquisadoras/es que investigam os significados e as
implicações da visibilidade social crescente de casais, com ou sem filhos, formados por
pessoas do mesmo sexo ou quando um dos cônjuges é transgênero. Com esta iniciativa
pioneira tem-se por objetivo ampliar os debates no meio acadêmico e na sociedade
brasileira acerca dos sentidos da família, nas dimensões conjugal e parental, como
instituição social capaz de proporcionar amparo e proteção aos indivíduos. Por outro
lado, busca-se publicizar os conflitos entre atores sociais distintos, os quais afirmam/negam
a legitimidade de arranjos familiares constituídos fora da norma heterocêntrica. Também
é importante dizer que esta publicação se insere no conjunto das atividades realizadas
pela Rede Brasileira de Pesquisadores sobre Conjugalidades e Parentalidades, coordenada
pelos organizadores deste Dossiê, os quais desenvolvem o projeto de pesquisa “Parceria
Civil, Conjugalidade e Homoparentalidade”, apoiado pelo CNPq.1 j g
p
p
p
q
Não há dúvidas de que a maior parte dos debates políticos e também acadêmicos
sobre conjugalidades e parentalidades de gays, lésbicas e transgêneros situa-se nos países
de capitalismo central, em especial norte-americanos e europeus. Nesse cenário, a reflexão
acadêmica tem sido feita dentro de dois grandes campos. De um lado, no campo de
estudos feministas/de gênero e de estudos queer, com autores como Judith Butler nos
Estados Unidos, Eric Fassin e Joan Scott na França ou Miguel Vale de Almeida em Portugal.2
De outro, no campo mais amplo do pensamento social contemporâneo, com autores
como Anthony Giddens, Elisabeth Roudinesco, Geneviève Delaisy, Manuel Castells, Maurice
Godelier, Pierre Bourdieu ou Zygmunt Bauman,3 que têm discutido a relevância política
das demandas de homossexuais e transgêneros, bem como sua centralidade para a
compreensão das possibilidades de redefinição das formas de organização das
sociedades humanas. Em lugares como a França, onde alguns de nós têm feito pesquisa
sobre essas questões, o debate reúne posições extremadas, e é curioso observar como
estas – contrárias ou favoráveis ao reconhecimento da parentalidade e da conjugalidade 481 Estudos Feministas, Florianópolis, 14(2): 248, maio-agosto/2006 481 482 Estudos Feministas, Florianópolis, 14(2): 481-487, maio-agosto/2006 CONJUGALIDADES E PARENTALIDADES DE GAYS, LÉSBICAS E TRANSGÊNEROS... obtido mais êxito ao tomar rumos semelhantes ao movimento de pessoas vivendo com
HIV-AIDS, ou seja, assegurar direitos individuais por meio de demandas judiciais, em que
os casos aparentemente isolados acabam por produzir conquistas gerais. Embora ainda
distante da formulação consistente de políticas, o resultado desse processo de luta é a
garantia de alguns direitos relativos à família.9 obtido mais êxito ao tomar rumos semelhantes ao movimento de pessoas vivendo com
HIV-AIDS, ou seja, assegurar direitos individuais por meio de demandas judiciais, em que
os casos aparentemente isolados acabam por produzir conquistas gerais. Embora ainda
distante da formulação consistente de políticas, o resultado desse processo de luta é a
garantia de alguns direitos relativos à família.9 g
g
Na falta de conceitos e de terminologia aceita socialmente no Brasil sobre a questão
da filiação e da parentalidade homoerótica, muitas vezes utiliza-se o termo
homoparentalidade, que foi cunhado na França, em meados dos anos 1990, por
integrantes da Associação de Pais e Mães Gays e Lésbicas (APGL). Ainda que careça de
uma definição mais específica, essa noção parece remeter-se à homossexualidade dos
pais, mesmo reconhecendo que este é um universo bastante diversificado – pais/mães
que se revelam homossexuais, homossexuais que decidem ser pais/mães, casais, pessoas
solteiras. O termo tem sido incorporado por vários pesquisadores que se debruçam sobre
a temática no Brasil atualmente e está presente também aqui neste Dossiê. Cabe ressaltar
ainda que o termo homoparentalidade pode fazer-nos cair em uma armadilha, que é
exatamente destacar o que as poucas pesquisas mostram e o que de certo modo é
importante combater: a idéia de que há algo de específico no exercício parental, marcado
pela orientação sexual dos pais. Por outro lado, pode ser interessante politicamente insistir
nesse termo, visto que concede visibilidade à questão. q
q
Com o objetivo de proporcionar novos elementos para o debate sobre a questão
no Brasil, este Dossiê reúne cinco artigos de pesquisadoras/es de distintas instituições de
ensino e pesquisa, sendo um dos autores Luiz Mott, originário da academia e também um
dos principais protagonistas do movimento de militância homossexual no Brasil. Nossa
idéia, ao organizarmos este Dossiê, era apresentar um painel diversificado de olhares
sobre a questão, contemplando abordagens específicas para diferenciadas experiências
de conjugalidade e parentalidade. Recebemos, após divulgação inicial de nosso projeto,
dez propostas de artigo, o que indica um crescente interesse na área. 9 Sobre a posição de militantes e simpatizantes do movimento, ver Sergio CARRARA e Silvia RAMOS, 2005. ANA PAULA UZIEL, LUIZ MELLO E MIRIAM PILAR GROSSI A participação de cientistas sociais e da área psi na produção de
conhecimento sobre esse (anti)familismo homossexual tem sido fundamental, muitas vezes
materializando-se em monografias, dissertações e teses, as quais apontam elementos
constituintes dos discursos, representações e práticas sociais relativas a formas não-
heterossexuais de organização dos laços conjugais e a vínculos parentais protagonizados
por gays, lésbicas e transgêneros.7 Se em 1995, quando da apresentação do projeto de lei da Parceria Civil Registrada
(PCR), o Brasil incluía-se no seletíssimo rol de países onde se discutia o amparo legal a
relações amorosas entre pessoas do mesmo sexo, em 2006, passados mais de dez anos,
o Congresso Nacional ainda não aprovou o projeto da PCR e não temos no país nenhum
indicativo de que os direitos parentais de casais de gays e de lésbicas venham a ser
assegurados a curto prazo. Enquanto aqui no Brasil a oposição religiosa e as posições
machistas, sexistas e homofóbicas dos parlamentares têm sido os principais responsáveis
pelos entraves à aprovação da PCR, quase vinte países já aprovaram leis que asseguram
amparo a uniões civis entre homossexuais, e Holanda (1.4.2001), Espanha (29.6.2005),
Bélgica (30.1.2003) e Canadá (28.6.2005) reconhecem a possibilidade de casamento
civil entre pessoas do mesmo sexo, com plenos direitos parentais no caso dos dois primeiros
países mencionados. De país na ponta das lutas sociais contemporâneas, o Brasil junta-
se aos países fundamentalistas no que diz respeito à garantia de direitos de ordem da
família para homossexuais e transgêneros. No entanto, na contramão da morosidade ou estagnação do Poder Legislativo, já
são significativas as conquistas de direitos por parte de casais ou pais homossexuais/
transgêneros, relativas ao reconhecimento da legitimidade da união conjugal e do
exercício parental, no âmbito do Poder Judiciário.8 Parece que o movimento LGBT tem 482 Estudos Feministas, Florianópolis, 14(2): 481-487, maio-agosto/2006 Estudos Feministas, Florianópolis, 14(2): 481-487, maio-agosto/2006 4 ANA PAULA UZIEL, LUIZ MELLO E MIRIAM PILAR GROSSI ANA PAULA UZIEL, LUIZ MELLO E MIRIAM PILAR GROSSI casamento/acasalamento seria o único caminho socialmente aceito para a legitimação
de uma certa homossexualidade, pautada na reprodução de valores heterossexistas, os
quais fundamentam, paradoxalmente, a injustiça erótica e a opressão sexual que atingem
gays e lésbicas e os excluem da norma familiar. Luiz Mott nos traz o artigo “Homo-afetividade e direitos humanos”, em que discute a
legitimidade do amparo legal à conjugalidade homossexual, a partir da crítica aos
fundamentos do preconceito anti-homossexual, destacando a importância da atuação
política organizada de gays, lésbicas e transgêneros no combate à homofobia no Brasil. Considerando os homossexuais a “última tribo romântica do mundo”, Mott afirma defender
não apenas a aprovação imediata do projeto de lei que institui a PCR, mas também do
casamento civil para casais de gays e de lésbicas, sob o argumento de que não haveria
razão histórica, lógica ou ética para negar amparo do Estado para os vínculos afetivo-
sexuais entre homossexuais. Após apresentar dez razões por que defende a legitimidade
do casamento entre pessoas do mesmo sexo, o autor conclui o artigo dizendo que nada,
além da ignorância e do preconceito, justifica qualquer discriminação contra gays, lésbicas
e transgêneros. O artigo seguinte, “Três casamentos e algumas reflexões: notas sobre conjugalidade
envolvendo travestis que se prostituem”, de Larissa Pelúcio, mostra como o universo conjugal
de seus informantes está pautado por perspectivas essencialistas quanto ao sistema sexo/
gênero, o que não permitiria ampliar o léxico dos vínculos afetivo-sexuais para além da
matriz heterocêntrica. A partir da compreensão das dinâmicas relacionais envolvendo
três tipos diferentes de casais – travesti/homem de classe média, travesti/travesti e travesti/
homem de classe popular – Pelúcio afirma que o estigma associado ao travestismo
contaminaria a relação conjugal, levando-a para a clandestinidade do gueto travesti. Por outro lado, constata que o recorrente apelo especular ao vínculo heterossexual pauta-
se por uma busca de inteligibilidade e legitimidade, por mais que o gênero transgrida o
sexo e que um “homem de verdade” se relacione com pessoas que nunca serão “mulheres
de verdade”. CONJUGALIDADES E PARENTALIDADES DE GAYS, LÉSBICAS E TRANSGÊNEROS... O dossiê inicia com o artigo “Acesso ao casamento no Brasil: uma questão de
cidadania sexual”, de Roberto Arriada Lorea, que advoga a tese de que a possibilidade
de casamento entre pessoas do mesmo sexo não depende de uma nova legislação
reguladora, já que a Constituição Federal veda qualquer modalidade de discriminação,
devendo ser asseguradas a gays e lésbicas as mesmas prerrogativas legais garantidas
aos demais cidadãos. Depois de destacar que a negação da laicidade do Estado brasileiro
é grave violação do texto constitucional, Lorea argumenta que os representantes do
Poder Judiciário não podem pautar suas decisões a partir de motivações religiosas, inclusive
no campo dos direitos sexuais e da cidadania sexual. Nesse contexto, o autor afirma que
não há, no ordenamento jurídico brasileiro, obstáculo que impeça a gays e lésbicas o
direito de solicitar a tutela do Estado para suas relações afetivo-sexuais, seja na forma de
união estável, seja na forma de casamento, inclusive no tocante a direitos parentais. Luiz Mello, em “Familismo (anti)homossexual e regulação da cidadania no Brasil”,
traz reflexões sobre os significados sociais e políticos da família e dos vínculos afetivo-
sexuais entre pessoas do mesmo sexo. Mesmo que o autor seja pouco otimista quanto às
possibilidades, a curto prazo, de reconhecimento legal pleno de direitos conjugais e
parentais para gays e lésbicas, a ênfase em um discurso familista é apontada por Mello
como uma nova forma de regulação da cidadania. Para o autor, a hipervalorização da
conjugalidade e da parentalidade é a expressão de um sentido integracionista e
conformista das relações afetivo-sexuais entre pessoas do mesmo sexo. Nesse cenário, o 483 Estudos Feministas, Florianópolis, 14(2): 481-487, maio-agosto/2006 484 Estudos Feministas, Florianópolis, 14(2): 481-487, maio-agosto/2006 RRRRReferências bibliográficas
eferências bibliográficas
eferências bibliográficas
eferências bibliográficas
eferências bibliográficas AGACINSKY, Sylviane. Política dos sexos. Rio de Janeiro: Nova Fronteira, 1998. ALMEIDA, Miguel Vale de. O casamento entre pessoas do mesmo sexo: sobre “gentes
remotas e estranhas” numa “sociedade decente”. Texto apresentado na VI Reunião
de Antropologia do Mercosul. Montevidéu, 2005. Mimeo. p
g
BAUMAN, Zygmunt. Amor líquido: sobre a fragilidade dos laços humanos. Rio de Janeiro:
Jorge Zahar Ed., 2004. BORRILLO, Daniel. “O indivíduo homossexual, o casal de mesmo sexo e as famílias
homoparentais: análise da realidade jurídica francesa no contexto internacional”. In:
LOYOLA, Maria Andréa (Org.). Bioética: reprodução e gênero na sociedade
contemporânea. Rio de Janeiro: ABEP; Brasília: Letras Livres, 2005. p. 175-211. BORRILLO, Daniel; FASSIN, Eric ; IACUB, Marcela (sous la direction de). Au-dela du PaCS –
L’expertise familiale à l’épreuve de l’homossexualité. 2eme. edition corrigée. Paris: PUF,
2001. BOURDIEU, Pierre. A dominação masculina. Rio de Janeiro: Bertrand Brasil, 1999. BUTLER, Judith. “O parentesco é sempre tido como heterossexual?” Cadernos Pagu,
Campinas: Unicamp, n. 21, p. 219-260, 2003. p
p,
, p
,
CASTELLS, Manuel. O poder da identidade. São Paulo: Paz e Terra, 2001. CADORET, Anne. Des Parents comme des autres: homossexualité et parente. Paris: Editions
Odile Jacob, 2002. CARRARA, Sergio; RAMOS, Silvia. Política, direitos, violência e homossexualidade. Pesquisa. CARRARA, Sergio; RAMOS, Silvia. Política, direitos, violência e homossexualidade. Pesquisa. 9ª Parada do Orgulho GLBT – Rio 2004 Rio de Janeiro: CLAM/IMS 2005 CARRARA, Sergio; RAMOS, Silvia. Política, direitos, violência e homossexualidade. Pesquisa. 9ª Parada do Orgulho GLBT – Rio 2004. Rio de Janeiro: CLAM/IMS, 2005. COSTA, Jurandir Freire. A inocência e o vício: estudos sobre o homoerotismo. Rio de Janeiro:
Relume Dumará 1992 9ª Parada do Orgulho GLBT – Rio 2004. Rio de Janeiro: CLAM/IMS, 2005. COSTA, Jurandir Freire. A inocência e o vício: estudos sobre o homoerotismo. Rio de Janeiro:
Relume-Dumará, 1992. DELAISY, Geneviève Parseval de. “Qu’est-ce qu’un parent suffisament bom?” In: GROSS,
Martine (Dir.). Homoparentalités, états des lieux: parentés et différence de sexes. Yssy-
les- Moulineux: ESF Editeur, 2000. p. 207-214. p
DIAS, Maria Berenice. “Vínculos hetero e homoafetivos”. In: IDEF – Instituto Interdisciplinar
de Direito de Família. Homossexualidade: discussões jurídicas e psicológicas. Curitiba:
Juruá, 2001. ______. Homoafetividade. O que diz a Justiça! As pioneiras decisões do Tribunal de Justiça
do Rio Grande do Sul que reconhecem direitos às uniões homossexuais. Porto Alegre:
Livraria do Advogado, 2003. g
DUBREUIL, Eric. Des Parents du même sexe. Paris: Editions Odile Jacob, 1998. ANA PAULA UZIEL, LUIZ MELLO E MIRIAM PILAR GROSSI Por fim, “‘Uma família de mulheres’: ensaio etnográfico sobre homoparentalidade
na periferia de São Paulo”, de Camila Pinheiro Medeiros, propõe uma estimulante leitura
sobre conjugalidade e parentalidade lésbica em camadas populares, construída a partir
de sua experiência de imersão etnográfica na casa de um casal de lésbicas, sendo uma
delas negra e a outra deficiente visual, e de suas duas filhas. Considerando a especificidade
do “ser lésbica” moradora de periferia, Medeiros mostra, por meio de análise comparativa
dos valores e práticas sociais de duas famílias homoparentais femininas, como a política
da lesbianidade repercute no viver em família – tanto no âmbito do vínculo conjugal
quanto no das relações com os filhos (e seus pais biológicos), com a família extensa e
com a vizinhança. A autora finaliza o artigo refletindo sobre os (des)encontros entre “teorias
nativas” e “teorias antropológicas”, expressos nos conflitos e impasses conceituais
vislumbrados depois de seus intensos contatos com “uma família de mulheres”. Como fio que conduz a discussão proposta pelas/os autoras/es, é consensual o
entendimento de que o direito às vivências conjugal e parental não pode ser
compreendido como monopólio das pessoas heterossexuais e que não há fundamento
ético que justifique a definição da família como instituição restrita ao universo da diferença
sexual. Para nós, trazer ao debate reflexões acerca das conjugalidades e parentalidades
de gays, lésbicas e transgêneros no Brasil é também uma maneira de aprofundar a
discussão sobre os significados da liberdade e da justiça. Esperamos que este Dossiê
possa contribuir para as lutas do movimento GLBTT no país, que seja um estímulo para que
mais pesquisadores se dediquem a compreender as dinâmicas relacionais entre diferentes ONJUGALIDADES E PARENTALIDADES DE GAYS, LÉSBICAS E TRANSGÊNEROS... CONJUGALIDADES E PARENTALIDADES DE GAYS, LÉSBICAS E TRANSGÊNEROS... grupos sexuais de nossa sociedade, e para que a universidade brasileira continue a
contribuir no processo de construção de uma sociedade mais justa, solidária e
democrática. Anna Paula Uziel
Universidade do Estado do Rio de Janeiro Luiz Mello
Universidade Federal de Goiás Luiz Mello
Universidade Federal de Goiás Miriam Grossi
Universidade Federal de Santa Catarina RRRRReferências bibliográficas
eferências bibliográficas
eferências bibliográficas
eferências bibliográficas
eferências bibliográficas 485 Estudos Feministas, Florianópolis, 14(2): 481-487, maio-agosto/2006 485 ANA PAULA UZIEL, LUIZ MELLO E MIRIAM PILAR GROSSI ANA PAULA UZIEL, LUIZ MELLO E MIRIAM PILAR GROSSI ERIBON, Didier. Sur cet instant fragile... Carnets, janvier-août 2004. Paris: Librairies Arthème
Fayard, 2004. FABRE, Clarisse; FASSIN, Eric. Liberte, Egalité, Sexualités: actualité politique des questions
sexuelles. Paris: Belfond, 2003. FASSIN, Eric. “Le Mariage des homosexuels: politique comparée des normes franco-
americaine”. French Politics, Culture and Society, New York, v. 17, n. 3-4, p. 165-179,
Summer/Fall, 1999. ______. “Usages de la science et science des usages: à propos des familles FASSIN, Eric. Le Mariage des homosexuels: politique comparée des normes franco-
americaine”. French Politics, Culture and Society, New York, v. 17, n. 3-4, p. 165-179,
Summer/Fall, 1999. ______. “Usages de la science et science des usages: à propos des familles
homoparentales”. L’Homme, Paris: Editions de l’Ecole des Hautes Etudes en Sciences
Sociales, n. 154-155 (Question de Parenté), p. 391-408, avril/sept. 2000. Summer/Fall, 1999. ______. “Usages de la science et science des usages: à propos des familles
homoparentales”. L’Homme, Paris: Editions de l’Ecole des Hautes Etudes en Sciences
Sociales, n. 154-155 (Question de Parenté), p. 391-408, avril/sept. 2000. FRY, Peter. Para inglês ver: identidade e política na cultura brasileira. Rio de Janeiro:
Zahar, 1982. GIDDENS, Anthony. A transformação da intimidade: sexualidade, amor e erotismo nas
sociedades modernas. São Paulo: Editora da Unesp, 1993. GODELIER, Maurice. Métamorphoses de la parenté. Paris: Editions Arthème Fayard, 2004. G OSS
C
É
f
Q
é GROSS, Martine; PEYCERÉ, Mathieu. Fonder une famille homoparentale (Questions éthiques,
juridiques, psychologiques... et quelques réponses pratiques). Paris: Editions Ramsay,
2005. GROSSI, Miriam. “Famílias homossexuais: novas famílias? Algumas reflexões sobre
paternidade gay e lésbica no Brasil e na França”. In: RIAL, Carmen; TONELLI, Juracy
(Orgs). Genealogias do silêncio: feminismo e gênero. Florianópolis: Editora Mulheres,
2004a. GROSSI, Miriam. “Famílias homossexuais: novas famílias? Algumas reflexões sobre
paternidade gay e lésbica no Brasil e na França”. In: RIAL, Carmen; TONELLI, Juracy
(Orgs). Genealogias do silêncio: feminismo e gênero. Florianópolis: Editora Mulheres,
2004a. ______. “Gênero e parentesco: famílias gays e lésbicas no Brasil”, Cadernos Pagu,
Campinas: Unicamp, n. 21, p .261-280, 2004b. ______. “Gênero e parentesco: famílias gays e lésbicas no Brasil”, Cadernos Pagu,
Campinas: Unicamp, n. 21, p .261-280, 2004b. GUIMARÃES, Carmen Dora. O homossexual visto por entendidos. Rio de Janeiro: Garamond,
2004. HEILBORN, Maria Luiza. Dois é par: gênero e identidade sexual em contexto igualitário. RRRRReferências bibliográficas
eferências bibliográficas
eferências bibliográficas
eferências bibliográficas
eferências bibliográficas Rio de Janeiro: Garamond, 2004. LOPES, Moisés. Debates, diálogos e confrontos: representações sociais das
homossexualidades nas discussões sobre a Parceria Civil Registrada. 2005. Dissertação
(Mestrado em Ciências Sociais) – Programa de Pós-Graduação em Ciências Sociais,
Universidade Estadual de Londrina. MACRAE, Edward. A construção da igualdade: identidade sexual e política no Brasil da
abertura. Campinas: Editora da Unicamp, 1990. MEDEIROS, Camila Pinheiro. Sobre deveres e prazeres: estudo acerca de mulheres que
se assumiram lésbicas depois de terem sido mães. 2004. Monografia de Graduação
em Ciências Sociais, Universidade Federal de Santa Catarina, Florianópolis. MELLO, Luiz. Novas famílias: conjugalidade homossexual no Brasil contemporâneo. Rio
de Janeiro: Garamond, 2005a. ______. “Outras famílias: a construção social da conjugalidade homossexual no Brasil”. Cadernos Pagu, Campinas: Unicamp, n. 24, p. 197-225, 2005b. MELLO, Luiz. Novas famílias: conjugalidade homossexual no Brasil contemporâneo. Rio
de Janeiro: Garamond, 2005a. ______. “Outras famílias: a construção social da conjugalidade homossexual no Brasil”. Cadernos Pagu, Campinas: Unicamp, n. 24, p. 197-225, 2005b. MOTT, Luiz. Sexo proibido: virgens, gays e escravos nas garras da Inquisição. Campinas:
Papirus, 1989. NADAUD, Stéphane. Homoparentalité: une nouvelle chance pour la famille? Paris: Editions
Arthème Fayard, 2002. PERLONGHER, Nestor. O negócio do michê: prostituição viril em São Paulo. São Paulo:
Brasiliense, 1987. 486 Estudos Feministas, Florianópolis, 14(2): 481-487, maio-agosto/2006 Estudos Feministas, Florianópolis, 14(2): 481-487, maio-agosto/2006 487 CONJUGALIDADES E PARENTALIDADES DE GAYS, LÉSBICAS E TRANSGÊNEROS... RALA, Luiz António. As tramas de um discurso: o sentido das práticas sexuais e afetivas
entre pessoas do mesmo sexo na tramitação do Projeto-Lei nº 1.151-A, de 1995. 1999. Dissertação (Mestrado em Psicologia Social) – Pontifícia Universidade Católica de São
Paulo. RIOS, Roger Raupp; GOLIN, Célio; POCAHY, Fernando Altair. A Justiça e os direitos de gays
e lésbicas: jurisprudência comentada. Porto Alegre: Editora Sulina/Nuances, 2003. ROUDINESCO, Elisabeth. A família em desordem. Rio de Janeiro: Jorge Zahar Ed., 2003. SCOTT, Joan. Parité! L´universel et la différence des sexes. Paris: Editions Albin Michel,
2005. SPENGLER, Fabiana. União homoafetiva: o fim do preconceito. Santa Cruz do Sul: EDUNISC,
2003. TARNOVSKI, Flávio Luiz. “Pais assumidos”: adoção e paternidade homossexual no Brasil
contemporâneo. 2002. Dissertação (Mestrado em Antropologia Social) – Programa de
Pós-Graduação em Antropologia Social, Universidade Federal de Santa Catarina,
Florianópolis. Publicado em Cadernos NIGS, Florianópolis, em 2004. ______. “‘Pai é tudo igual?’ Significados da paternidade para homens que se autodefinem
como homossexuais”. In: PISCITELLI, Adriana; GREGORI, Maria Filomena; CARRARA, Sérgio
(Orgs.). Sexualidade e saberes: convenções e fronteiras. Rio de Janeiro: Garamond,
2004. p. 385-414. TASKER, Fione; GOLOMBOK, Susan. Grandir dans une famille lesbienne: quels effets sur le
développement de l´enfant? Issy Les Moulineux: ESF Editeur, 2002. UZIEL, Anna Paula. Família e homossexualidade: velhas questões, novos problemas. 2002. Tese (Doutorado em Ciências Sociais), Unicamp, Campinas. UZIEL, Anna Paula. Família e homossexualidade: velhas questões, novos problemas. 2002. Tese (Doutorado em Ciências Sociais), Unicamp, Campinas. ______. “Homossexualidade e parentalidade: ecos de uma conjugação”. In: HEILBORN,
Maria Luiza (Org.). Família e sexualidade. Rio de Janeiro: FGV Editora, 2004. p. 87-117. (Coleção Família, Geração e Cultura). ______. “Homossexualidade e parentalidade: ecos de uma conjugação”. In: HEILBORN,
Maria Luiza (Org.). Família e sexualidade. Rio de Janeiro: FGV Editora, 2004. p. 87-117. (Coleção Família, Geração e Cultura).
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The role of the category of chance in the analysis of social relations
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Abstract In the article, a number of opinions expressed by Abu Nasr Farabi and 20th
century philosopher D.I. Shirokanov, among the ancient Central Asian
philosophers, about the phenomenon of coincidence, as well as the study of
coincidence together with the phenomena of necessity, causation and probability in
synergetics, which is considered one of the modern philosophical teachings,
because these phenomena are constantly one - some claim to be in touch. Also,
specific conclusions were drawn regarding the concepts of synergetics, fluctuation,
and bifurcation. Key words: coincidence, necessity, synergetics, forecasting, fluctuation,
bifurcation. ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS
The role of the category of chance in the analysis of social relations ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS
The role of the category of chance in the analysis of social relations ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS | J | life of a person and society: "If there were no random events in the world, the
causes of which are unknown, there would be no feelings of fear, hope, and
confidence (in a person). In turn, the absence of these leads to the complete loss of
order and discipline both in human life and in the legal functioning of politics. Subjects who will not have anything tomorrow due to fear and lack of hope and
trust will not obey their governors, and governors will not care about their
subordinates. No one listens to Allah and (people) do not show kindness and mercy
to each other. Because he who knows exactly what will happen tomorrow will try
hard not to take advantage of that thing.1 1 Cалимов Бахриддин Лутфуллаевич (2020). ФИЛОСОФСКАЯ РОЛЬ ДИАЛЕКТИЧЕСКИХ
КАТЕГОРИЙ В ЖИЗНИ ЧЕЛОВЕКА. Историческая психология и социология истории, 13 (1),
111-119.
2 Cалимов Бахриддин Лутфуллаевич (2020). ФИЛОСОФСКАЯ РОЛЬ ДИАЛЕКТИЧЕСКИХ
КАТЕГОРИЙ В ЖИЗНИ ЧЕЛОВЕКА. Историческая психология и социология истории, 13 (1),
111-119. INTRODUCTION Among the Eastern or Central Asian thinkers, Abu Nasr Farabi analyzes the
category of chance in a meaningful way. The peculiarity of Farobi's worldview is
that the scholar recognizes that random events are necessary and positive for the ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS
life of a person and society: "If there were no random events in the world, the
causes of which are unknown, there would be no feelings of fear, hope, and
confidence (in a person). In turn, the absence of these leads to the complete loss of
order and discipline both in human life and in the legal functioning of politics. Subjects who will not have anything tomorrow due to fear and lack of hope and
trust will not obey their governors, and governors will not care about their
subordinates. No one listens to Allah and (people) do not show kindness and mercy
to each other. Because he who knows exactly what will happen tomorrow will try
hard not to take advantage of that thing.1 ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS 3 Salimov, Bakhriddin Lutfullaevich (2023). OPINIONS OF CENTRAL ASIAN SCHOLARS ON SOCIAL
RELATIONS. Oriental renaissance: Innovative, educational, natural and social sciences, 3 (1-2), 178-182. 3 Salimov, Bakhriddin Lutfullaevich (2023). OPINIONS OF CENTRAL ASIAN SCHOLARS ON SOCIAL
RELATIONS Oriental renaissance: Innovative educational natural and social sciences 3 (1-2) 178-182 (
)
RELATIONS. Oriental renaissance: Innovative, educational, natural and social sciences, 3 (1-2), 178-18 MAIN PART It should be noted that this confession of Farabi helps us to understand the
importance of coincidence and draw theoretical conclusions. If there were no
accidents in the world, if everything was subject to legal necessity, the meaning of
life would change. Life, according to Farobi, consists of contradictions,
coincidences and contradictions. A person who overcomes these conditions can
live in society. In particular, the activity of the mayor should be an example to
others. If it has 12 senses, the management is good and accidents are avoided2. Humans have always tried to overcome difficulties and problems. Although most
of them could not get out of the hardships of life, but they connected the content of .2023 2 ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS
their whole life to the struggle against life's eddies. It is the faith and hope to get
rid of the whirlwinds of life that inspired them to fight and live. Most of the people
also hope that their lives will be improved by some coincidences. ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS 3 Salimov, Bakhriddin Lutfullaevich (2023). OPINIONS OF CENTRAL ASIAN SCHOLARS O ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS | J this topic: "If there is a need for a famous person, he is always found. The fact that
he (the famous person) is that person, that he was born there and became a famous
person is a coincidence. However, if there was no such famous person, surely
another person would be found and could take his place, better or worse than him4. This opinion of D. I. Shirokanov corresponds to the process of self-organization in
society in terms of its content. In our view, bringing a society from instability to a
state of stability by celebrities is a form of self-organization. One can agree with
many aspects of D.I. Shirokanov's opinion. After all, coincidences play an
important role in the birth of any person, his career and the process of becoming a
famous person. But in the absence of that well-known and famous person, we
cannot say that someone else will definitely take his place. True, substitutes are
always found to take the place of any famous person. But what will be the activity
of these substitutes, there is a doubt that they will take the place of a famous person
and become famous people themselves. In our opinion, D.I. Shirokanov took a
model from the following opinion of F. Engels, one of the ideological leaders of
the system he lived and created: "The appearance of this same famous person in a
certain country at a certain time is nothing more than a pure coincidence," writes F. .Engels. - But if we reject this person, there will be a need to replace him with
another person, and such a replacement will be found immediately. It was
something of a coincidence that Napoleon's ancestor of Corsica became the
military dictator needed by the war-weary French Republic. But, if it were not for
Napoleon, someone else would have played his role. This is proven by the fact that this topic: "If there is a need for a famous person, he is always found. The fact that
he (the famous person) is that person, that he was born there and became a famous
person is a coincidence. However, if there was no such famous person, surely
another person would be found and could take his place, better or worse than him4. р
ф
ўр
ф ф
р
диссертацияси. Алишер Навоий номли Ўзбекистон Миллий кутубхонаси(№417. Ю-34272/0).
Тошкент. 2008, 144 б. Салимов Б.Л. Илмий билиш жараёнида тасодифнинг ўрни.
Фалсафа фанлари номзоди
диссертацияси. Алишер Навоий номли Ўзбекистон Миллий кутубхонаси(№417. Ю-34272/0).
Тошкент 2008 144 б 4 Салимов Б.Л. Илмий билиш жараёнида тасодифнинг ўрни.
Фалсафа фанлари н
Ў ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS | J their whole life to the struggle against life's eddies. It is the faith and hope to get
rid of the whirlwinds of life that inspired them to fight and live. Most of the people
also hope that their lives will be improved by some coincidences. their whole life to the struggle against life's eddies. It is the faith and hope to get
rid of the whirlwinds of life that inspired them to fight and live. Most of the people
also hope that their lives will be improved by some coincidences. Now, if we think about the negative consequences that occur due to the lack
of fear mentioned by Farobi, it should be mentioned that the abstractness of the
causes of many things in human life, including death, certain diseases and various
disasters, makes a person alert in a sense. People fear Allah. Because no one knows
what kind of days Allah will bring upon us tomorrow. All this ultimately leads
people to respect the state system in which they live. D.I., one of the philosophers who conducted significant scientific research
on the role of the category of chance in scientific knowledge. Shirokanov has some
interesting ideas about the category of chance. In particular, D.I. Shirokanov's
"chance will always have its place in society. Because different and relatively
independent events in it (society) inevitably crossed each other3 His conclusion is
commendable. In fact, in all processes in all areas of society, one can see the
phenomenon of chance to one degree or another. If we take as an example the
political life of the society, or rather the activities of famous people who play a
decisive role in political life, then it is necessary to note that, first of all, the
appearance of every famous person is a random event. After all, any famous person
is, first of all, a human being. We have analyzed in the previous parts of our
scientific work that chance haunts every person throughout his life. So, chance
shows its influence on society through human activities. D.I. Shirokanov writes on .2023 .2023 3 ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS such a person was always found when needed."5 From these thoughts of F.Engels,
it seems that the wrong conclusion was drawn that the deputy who would take
Napoleon's place would also carry out Napoleon's policy. However, it is difficult to
believe that Napoleon's successor, who by chance is never exactly the same in this
world, could repeat Napoleon's actions in full, including re-establishing the
monarchy in France or declaring war on Russia. In our opinion, when Napoleon
Bonaparte did not take power in France in 1799, he was definitely replaced by
another deputy. In it, we could see a relatively different image of France, Europe,
and the whole world today. Our conclusion is that in place of any famous person,
another substitute, maybe another famous person will be found. But then the
historical development in that society, in that country, then in that region, and even
in the whole world will take a different shape. According to V. P. Bransky, "there
is also a possibility of turning prediction (prediction) into foretelling. In the
language of physicists, it is also possible to confirm predictions on the basis of
experiment, that is, scientific experience. If theory and practice coincide, then it
can be considered that prophecy is confirmed. In this case, no one can prove that
this is a coincidence. It is a social experiment and the whole history of the world
embodies a chain of such experiments"6. 5 Lutfullaevich, Salimov B. "The Manifestation of Dialectical Principles in the Life of the Human and
Society." JournalNX, vol. 6, no. 05, 2020, pp. 112-117. 6 Салимов Б.Л. Илмий билиш жараёнида тасодифнинг ўрни.
Фалсафа
фанлари
номзоди
диссертацияси. Алишер Навоий номли Ўзбекистон Миллий кутубхонаси(№417. Ю-34272/0). Тошкент. 2008,
144 б. 6 Салимов Б.Л. Илмий билиш жараёнида тасодифнинг ўрни.
Фалсафа
фанлари
номзоди
диссертацияси. Алишер Навоий номли Ўзбекистон Миллий кутубхонаси(№417. Ю-34272/0). Тошкент. 2008,
144 б y
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Салимов Б.Л. Илмий билиш жараёнида тасодифнинг ўрни.
Фалсафа
фанлари
Ў р
д
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иссертацияси. Алишер Навоий номли Ўзбекистон Миллий кутубхонаси(№417. Ю-34272/0). Тошкен
4 б. 5 Lutfullaevich, Salimov B. "The Manifestation of Dialectical Principles in the Life of the Human and
Society." JournalNX, vol. 6, no. 05, 2020, pp. 112-117.
6 Салимов Б.Л. Илмий билиш жараёнида тасодифнинг ўрни.
Фалсафа
фанлари
номзоди ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS this topic: "If there is a need for a famous person, he is always found. The fact that
he (the famous person) is that person, that he was born there and became a famous
person is a coincidence. However, if there was no such famous person, surely
another person would be found and could take his place, better or worse than him4. This opinion of D. I. Shirokanov corresponds to the process of self-organization in
society in terms of its content. In our view, bringing a society from instability to a
state of stability by celebrities is a form of self-organization. One can agree with
many aspects of D.I. Shirokanov's opinion. After all, coincidences play an
important role in the birth of any person, his career and the process of becoming a
famous person. But in the absence of that well-known and famous person, we
cannot say that someone else will definitely take his place. True, substitutes are
always found to take the place of any famous person. But what will be the activity
of these substitutes, there is a doubt that they will take the place of a famous person
and become famous people themselves. In our opinion, D.I. Shirokanov took a
model from the following opinion of F. Engels, one of the ideological leaders of
the system he lived and created: "The appearance of this same famous person in a
certain country at a certain time is nothing more than a pure coincidence," writes F. .Engels. - But if we reject this person, there will be a need to replace him with
another person, and such a replacement will be found immediately. It was
something of a coincidence that Napoleon's ancestor of Corsica became the
military dictator needed by the war-weary French Republic. But, if it were not for
Napoleon, someone else would have played his role. This is proven by the fact that .2023 .2023 4 ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS цияси. Алишер Навоий номли Ўзбекистон Миллий кутубхонаси(№417. Ю-34272/0). Тошкент. 200 .2023
6
7 Салимов Б.Л. Илмий билиш жараёнида тасодифнинг ўрни.
Фалсафа
фанлари
номзоди
диссертацияси. Алишер Навоий номли Ўзбекистон Миллий кутубхонаси(№417. Ю-34272/0). Тошкент. 2008,
144 б. ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS | J | acquire a positive character. But we must not forget that theory and practice do not
always match. Their compatibility depends, first of all, on the extent to which that
theory is developed. However, we must also remember that the complete
predictability of one's life makes people bored. Because their lifestyles are the
same. "If all the events in the world were fully predicted, there would be no
uncertainties. As a result, there is no need to take any risk in the movement of
people. Such a world would be defined in a strictly relational or homogeneous
sense. Nothing new can be created in it. Everything is doomed to repeat itself
forever in the same way".7 In short, the phenomenon of chance can be seen in the
life of the society. Chance affects society through human activities. CONCLUSION The application of theory to practice opens the door to not only telling, but also
to the flourishing of human society. Of course, both theory and practice should .2023 5 ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS .2023
6
REFERENCES
1. Салимов Б.Л. Илмий билиш жараёнида тасодифнинг ўрни.
Фалсафа
фанлари номзоди диссертацияси. Алишер Навоий номли Ўзбекистон
Миллий кутубхонаси(№417. Ю-34272/0). Тошкент. 2008, 144 б.
2. Lutfullaevich, Salimov B. "The Manifestation of Dialectical Principles in the
Life of the Human and Society." JournalNX, vol. 6, no. 05, 2020, pp. 112-117.
3. Salimov, Bakhriddin Lutfullaevich (2023). OPINIONS OF CENTRAL ASIAN
SCHOLARS ON SOCIAL RELATIONS. Oriental renaissance: Innovative,
educational, natural and social sciences, 3 (1-2), 178-182.
7 Салимов Б.Л. Илмий билиш жараёнида тасодифнинг ўрни.
Фалсафа
фанлари
номзоди
диссертацияси. Алишер Навоий номли Ўзбекистон Миллий кутубхонаси(№417. Ю-34272/0). Тошкент. 2008,
144 б. 1. Салимов Б.Л. Илмий билиш жараёнида тасодифнинг ўрни. Фалсафа
фанлари номзоди диссертацияси. Алишер Навоий номли Ўзбекистон
Миллий кутубхонаси(№417. Ю-34272/0). Тошкент. 2008, 144 б. 1. Салимов Б.Л. Илмий билиш жараёнида тасодифнинг ўрни. Фалсафа
фанлари номзоди диссертацияси. Алишер Навоий номли Ўзбекистон
Миллий кутубхонаси(№417. Ю-34272/0). Тошкент. 2008, 144 б. 2. Lutfullaevich, Salimov B. "The Manifestation of Dialectical Principles in the
Life of the Human and Society." JournalNX, vol. 6, no. 05, 2020, pp. 112-117. 2. Lutfullaevich, Salimov B. "The Manifestation of Dialectical Principles in the
Life of the Human and Society." JournalNX, vol. 6, no. 05, 2020, pp. 112-117. 3. Salimov, Bakhriddin Lutfullaevich (2023). OPINIONS OF CENTRAL ASIAN
SCHOLARS ON SOCIAL RELATIONS. Oriental renaissance: Innovative,
educational, natural and social sciences, 3 (1-2), 178-182. 6 ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS
4. Cалимов Бахриддин Лутфуллаевич (2020). ФИЛОСОФСКАЯ РОЛЬ
ДИАЛЕКТИЧЕСКИХ
КАТЕГОРИЙ
В
ЖИЗНИ
ЧЕЛОВЕКА.
Историческая психология и социология истории, 13 (1), 111-119. ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS
4. Cалимов Бахриддин Лутфуллаевич (2020). ФИЛОСОФСКАЯ РОЛЬ
ДИАЛЕКТИЧЕСКИХ
КАТЕГОРИЙ
В
ЖИЗНИ
ЧЕЛОВЕКА. Историческая психология и социология истории, 13 (1), 111-119. ECONOMICS AND LAW | JOURNAL COLLECTION OF MATERIALS 4. Cалимов Бахриддин Лутфуллаевич (2020). ФИЛОСОФСКАЯ РОЛЬ
ДИАЛЕКТИЧЕСКИХ
КАТЕГОРИЙ
В
ЖИЗНИ
ЧЕЛОВЕКА. Историческая психология и социология истории, 13 (1), 111-119. 4. Cалимов Бахриддин Лутфуллаевич (2020). ФИЛОСОФСКАЯ РОЛЬ
ДИАЛЕКТИЧЕСКИХ
КАТЕГОРИЙ
В
ЖИЗНИ
ЧЕЛОВЕКА. Историческая психология и социология истории, 13 (1), 111-119. .2023 7
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https://openalex.org/W2018781231
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https://www.cdc.gov/pcd/issues/2014/pdf/14_0047.pdf
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English
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Characteristics of the Built Environment in Relation to Objectively Measured Physical Activity Among Mexican Adults, 2011
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Preventing chronic disease
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public-domain
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Introduction Our findings contrast with those from high-income countries, sug-
gesting that environmental programs and policies to increase phys-
ical activity in Mexican cities cannot be adapted from high-in-
come countries without considering the local context. The built environment correlates of physical activity are docu-
mented in high-income countries but have yet to be studied among
Mexican adults. Our objectives were to assess the associations
between characteristics of the built environment and physical
activity among adults in Cuernavaca, Mexico, and to examine po-
tential moderation by perceived park and neighborhood safety. Introduction Physical inactivity contributes to 5.3 million annual deaths world-
wide and is a risk factor for obesity, diabetes, cardiovascular dis-
ease, and cancer (1). Evidence links the built environment and
physical activity (2). Walkability (an index incorporating residen-
tial density, retail area-to-land ratio, connectivity [ie, number of 3-
and 4-way intersections], and land-use mix) is positively associ-
ated with physical activity (3), but most evidence is from high-in-
come countries (2). Correlate studies from low-to-middle income
countries have recently emerged, and initial findings suggest dif-
ferences from findings for high-income countries (4,5). Results Walkability was inversely related to total weekly minutes of
MVPA (1-km buffer, −46.9 [standard error, 20.0]; P = .03) and
weekly minutes of MVPA within bouts (500-m buffer, −31.5
[12.9]; P = .02). The number of transit routes in the 500-m buffer
was inversely related to total weekly minutes of MVPA (−23.8
[10.6]; P = .04). Perception of park safety moderated the associ-
ation between physical activity and having a park intersect the
500-m buffer. Deborah Salvo, PhD; Rodrigo S. Reis, PhD; Areyh D. Stein, PhD; Juan Rivera, PhD;
Reynaldo Martorell, PhD; Michael Pratt, MD, MPH Suggested citation for this article: Salvo D, Reis RS, Stein AD,
Rivera J, Martorell R, Pratt M. Characteristics of the Built
Environment in Relation to Objectively Measured Physical
Activity Among Mexican Adults, 2011. Prev Chronic Dis 2014;
11:140047. DOI: http://dx.doi.org/10.5888/pcd11.140047. ORIGINAL RESEARCH
Characteristics of the Built Environment in
Relation to Objectively Measured Physical
Activity Among Mexican Adults, 2011 Deborah Salvo, PhD; Rodrigo S. Reis, PhD; Areyh D. Stein, PhD; Juan Rivera, PhD;
Reynaldo Martorell, PhD; Michael Pratt, MD, MPH The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health
and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions.
www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Prevention 1 Methods We conducted a population-based study of adults in Cuernavaca,
Mexico, in 2011 (N = 677). Participants wore Actigraph GT3X ac-
celerometers for 7 days. We used geographic information systems
(GIS) to generate 500-m- and 1-km-buffer–based measures of net
residential density, proportion of commercial land use, land-use
mix, connectivity, walkability, and number of parks and transit
routes. We also obtained data on distance to the nearest park with
GIS. Perceived neighborhood and park safety were self-reported. We created quartile-based categories for all built environment
characteristics and ran linear regression models to estimate the as-
sociation between each characteristic and total weekly moderate-
to-vigorous physical activity (MVPA) and MVPA within 10-
minute bouts. Studies that rely on self-report (as most studies do [2]) are valu-
able in identifying domains of activity (eg, leisure vs transporta-
tion) and environmental perceptions, but objective measures
provide more credible evidence for both research and policy (2,6). New technologies, such as accelerometry and geographic informa-
tion systems (GIS), allow for a more precise estimation of physic-
al activity and the built environment (2,7). www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Prevention 1 Physical activity measurement and outcomes Physical activity was measured with ActiGraph GT3X accelero-
meters (ActiGraph, LLC) using 60-second epochs (counts per
minute). Participants wore an accelerometer on their right hip for 7
days at all times except when sleeping, showering, or swimming. Verbal (in person and by demonstration) and written instructions
on how to wear the accelerometer were provided. To further en-
sure protocol compliance, we made 2 telephone calls during the
week, and participants indicated their start- and end-time of use
per day in an accelerometer log. After 7 days, we made a second
home visit to verify wear time. Nonwear time was defined as 60 or
more consecutive zeros (1 hour). A valid wear-day was defined as
10 or more valid hours. We validated wear time using MeterPlus
4.2 software (Santech, Inc) and comparing data from the accelero-
meter log and the telephone calls. Additional information was re-
quested on site from the participant if unusual patterns of daily
wear time were observed. If fewer than 5 valid days were recor-
ded, the participant was asked to wear the device for additional
days, and a third home visit was scheduled. Days on which we de-
livered and recovered the accelerometer were considered non-val-
id. Data were scored with MeterPlus 4.2 using Freedson’s cut-
points for adults (14). The objective of this study was to identify associations between
objectively measured physical activity and objectively measured
aspects of the built environment among adults in Cuernavaca,
Mexico. We hypothesized that the US-based walkability index
would not be positively associated with moderate-to-vigorous
physical activity (MVPA) in Cuernavaca and that the associations
between the built environment and physical activity would be
moderated by safety perception. PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 11, E147
AUGUST 2014 cluded to avoid bias. When a household declined to participate or
there was no eligible participant, the household immediately to the
right was selected. cluded to avoid bias. When a household declined to participate or
there was no eligible participant, the household immediately to the
right was selected. Mexico has an epidemic of obesity and chronic disease (8). The
2012 National Health and Nutrition Survey reported that 71.2% of
Mexican adults are overweight or obese, and diabetes and cardi-
ovascular disease are the leading causes of death (8,9). In 2004,
physical inactivity accounted for an estimated 4.4% of deaths and
1.2% of disability-adjusted life years among Mexicans (10). However, only 17.2% of adults reported being inactive in 2012
(8). The built environment correlates of physical activity in Mex-
ico are unexplored. Trained field workers recruited 1 participant per household during
a home visit. Eligible participants were adults aged 20 to 65 who
lived permanently in the household for at least 6 months and had
no disability that precluded walking. Participants provided written
informed consent; the study was approved by the institutional re-
view boards of Emory University and the Mexican National Insti-
tute of Public Health. Designing and implementing environmental strategies to increase
physical activity requires context-specific studies (2,6,11). Crime
and safety may influence physical activity, but studies report in-
consistent results (2). Given the current high crime rates in Mex-
ico (9), safety perception may moderate the association of physic-
al activity with objectively measured environmental variables. Sampling We collected data in 2011. A representative sample was identified
using census tracts as the primary sampling units, which were then
stratified into 4 levels of socioeconomic status (SES) based on
quartiles and 2 levels of walkability (3) stratified by the median. Eight census tracts per stratum were randomly selected, yielding
32 (of 123 in Cuernavaca) study census tracts. Seven blocks per
census tract were randomly selected, and 2 to 4 households per
block were randomly selected. Blocks immediately proximal to a
census tract with a different SES–walkability stratum were ex- Study design and site This was a population-based, multistage cluster study in Cuerna-
vaca, a city of 365,000 inhabitants in central Mexico. Although it
is wealthier than the average Mexican city (Human Development
Index of 0.86 vs 0.78 for Mexico [12]), Cuernavaca has the so-
cioeconomic, structural, political, and cultural characteristics typ-
ical of a low-to-middle income city (12). Crime in Cuernavaca in-
creased during the past decade; homicides increased by 277%
(9,13). This study is part of the International Physical Activity and
the Environment Network (IPEN)–Mexico project (6). We calculated minutes per week of total MVPA and minutes per
week of MVPA within 10-minute bouts (Appendix). Bouts were
defined as having a minimum duration of 10 minutes, with at least
80% of the bout corresponding to MVPA (ie, all breaks totaled
<20% of the bout). If a single break lasted more than 2 minutes,
the bout was considered to have been interrupted. A similar defini-
tion has been reported (15). Data on bouts were generated in Mat-
Lab 7.7 (The MathWorks Inc). 2 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. Objective measurement of the built environment We assessed the built environment using GIS-derived attributes. The location of each participant’s residence was geocoded using
ArcGIS 9.3 (ESRI Inc). One kilometer and 500-m street-network
buffers were generated around each participant’s household. Al- The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 2 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm ontrol and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm Perception of safety Trained field workers administered the Neighborhood Environ-
ment Walkability Scale–Abbreviated (NEWS-A) (18) during the
second home visit. Two variables for perceived safety were gener-
ated: perception of neighborhood safety and perception of park
safety. Both variables were dichotomized as safe or unsafe (Ap-
pendix). PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 11, E147
AUGUST 2014 though other studies used a 1-km buffer (16), the greater density
and urbanization in some Latin American cities compared with cit-
ies in high-income countries (17) suggest that certain features of a
built environment in a smaller buffer (500 m) may be more relev-
ant to our study. (20). Regression coefficients are represented as weekly minutes
(standard error [SE]) of total MVPA or bouts of MVPA. We used
likelihood ratio tests to identify potential interactions between the
built environment and perceived safety. If an interaction was sig-
nificant at P < .05, linear regression models were run to estimate
the association of the built environment variable with each physic-
al activity outcome in each stratum of perceived safety. The built
environment characteristics were also modeled as continuous vari-
ables. Statistical significance for all regression analyses, likeli-
hood ratio tests, and tests for linear trend was defined as P < .05. We generated the following built environment variables: net resid-
ential density, proportion of commercial land use, connectivity
(intersection density), land-use mix, walkability index (3), num-
ber of parks intersecting the buffer, and number of public transit
(bus) routes intersecting the buffer. Because no bus route intersec-
ted any 1-km buffer without also intersecting the corresponding
500-m buffer, we used a single variable. We measured the dis-
tance to the nearest park by using the street network. We categor-
ized each variable according to city-wide tertiles (for the number
of parks intersecting buffer) or quartiles (for all other built envir-
onment variables). The category with the smallest value was used
as the reference value for each variable (Appendix). GIS data were
provided by the Mexican National Institute of Statistics and Geo-
graphy and the Land Use Registry Department of Cuernavaca. We
generated all GIS variables through ArcGIS 9.3. Covariates Having 8 or more bus routes intersecting the 500-m buffer was in-
versely related to total MVPA (−23.8 [0.6] min; P = .04) (Table 3)
but not to MVPA within bouts (−7.0 [12.3] min; P = .58) (Table
4). We found no significant linear trend for the relationship
between the number of transit routes and total MVPA or MVPA
within bouts; neither did we find any significant association
between distance to the closest park and total MVPA or MVPA
within bouts. Age, sex, education, marital status, individual-level SES (based on
25 questions on household characteristics and assets used by the
National Health and Nutrition Survey of Mexico [8]), and motor-
vehicle ownership were ascertained by questionnaire during the
second home visit. Body mass index (BMI) was measured using
Tanita scales and fixed stadiometers (Tanita Corporation of Amer-
ica, Inc), following standardized procedures (19). Results The study response rate was 58.9%, calculated on the basis of the
number of selected households that had an eligible adult. Eight
participants were excluded because of invalid accelerometry data,
and 7 were excluded because of geocoding problems; the final
analytic sample was 662 participants. We found no significant dif-
ferences in sociodemographic characteristics between the final
sample and the excluded participants. The mean age of the sample
was 42.0 years; 48.1% were male, 32.7% had education beyond
high school, 55.8% were motor vehicle owners, and 31.8% were
obese (Table 1); 41.3% perceived their neighborhood as unsafe,
and 39.9% perceived their parks as unsafe (Table 2). Participants
engaged in MVPA an average 221.3 (standard deviation [SD],
10.1) minutes per week and 63.4 (SD, 4.3) minutes per week with-
in bouts of MVPA; 58.5% met the 150 minutes per week of phys-
ical activity recommended by the World Health Organization (21)
when considering total MVPA, but 13.3% met the requirement
when considering bouts of MVPA. PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 11, E147
AUGUST 2014 The walkability index in the 1-km buffer was inversely associated
with total MVPA (−46.9 [20.0] min; P = .03 for highest vs lowest
quartile of walkability). For the 500-m buffer, a significant in-
verse linear trend (P = .02) was identified for the association of
walkability with total MVPA. High walkability in the 500-m buf-
fer was related to 31.5 (12.9) fewer minutes per week of MVPA
within bouts (P = .02) when compared with low walkability
(quartile 1), and the inverse linear trend between walkability and
bouts of MVPA persisted (P = .01). For walkability within the 1-
km buffer, quartile 2 (medium, −22.3 [10.2] min; P = .04) and
quartile 3 (medium-high, −34.2 [10.01] min; P = <.001) but not
quartile 4 were negatively associated with bouts of MVPA, using
quartile 1 (low walkability) as the reference. The linear trend was
not significant for the relationship between walkability in the 1-km
buffer and MVPA within bouts. We found similar significant in-
verse associations between both physical activity outcome vari-
ables (total MVPA and MVPA within bouts) and the individual
components of the walkability index as defined for high-income
countries (3). associations with physical activity were found for proximity to bus
stops more than 500 meters apart in Bogotá (23), suggesting that if
norms for use of designated bus stops changed, physical activity
among users might increase. This is relevant for Cuernavaca be-
cause about half of adults do not own motor vehicles. associations with physical activity were found for proximity to bus
stops more than 500 meters apart in Bogotá (23), suggesting that if
norms for use of designated bus stops changed, physical activity
among users might increase. This is relevant for Cuernavaca be-
cause about half of adults do not own motor vehicles. Despite high crime rates in Cuernavaca (9,13), neighborhood
safety perception was not associated with physical activity, nor did
it moderate any of the relationships with built environment vari-
ables. However, park safety perception elucidated the negative as-
sociation between having 1 park in the 500-m buffer and having
lower levels of weekly physical activity. It was only when parks
were perceived as unsafe that the presence of a park in the 500-m
buffer was inversely related to physical activity; no association
was found when parks were perceived as safe. Statistical analysis Participants who had 1 park intersecting the 500-m buffer en-
gaged in 27.9 (14.9) fewer minutes per week of total MVPA (P =
.05) and 16.8 (8.2) fewer minutes of MVPA within bouts (P = .03)
than participants with no parks intersecting the 500-m buffer. We
found no significant association for participants with 2 or more in-
tersecting parks for total MVPA or MVPA within bouts (using 0
parks as reference), and no significant linear trends were found for
this relationship. All analyses accounted for the multistage clustered design and
were weighted for probability of selection and nonresponse by
sex, using the survey procedures of SAS 9.3 (SAS Institute Inc). Unadjusted linear regression models for total MVPA and bouts of
MVPA were run using each GIS and safety perception variable as
independent variables. We ran models adjusting for all covariates. We used SAS’s SURVEYREG procedure, which uses a design-
based approach instead of a model-based approach, therefore al-
lowing for linear modeling of non-normally distributed outcomes g to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
ealth Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Prevention 3 PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY Our findings on the walkability index (3) have research and pub-
lic health implications for low-to-middle income countries. Stud-
ies from the United States, Europe, and Australia showed positive
associations between physical activity and intersection density
(connectivity), land-use mix, and residential density (3,24–26). These elements were combined into a walkability index (3). Our
results show an inverse association between this index (or its indi-
vidual components) and physical activity. Perceived neighborhood safety was not significantly associated
with total MVPA or MVPA within bouts. Unsafe park perception
was inversely associated with total MVPA (−23.2 [9.2] min; P =
.08) and MVPA within bouts (−12.0 [7.0] min; P = .05). The asso-
ciation between number of parks intersecting the 500-m buffer
with both total MVPA and MVPA within bouts was moderated by
perceived park safety (tests for interaction, total MVPA, P = .04;
bouts of MVPA, P = .02). When parks were perceived as unsafe,
having 1 park intersect the 500-m buffer was associated with 30.8
(14.9) (P = .047) and 19.2 (7.2) (P = .03) fewer weekly minutes of
total MVPA and bouts of MVPA respectively, whereas no signi-
ficant association was found when parks were perceived as safe. Although findings from Curitiba, Brazil, show associations
between physical activity and walkability consistent with those
from high-income countries (27), in Bogotá, Colombia, research-
ers using GIS-derived data and self-reported physical activity re-
ported no associations between residential density or land-use mix
and physical activity, which may reflect scale differences because
almost all neighborhoods were dense with mixed-use (23,28). Similar results to ours were reported in Bangladesh (29). Studies
used sample-specific definitions of high and low walkability, but
low density, low connectivity, or low land-use mix in Cuernavaca
may be equivalent to high density, high land-use mix, and high
connectivity in high-income countries. Perhaps neighborhoods that
are too dense, mixed, or connected represent a barrier for physical
activity, and the association of physical activity with walkability
may be of an inverse U-shape rather than linear. Our data are in-
sufficient to test this hypothesis. Analyses of the full IPEN data set
could help address this question. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions.
4
Centers for Disease Control and Prevention • www cdc gov/pcd/issues/2014/14 0047 htm 4 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm Discussion The associations of objectively measured physical activity with
objectively measured aspects of the built environment for a repres-
entative sample of adults in Cuernavaca differ markedly from as-
sociations reported for high-income countries. Our results suggest
that the relationship between the built environment and physical
activity may be context-specific, and that the context in Mexico
differs markedly from that in high-income countries. Our results highlight the complexity of the relationships between
the built environment and physical activity among Mexican adults. Medium and medium-high but not high net residential density in
the 1-km buffer were associated with lower levels of total MVPA
and bouts of MVPA when compared with low density, suggesting
a threshold for this relationship. We observed similar nonlinear re-
lationships for other built environment characteristics. Meanwhile, In contrast to reports from high-income countries (22), the num-
ber of bus routes in our study was inversely associated with phys-
ical activity. The exact location of bus stops is not provided by
transportation authorities, which is characteristic of public transit
in Mexico, where it is common practice to signal a bus to stop
anywhere along its route. Our results are consistent with a study in
Bogotá, Colombia, which has a similar transit system (5). Positive The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. ontrol and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm Acknowledgments inverse linear trends were found for the association of both total
MVPA and bouts of MVPA with the US-based walkability index
and the proportion of commercial land-use in the 500-m buffer. Some characteristics showed stronger associations with physical
activity for the 500-m buffer only (number of parks per buffer,
commercial land-use proportion), while for others the 1-km buffer
yielded more significant associations (residential density, land-use
mix), suggesting that some environmental features are related to
physical activity only in a microenvironment. Some features were
more strongly associated with bouts of MVPA (residential density,
land-use mix, walkability, number of parks) and others with total
MVPA (number of transit routes). More studies are needed to elu-
cidate these complex relationships. This work was supported by the CDC Foundation, which received
an unrestricted training grant from The Coca-Cola Company. We
thank Catalina Torres, Hugo Rodríguez, Lilian Perez, Andrea
Ramirez, and our field workers for essential logistic support. Author Information Corresponding Author: Deborah Salvo, PhD, Nutrition and Health
Research Center, National Institute of Public Health of Mexico,
Av. Universidad 655, Col. Sta. Maria Ahuacatitlan, Cuernavaca,
Morelos, Mexico, CP 62100. Telephone: 408-829-0005. E-mail:
deborah.salvo@insp.mx. Out study has several limitations, including a cross-sectional
design, self-reported data, and a low response rate (but consistent
with other studies in Mexico [8]). We did not examine the associ-
ation of park type with physical activity. Objective data on neigh-
borhood crime were not available. Only land-cover (vs parcel-
level) data were available for land use, possibly decreasing preci-
sion for these variables. GIS pedestrian-enhanced street layers
were not available. More accessible GIS data are required to bet-
ter study built environment associations in Mexico. Objective
measures provide excellent estimates of physical activity and the
built environment but do not allow the study of physical activity
domains and neighborhood perceptions that self-report tools can
allow. This shortcoming of objective measures was demonstrated
by the significant moderation by park safety perception of the as-
sociation of number of parks with physical activity. Analyses of
transportation and leisure-time physical activity might better elu-
cidate these relationships. Author Affiliations: Rodrigo S. Reis, Pontificia Universidade
Catolica do Parana, Curitiba, Brazil, and Federal University of
Parana, Curitiba, Brazil; Areyh D. Stein, Reynaldo Martorell,
Emory University, Atlanta, Georgia; Juan Rivera, Nutrition and
Health Research Center, National Institute of Public Health,
Cuernavaca, Mexico; Michael Pratt, Emory University, Atlanta,
Georgia, and Centers for Disease Control and Prevention, Atlanta,
Georgia. Dr Salvo is also affiliated with the Stanford Prevention
Research Center, Stanford University School of Medicine,
Stanford, California. At the time of the research, she was a
doctoral student at Emory University, Atlanta, Georgia. References Lee IM, Shiroma EJ, Lobelo F, Puska P, Blair SN, Katzmarzyk
PT, et al. Effect of physical inactivity on major non-
communicable diseases worldwide: an analysis of burden of
disease and life expectancy. Lancet 2012;380(9838):219–29. 1. Our study has many strengths. It is the first Latin American study
to examine physical activity and the built environment using ob-
jective measures for both dependent and independent variables. As
part of IPEN, state-of-the-art methods, measures, and instruments
were used (6). The use of a non-normalized physical activity out-
come variable responded to the need for studies that treat physical
activity as a continuous variable (2,21,30). By using quartiles for
the environmental variables instead of dichotomizing or assigning
z scores, we identified more associations and observed complex
relationships between physical activity and the built environment. Our study is the first to provide estimates of the built environment
correlates of physical activity among urban Mexican adults. Our
findings have public health implications for Mexico and poten-
tially other low-to-middle income countries, showing associations
of physical activity with walkability that are discordant with those
observed in high-income countries (3) and suggesting that caution
should be taken when translating evidence from high-income
countries to low-to-middle income countries (4,5,11). Bauman AE, Reis RS, Sallis JF, Wells JC, Loos RJ, Martin
BW, et al. Correlates of physical activity: why are some people
physically active and others not? Lancet 2012;
380(9838):258–71. 2. Frank LD, Sallis JF, Saelens BE, Leary L, Cain K, Conway
TL, et al. The development of a walkability index: application
to the Neighborhood Quality of Life Study. Br J Sports Med
2010;44(13):924–33. 3. Hino AA, Reis RS, Sarmiento OL, Parra DC, Brownson RC. The built environment and recreational physical activity among
adults in Curitiba, Brazil. Prev Med 2011;52(6):419–22. 4. Sarmiento OL, Schmid TL, Parra DC, Diaz-del-Castillo A,
Gomez LF, Pratt M, et al. Quality of life, physical activity, and
built environment characteristics among Colombian adults. J
Phys Act Health 2010;7(Suppl 2):S181–95. 5. opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. g to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
ealth Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions.
www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Prevention 5 PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 11, E147
AUGUST 2014 VOLUME 11, E147
AUGUST 2014 VOLUME 11, E147
AUGUST 2014 Cerin E, Conway TL, Cain KL, Kerr J, De Bourdeaudhuij I,
Owen N, et al. Sharing good NEWS across the world:
developing comparable scores across 12 countries for the
Neighborhood Environment Walkability Scale (NEWS). BMC
Public Health 2013;13:309. 18. Kerr J, Sallis J, Owen N, De Bourdeaudhuij I, Cerin E, Reis
RS, et al. Advancing science and policy through a coordinated
international study of physical activity and built environments:
IPEN adult methods. J Phys Act Health 2013;10(4):581–601. 6. Frank LD, Schmid TL, Sallis JF, Chapman J, Saelens BE. Linking objectively measured physical activity with
objectively measured urban form: findings from SMARTRAQ. Am J Prev Med 2005;28(Suppl 2):117–25. 7. Gibson RS. Principles of nutritional assessment. 2nd edition. New York (NY): Oxford University Press; 2005. Chapter 10,
Anthropometric assessment of body size. 19. Gutiérrez JP, Rivera-Dommarco J, Shamah-Levy T,
Villalpando-Hernández S, Franco A, Cuevas-Nasu L, et
al.Encuesta Nacional de Salud y Nutrición 2012. Resultados
Nacionales. Cuernavaca (MX): Instituto Nacional de Salud
Pública; 2012. 8. Cassell D. Wait wait, don’t tell me . . . you’re using the wrong
proc!. SAS Users Group International (SUGI). 2006. http://
www2.sas.com/proceedings/sugi31/193-31.pdf. Accessed June
17, 2014. 20. World Health Organization. Global recommendations on
p h y s i c a l a c t i v i t y f o r h e a l t h , 2 0 1 0 . h t t p : / /
www.who.int.proxy.library.emory.edu/dietphysicalactivity/
factsheet_recommendations/en/index.html. Accessed October
10, 2012. 21. Instituto Nacional de Estadistica y Geografia. Causas de
defunción en México: defunciones generales totales por
principales causas de mortalidad, 2011. http://
w w w . i n e g i . o r g . m x / s i s t e m a s / s i s e p t /
default.aspx?t=mdemo107&s=est&c=23587. Accessed
October 24, 2013. 9. Besser LM, Dannenberg AL. Walking to public transit: steps to
help meet physical activity recommendations. Am J Prev Med
2005;29(4):273–80. 22. Stevens G, Dias RH, Thomas KJ, Rivera JA, Carvalho N,
Barquera S, et al. Characterizing the epidemiological transition
in Mexico: national and subnational burden of diseases,
injuries, and risk factors. PLoS Med 2008;5(6):e125. 10. Cervero R, Sarmiento OL, Jacoby E, Gomez LF, Neiman A. Influences of built environments on walking and cycling:
lessons from Bogotá. Int J Sustain Transport 2009;
3(4):203–26. 23. Ebrahim S, Pearce N, Smeeth L, Casas JP, Jaffar S, Piot P. References www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Prevention 5 5 PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 11, E147
AUGUST 2014 6 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. VOLUME 11, E147
AUGUST 2014 Tackling non-communicable diseases in low- and middle-
income countries: is the evidence from high-income countries
all we need? PLoS Med 2013;10(1):e1001377. 11. Sundquist K, Eriksson U, Kawakami N, Skog L, Ohlsson H,
Arvidsson D. Neighborhood walkability, physical activity, and
walking behavior: the Swedish Neighborhood and Physical
Activity (SNAP) study. Soc Sci Med 2011;72(8):1266–73. 24. Lemaresquier T, Santizo R. Índice de desarrollo humano
municial en México. Mexico City (MX): Programa de las
Naciones Unidas para el Desarrollo; 2004. 12. Owen N, Cerin E, Leslie E, duToit L, Coffee N, Frank LD, et
al. Neighborhood walkability and the walking behavior of
Australian adults. Am J Prev Med 2007;33(5):387–95. 25. Morelos CIdlDHd. Criminalidad, violencia y derechos
humanos en Morelos 2007: http://cidhmorelos.wordpress.com/. 13. Van Dyck D, Cardon G, Deforche B, Owen N, Sallis JF, De
Bourdeaudhuij I. Neighborhood walkability and sedentary time
in Belgian adults. Am J Prev Med 2010;39(1):25–32. 26. Freedson PS, Melanson E, Sirard J. Calibration of the
Computer Science and Applications, Inc. accelerometer. Med
Sci Sports Exerc 1998;30(5):777–81. 14. Siqueira Reis RS, Hino AA, Kerr J, Hallal PC. Walkability and
physical activity: findings from Curitiba, Brazil. Am J Prev
Med 2013;45(3):269–75. 27. Holman RM, Carson V, Janssen I. Does the fractionalization of
daily physical activity (sporadic vs. bouts) impact
cardiometabolic risk factors in children and youth? PLoS ONE
2011;6(10):e25733. 15. Gomez LF, Sarmiento OL, Parra DC, Schmid TL, Pratt M,
Jacoby E, et al. Characteristics of the built environment
associated with leisure-time physical activity among adults in
Bogota, Colombia: a multilevel study. J Phys Act Health 2010;
7(Suppl 2):S196–203. 28. Oliver LN, Schuurman N, Hall AW. Comparing circular and
network buffers to examine the influence of land use on
walking for leisure and errands. Int J Health Geogr 2007;6:41. 16. World urbanization prospects: the 2011 revision: data tables
and highlights. United Nations Department of Economic and
Social Affairs, Population Division; 2012. http://esa.un.org/
wpp/Documentation/pdf/WPP2012_HIGHLIGHTS.pdf. Accessed October 24, 2013. 17. Zakiul M. Children and urban neighborhoods: relationships
between outdoor activities of children and neighborhood
physical characteristics in Dhaka, Bangladesh [doctoral
dissertation]. Raleigh (NC): North Carolina State University;
2012 http://www.lib.ncsu.edu/resolver/1840.16/3963. 29. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. VOLUME 11, E147
AUGUST 2014 ontrol and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm 6 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm 6 Haskell WL, Lee I, Pate RR, Powell KE, Blair SN, Franklin
BA, et al. Physical activity and public health: updated
recommendation for adults from the American College of
Sports Medicine and the American Heart Association. Med Sci
Sports Exerc 2007;39(8):1423.
30. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions.
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the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. p
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the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 8 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm VOLUME 11, E147
AUGUST 2014 Table 2. Characteristics of the Built Environment and Perceptions of Safety Among Adults, Overall and by Buffer, Cuernavaca, Mex-
ico, 2011
Variable
No. of Respondents (% [SE])a (N = 662)b
Distance to closest park
Very close (<313 m)
165 (16.3 [4.4])
Medium (313 to <771 m)
166 (25.6 [5.0])
Far (771 to <1,357 m)
166 (30.9 [5.4])
Very far (≥1,357 m)
165 (27.2 [6.6])
No. of public bus routes per buffer
0
81 (11.0 [4.9])
1 or 2
228 (35.8 [6.9])
3–7
172 (24.4 [6.3])
≥8
181 (28.8 [7.1])
Neighborhood safety perception (score range, 0–5)
Safe (score <3)
387 (58.7 [2.8])
Unsafe (score ≥3)
275 (41.3 [2.8])
Park safety perception (score range, 0–5)
Safe (score <3)
382 (60.1 [2.9])
Unsafe (≥3)
280 (39.9 [2.9])
Variable
By Bufferc
500 m
1 km
n (% [SE])
n (% (SE])
Net residential density (no. of single family units/km2 of residential land use in buffer)
Low (<1,583)
190 (27.9 [6.6])
165 (23.6 [4.4])
Medium (1,583 to <2,174)
115 (20.6 [4.4])
173 (30.3 [5.9])
Medium-high (2,174 to <2,730)
115 (20.6 [5.2])
166 (25.0 [4.4])
High (≥2,730)
242 (31.0 [5.7])
158 (21.1 [4.5])
Proportion of commercial land use (km2 designated to commercial land use/km2 of buffer)
Low (0%)
249 (37.8 [6.4])
154 (25.0 [5.4])
Medium (0% to <15%)
119 (18.1 [4.8])
183 (27.5 [4.7])
Medium-high (15% to <25%)
132 (22.5 [4.4])
162 (26.4 [4.2])
High (≥25%)
162 (21.6 [4.4])
163 (21.1 [4.6])
Land-use mixd Table 2. Characteristics of the Built Environment and Perceptions of Safety Among Adults, Overall and by Buffer, Cuernavaca, Mex-
ico, 2011
Variable
No. of Respondents (% [SE])a (N = 662)b
Distance to closest park
Very close (<313 m)
165 (16.3 [4.4])
Medium (313 to <771 m)
166 (25.6 [5.0])
Far (771 to <1,357 m)
166 (30.9 [5.4])
Very far (≥1,357 m)
165 (27.2 [6.6])
No. of public bus routes per buffer
0
81 (11.0 [4.9])
1 or 2
228 (35.8 [6.9])
3–7
172 (24.4 [6.3])
≥8
181 (28.8 [7.1])
Neighborhood safety perception (score range, 0–5)
Safe (score <3)
387 (58.7 [2.8])
Unsafe (score ≥3)
275 (41.3 [2.8])
Park safety perception (score range, 0–5)
Safe (score <3)
382 (60.1 [2.9])
Unsafe (≥3)
280 (39.9 [2.9])
Variable
By Bufferc
500 m
1 km
n (% [SE])
n (% (SE])
Net residential density (no. Tables Table 1. Characteristics of Study Participants, Cuernavaca, Mexico, 2011
Sociodemographic variables
n (%a) (N = 662)b
Male
297 (48.1)
Age, y
≤35
217 (33.4)
>35 to ≤50
260 (40.0)
>50 to ≤65
185 (26.6)
Socioeconomic statusc
Low
196 (31.8)
Medium
163 (24.0)
Medium-high
192 (28.0)
High
111 (16.2)
Education
More than high school
216 (32.7)
Marital status
Single
162 (25.0)
Marriedd
434 (65.3)
Divorcede
66 (9.7)
Motor vehicle ownership
370 (55.8)
Body mass index (kg/m2)
Overweight (25 to <30)
275 (41.2)
Obese (≥30)
210 (31.8)
a Percentages are weighted for probability of selection and nonresponse by sex. b Final sample (N = 662) excluded 15 participants for whom accelerometry or GIS data were not available; no differences in sociodemographic characteristics were
found between the final sample and excluded participants. c Classifications based on quartiles of index of socioeconomic status; index based on household characteristics and assets. d Includes living with someone. e Includes separated and widows. hors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Hum
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions 8 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm 8 PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 11, E147
AUGUST 2014 VOLUME 11, E147
AUGUST 2014 of single family units/km2 of residential land use in buffer)
Low (<1,583)
190 (27.9 [6.6])
165 (23.6 [4.4])
Medium (1,583 to <2,174)
115 (20.6 [4.4])
173 (30.3 [5.9])
Medium-high (2,174 to <2,730)
115 (20.6 [5.2])
166 (25.0 [4.4])
High (≥2,730)
242 (31.0 [5.7])
158 (21.1 [4.5])
Proportion of commercial land use (km2 designated to commercial land use/km2 of buffer)
Low (0%)
249 (37.8 [6.4])
154 (25.0 [5.4])
Medium (0% to <15%)
119 (18.1 [4.8])
183 (27.5 [4.7])
Medium-high (15% to <25%)
132 (22.5 [4.4])
162 (26.4 [4.2])
High (≥25%)
162 (21.6 [4.4])
163 (21.1 [4.6])
Land-use mixd
Abbreviations: SE standard error www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Prevention 9 9 PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 11, E147
AUGUST 2014 (continued)
Table 2. Characteristics of the Built Environment and Perceptions of Safety Among Adults, Overall and by Buffer, Cuernavaca, Mex-
ico, 2011
Variable
No. of Respondents (% [SE])a (N = 662)b
Low (<0.25)
224 (34.4 [5.9])
135 (21.7 [5.9])
Medium (0.25 to <0.40)
208 (31.7 [4.1])
224 (29.1 [4.6])
Medium-high (0.40 to <0.50)
135 (17.8 [3.9])
169 (27.2 [5.3])
High (≥0.50)
95 (16.0 [4.7])
134 (22.0 [5.0])
Connectivity (no. of 3- and 4-way intersections/km2 of buffer)
Low (<111)
166 (25.7 [4.2])
166 (25.7 [5.6])
Medium (111 to <135)
167 (26.2 [3.7])
161 (22.1 [3.9])
Medium-high (135 to <167)
166 (26.0 [2.8])
166 (25.8 [7.0])
High (≥167)
163 (22.1 [6.1])
169 (26.4 [5.8])
Walkability indexd
Low (less than −45)
148 (25.4 [6.6])
155 (23.3 [6.5])
Medium (−45 to <15)
231 (33.4 [4.3])
174 (30.3 [5.1])
Medium-high (15 to <50)
135 (20.4 [3.7])
187 (27.3 [4.0])
High (≥50)
148 (20.8 [4.2])
146 (19.1 [4.9])
No. of parks per buffer
0
415 (72.9 [7.7])
236 (40.9 [6.5])
1
165 (20.9 [6.8])
231 (36.5 [5.9])
≥2
82 (6.3 [5.8])
195 (22.5 [6.3])
Abbreviations: SE, standard error. a Percentages were weighted for probability of selection and nonresponse by sex. b Final sample (N = 662) excluded 15 participants for whom accelerometry or GIS data were not available; no differences in sociodemographic characteristics were
found between the final sample and excluded participants. c For these variables, 1-km and 500-m street-network buffers were generated around each participant’s household. d See Appendix for definition. (continued)
Table 2. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 11, E147
AUGUST 2014 Table 3. Association of Total Minutes Per Week of Moderate-to-Vigorous Physical Activity With Environmental Variables Among Mex-
ican Adults, Overall and by Buffer, Cuernavaca, Mexico, 2011
Variable
Regression Estimate (SE) [P Value]
Unadjusted
Adjusteda
Neighborhood safety
Safe
0
0
Unsafe
0.23 (16.38) [.99]
−1.33 (18.50) [.94]
Park safety
Safe
0
0
Unsafe
−17.19 (10.11) [.11]
−23.17 (9.16) [.08]
Distance to park
Very close (<313 m)
0
0
Medium (313 to <771 m)
−2.43 (16.86) [.87]
12.93 (16.18) [.43]
Far (771 to <1,357 m)
−4.81 (22.20) [.83]
6.13 (16.61) [.72]
Very far (≥1,357 m)
10.59 (24.19) [.67]
15.56 (18.02) [.40]
No. of public bus routes per buffer
0
0
0
1
−12.69 (19.50) [.52]
−7.76 (20.02) [.70]
2–7
−17.99 (19.43) [.36]
−15.55 (23.82) [.52]
≥8
−51.72 (15.62) [.002]
−23.78 (10.61) [.04]
Variable
By Bufferb
500 m
1 km
Unadjusted
Adjusteda
Unadjusted
Adjusteda
Net residential density (no. of single family units/km2 of residential land use in buffer)
Low (<1,583)
0
0
0
0
Medium (1,583 to <2,174)
1.02 (27.01) [.87]
−4.72 (23.1) [.84]
−37.60 (17.02) [.04]
−39.88 (18.20) [.04]
Medium-high (2,174 to
<2,730)
−9.75 (26.88) [.72]
−39.10 (22.65) [.16]
−30.38 (21.45) [.24]
−40.42 (22.61) [.08]
High (≥2,730)
−16.12(23.56) [.50]
−30.58 (18.69) [.20]
6.20 (12.31) [.81]
−15.99 (18.14) [.49]
Proportion of commercial land use (km2 designated to commercial land use/km2 of buffer)
Low (0%)
0
0
0
0
Medium (0% to <15%)
0.67 (21.47) [.98]
−2.77 (20.55) [.89]
−9.00 (25.80) [.73]
−0.37 (24.17) [.99]
Medium-high (15% to <25%)
−7.88 (24.93) [.75]
−9.48 (21.06) [.66]
−44.31 (20.64) [.04]
−26.26 (20.43) [.21]
High (≥25%)
−73.18 (16.92) [<.001] −54.51 (15.53) [<.001]c
−35.53 (22.13) [.12]
−20.27 (20.85) [.34]
a Adjusted models control for total accelerometer wear time, sex, age, individual socioeconomic status, education, marital status, motor vehicle ownership, and
body mass index
,
, Table 3. VOLUME 11, E147
AUGUST 2014 Characteristics of the Built Environment and Perceptions of Safety Among Adults, Overall and by Buffer, Cuernavaca, Mex-
ico, 2011 10 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm 10 www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Prevention 11 body mass index.
b For these variables, 1-km and 500-m street-network buffers were generated around each participant’s household.
c Significant linear trend (P < .05).
d See Appendix for definition PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY Association of Total Minutes Per Week of Moderate-to-Vigorous Physical Activity With Environmental Variables Among Mex-
ican Adults, Overall and by Buffer, Cuernavaca, Mexico, 2011
Variable
Regression Estimate (SE) [P Value]
Unadjusted
Adjusteda
Neighborhood safety
Safe
0
0
Unsafe
0.23 (16.38) [.99]
−1.33 (18.50) [.94]
Park safety
Safe
0
0
Unsafe
−17.19 (10.11) [.11]
−23.17 (9.16) [.08]
Distance to park
Very close (<313 m)
0
0
Medium (313 to <771 m)
−2.43 (16.86) [.87]
12.93 (16.18) [.43]
Far (771 to <1,357 m)
−4.81 (22.20) [.83]
6.13 (16.61) [.72]
Very far (≥1,357 m)
10.59 (24.19) [.67]
15.56 (18.02) [.40]
No. of public bus routes per buffer
0
0
0
1
−12.69 (19.50) [.52]
−7.76 (20.02) [.70]
2–7
−17.99 (19.43) [.36]
−15.55 (23.82) [.52]
≥8
−51.72 (15.62) [.002]
−23.78 (10.61) [.04]
Variable
By Bufferb
500 m
1 km
Unadjusted
Adjusteda
Unadjusted
Adjusteda
Net residential density (no. of single family units/km2 of residential land use in buffer)
Low (<1,583)
0
0
0
0
Medium (1,583 to <2,174)
1.02 (27.01) [.87]
−4.72 (23.1) [.84]
−37.60 (17.02) [.04]
−39.88 (18.20) [.04]
Medium-high (2,174 to
<2,730)
−9.75 (26.88) [.72]
−39.10 (22.65) [.16]
−30.38 (21.45) [.24]
−40.42 (22.61) [.08]
High (≥2,730)
−16.12(23.56) [.50]
−30.58 (18.69) [.20]
6.20 (12.31) [.81]
−15.99 (18.14) [.49]
Proportion of commercial land use (km2 designated to commercial land use/km2 of buffer)
Low (0%)
0
0
0
0
Medium (0% to <15%)
0.67 (21.47) [.98]
−2.77 (20.55) [.89]
−9.00 (25.80) [.73]
−0.37 (24.17) [.99]
Medium-high (15% to <25%)
−7.88 (24.93) [.75]
−9.48 (21.06) [.66]
−44.31 (20.64) [.04]
−26.26 (20.43) [.21]
High (≥25%)
−73.18 (16.92) [<.001] −54.51 (15.53) [<.001]c
−35.53 (22.13) [.12]
−20.27 (20.85) [.34]
a Adjusted models control for total accelerometer wear time, sex, age, individual socioeconomic status, education, marital status, motor vehicle ownership, and
body mass index. b For these variables, 1-km and 500-m street-network buffers were generated around each participant’s household. ted models control for total accelerometer wear time, sex, age, individual socioeconomic status, education, marital status, motor vehicle ownership, and
ass index. ese variables, 1-km and 500-m street-network buffers were generated around each participant’s household. ficant linear trend (P < .05). ppendix for definition. (continued on next page)
nions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Prevention 11 www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Preven PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 11, E147
AUGUST 2014 Table 4. Association of Minutes Per Week of 10-Minute Boutsa of Moderate-to-Vigorous Physical Activity With Environmental Vari-
ables Among Mexican Adults, Overall and by Buffer, Cuernavaca, Mexico, 2011
Variable
Regression Estimate (SE) [P Value]
Unadjusted
Adjustedb
Neighborbood safety
Safe
0
0
Unsafe
−5.00 (6.55) [.45]
−2.61 (7.38) [.73]
Park safety
Safe
0
0
Unsafe
−10.60 (6.56) [.09]
−12.00 (7.01) [.05]
Distance to park
Very close (<313 m)
0
0
Medium (313 to <771 m)
5.17 (6.76) [.45]
9.91 (7.00) [.17]
Far (771 to <1,357 m)
4.92 (8.68) [.58]
8.16 (7.72) [.30]
Very far (≥1,357 m)
4.44 (10.18) [.67]
8.24 (11.51) [.48]
No. of public bus routes per buffer
0
0
0
1
2.66 (0.26) [.80]
−1.59 (11.09) [.89]
2–7
−5.39 (8.13) [.51]
−4.51 (11.42) [.70]
≥8
−10.70 (11.29) [.35]
−6.95 (12.29) [.58]
Variable
By Bufferc
500 m
1 km
Unadjusted
Adjustedb
Unadjusted
Adjustedb
Net residential density (no. of single family units/km2 of residential land use in buffer)
Low (<1,583)
0
0
0
0
Medium (1,583 to <2,174)
−3.87(11.59) [.74]
−7.97 (12.00) [.51]
−21.28 (7.12) [.04]
−24.32 (6.24) [.03]
Medium-high (2,174 to
<2,730)
−15.87 (7.72) [.17]
−22.87 (10.55) [.05]
−16.95 (12.80) [.15]
−23.34 (10.88) [.05]
High (≥2,730)
−10.46 (9.74) [.40]
−21.68 (9.32) [.04]
−1.55 (15.22) [.91]
−8.92 (10.30) [.50]
Proportion of commercial land use (km2 designated to commercial land use/km2 of buffer)
Low (0%)
0
0
0
0
Medium (0% to <15%)
3.20 (10.70 [.77]
−0.82 (10.50) [.94]
−0.91 (9.92) [.93]
1.56 (9.75) [.87]
Medium-high (15% to <25%)
−4.12 (9.07) [.65]
−8.59 (9.48) [.37]
−7.46 (11.07) [.51]
−5.70 (10.84) [.60]
a Only activity recorded as moderate-to-vigorous bouts of at least 10 minutes; at least 80% of bout had to be moderate-to-vigorous activity (ie, no breaks of ≥2
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY AUGUST 2014 Table 4. PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 11, E147
AUGUST 2014 (continued)
Table 3. Association of Total Minutes Per Week of Moderate-to-Vigorous Physical Activity With Environmental Variables Among Mex-
ican Adults, Overall and by Buffer, Cuernavaca, Mexico, 2011
Variable
Regression Estimate (SE) [P Value]
Unadjusted
Adjusteda
Land-use mixd
Low (<0.25)
0
0
0
0
Medium (0.25 to <0.40)
11.03 (18.18) [.55]
6.29 (18.55) [.74]
10.92 (19.07) [.57]
14.44 (19.32) [.46]
Medium-high (0.40 to <0.50)
−36.34 (24.06) [.14]
−25.61 (20.89) [.23]
−18.09 (15.34) [.25]
−11.20 (15.24) [.47]
High (≥0.50)
−4.33 (26.73) [.87]
−12.68 (19.76) [.53]
−1.14 (24.20) [.96]
−0.39 (21.75) [.99]
Connectivity (no. of 3- and 4-way intersections/km2 of buffer)
Low (<111)
0
0
0
0
Medium (111 to <135)
−31.87 (29.88) [.30]
−36.14 (22.05) [.11]
−6.93 (21.90) [.75]
−21.55 (16.94) [.21]
Medium-high (135 to <167)
−16.58 (30.20) [.59]
−22.63 (31.40) [.48]
−41.20 (18.82) [.04]
−35.51 (14.67) [.02]
High (≥167)
−3.71 (18.49) [.84]
−18.68 (18.25) [.32]
−16.71 (20.40) [.42]
−32.07 (17.49) [.09]
Walkability indexd
Low (less than −45)
0
0
0
0
Medium (−45 to <15)
−23.78 (22.19) [.29]
−24.35 (18.31) [.20]
−8.35 (18.02) [.65]
−20.42 (18.91) [.29]
Medium-high (15 to <50)
−19.92 (30.83) [.52]
−27.11 (26.53) [.32]
−36.07 (21.03) [.10]
−12.89 (21.54) [.55]
High (≥50)
−25.88 (26.16) [.33]
−34.30 (21.69) [.13]c
2.59 (23.42) [.91]
−46.91 (20.04) [.03]
No. of parks
0
0
0
0
0
1
−13.23 (14.96) [.38]
−27.87 (14.90) [.05]
−19.25 (19.03) [.32]
−10.82 (16.98) [.53]
>2
45.03 (43.78) [.31]
31.61 (35.60) [.38]
5.25 (18.58) [.78]
−3.27 (17.73) [.86]
a Adjusted models control for total accelerometer wear time, sex, age, individual socioeconomic status, education, marital status, motor vehicle ownership, and
body mass index. b For these variables, 1-km and 500-m street-network buffers were generated around each participant’s household. c Significant linear trend (P < .05). d See Appendix for definition. Per Week of Moderate-to-Vigorous Physical Activity With Environmental Variables Among Mex-
rnavaca, Mexico, 2011 The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 12 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm 12 PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY Association of Minutes Per Week of 10-Minute Boutsa of Moderate-to-Vigorous Physical Activity With Environmental Vari-
ables Among Mexican Adults, Overall and by Buffer, Cuernavaca, Mexico, 2011
Variable
Regression Estimate (SE) [P Value]
Unadjusted
Adjustedb
Neighborbood safety
Safe
0
0
Unsafe
−5.00 (6.55) [.45]
−2.61 (7.38) [.73]
Park safety
Safe
0
0
Unsafe
−10.60 (6.56) [.09]
−12.00 (7.01) [.05]
Distance to park
Very close (<313 m)
0
0
Medium (313 to <771 m)
5.17 (6.76) [.45]
9.91 (7.00) [.17]
Far (771 to <1,357 m)
4.92 (8.68) [.58]
8.16 (7.72) [.30]
Very far (≥1,357 m)
4.44 (10.18) [.67]
8.24 (11.51) [.48]
No. of public bus routes per buffer
0
0
0
1
2.66 (0.26) [.80]
−1.59 (11.09) [.89]
2–7
−5.39 (8.13) [.51]
−4.51 (11.42) [.70]
≥8
−10.70 (11.29) [.35]
−6.95 (12.29) [.58]
Variable
By Bufferc
500 m
1 km
Unadjusted
Adjustedb
Unadjusted
Adjustedb
Net residential density (no. of single family units/km2 of residential land use in buffer)
Low (<1,583)
0
0
0
0
Medium (1,583 to <2,174)
−3.87(11.59) [.74]
−7.97 (12.00) [.51]
−21.28 (7.12) [.04]
−24.32 (6.24) [.03]
Medium-high (2,174 to
<2,730)
−15.87 (7.72) [.17]
−22.87 (10.55) [.05]
−16.95 (12.80) [.15]
−23.34 (10.88) [.05]
High (≥2,730)
−10.46 (9.74) [.40]
−21.68 (9.32) [.04]
−1.55 (15.22) [.91]
−8.92 (10.30) [.50]
Proportion of commercial land use (km2 designated to commercial land use/km2 of buffer)
Low (0%)
0
0
0
0
Medium (0% to <15%)
3.20 (10.70 [.77]
−0.82 (10.50) [.94]
−0.91 (9.92) [.93]
1.56 (9.75) [.87]
Medium-high (15% to <25%)
−4.12 (9.07) [.65]
−8.59 (9.48) [.37]
−7.46 (11.07) [.51]
−5.70 (10.84) [.60]
a Only activity recorded as moderate-to-vigorous bouts of at least 10 minutes; at least 80% of bout had to be moderate-to-vigorous activity (ie, no breaks of ≥2
minutes). b Adjusted models control for total accelerometer wear time, sex, age, individual socioeconomic status, education, marital status, motor vehicle ownership, and
BMI status a Only activity recorded as moderate-to-vigorous bouts of at least 10 minutes; at least 80% of bout had to be moderate-to-vigorous activity (ie, no breaks of ≥2
minutes). b Adjusted models control for total accelerometer wear time, sex, age, individual socioeconomic status, education, marital status, motor vehicle ownership, and
BMI status. c For these variables, 1-km and 500-m street-network buffers were generated around each participant’s household. d Significant linear trend (P < .05). The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY (continued on next page)
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Prevention 13 13 PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY a Only activity recorded as moderate-to-vigorous bouts of at least 10 minutes; at least 80% of bout had to be moderate-to-vigorous activity (ie, no breaks of ≥2
minutes).
b Adjusted models control for total accelerometer wear time, sex, age, individual socioeconomic status, education, marital status, motor vehicle ownership, and
BMI status.
c For these variables, 1-km and 500-m street-network buffers were generated around each participant’s household.
d Significant linear trend (P < .05). VOLUME 11, E147
AUGUST 2014 Appendix. Definitions of Physical Activity Outcomes, Environmental Variables, and Covariates
Variable
Type
Definition
Physical activity outcomes
Total minutes of
MVPA per week
Continuous Total minutes per week of activity ≥1,952 counts per minutea. Minutes of MVPA
within bouts
Continuous Minutes per week of activity ≥1,952 counts per minutea, recorded within bouts of MVPA as
defined for this study
MVPA bout
—
All of the following characteristics were required for a bout of MVPA: 1) duration of at least 10
minutes; 2) intensity of activity is moderate to vigorous (≥1,952 counts per minute); 3) ≥80%
of bout consists of moderate-to-vigorous intensity of activity (≥1,952 counts per minute);
therefore, ≤20% of the bout could correspond to breaks of ≤1,952 counts per minute; 4) each
break of ≤1,952 counts per minutea has a maximum duration of 2 minutes. If requirement
no.3 or no. 4 was not met, the bout was considered interrupted. Environmental variables
Net residential
density (500 m and 1
km)
Categorical Number of single family units per buffer area/total squared kilometers of residential land use
in buffer area. Categoriesb were defined as low (reference), <1,582.99; medium, 1,582.99 to
<2,174.19; medium-high, 2,174.19 to <2,729.75; high, ≥2,729.75. These values were
rounded to whole numbers for reporting purposes. Proportion of
commercial land use
(500 m and 1 km)
Categorical Squared kilometers designated to commercial land use in buffer area/total squared
kilometers of buffer area. Categoriesb were defined as low (reference); 0%; medium, 0% to
less than15%: medium-high, 15% to less than 25%; high, ≥25%. Connectivity (500 m
and 1 km)
Categorical Intersection density defined as the number of 3- and 4-way intersections in a buffer area/total
buffer area in squared kilometers. Categoriesb were defined as low (reference), <111.03;
medium, 111.03 to <135.33; medium-high, 135.33 to <166.59; high: ≥166.59. Values were
rounded to whole numbers for reporting purposes. Land-use mix (500 m
and 1 km])
Categorical −[∑i (pi)(ln pi)]/(ln k) where p = proportion of total land uses, i = land use category, ln =
natural logarithm, k = number of land uses. Range is 0 to 1. Categoriesb are defined as low
(reference), <0.25; medium, 0.25 to <0.40; Medium-high, 0.40 to <0.50; high, ≥0.50. Walkability index
Categorical z-scored net residential density + z-scored commercial land use proportion + 2(z-scored
connectivity) + z-scored land-use mixc. Categoriesb are defined as low (reference), less than
−45; medium, −45 to <15, medium-high, 15 to <50; high, ≥50. PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 11, E147
AUGUST 2014 (continued)
Table 4. Association of Minutes Per Week of 10-Minute Boutsa of Moderate-to-Vigorous Physical Activity With Environmental Vari-
ables Among Mexican Adults, Overall and by Buffer, Cuernavaca, Mexico, 2011
Variable
Regression Estimate (SE) [P Value]
Unadjusted
Adjustedb
High (≥25%)
−32.64 (8.32) [<.001]d
−33.96 (8.28) [.001]d
−16.35 (10.79) [.14]
−20.31 (13.42) [.11]
Land-use mixd
Low (<0.25)
0
0
0
0
Medium (0.25 to <0.40)
0.73 (8.60) [.93]
−5.49 (9.06) [.55]
2.93 (10.34) [.78]
1.03 (11.17) [.93]
Medium-high (0.40 to <0.50)
−19.58 (14.46) [.19]
−20.65 (12.96) [.12]
−20.72 (8.57) [.02]
−22.33 (9.64) [.03]
High (≥0.50)
−16.44 (11.62) [.17]
−19.17 (11.63) [.11]
−12.42 (13.10) [.35]
−17.34 (8.27) [.05]
Connectivity (number of 3- and 4-way intersections/km2 of buffer)
Low (<111)
0
0
0
0. Medium (111 to <135)
−8.79 (11.98) [.47]
−8.68 (10.15) [.40]
−6.58 (10.32) [.53]
−9.24 (10.63) [.39]
Medium-high (135 to <167)
−8.33 (17.49) [.64]
−11.54 (16.65) [.49]
−13.75 (9.10) [.14]
−18.33 (10.37) [.09]
High (≥167)
−7.95 (9.93) [.43]
−10.35 (10.63) [.34]
−5.95 (9.92) [.55]
−9.48 (6.91) [.12]
Walkability indexd
Low (less than −45)
0
0
0
0
Medium (−45 to <15)
−9.84 (12.18) [.43]
−10.22 (11.47) [.38]
−16.79 (9.48) [.09]
−22.25 (10.22) [.04]
Medium-high (15 to <50)
−16.54 (12.23) [.19]
−18.31 (12.94) [.17]
−28.23 (9.31) [.01] −34.21 (10.01) [<.001]
High (≥50)
−29.89 (11.96) [.02]d
−31.49 (12.93) [.02]d
−6.25 (12.47) [.62]
−12.65 (13.19) [.35]
No. of parks
0
0
0
0
0
1
−11.96 (6.24) [.07]
−16.84 (8.19) [.03]
−0.02 (11.80) [.99]
0.24 (12.38) [.98]
≥2
25.91 (27.18) [.35]
20.51 (26.16) [.44]
1.15 (10.44) [.91]
−2.17 (11.13) [.85]
a Only activity recorded as moderate-to-vigorous bouts of at least 10 minutes; at least 80% of bout had to be moderate-to-vigorous activity (ie, no breaks of ≥2
minutes). The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 14 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm 14 VOLUME 11, E147
AUGUST 2014 Distance to closest
park
Categorical Distance in meters to the nearest park. Categoriesb are defined as near (reference),
<313.116; medium, 313.116 to <771.156; far, 771.156 to <1,356.51; very far, ≥1,356.51. These values were rounded to whole numbers for reporting purposes. Number of parks
(500 m and 1 km])
Categorical Number of parks intersecting a buffer (ie, includes parks fully in and parks partially in a
buffer). Categories are 0 parks in buffer (reference); 1 park in buffer; ≥2 parks in buffer. Public transportation
routes
Categorical Number of bus public transit routes intersecting the buffer. No difference was found between
500-m and 1-km buffers (ie, no public transit route intersected the 1-km buffer while not
intersecting the 500-m buffer). Therefore, a unique variable was used. Categories were based
on quartiles: 0 routes (reference), 1 or 2 routes, 3–7 routes, ≥8 routes. Neighborhood safety
perception
Categorical Binary: 1 = unsafe neighborhood, 0 = safe neighborhood. Based on average score (score
range, 1–4]) of 5 NEWS–Ad items (1 = lowest agreement, 4 = highest agreement]): The crime
rate in my neighborhood is high, the crime rate in my neighborhood makes it unsafe to walk
during the day, the crime rate in my neighborhood makes it unsafe to walk during the night,
a Source: Freedson et al (14). Number of single family units per buffer area/total squared kilometers of residential land use
in buffer area. Categoriesb were defined as low (reference), <1,582.99; medium, 1,582.99 to
<2,174.19; medium-high, 2,174.19 to <2,729.75; high, ≥2,729.75. These values were
rounded to whole numbers for reporting purposes. Number of bus public transit routes intersecting the buffer. No difference was found between
500-m and 1-km buffers (ie, no public transit route intersected the 1-km buffer while not
intersecting the 500-m buffer). Therefore, a unique variable was used. Categories were based
on quartiles: 0 routes (reference), 1 or 2 routes, 3–7 routes, ≥8 routes. a Source: Freedson et al (14). b Defined according to city-wide quartiles using census-tract–level data for Cuernavaca. c Source: Frank et al (3). d Neighborhood Environment Walkability Scale–Abbreviated (NEWS-A) (18). (continued on next page)
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. PREVENTING CHRONIC DISEASE
PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 11, E147
AUGUST 2014 VOLUME 11, E147
AUGUST 2014 6 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services,
the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. VOLUME 11, E147
AUGUST 2014 www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Prevention 15 www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Prevention 15 www.cdc.gov/pcd/issues/2014/14_0047.htm • Centers for Disease Control and Preven VOLUME 11, E147
AUGUST 2014 VOLUME 11, E147
AUGUST 2014 (continued)
Appendix. Definitions of Physical Activity Outcomes, Environmental Variables, and Covariates
Variable
Type
Definition
the parks and plazas in my neighborhood are unsafe to visit during the day, the parks and
plazas in my neighborhood are unsafe to visit during the night. A score ≥3 was categorized as
unsafe; a score <3, safe (reference). Park safety
perception
Categorical Binary: 1 = unsafe park, 0 = safe park. Based on average score (score range 1–4) of 2
NEWS–Ad items (1 = lowest agreement, 4 = highest agreement]): The parks and plazas in my
neighborhood are unsafe to visit during the day, the parks and plazas in my neighborhood are
unsafe to visit during the night. A score ≥3 was categorized as unsafe; a score <3, safe
(reference). Covariates
Sex
Categorical Binary: 1 = male, 0 = female (reference). Age
Continuous Range, 20–65 years. Individual
socioeconomic status
(SES)
Categorical Low (reference), medium, medium-high, high. Based on quartiles of individual SES index,
constructed using centralized z scores from a set of 25 questions on household
characteristics and assets per participant. The index excluded motor vehicle ownership and
education. Education level
Categorical Binary: 0 = high school or less (≤12 y of education) (reference), 1 = more than high school
(>12 y of education). Marital status
Categorical Single (not living with a partner) (reference); married (includes living with a partner); divorced
(includes separated and widows. Motor vehicle
ownership
Categorical Binary: 1 = yes (owning at least 1 car or motorcycle), 0 = no (reference). Body mass index
(BMI)
Categorical BMI (kg/m2) <25 (normal) (reference); 25 to <30 (overweight); ≥30 (obese). a Source: Freedson et al (14). b Defined according to city-wide quartiles using census-tract–level data for Cuernavaca. c Source: Frank et al (3). d Neighborhood Environment Walkability Scale–Abbreviated (NEWS-A) (18). 16 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2014/14_0047.htm 16
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Use of Polyamidoamine Dendrimers in Brain Diseases
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molecules
Review
Use of Polyamidoamine Dendrimers in
Brain Diseases
Maria Florendo 1,2,† , Alexander Figacz 1,2,† , Bhairavi Srinageshwar 1,2,3 , Ajit Sharma 4, *,
Douglas Swanson 4 , Gary L. Dunbar 2,3,5,6 and Julien Rossignol 1,2,3, * ID
1
2
3
4
5
6
*
†
College of Medicine, Central Michigan University, Mt. Pleasant, MI 48859, USA;
flore1mv@cmich.edu (M.F.); figac1am@cmich.edu (A.F.); srina1b@cmich.edu (B.S.)
Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University,
Mt. Pleasant, MI 48859, USA; dunba1g@cmich.edu
Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI 48859, USA
Department of Chemistry & Biochemistry, Central Michigan University, Mt. Pleasant, MI 48859, USA;
swans1d@cmich.edu
Department of Psychology, Central Michigan University, Mt. Pleasant, MI 48859, USA
Field Neurosciences Institute, St. Mary’s of Michigan, Saginaw, MI 48604, USA
Correspondence: sharm1a@cmich.edu (A.S.); rossi1j@cmich.edu (J.R.); Tel.: +1-989-774-3303 (A.S.);
+1-989-774-3405 (J.R.)
These authors contributed equally to this work.
Received: 30 July 2018; Accepted: 28 August 2018; Published: 3 September 2018
Abstract: Polyamidoamine (PAMAM) dendrimers are one of the smallest and most precise
nanomolecules available today, which have promising applications for the treatment of brain diseases.
Each aspect of the dendrimer (core, size or generation, size of cavities, and surface functional groups)
can be precisely modulated to yield a variety of nanocarriers for delivery of drugs and genes to
brain cells in vitro or in vivo. Two of the most important criteria to consider when using PAMAM
dendrimers for neuroscience applications is their safety profile and their potential to be prepared in a
reproducible manner. Based on these criteria, features of PAMAM dendrimers are described to help
the neuroscience researcher to judiciously choose the right type of dendrimer and the appropriate
method for loading the drug to form a safe and effective delivery system to the brain.
Keywords: PAMAM dendrimers; nanoparticles; blood-brain barrier; DNA delivery; drug delivery;
neurodegenerative diseases
1. Introduction
The major brain diseases that will be the focus of this review include neurodegenerative
diseases and an aggressive form of primary brain cancer known as glioblastoma multiforme
(GBM). Neurodegenerative diseases, such as Huntington’s disease (HD), Alzheimer’s disease (AD),
or Parkinson’s disease (PD), are pathologies consisting of neuronal degeneration causing loss of
function of the affected part of the brain. Since most types of neurons are unable to regenerate,
the death of these cells results in an irreversible pathology with detrimental outcomes [1]. Disease
presentations for neurodegenerative diseases vary from memory loss to lack of motor coordination,
depending on the area of the brain that is involved. Despite the difference in clinical symptoms,
all neurodegenerative diseases progress to the same outcome: irreversible cell death. Patients who
suffer from neurodegenerative diseases must live with their debilitating effects, making developments
for treatments increasingly important. The causes of neurodegenerative diseases are mostly
unknown [2]. However, inflammation seems to be closely associated with these diseases. Under the
broad category of different forms of glioma that arise from the glial cells, the most aggressive form
Molecules 2018, 23, 2238; doi:10.3390/molecules23092238
www.mdpi.com/journal/molecules
Molecules 2018, 23, 2238
2 of 18
of tumor is the GBM or grade-4 astrocytoma. This form of cancer does not have a cure and the
patients diagnosed with glioblastoma have a high rate of mortality and morbidity. GBM is also highly
associated with brain inflammation [3].
Many excellent reviews on the chemistry and biomedical applications of polyamidoamine
(PAMAM) dendrimers are available [4–9]. PAMAM dendrimers are by themselves complex
macromolecules and their appropriate use for a specific application can be confusing to many
non-chemists. Excellent recent reviews on the use of dendrimers for the central nervous system are
available [10].The intent of this review is not to summarize all of the work related to the applications of
PAMAM dendrimers to brain diseases. Instead, we shall look at the physical and chemical features of
PAMAM dendrimers that are beneficial for their potential uses in neuroscience research and eventually
in the clinic, keeping two critical criteria in mind:
•
•
Safety of the dendrimer formulation
Reproducibility in preparing the dendrimer formulation
Safety is always an essential requirement for all drugs, which is obvious and deserves no further
comments. However, reproducibility of dendrimer preparation is an important consideration for
clinical use of macromolecular drugs or macromolecular drug excipients. Preparation of large
[molecular weight (MW) > 1000 Da] synthetic macromolecules such as PAMAM dendrimers and
other nanomolecules, unlike small molecule drugs (MW < 500–1000 Da), is a challenging task for
the organic chemist. Besides the target compound, closely related chemical species are often formed
due to side reactions and are difficult to remove by well-established purification methods. The final
macromolecule formulation is typically plagued with large batch-to-batch variations that may not
meet the stringent requirements for regulatory approvals.
2. Features of PAMAM Dendrimers Useful for Neuroscience Applications
Dendrimers are branched nanomolecules. There are many different types of dendrimers that
have been synthesized with potential uses in the treatment of brain diseases. Examples include
carbosilane dendrimers, polylysine dendrimers, phosphorus-containing dendrimers, and PAMAM
dendrimers [11–13].
PAMAM dendrimers are one of the earliest dendrimers synthesized and made commercially
available [8]. As shown in Figure 1B, a PAMAM dendrimer consists of: (1) a core; (2) branches
composed of amide groups emanating from a branching point (tertiary amine) that form the walls of
cavities; and (3) terminal groups. The earliest core reported was ethylenediamine (EDA). Subsequently
PAMAM dendrimers were synthesized from a variety of other cores such as diaminobutane (DAB),
diaminohexane, diaminododecane and cystamine [14]. As with disulfide-containing proteins,
the disulfide bonds in cystamine-core dendrimers may be cleaved by reducing agents such as
glutathione and beta-mercaptoethanol to yield two half-dendrimers known as dendrons [15].
The branches of PAMAM dendrimers contain amide bonds that are similar to the peptide backbones
of proteins. Dendrimers with termini composed of primary amines (−NH2 ), hydroxyls (−OH)
or carboxyls (−COOH) were the earliest type of PAMAM dendrimers reported and commercially
available. The amine-terminated PAMAM dendrimers became popular among biologists and
biomedical researchers since they were commercially available and, more importantly, could easily
undergo bio-conjugation reactions with other molecules, using protocols and reagents that were
well-established for proteins. For example, these dendrimers are easily conjugated to fluorescent dyes
using reactive fluorescent reagents made for protein-labeling such as fluorescein isothiocyanate (FITC)
or NHS-Cyanine 5.5 (Cy5.5) for tracking purposes in biological systems.
Some of the important features of PAMAM dendrimers for neuroscience applications include:
•
•
•
High stability
High water-solubility
Small size
Molecules 2018, 23, 2238
•
•
•
3 of 18
Precision
Presence of cavities
Surfaces that can be readily modified
Thermogravimetric analysis data shows that PAMAM dendrimers are stable to heat; they start
degrading over 200 ◦ C [16,17]. Proteins rely on weak interactions such as salt bridges, hydrogen
bonds and hydrophobic forces for their shape. However, the three-dimensional shapes of PAMAM
dendrimers are mostly due to their dendritic architecture with some contribution by weak forces.
For this reason, PAMAM dendrimers may be stored frozen (dried or as solutions) and thawed for use
without any significant denaturation or aggregation. The use and storage of protein drugs requires
many precautions. However, this is not the case for PAMAM dendrimers if they become routinely
used in drug formulations. This is especially important for use in neurodegenerative diseases due to
the chronic treatment involved and patients that often have some degree of memory loss who may
leave the drugs at room temperature instead of in the refrigerator.
Like non-membrane proteins, PAMAM dendrimers are highly water-soluble if their surface is
composed of functional groups (e.g., −NH2 , −OH, COOH) that hydrogen-bond with water. Aqueous
solutions as high as 100–200 mg/mL dendrimer are readily formed. The water solubility of chemicals
is an important factor that affects their absorption, distribution, metabolism and excretion (ADME).
Nanomolecules with hydrophilic surfaces are less likely to become attracted to plasma proteins
(opsonization) and thus less prone to be cleared from the bloodstream via engulfment by cells of
the reticuloendothelial system. It is therefore important to keep in mind that attaching chemicals
such as drugs and fluorescent dyes to the surface of dendrimers will decrease the solubility of the
resulting nanomolecules and also affect their ADME. This is a serious practical problem since academic
research requires attachment of fluorescent dyes or other imaging agents to understand the biological
behavior of the nanomolecules. An example of the effect of dendrimer solubility on its biological
properties was demonstrated by Dougherty and colleagues [18]. The reaction of attaching fluorescent
dye to PAMAM dendrimers (or any macromolecules with numerous reactive sites) not only gives a
Poisson distribution of dye:dendrimer ratios, but the cellular uptake increases with the number of
dye molecules on the surface of the dendrimer. Since dyes are hydrophobic, their attachment on the
hydrophilic dendrimer surface creates hydrophobic sites that interact with the plasma membrane and
increase their cellular uptake via adsorptive endocytosis. Another example is evident in an elegant
report by Albertazzi and co-workers. Using two-photon microscopy, they showed that after injection
into the brain cortex of live animals, amine-terminated PAMAM G4 was able to diffuse from the
injection site while the less water-soluble G4-C12 (similar dendrimer with 25% of surface coated with
C12 chains) remained at the injection site. Clearly, good water solubility will be important for the
dendrimer to distribute its drug payload throughout the brain. In addition, the G4-C12 PAMAM
dendrimers were over three-fold more toxic to neurons than the G4 amine-terminated dendrimer. Due
to its water solubility, the G4 dendrimer readily diffused in the brain of living animals and therefore
did not form high local concentrations of the dendrimer to be toxic; neurons with the dendrimer
were still shown to be physiologically active. However, the poor water solubility of the G4-C12 led
to poor diffusion and high local concentration at the site of injection, resulting in neuronal death via
apoptosis. Even glial cell activation was none to minimal after exposure to the G4 amine-terminated
PAMAM dendrimer. This is an important concept to consider when trying to extrapolate cell culture
data to in vivo conditions. Since concentration drives many biological processes, good water solubility
diminishes the chances of highly localized concentrations of the nanomolecules. Thus, although G4
amine-terminated dendrimers may show toxicity under cell culture conditions (typically at micromolar
concentrations),their effect in the dynamic and changing in vivo conditions (e.g., rapid dilution to
nanomolar concentrations due to diffusion) might be surprisingly different [19].
Another critical feature of chemicals that affect their ADME is size. Commonly used PAMAM
dendrimers such as G4, G5 and G6 have molecular weights in the range of approximately 14–56 kDa.
These sizes are comparable to common proteins such as cytochrome c (MW ~12 kDa), lysozyme
Molecules 2018, 23, 2238
4 of 18
(MW ~14 kDa) and albumin (MW ~67 kDa). In terms of dimensions, the PAMAM dendrimers
(G1–G10) range from about 1–15 nm. Compared to other nanomolecules such as liposomes and those
made from polymers, PAMAM dendrimers are the smallest in size. Since the molecular weight cut-off
(MWCO) for the glomerular filtration membrane in healthy kidneys is about 45,000 Da or 6–8 nm for
nanoparticles [20,21], one can expect the commonly-used PAMAM dendrimers, especially G4 and G5,
to be eventually excreted out of the body after administration. This is important to avoid potential
accumulation and toxicity of the nanomolecules, especially with chronic use which is often the case for
treatment of neurodegenerative diseases. However, this also means that the dendrimers have shorter
half-lives when injected into the body. Larger generations >G6 or PEGylated dendrimers tend to stay
longer in the bloodstream for distribution to the organs [22,23]. The size of the nanomolecule is also
important for its ability to leave the bloodstream and enter the tissue. The distance between endothelial
cells lining blood vessels varies considerably from as large as over 100 nm in liver capillaries to less
than 1 nm in the blood brain barrier (BBB). This implies that it will be difficult for G4 or G5 PAMAM
dendrimers (or other nanomolecules) to simply travel between endothelial cells of the BBB unless the
barrier is compromised due to neuroinflammation [24]. Sarin and colleagues showed that dendrimers
smaller than 11.7–11.9 nm were able to cross the compromised blood-brain barrier of rodents with
malignant gliomas [25]. Nance and colleagues showed that neutral G4 PAMAM dendrimers composed
of 100% −OH (G4-OH) are able to cross the compromised BBB and diffuse into the brain parenchyma
to localize to activated glia of diseased mouse models [26]. In fact, this difference in size between
the healthy and disrupted BBB barrier offers PAMAM dendrimers the advantage of traversing the
compromised barrier compared to other nanomolecules such as liposomes, which are much larger.
Macromolecules made by nature such as proteins and nucleic acids are precise molecules with
well-defined molecular weights. On the other hand, laboratory synthesis of macromolecules is
challenging and often yields contaminating macromolecules with varying molecular weights besides
the target compound; this is often due to side reactions and the difficulty in purifying closely related
species. Reproducible synthesis of nanomolecules and good characterization data are critical for
their successful use in humans. There must be minimal lot-to-lot variation between batches of
dendrimers or other nanomolecules. The methods for PAMAM synthesis and characterization are
well-established. By using excess reagents (such as ethylenediamine) during PAMAM dendrimer
synthesis, the formation of side products (such as oligomers and looped surfaces) is dramatically
reduced. As previously mentioned, it is important to note that although the parent dendrimers may
be precise (low polydispersity), attachment of fluorescent dyes or other chemicals to the surface
(carried out without using reagent excess) often yields a heterogenous mixture of products with
varying ADME profiles. In addition, when administered into the body, such macromolecules may
become altered as their surface groups become detached via metabolism, potentially resulting in the
formation of toxic products. For example, a PAMAM dendrimer with 10 drugs attached to its surface
via hydrolysable ester bonds is synthesized and tested for safety. After administration to an animal,
the dendrimer inside the bloodstream or cells may be enzymatically degraded to yield a dendrimer
with six drugs attached. The ADME or safety profile of this new product is unpredictable and may
lead to long-term complications. Evidence for such a phenomenon is well-known among dendrimer
researchers. For example, as mentioned earlier, even the attachment of a few fluorescent dye molecules
to the surface of a dendrimer alters its cellular uptake [18].
The precision of PAMAM dendrimers and their derivatives is best appreciated by high-resolution
separation techniques that have been well-established for precise macromolecules such as proteins.
These techniques include reverse-phase high performance liquid chromatography (RP-HPLC) and
electrophoresis [27,28]. Although non-separation techniques such as Nuclear magnetic resonance
(NMR), infra-red (IR) or UV-visible spectrophotometry yield valuable characterization data, they do
not show the purity of the nanomolecules. When analyzed by electrophoresis on polyacrylamide gels
(typically at 1 mg/mL concentration), PAMAM dendrimers appear as discrete bands similar to proteins.
Polymers, on the other hand, are seen as smears on these gels [29]. Although electrophoresis has been
Molecules 2018, 23, 2238
5 of 18
frequently used for the characterization of amine-surface dendrimers, the technique also works very
well for separation of all types of PAMAM dendrimers (and their derivatives), irrespective of their
cores or surface groups, since all PAMAM dendrimers have tertiary amines (at the branching points,
Figure 1A) that are positively charged under acidic conditions. Charges are necessary for migration
in electrophoresis gels. Electrophoresis has been a gold standard for determination of protein purity
for several decades and is clinically an important tool for detection of various macromolecules in
blood such as serum proteins, isozymes, hemoglobin variants and lipoproteins. Unfortunately, many
reports on the synthesis and biomedical applications of PAMAM dendrimers do not show the purity
of their parent or modified dendrimers by electrophoresis. For example, we have run a variety of
PAMAM dendrimers by polyacrylamide gel electrophoresis (PAGE) and found that EDA-core PAMAM
dendrimers are less pure than DAB-core dendrimers, yet many neuroscience researchers still use the
former nanomolecules.
Another useful feature of PAMAM dendrimers for neuroscience applications is the presence
of cavities. When placed in aqueous solutions, these cavities are hydrophobic and offer sites for
loading small molecule drugs by a process known as encapsulation. Since the PAMAM dendrimer
itself is extremely water-soluble, encapsulation within the cavities is an ideal way to solubilize poorly
water-soluble drugs in aqueous solutions. For example, Igartúa and colleagues found that a G4
PAMAM dendrimer with an amine surface (or a G4.5 PAMAM dendrimer with a carboxyl surface)
could encapsulate about 20 molecules of the anti-epileptic drug, carbamazepine, which has potential
for treating neurodegenerative diseases [30]. Recent reviews on the use of PAMAM dendrimers
for encapsulating drugs are available [5,31]. Encapsulation is also how cyclodextrins (cyclic sugar
molecules popular in the drug industry) help to solubilize drugs. A cyclodextrin has one cavity
per molecule while a G4 PAMAM dendrimer has several cavities per molecule [32]. Many drugs,
including those used for treatment of neurodegenerative diseases, have limited solubility in water.
Their loading into PAMAM dendrimers will not only dramatically increase their solubility but will
also allow them to be taken up via endocytosis to cross the BBB. This feature of PAMAM dendrimers
is a major advantage over the use of proteins as nanocarriers, since proteins are highly compact
nanostructures with negligible empty space in their interiors. Although the water solubility of the
drug-encapsulated dendrimer may decrease compared to the free dendrimer, a major advantage of
encapsulation (compared to surface attachment of drugs discussed below) is that the purity, size and
surface of the dendrimer is unchanged; as previously mentioned, this is important for safety and
predictable ADME profiles.
Among all the components of a PAMAM dendrimer, the surface has drawn the most attention.
This is because macromolecules such as PAMAM dendrimers interact with cells and biomolecules
predominantly via surface interactions. The surface therefore dictates the safety profile, which is
one of the most important requirements for any therapeutic agent. Numerous studies have shown a
strong correlation between the toxicity of PAMAM dendrimers and their surface functional groups.
The amine-terminated dendrimer has been found to be the most toxic to cells compared to dendrimers
with –OH or –COOH groups. This is because amines are positively charged under physiological
conditions. Although amines are found on the surface of proteins, the toxicity of an amine-terminated
dendrimer is due to the very high amounts of amines per given surface area (charge density), a property
that results from its dendritic architecture. The high positive charge density of the amine-terminated
dendrimer is responsible for its rapid and strong interaction with most biomolecules and cells (which
have many negative charges on their surfaces), resulting in dendrimer toxicity [33]. For example,
when mixed with red blood cells (RBCs) in vitro, these dendrimers form holes in the RBCs and cause
hemolysis. When injected into the bloodstream, they would also readily attract complex acidic proteins
(with isoelectric point (pI) values less than seven) that are abundantly present in the extracellular
medium, resulting in altered ADME profiles. Several strategies have been used to alleviate this problem.
Chemicals such as PEG and amino acids have been attached to the surface amines in order to reduce
the charge density of the dendrimer. However, as noted above, such a strategy results in a mixture of
Molecules 2018, 23, 2238
6 of 18
products with different ADME profiles. In addition, altering the surface of the nanomolecule with PEG
or amino acids will form unnatural surfaces that may be recognized by the immune system, resulting in
theMolecules
production
of xantibodies,
loss of therapeutic efficacy and more importantly, adverse immune effects
2018, 23,
FOR PEER REVIEW
6 of 18
such as anaphylaxis [34]. On the other hand, parent PAMAM dendrimers with unmodified surfaces
immunogenic
The amines on[35].
theThe
surface
of on
thethe
dendrimer
readily
attach
were
found to be[35].
non-immunogenic
amines
surface ofare
thenecessary
dendrimertoare
necessary
dyes
(or other imaging
agents)
for tracking
and
complexand
andtodeliver
nucleic
acids for
to fluorescent
readily attach
fluorescent
dyes (or other
imaging
agents)
fortotracking
complex
and deliver
gene acids
therapy,
but are
also toxic
to also
cells.toxic
In order
to solve
thistodilemma,
important
question to
nucleic
for gene
therapy,
but are
to cells.
In order
solve this an
dilemma,
an important
answertois:answer
how many
amines
are
needed
dendrimer
so that they
safe
but
bebut
functional?
question
is: how
many
amines
areper
needed
per dendrimer
so can
thatbe
they
can
beyet
safe
yet be
A major step
towards
this objective
was recently
reported
by Srinageshwar
and colleagues.
They
functional?
A major
step towards
this objective
was recently
reported
by Srinageshwar
and colleagues.
designed
andand
synthesized
a mixed-surface
G4 PAMAM
dendrimer
composed
of 58 of
hydroxyl
groups
They
designed
synthesized
a mixed-surface
G4 PAMAM
dendrimer
composed
58 hydroxyl
(90%)(90%)
and six
amines
(10%),(10%),
known
as theasG4-90/10
dendrimer.
In comparison,
the parent
G4 aminegroups
and
six amines
known
the G4-90/10
dendrimer.
In comparison,
the parent
G4
terminated PAMAM
dendrimer
has 64
amine
groups
on itson
surface
and no
groups.
These
amine-terminated
PAMAM
dendrimer
has
64 amine
groups
its surface
andhydroxyl
no hydroxyl
groups.
mixed-surface
PAMAM
dendrimers,
whichwhich
can becan
prepared
by well-established
dendrimer
synthesis
These
mixed-surface
PAMAM
dendrimers,
be prepared
by well-established
dendrimer
protocols
(using excess
at different
hydroxyl/amine
ratios, were
found
to be
as pure
synthesis
protocols
(usingreagents)
excess reagents)
at different
hydroxyl/amine
ratios,
were
found
to beasasthe
parent
dendrimers
(as observed
on electrophoresis
gels and by
In addition,
the simple
pure
as the
parent dendrimers
(as observed
on electrophoresis
gelsRP-HPLC).
and by RP-HPLC).
In addition,
the dendrimer,
which is which
composed
of mostlyof−OH
and−aOH
fewand
−NH
2 groups,
probably
thesurface
simpleofsurface
of the dendrimer,
is composed
mostly
a few
−NHis
2 groups,
for its significantly
improved improved
safety profile.
The
G4-90/10
were also found
is responsible
probably responsible
for its significantly
safety
profile.
Thedendrimers
G4-90/10 dendrimers
were to
cross
the toBBB
when
injected
the carotid
rodents
[36]. Mixed-surface
PAMAM
also
found
cross
the BBB
wheninto
injected
into theartery
carotidofartery
of rodents
[36]. Mixed-surface
dendrimers
may therefore
offeroffer
an an
excellent
alternative
modified dendrimers
dendrimersas as
PAMAM
dendrimers
may therefore
excellent
alternativetoto surface
surface modified
nanocarriers
drugs
and
genes
treating
neurodegenerative
diseases.
nanocarriers
of of
drugs
and
genes
forfor
treating
neurodegenerative
diseases.
(A)
(B)
Figure
1. (A)
polyamidoamine
(PAMAM)
dendrimer
[37].(B)
Every
component
a dendrimer
Figure
1. (A)
AA
G4G4
polyamidoamine
(PAMAM)
dendrimer
[37].(B)
Every
component
of of
a dendrimer
can
be
controlled
including
its
core
composition,
size,
shape,
and
surface
(adapted
from
[38]).
can be controlled including its core composition, size, shape, and surface (adapted from [38]).
With these properties of PAMAM dendrimers in mind (especially safety as the top priority), let
With these properties of PAMAM dendrimers in mind (especially safety as the top priority), let us
us look at the potential use of PAMAM dendrimers for the treatment of neurodegenerative diseases.
look at the potential use of PAMAM dendrimers for the treatment of neurodegenerative diseases.
PAMAM
Dendrimers
and
Delivery
Small
Molecule
Drugs
and
Genes
across
Blood
3. 3.
PAMAM
Dendrimers
and
thethe
Delivery
of of
Small
Molecule
Drugs
and
Genes
across
thethe
Blood
Brain
Barrier
Brain
Barrier
The
BBB
refers
selective
membrane
that
forms
physical
and
chemical
barriers
between
The
BBB
refers
to to
thethe
selective
membrane
that
forms
thethe
physical
and
chemical
barriers
between
the
blood
and
brain
parenchyma.
It
has
evolved
to
regulate
the
chemical
environment
of
the
central
the blood and brain parenchyma. It has evolved to regulate the chemical environment of the central
nervous
system
(CNS)
through
a
variety
of
specialized
cells
and
transporters.
The
BBB
is
composed
nervous system (CNS) through a variety of specialized cells and transporters. The BBB is composed
specializedcapillary
capillaryendothelial
endothelialcells
cellsjoined
joinedbyby
tight
junctions
surrounded
extravascular
of of
specialized
tight
junctions
surrounded
byby
extravascular
components including the basal lamina, astrocyte foot processes, pericytes, and interneurons [39].
These components form a physical transport barrier that regulates the flow of nutrients in and waste
out of the CNS. Additionally, a collection of efflux proteins and enzymes, including intracellular
monoamine oxidase, cytochrome P450s, extracellular nucleases, and peptidases help make an active
chemical barrier to protect the CNS from potentially toxic chemicals [40]. Notably, the endothelial
cells of the BBB play a major role in regulating chemical homeostasis of the CNS and have unique
Molecules 2018, 23, 2238
7 of 18
components including the basal lamina, astrocyte foot processes, pericytes, and interneurons [39].
These components form a physical transport barrier that regulates the flow of nutrients in and waste
out of the CNS. Additionally, a collection of efflux proteins and enzymes, including intracellular
monoamine oxidase, cytochrome P450s, extracellular nucleases, and peptidases help make an active
chemical barrier to protect the CNS from potentially toxic chemicals [40]. Notably, the endothelial cells
of the BBB play a major role in regulating chemical homeostasis of the CNS and have unique properties
compared to other systemic vascular endothelial cells. These cells contain a higher number of tight
junctions (lack of fenestrations) which reduce the amount of paracellular movement of solutes and
experience far lower rates of transcytosis compared to peripheral tissue, reducing vesicle-mediated
transport of solutes [41].
The BBB does not completely halt the transport of substances into the brain. To function
properly, the brain needs key nutrients, such as glucose, amino acids, nucleic acids, fatty acids,
vitamins, and electrolytes, which are transported into the brain by numerous transporters and channels
located on the endothelial membranes [42]. In addition to protein transporters, certain endogenous
peptides and hormones, such as transferrin and insulin, may cross the BBB via receptor-mediated
endocytosis [43]. Additionally, other types of BBB crossings exist, including: (1) small, water-soluble
agents such as ions, which may cross by paracellular transport between tight junctions; (2) lipid-soluble
chemicals, which may cross transcellularly, directly through the endothelial membrane; and (3) plasma
proteins, which may cross via adsorptive endocytosis [40,44] or via receptor-mediated endocytosis.
Examples of receptor-ligand interactions observed and used for receptor-mediated trafficking of
nanomolecules across the BBB endothelial cells include transferrin [45], insulin [46], insulin-like growth
factor II [47], low-density lipoprotein receptor-related protein (LRP) receptors [48], and diphtheria toxin
receptors [49]. Since about 98% of small-molecule drugs fail to cross the BBB and few large-molecule
drugs (e.g., insulin) cross, finding ways to deliver drugs across the BBB has been the focus of much
research, which has, thus far, produced only mixed results [50].
How are PAMAM dendrimers used for delivery across the BBB? In order to answer this question,
we must separate small molecule delivery (such as drugs < MW 500 Da) and macromolecule
delivery (e.g., gene delivery MW > 500 Da). This is due to the significant differences in the size
and surface characteristics of the products formed when dendrimers are loaded with drugs compared
to dendrimers loaded with DNA or RNA (known as dendriplexes).
4. Delivery of Small Molecules
We shall discuss small molecule drugs first. These chemicals are either loaded into the cavities
or attached to the surface of PAMAM dendrimers [51]. The size of the final dendrimer-drug product
does not change appreciably (typically < 15 nm). Several chemotherapy agents have been loaded
into PAMAM dendrimer cavities for treatment of a variety of cancers and diseases of other organs.
However, surprisingly, there are hardly any reports about loading drugs within PAMAM dendrimer
cavities for brain diseases. A few examples of this are given below:
•
•
Carbamazepine (CBZ), an anti-epileptic drug, was shown to enhance autophagy and protect
against neurodegeneration in vivo. However, it is poorly soluble in water and shows
unpredictable pharmacokinetic profiles. Generation 4.5 carboxyl-terminated dendrimers were
loaded with CBZ to yield stable, soluble and safe (as tested on RBCs and zebrafish) formulations
for potential applications in the treatment of neurodegenerative diseases associated with
toxicity of aggregated proteins such as Alzheimer’s disease, amyotrophic lateral sclerosis,
Huntington’s disease and Parkinson’s disease [30]. No further studies were performed to see if
the CBZ-dendrimer formulations were effective for the treatment of these diseases.
He and co-workers encapsulated doxorubicin into G4 PAMAM dendrimers with surface PEG
(PEGylated dendrimers) and the targeting ligands wheat germ agglutinin (WGA) and transferrin
(Tf). The BBB-targeting ligands WGA and Tf increased the BBB permeability of the dendrimers.
These nanoparticles were < 20 nm in size as measured by electron microscopy and dynamic light
Molecules 2018, 23, 2238
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scattering. The PAMAM-PEG-WGA-Tf delivered more payload (doxorubicin) at brain tumor sites
compared to free drug or dendrimers without Tf and WGA [52].
Swami and colleagues loaded docetaxel (DTX) into G4 PAMAM dendrimers and covalently
attached p-hydroxyl benzoic acid to the surface (pHBA). The pHBA has high affinity to sigma
receptors that are predominant in the central nervous system. The G4-pHBA-DTX dendrimers
were more effective in killing glioblastoma cells and delivered more DTX to the brain compared
to free drug [53].
Compared to cavity loading, most researchers prefer covalent attachment of the drug to the
surface of PAMAM dendrimers via hydrolysable amide or ester bonds. One reason for this may be due
to the rapid release of encapsulated drugs compared to covalently linked drugs. For example, Patri and
colleagues observed rapid release of PAMAM G5 encapsulated methotrexate in phosphate buffered
saline compared to covalently coupled drug [54]. Clearly, a better way to encapsulate drugs within the
dendrimer cavity is required to avoid changing the surface of the macromolecule. An example would
be the entrapment of drugs within the dendritic branches of the dendrimers, which was demonstrated
by Jansen and colleagues who introduced guest molecules during the construction phase of the
dendrimer. This approach resulted in a significantly slower release of the guest molecules into solution,
due to the dense packing of the dendrimer shell [55].
A few examples of the use of drugs covalently attached to PAMAM dendrimers for brain diseases
are listed below:
•
•
•
•
•
•
Microtubule inhibitors (estramustine and podophyllotoxin), covalently attached to PAMAM
dendrimers, were found to be more effective in killing glioma cells compared to free drug [56].
Teow and colleagues conjugated paclitaxel and lauryl chains on the surface of a G3 PAMAM
dendrimer. The conjugates showed increased cytotoxicity and permeability across porcine brain
endothelial cells [57].
Sharma and colleagues found that minocycline, conjugated to G6 hydroxyl-terminated PAMAM
dendrimers via amide linkages, reduced neuroinflammation in vivo when compared to free
minocycline and did so at lower dosages, thus reducing potential drug toxicity [58].
Kannan and colleagues conjugated N-acetyl-L-cysteine to a G4 hydroxylated PAMAM dendrimer
via disulfide linkages, which could be cleaved by intracellular glutathione (GSH). This formulation
was shown to reduce motor dysfunction in rabbit models with cerebral palsy when administered
postnatally [59].
Yang and Lopina showed that attaching venlafaxine, a SNRI antidepressant, to PAMAM
dendrimer-PEG hydrogels via ester linkages provided an extended release formulation that
may help patients with poor drug compliance [60].
Gamage and colleagues showed that curcumin was conjugated onto a G3-succinamic acid surface
dendrimer via ester bonds. This formulation was administered to rats implanted with human
glioma cells. The G3-curcumin showed tumor specific distribution, suggesting a potential use for
the treatment of brain cancer [61].
A combination of cavity loading and surface attachment has also been used. Li and co-workers
prepared G4 PAMAM dendrimers with doxorubicin, PEG and transferrin attached to the surface
while tamoxifen was encapsulated within the cavities of the dendrimer. The dendrimer conjugate
crossed a BBB model via transferrin-mediated endocytosis and accumulated within glioma cells as
tamoxifen-inhibited drug efflux proteins [62].
These studies on the use of PAMAM dendrimers for small molecule drug delivery, although
still in its infancy, suggest the promise of PAMAM dendrimers for treating brain diseases. PAMAM
dendrimers have also been used as drugs instead of carriers for drugs. The dendrimers by themselves
decrease fibrillation of alpha-synuclein, with the anti-fibrillation effect increasing with the generation
number and concentration of the dendrimer [63]. In addition, PAMAM dendrimers were found
to promote the degradation of pre-existing alpha-synuclein fibers, providing potential benefit to
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Parkinson’s disease patients [64]. This anti-aggregation effect is also seen in Alzheimer’s disease
models, where dendrimers have been shown to disrupt Aβ amyloid plaque formation and disruption
of protease-resistant prion protein (PrPSc) in prion diseases, where protein aggregation is thought to
play a key role in functional decline [65,66].
A summary of the various types of drugs delivered by PAMAM dendrimers for brain diseases is
shown in Table 1.
Table 1. Applications of PAMAM Dendrimers for Brain Disease.
Drug(s)
Carbamazepine
Loading Method
Encapsulation within a G4.5
carboxyl-terminated dendrimer
Application
Results
Neurodegenerative
diseases
Decreased neurodegeneration
in vivo, decreased protein
aggregation, enhanced
autophagy, and increased drug
solubility [30]
Increased delivery in a
tumor-specific distribution [61]
Curcumin
Covalent linkage to a
G3-succinamic acid surface
dendrimer via ester bonds
Glioma
Docetaxel
Encapsulation within G4
PAMAM dendrimers with
covalently attached pHBA
Glioblastoma
Increased glioblastoma-cell
death, and increased drug
delivery to the brain [53]
Encapsulation within PEGylated
G4 PAMAM dendrimers with
WGA and Tf targeting ligands
Brain tumors
Increased doxorubicin payload
at tumor sites [52]
Glioma
More effective killing of glioma
cells [56]
Psychiatric
Increased brain and plasma
concentrations of haloperidol
compared to control formulation
in a rat model [67]
Doxorubicin
Estramustine and
podophyllotoxin
Covalent linkage to
PAMAM dendrimers
Haloperidol
Encapsulation within a G5
PAMAM dendrimer with
1,4-diaminobutane core
Minocycline
Covalent linkage to G6
hydroxyl-terminated PAMAM
dendrimers via amide linkages
Stroke
N-acetyl-L-cysteine
Covalent linkage to a G4
hydroxylated PAMAM
dendrimer via disulfide linkages
Cerebral palsy
Reduced motor dysfunction in
rabbit models [59]
Paclitaxel
Covalent linkage to G3 PAMAM
dendrimers with added lauryl
chains
Brain tumors
Increased cytotoxicity and
permeability across porcine
brain endothelial cells [57]
Reduced neuroinflammation
in vivo at lower doses [58]
Risperidone
Encapsulation within a G4
PAMAM dendrimer
Psychiatric
Increased aqueous solubility of
risperidone without significant
hemolysis or morphological
changes to human red blood
cells [68]
Tamoxifen and
doxorubicin
Combination encapsulation
(tamoxifen) and covalent
linkage (doxorubicin) to G4
PAMAM dendrimers with
added PEG and Tf
Glioma
Increased accumulation within
glioma cells [62]
Venlafaxine
Covalent linkage to PAMAM
dendrimers-PEG hydrogels via
ester linkages
Psychiatric
Extended release [60]
PAMAM—polyamidoamine; PEG—polyethylene glycol; pHBA—p-hydroxyl benzoic acid; Tf—transferrin;
WGA—wheat germ antigen.
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5. Delivery of Genes
The interaction of dendrimers with nucleic acids is very different than small molecule drugs.
Practically, only dendrimers with surface amines can be used for the delivery of nucleic acids
(DNA or RNA). The amines form positive charges under physiological conditions that interact
electrostatically with the negative charges on nucleic acids to form dendrimer-nucleic acid dendriplexes.
Under physiological conditions (especially pH ~7), hydroxyls are neutral and carboxyls are anionic;
thus, dendrimers with these surface groups will not bind and complex nucleic acids. Due to the
multiple charges on both the amine-terminated dendrimers and nucleic acids, the dendriplexes
formed can vary significantly in size and structure [69]. Several factors affect the nature of the
dendriplexes including the size (generation) of the dendrimer, the size of the nucleic acid, the ratio of
dendrimer amines (N) to nucleic acid phosphates (P) (also known as N/P or charge ratio), as well as
solvent properties [70,71]. Dendrimer binding affinity to DNA increases with increasing generation.
The complexes typically have low stability and tend to aggregate and precipitate at charge ratios
close to one. In addition, higher dendrimer generations and charge ratios greater than 2 (twice as
many amines as phosphates in the dendriplex) tend to form complexes with smaller size distributions
and with better transfection efficiencies. In general, stable, uniform dendriplexes with nanometer
dimensions are formed with higher generations and higher N/P ratios [69,71]. The transfection
efficiency of dendrimer-nucleic acid complexes is difficult to predict, since it depends not only on the
nature of the complexes formed, but also varies between different cell lines and even cell densities [70].
If preliminary biological results using dendriplexes are promising, it is advisable for researchers to
carefully characterize the complexes formed by a variety of analytical techniques such as dynamic
light scattering (DLS), agarose gel electrophoresis, fourier-transform infrared spectroscopy (FTIR),
isothermal titration calorimetry (ITC), circular dichroism (CD )and differential scanning calorimetry
(DSC) in order to ensure that similar complexes can be reproducibly prepared [69].
Several researchers have studied the delivery of genes with dendrimers. Excellent reviews exist on
this subject [6,70]. Examples of PAMAM dendrimers used in gene delivery relevant to brain diseases
are given below:
•
•
One method of attenuating neurodegenerative disease progression is by slowing down the rate
of neuronal death. This can be achieved by providing growth factors to the regions of neuronal
degeneration, such as brain-derived neurotrophic factor (BDNF), glial-derived neurotropic factor
(GDNF) and nerve growth factor (NGF) which are needed for the survival of particular neurons
in the brain [72,73]. In HD, lack of BDNF results in the loss of medium spiny neurons of the
striatum, a brain region involved in motor, cognitive, and emotional functions. As a result,
HD patients present with deterioration in all three of these domains. Examples of this include
chorea (an involuntary dance like movement), learning, and memory impairments as well as
psychiatric problems. Since the lack of BDNF in the striatum results in the death of these cells,
our laboratory has been involved in developing methods to increase BDNF production in the
brains of HD rodent models. We have shown that increasing BDNF in HD mice (such as YAC128
and R6/2 models) resulted in the attenuation of motor and cognitive loss [73,74]. Shakhbazau and
colleagues found that PAMAM G4 complexed with a plasmid for BDNF significantly increased the
secretion of BDNF protein in human bone marrow mesenchymal stem cells (hMSCs), which can
be implanted into diseased brains [75]. Although most researchers use charge ratios greater than
one, it is interesting that these authors used a charge ratio of 1:1, giving a dendriplex size of about
150–200 nm.
Similar complexes, formed between G4 and a plasmid for neurotrophin, were successfully used
for transfecting human and rodent stem cells [75].
Unlike direct transfection of cells in culture, PAMAM-mediated delivery of genes in vivo often
uses targeting ligands that are predominant in the brain. Examples of targeted delivery include:
Molecules 2018, 23, 2238
•
•
•
•
•
•
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Huang and colleagues have shown that transferrin conjugated to PAMAM dendrimer-DNA
dendriplexes increases gene expression approximately two-fold in the brain compared to
dendriplexes that were not conjugated to transferrin [76].
Additionally, the same researchers have shown in BALB/c mice that conjugating lactoferrin
to PAMAM dendrimers with PEG spacers increased brain uptake of the dendrimer 4.6-fold,
compared to non-conjugated PAMAM dendrimers and by a 2.2-fold increase compared to
dendrimers conjugated to transferrin [77].
LRP receptors have also been shown to be abundantly expressed in mammalian neuronal
cells [78]. Angiopep has been used to target LRP receptors with some specificity [79]. Researchers
have shown that gene expression was significantly increased in the cortex, caudate putamen,
hippocampus and substantia nigra of BALB/c mice when Angiopep-PEG-PAMAM loaded with
DNA was administered, as compared to unconjugated PAMAM loaded with DNA [80].
Another receptor found on the BBB, the mannose 6-phosphate/insulin-like growth factor II
receptor (M6P/IGFR2R), binds to M6P and IGFR2 at distinct sites. After the binding of
M6P-tagged proteins, as well as IGF-2 on those receptors, the molecules may be internalized and
sent to lysosomes for degradation [81]. Urayama and colleagues found that this receptor is highly
expressed in neonatal mice compared to adult mice. Their findings showed increased uptake
of radiolabeled β-glucuronidase in neonatal mice compared to adults, and that this uptake was
inhibited by M6P via competitive inhibition [82]. Potentially, conjugating a dendrimer to one of
the ligands of the M6P/IGFR2R could be therapeutic for neonatal diseases of the CNS.
Another targeting ligand for CNS-enhanced drug delivery is the 29 amino-acid rabies virus
glycoprotein (RVG29), which allows viral entry into the CNS by binding to nicotinic acetylcholine
receptors on neurons [83]. Liu and colleagues have successfully exploited this interaction
when RVG29 was conjugated to a PAMAM dendrimer loaded with DNA. They found that this
conjugation crossed the BBB more efficiency in vitro, and had a preferential brain accumulation
in vivo [84].
Serramía and colleagues delivered a small siRNA systemically using carbosilane dendrimers
targeting astrocytes in BALB/c mice and detected the presence of the dendrimers and the
dendriplexes in the brain one hour and 24 hours following injection [85].
6. Fate of PAMAM Dendrimers in Cells
Crossing the blood brain barrier or brain cells requires that the drug-loaded dendrimer or the
dendriplex attaches to cell membranes. If the dendrimer has a surface receptor, entry would be
via receptor-mediated endocytosis. Otherwise, cells will use adsorptive endocytosis for uptake.
The presence of a positive charge on the drug-loaded dendrimer or the dendriplex will enhance
adsorptive endocytosis and subsequent cellular uptake. This is why dendrimers with positively
charged surfaces readily cross the cell membrane and are more effective in delivering DNA/drugs
to the cell in vitro and in vivo [33]. On the other hand, a completely neutral dendrimer having
a 100% −OH surface will be less efficient to cross the hydrophobic cell membrane in order to
deliver DNA/drugs [86]. This is also why dendriplexes with charge ratios greater than one are
used for transfection.
The precise mechanism of how the dendrimer enters the cell, especially neurons and astrocytes,
and how they release their drug and/or gene is not known. A few mechanisms for dendrimer uptake
by cells have been proposed and include: (1) clathrin-mediated endocytosis; (2) caveolae-mediated
endocytosis (specifically for G4 dendrimers); and (3) macropinocytosis [87]. Once inside the cytoplasm,
the drug-loaded dendrimer or the dendriplex are enclosed within vesicles (endosomes). Normally,
the endosomes mature as they become acidic and finally fuse with lysosomes. According to the proton
sponge mechanism, it is believed that the acidification of the endosomes is suppressed, especially by
the tertiary amines within the interior of the PAMAM dendrimer. This results in the entry of chloride
ions and water into the endosomes (osmotic swelling). The swollen endosomes burst, releasing the
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dendrimer with its cargo. Under acidic conditions, most of the dendrimer tertiary amines become
positively charged and repel the branches of the nanomolecule. This increases the dendrimer size and
also helps in dislodging its bound drug or gene cargo [34,88].
7. Routes of Dendrimer Administration
The route of administration is also an important factor to consider when delivering dendrimers
to the brain. The major challenge to brain delivery is to find an ideal route of administration that
can maximize the effects of administered dendrimers on the CNS without causing systemic toxicity.
Intravenous injection is the simplest method for achieving a rapid increase in blood concentration of
the dendrimer and its payload. While directly injecting the dendrimer into the blood has the fastest
pharmacokinetic properties, there are some undesirable aspects to this method. It has been shown
by Kurokawa and colleagues that BBB penetration may not be effective. These authors found that
intravenous administration of a G4 amine-terminated PAMAM dendrimer had poor CNS penetration
due to their tendency to aggregate via non-Derjaguin-Landau-Verwey-Overbeek attractive forces
originating from the surrounding divalent ions [88]. Neutral surface PAMAM dendrimers are better
in this respect. Zhang and colleagues demonstrated that intravenous injection of G4-OH PAMAM
dendrimers effectively penetrated CNS tumors in a rodent gliosarcoma model (with a compromised
BBB). Additionally, they were able to reveal the homogeneous distribution of dendrimers throughout
the solid tumor surrounding tissue fifteen minutes after injection. In a later study, Zhang and
colleagues demonstrated in a canine model of neuro-inflammation that intravenously delivered
G6-OH dendrimers showed extensive blood circulation and an extended half-life in cerebrospinal
fluid (CSF) compared to the smaller G4-OH dendrimers administered intravenously. The authors also
showed that brain penetration (concentration of drug in CSF/serum) correlated with the severity of
neuro-inflammation and that the dendrimers preferentially targeted microglia and injured neuronal
cells. Their research suggests that intravenous administration of larger dendrimers could provide
extended therapy with significant CNS penetration [89]. Another way of increasing delivery to the
brain is to attach a targeting ligand specific to, and/or abundantly found specifically on or abundant on
brain cells. Unfortunately, no such ligand is available today that is unique to normal or diseased brain
cells. Ligands such as transferrin [45], insulin [46], insulin-like growth factor II [47] and low-density
lipoprotein receptor-related protein (LRP) receptors [48] are also found in other cells of the body.
Another way of enhancing the delivery of dendrimers to the brain is injection close to the brain
via the carotid artery. Administration into the carotid artery increases the concentration of dendrimer
reaching the brain. Srinageshwar and colleagues demonstrated that G4 PAMAM mixed-surface
dendrimers composed of 90% −OH and 10% −NH2 surface molecules are taken up by neurons in vitro,
cross the BBB following carotid artery administration, and integrate into neurons and glial cells of
healthy mouse models with intact BBBs. No detectable amount of dendrimers were found in the
peripheral organs (liver, lungs, and spleen) [35]. The small amount of positive charge on the G4-90/10
dendrimer (six amines/dendrimer) was also responsible for its improved safety profile (compared
to an amine-terminated dendrimer, which has 64 amines/dendrimer) and allowed the dendrimer to
stick to the endothelial cells for subsequent adsorptive endocytosis across the BBB. Although carotid
injections are challenging and must be performed by highly trained professionals, this route of injection
offers an excellent alternative for sending high amounts of drugs to the brain in case of life-threatening
diseases such as glioblastoma multiforme.
Other than vascular injection, oral and intranasal dendrimer preparations have also been
administered for brain uptake studies. Besides being the most common route of administration
in the pharmaceutical market today, oral administration has the benefit of convenience and increased
patient compliance. Challenges to oral delivery of all drugs has been intestinal epithelial penetration
and first-pass metabolism in the liver [90]. In regard to the former barrier, dendrimers may actually
increase intestinal absorption of therapeutics. In a previous study, it was found that doxorubicin levels
in serum were 200-fold higher when orally administered with PAMAM dendrimers compared to free
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doxorubicin [91]. It was also found that there was a significantly higher transport of doxorubicin
when using a doxorubicin–PAMAM complex compared to free doxorubicin in Caco-2 monolayers [92].
Studies with everted rat intestinal sac systems also showed high transport of PAMAM dendrimers,
especially those with anionic surfaces [93], while other studies found less promising results using
isolated human intestinal epithelium [94]. Although there is much work to be done in this area,
oral administration of dendrimer-drug formulations could potentially provide an efficacious and
convenient way of delivering drugs to the CNS. If the targeting ligand (mostly proteins) can withstand
destruction in the gastrointestinal system, then oral formulations of dendrimers conjugated to a
BBB-targeting moiety may help enhance delivery to the brain [91].
Perhaps more promising than oral formulations, intranasal preparations of dendrimers offer the
benefits of being non-invasive without the same metabolic issues of oral drugs [95]. Win-Shwe and
colleagues found that a single dose of PAMAM dendrimers intranasally administered to eight-week-old
BALB/c mice upregulated BDNF mRNA in the hippocampus and cerebral cortex of treated mice.
This study suggests that the dendrimers entered the CNS via systemic circulation or through olfactory
nerve routes to alter gene expression, however no analyses were done to confirm the presence of
dendrimers in the affected brain areas [96]. Katare and colleagues took this approach one step further
and complexed water-insoluble haloperidol with a dendrimer formulation and found a 100-fold
increase in aqueous solubility. The dendrimer formulation was seven times more efficient at targeting
haloperidol to the striatum when administered intranasally, compared to intraperitoneal injection.
Additionally, an intranasal dose 6.7 times lower than the intraperitoneal dose produced comparable
cataleptic and locomotor behavioral responses. However, while a 6.7 times lower dose produced
similar responses, intranasal administration produced a haloperidol concentration two to three times
lower in the striatum compared to intraperitoneal injection without dendrimers [67]. While the striatal
concentrations were lower, an increased efficiency and lower dose that produce the intended response
should not be discounted.
8. Conclusions
Keeping safety and reproducibility in preparation of dendrimer-drug formulations as the top two
priorities for the potential use of these nanomolecules for treating brain diseases, PAMAM dendrimers
with −OH surfaces are the preferred choice since they do not stick to surfaces, diffuse well throughout
the brain, are non-immunogenic and are highly water-soluble. Although cellular uptake is better for
amine-surface dendrimers, the compromised BBB associated with the diseased state can facilitate
targeting the hydroxyl surface PAMAM dendrimers to the affected sites of the brain. The use of
mixed-surface dendrimers with limited amines on the dendrimer surface, such as the G4 90/10,
is another option that provides safety, the ability to bind drugs and DNA, improved BBB penetration
and cellular uptake. It is also advisable to limit surface changes to the dendrimer to maintain its
benefits, such as purity and safety, and avoid unpredictable ADME profiles. For this reason, dendrimer
cavities should be used for loading drugs by encapsulation rather than by attaching them to the surface.
The rapid release of encapsulated drugs in vivo can be solved by entrapment, which results in a much
slower release of the payload. In the case of gene delivery, where amines are required for DNA binding
and compaction, use of mixed-surface dendrimers that have the minimal amines required to carry the
gene and with the dendrimer surface decorated with the more biocompatible hydroxyl groups should
be considered. If safe and high purity dendrimer formulations are used, high doses may be used to
deliver sufficient drug or gene to the brain. The compromised BBB associated with the diseased state
can facilitate targeting the payload to only the affected sites of the brain.
Author Contributions: A.S. and D.S. contributed to the chemistry portion while M.F. and A.F. drafted the
manuscript. B.S. drafted and edited the manuscript. A.S.; G.L.D. and J.R. performed the final proof of the
manuscript. All authors read and approved the final manuscript.
Funding: This research received no external funding.
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Acknowledgments: Support for this study was provided by the College of Medicine at Central Michigan
University, the Field Neurosciences Institute, and the John G. Kulhavi Professorship in Neuroscience at Central
Michigan University
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Huebner, E.A.; Strittmatter, S.M. Axon regeneration in the peripheral and central nervous systems.
Results Probl. Cell Differ. 2009, 48, 339–351. [CrossRef] [PubMed]
Young, A.B. Four decades of neurodegenerative disease research: How far we have come! J. Neurosci. 2009,
29, 12722–12728. [CrossRef] [PubMed]
Holland, E.C. Glioblastoma multiforme: The terminator. Proc. Natl. Acad. Sci. USA 2000, 97, 6242–6244.
[CrossRef] [PubMed]
Kannan, R.M.; Nance, E.; Kannan, S.; Tomalia, D.A. Emerging concepts in dendrimer-based nanomedicine:
From design principles to clinical applications. J. Intern. Med. 2014, 276, 579–617. [CrossRef] [PubMed]
Otto, D.P.; de Villiers, M.M. Poly(amidoamine) Dendrimers as a Pharmaceutical Excipient. Are We There
yet? J. Pharm. Sci. 2018, 107, 75–83. [CrossRef] [PubMed]
Palmerston Mendes, L.; Pan, J.; Torchilin, V.P. Dendrimers as Nanocarriers for Nucleic Acid and Drug
Delivery in Cancer Therapy. Mol. Basel Switz. 2017, 22. [CrossRef] [PubMed]
Menjoge, A.R.; Kannan, R.M.; Tomalia, D.A. Dendrimer-based drug and imaging conjugates: Design
considerations for nanomedical applications. Drug Discov. Today 2010, 15, 171–185. [CrossRef] [PubMed]
Esfand, R.; Tomalia, D.A. Poly(amidoamine) (PAMAM) dendrimers: From biomimicry to drug delivery and
biomedical applications. Drug Discov. Today 2001, 6, 427–436. [CrossRef]
Zhang, F.; Lin, Y.-A.; Kannan, S.; Kannan, R.M. Targeting specific cells in the brain with nanomedicines for
CNS therapies. J. Control. Release 2016, 240, 212–226. [CrossRef] [PubMed]
Somani, S.; Dufès, C. Applications of dendrimers for brain delivery and cancer therapy. Nanomedicine 2014,
9, 2403–2414. [CrossRef] [PubMed]
Boyd, B.J.; Kaminskas, L.M.; Karellas, P.; Krippner, G.; Lessene, R.; Porter, C.J.H. Cationic poly-L-lysine
dendrimers: Pharmacokinetics, biodistribution, and evidence for metabolism and bioresorption after
intravenous administration to rats. Mol. Pharm. 2006, 3, 614–627. [CrossRef] [PubMed]
Kazmierczak-Baranska, J.; Pietkiewicz, A.; Janicka, M.; Wei, Y.; Turrin, C.-O.; Majoral, J.-P.; Nawrot, B.;
Caminade, A.-M. Synthesis of a fluorescent cationic phosphorus dendrimer and preliminary biological
studies of its interaction with DNA. Nucleosides Nucleotides Nucleic Acids 2010, 29, 155–167. [CrossRef]
[PubMed]
Wrobel, D.; Kubikova, R.; Müllerová, M.; Strašák, T.; RůŽička, K.; Fulem, M.; Maly, J. Phosphonium
carbosilane dendrimers—Interaction with a simple biological membrane model. Phys. Chem. Chem. Phys.
2018, 20, 14753–14764. [CrossRef] [PubMed]
Abbasi, E.; Aval, S.F.; Akbarzadeh, A.; Milani, M.; Nasrabadi, H.T.; Joo, S.W.; Hanifehpour, Y.;
Nejati-Koshki, K.; Pashaei-Asl, R. Dendrimers: Synthesis, applications, and properties. Nanoscale Res. Lett.
2014, 9, 247. [CrossRef] [PubMed]
Nwe, K.; Milenic, D.E.; Ray, G.L.; Kim, Y.-S.; Brechbiel, M.W. Preparation of cystamine core dendrimer and
antibody-dendrimer conjugates for MRI angiography. Mol. Pharm. 2012, 9, 374–381. [CrossRef] [PubMed]
Caminade, A.-M.; Laurent, R.; Majoral, J.-P. Characterization of dendrimers. Adv. Drug Deliv. Rev. 2005, 57,
2130–2146. [CrossRef] [PubMed]
Dendrimers and Other Dendritic Polymers. Available online: https://www.wiley.com/en-us/Dendrimers+
and+Other+Dendritic+Polymers-p-9780471638506 (accessed on 18 July 2018).
Dougherty, C.A.; Vaidyanathan, S.; Orr, B.G.; Banaszak Holl, M.M. Fluorophore:dendrimer ratio impacts
cellular uptake and intracellular fluorescence lifetime. Bioconjug. Chem. 2015, 26, 304–315. [CrossRef]
[PubMed]
Albertazzi, L.; Gherardini, L.; Brondi, M.; Sulis Sato, S.; Bifone, A.; Pizzorusso, T.; Ratto, G.M.; Bardi, G.
In vivo distribution and toxicity of PAMAM dendrimers in the central nervous system depend on their
surface chemistry. Mol. Pharm. 2013, 10, 249–260. [CrossRef] [PubMed]
Molecules 2018, 23, 2238
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
15 of 18
Yu, M.; Zheng, J. Clearance Pathways and Tumor Targeting of Imaging Nanoparticles. ACS Nano 2015, 9,
6655–6674. [CrossRef] [PubMed]
Sharma, A. Medical Biochemistry: Molecules to Disease, Understanding & Applying Biochemistry in Healthcare;
Educational Life Designs, 2016, 1st ed. Available online: https://www.pinterest.com/pin/183943966009628878/
(accessed on 18 June 2018).
Lesniak, W.G.; Mishra, M.K.; Jyoti, A.; Balakrishnan, B.; Zhang, F.; Nance, E.; Romero, R.; Kannan, S.;
Kannan, R.M. Biodistribution of fluorescently labeled PAMAM dendrimers in neonatal rabbits: Effect of
neuroinflammation. Mol. Pharm. 2013, 10, 4560–4571. [CrossRef] [PubMed]
Mishra, M.K.; Beaty, C.A.; Lesniak, W.G.; Kambhampati, S.P.; Zhang, F.; Wilson, M.A.; Blue, M.E.;
Troncoso, J.C.; Kannan, S.; Johnston, M.V.; et al. Dendrimer brain uptake and targeted therapy for brain
injury in a large animal model of hypothermic circulatory arrest. ACS Nano 2014, 8, 2134–2147. [CrossRef]
[PubMed]
Posadas, I.; Monteagudo, S.; Ceña, V. Nanoparticles for brain-specific drug and genetic material delivery,
imaging and diagnosis. Nanomedicine 2016, 11, 833–849. [CrossRef] [PubMed]
Sarin, H.; Kanevsky, A.S.; Wu, H.; Brimacombe, K.R.; Fung, S.H.; Sousa, A.A.; Auh, S.; Wilson, C.M.;
Sharma, K.; Aronova, M.A.; et al. Effective transvascular delivery of nanoparticles across the blood-brain
tumor barrier into malignant glioma cells. J. Transl. Med. 2008, 6, 80. [CrossRef] [PubMed]
Nance, E.; Zhang, F.; Mishra, M.K.; Zhang, Z.; Kambhampati, S.P.; Kannan, R.M.; Kannan, S. Nanoscale
effects in dendrimer-mediated targeting of neuroinflammation. Biomaterials 2016, 101, 96–107. [CrossRef]
[PubMed]
Park, E.J.; Cho, H.; Kim, S.W.; Na, D.H. Chromatographic methods for characterization of poly(ethylene
glycol)-modified polyamidoamine dendrimers. Anal. Biochem. 2014, 449, 42–44. [CrossRef] [PubMed]
Park, E.J.; Na, D.H. Difference in microchip electrophoretic mobility between partially and fully PEGylated
poly(amidoamine) dendrimers. Anal. Biochem. 2015, 488, 9–11. [CrossRef] [PubMed]
Sharma, A.; Desai, A.; Ali, R.; Tomalia, D. Polyacrylamide gel electrophoresis separation and detection
of polyamidoamine dendrimers possessing various cores and terminal groups. J. Chromatogr. A 2005,
1081, 238–244. [CrossRef] [PubMed]
Igartúa, D.E.; Martinez, C.S.; Temprana, C.F.; Alonso, S.D.V.; Prieto, M.J. PAMAM dendrimers as a
carbamazepine delivery system for neurodegenerative diseases: A biophysical and nanotoxicological
characterization. Int. J. Pharm. 2018, 544, 191–202. [CrossRef] [PubMed]
Choudhary, S.; Gupta, L.; Rani, S.; Dave, K.; Gupta, U. Impact of Dendrimers on Solubility of Hydrophobic
Drug Molecules. Front. Pharmacol. 2017, 8, 261. [CrossRef] [PubMed]
Arima, H.; Motoyama, K.; Higashi, T. Cyclodextrin/Dendrimer conjugates as DNA and oligonucleotide
carriers. Curr. Top. Med. Chem. 2014, 14, 465–477. [CrossRef] [PubMed]
Vidal, F.; Guzman, L. Dendrimer nanocarriers drug action: Perspective for neuronal pharmacology.
Neural Regen. Res. 2015, 10, 1029–1031. [CrossRef] [PubMed]
Zhang, P.; Sun, F.; Liu, S.; Jiang, S. Anti-PEG antibodies in the clinic: Current issues and beyond PEGylation.
J. Control. Release 2016, 244, 184–193. [CrossRef] [PubMed]
Roberts, J.C.; Bhalgat, M.K.; Zera, R.T. Preliminary biological evaluation of polyamidoamine (PAMAM)
Starburst dendrimers. J. Biomed. Mater. Res. 1996, 30, 53–65. [CrossRef]
Srinageshwar, B.; Peruzzaro, S.; Andrews, M.; Johnson, K.; Hietpas, A.; Clark, B.; McGuire, C.; Petersen, E.;
Kippe, J.; Stewart, A.; et al. PAMAM Dendrimers Cross the Blood–Brain Barrier When Administered through
the Carotid Artery in C57BL/6J Mice. Int. J. Mol. Sci. 2017, 18, 628. [CrossRef] [PubMed]
Kesharwani, P.; Banerjee, S.; Gupta, U.; Mohd Amin, M.C.I.; Padhye, S.; Sarkar, F.H.; Iyer, A.K. PAMAM
dendrimers as promising nanocarriers for RNAi therapeutics. Mater. Today 2015, 18, 565–572. [CrossRef]
Arseneault, M.; Wafer, C.; Morin, J.-F. Recent advances in click chemistry applied to dendrimer synthesis.
Mol. Basel Switz. 2015, 20, 9263–9294. [CrossRef] [PubMed]
Xu, L.; Zhang, H.; Wu, Y. Dendrimer Advances for the Central Nervous System Delivery of Therapeutics.
ACS Chem. Neurosci. 2013, 5, 2–13. [CrossRef] [PubMed]
Abbott, N.J.; Patabendige, A.A.K.; Dolman, D.E.M.; Yusof, S.R.; Begley, D.J. Structure and function of the
blood-brain barrier. Neurobiol. Dis. 2010, 37, 13–25. [CrossRef] [PubMed]
Coomber, B.L.; Stewart, P.A. Morphometric analysis of CNS microvascular endothelium. Microvasc. Res.
1985, 30, 99–115. [CrossRef]
Molecules 2018, 23, 2238
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
16 of 18
Simpson, I.A.; Carruthers, A.; Vannucci, S.J. Supply and demand in cerebral energy metabolism: The role of
nutrient transporters. J. Cereb. Blood Flow Metab. 2007, 27, 1766–1791. [CrossRef] [PubMed]
Lajoie, J.M.; Shusta, E.V. Targeting receptor-mediated transport for delivery of biologics across the
blood-brain barrier. Annu. Rev. Pharmacol. Toxicol. 2015, 55, 613–631. [CrossRef] [PubMed]
Pardridge, W.M. Blood-brain barrier drug targeting: The future of brain drug development. Mol. Interv.
2003, 3, 90–105. [CrossRef] [PubMed]
Hervé, F.; Ghinea, N.; Scherrmann, J.-M. CNS delivery via adsorptive transcytosis. AAPS J. 2008, 10, 455–472.
[CrossRef] [PubMed]
Wu, D.; Yang, J.; Pardridge, W.M. Drug targeting of a peptide radiopharmaceutical through the primate
blood-brain barrier in vivo with a monoclonal antibody to the human insulin receptor. J. Clin. Investig. 1997,
100, 1804–1812. [CrossRef] [PubMed]
Reinhardt, R.R.; Bondy, C.A. Insulin-like growth factors cross the blood-brain barrier. Endocrinology 1994,
135, 1753–1761. [CrossRef] [PubMed]
Kreuter, J.; Ramge, P.; Petrov, V.; Hamm, S.; Gelperina, S.E.; Engelhardt, B.; Alyautdin, R.; von Briesen, H.;
Begley, D.J. Direct evidence that polysorbate-80-coated poly(butylcyanoacrylate) nanoparticles deliver drugs
to the CNS via specific mechanisms requiring prior binding of drug to the nanoparticles. Pharm. Res. 2003,
20, 409–416. [CrossRef] [PubMed]
Wang, P.; Xue, Y.; Shang, X.; Liu, Y. Diphtheria toxin mutant CRM197-mediated transcytosis across
blood-brain barrier in vitro. Cell. Mol. Neurobiol. 2010, 30, 717–725. [CrossRef] [PubMed]
Pardridge, W.M. Drug and gene delivery to the brain: The vascular route. Neuron 2002, 36, 555–558.
[CrossRef]
Kim, Y.; Park, E.J.; Na, D.H. Recent progress in dendrimer-based nanomedicine development. Arch. Pharm. Res.
2018, 41, 571–582. [CrossRef] [PubMed]
He, H.; Li, Y.; Jia, X.-R.; Du, J.; Ying, X.; Lu, W.-L.; Lou, J.-N.; Wei, Y. PEGylated Poly(amidoamine)
dendrimer-based dual-targeting carrier for treating brain tumors. Biomaterials 2011, 32, 478–487. [CrossRef]
[PubMed]
Swami, R.; Singh, I.; Kulhari, H.; Jeengar, M.K.; Khan, W.; Sistla, R. Correction to: p-Hydroxy benzoic
acid-conjugated dendrimer nanotherapeutics as potential carriers for targeted drug delivery to brain:
An in vitro and in vivo evaluation. J. Nanopart. Res. 2017, 19, 358. [CrossRef]
Patri, A.K.; Kukowska-Latallo, J.F.; Baker, J.R. Targeted drug delivery with dendrimers: Comparison of
the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex. Adv. Drug
Deliv. Rev. 2005, 57, 2203–2214. [CrossRef] [PubMed]
Jansen, J.F.; de Brabander-van den Berg, E.M.; Meijer, E.W. Encapsulation of guest molecules into a dendritic
box. Science 1994, 266, 1226–1229. [CrossRef] [PubMed]
Sk, U.H.; Dixit, D.; Sen, E. Comparative study of microtubule inhibitors—Estramustine and natural
podophyllotoxin conjugated PAMAM dendrimer on glioma cell proliferation. Eur. J. Med. Chem. 2013,
68, 47–57. [CrossRef] [PubMed]
Teow, H.M.; Zhou, Z.; Najlah, M.; Yusof, S.R.; Abbott, N.J.; D’Emanuele, A. Delivery of paclitaxel across
cellular barriers using a dendrimer-based nanocarrier. Int. J. Pharm. 2013, 441, 701–711. [CrossRef] [PubMed]
Sharma, R.; Kim, S.-Y.; Sharma, A.; Zhang, Z.; Kambhampati, S.P.; Kannan, S.; Kannan, R.M.
Activated Microglia Targeting Dendrimer-Minocycline Conjugate as Therapeutics for Neuroinflammation.
Bioconjug. Chem. 2017, 28, 2874–2886. [CrossRef] [PubMed]
Kannan, S.; Dai, H.; Navath, R.S.; Balakrishnan, B.; Jyoti, A.; Janisse, J.; Romero, R.; Kannan, R.M.
Dendrimer-based postnatal therapy for neuroinflammation and cerebral palsy in a rabbit model. Sci. Transl. Med.
2012, 4, 130ra46. [CrossRef] [PubMed]
Yang, H.; Lopina, S.T. Extended release of a novel antidepressant, venlafaxine, based on anionic
polyamidoamine dendrimers and poly(ethylene glycol)-containing semi-interpenetrating networks. J. Biomed.
Mater. Res. A 2005, 72, 107–114. [CrossRef] [PubMed]
Gamage, N.; Jing, L.; Worsham, M.; Ali, M. Targeted Theranostic Approach for Glioma Using
Dendrimer-Based Curcumin Nanoparticle. J. Nanomed. Nanotechnol. 2016, 7, 393. [CrossRef] [PubMed]
Li, Y.; He, H.; Jia, X.; Lu, W.-L.; Lou, J.; Wei, Y. A dual-targeting nanocarrier based on poly(amidoamine)
dendrimers conjugated with transferrin and tamoxifen for treating brain gliomas. Biomaterials 2012,
33, 3899–3908. [CrossRef] [PubMed]
Molecules 2018, 23, 2238
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
17 of 18
Milowska, K.; Malachowska, M.; Gabryelak, T. PAMAM G4 dendrimers affect the aggregation of α-synuclein.
Int. J. Biol. Macromol. 2011, 48, 742–746. [CrossRef] [PubMed]
Rekas, A.; Lo, V.; Gadd, G.E.; Cappai, R.; Yun, S.I. PAMAM dendrimers as potential agents against fibrillation
of alpha-synuclein, a Parkinson’s disease-related protein. Macromol. Biosci. 2009, 9, 230–238. [CrossRef]
[PubMed]
Klajnert, B.; Cladera, J.; Bryszewska, M. Molecular interactions of dendrimers with amyloid peptides:
pH dependence. Biomacromolecules 2006, 7, 2186–2191. [CrossRef] [PubMed]
Cordes, H.; Boas, U.; Olsen, P.; Heegaard, P.M.H. Guanidino- and urea-modified dendrimers as potent
solubilizers of misfolded prion protein aggregates under non-cytotoxic conditions. dependence on dendrimer
generation and surface charge. Biomacromolecules 2007, 8, 3578–3583. [CrossRef] [PubMed]
Katare, Y.K.; Daya, R.P.; Sookram Gray, C.; Luckham, R.E.; Bhandari, J.; Chauhan, A.S.; Mishra, R.K. Brain
Targeting of a Water Insoluble Antipsychotic Drug Haloperidol via the Intranasal Route Using PAMAM
Dendrimer. Mol. Pharm. 2015, 12, 3380–3388. [CrossRef] [PubMed]
Prieto, M.J.; Temprana, C.F.; del Río Zabala, N.E.; Marotta, C.H.; del Valle Alonso, S. Optimization and
in vitro toxicity evaluation of G4 PAMAM dendrimer-risperidone complexes. Eur. J. Med. Chem. 2011,
46, 845–850. [CrossRef] [PubMed]
Braun, C.S.; Vetro, J.A.; Tomalia, D.A.; Koe, G.S.; Koe, J.G.; Middaugh, C.R. Structure/function relationships
of polyamidoamine/DNA dendrimers as gene delivery vehicles. J. Pharm. Sci. 2005, 94, 423–436. [CrossRef]
[PubMed]
Dufès, C.; Uchegbu, I.F.; Schätzlein, A.G. Dendrimers in gene delivery. Adv. Drug Deliv. Rev. 2005, 57,
2177–2202. [CrossRef] [PubMed]
Shen, X.-C.; Zhou, J.; Liu, X.; Wu, J.; Qu, F.; Zhang, Z.-L.; Pang, D.-W.; Quéléver, G.; Zhang, C.-C.;
Peng, L. Importance of size-to-charge ratio in construction of stable and uniform nanoscale RNA/dendrimer
complexes. Org. Biomol. Chem. 2007, 5, 3674–3681. [CrossRef] [PubMed]
Deng, X.; Liang, Y.; Lu, H.; Yang, Z.; Liu, R.; Wang, J.; Song, X.; Long, J.; Li, Y.; Lei, D.; et al. Co-transplantation
of GDNF-overexpressing neural stem cells and fetal dopaminergic neurons mitigates motor symptoms in a
rat model of Parkinson’s disease. PLoS ONE 2013, 8, e80880. [CrossRef] [PubMed]
Dey, N.D.; Bombard, M.C.; Roland, B.P.; Davidson, S.; Lu, M.; Rossignol, J.; Sandstrom, M.I.; Skeel, R.L.;
Lescaudron, L.; Dunbar, G.L. Genetically engineered mesenchymal stem cells reduce behavioral deficits
in the YAC 128 mouse model of Huntington’s disease. Behav. Brain Res. 2010, 214, 193–200. [CrossRef]
[PubMed]
Rossignol, J.; Fink, K.D.; Crane, A.T.; Davis, K.K.; Bombard, M.C.; Clerc, S.; Bavar, A.M.; Lowrance, S.A.;
Song, C.; Witte, S.; et al. Reductions in behavioral deficits and neuropathology in the R6/2 mouse model
of Huntington’s disease following transplantation of bone-marrow-derived mesenchymal stem cells is
dependent on passage number. Stem Cell Res. Ther. 2015, 6, 9. [CrossRef] [PubMed]
Shakhbazau, A.; Shcharbin, D.; Seviaryn, I.; Goncharova, N.; Kosmacheva, S.; Potapnev, M.; Gabara, B.;
Ionov, M.; Bryszewska, M. Use of polyamidoamine dendrimers to engineer BDNF-producing human
mesenchymal stem cells. Mol. Biol. Rep. 2010, 37, 2003–2008. [CrossRef] [PubMed]
Huang, R.-Q.; Qu, Y.-H.; Ke, W.-L.; Zhu, J.-H.; Pei, Y.-Y.; Jiang, C. Efficient gene delivery targeted to the brain
using a transferrin-conjugated polyethyleneglycol-modified polyamidoamine dendrimer. FASEB J. 2007, 21,
1117–1125. [CrossRef] [PubMed]
Huang, R.; Ke, W.; Liu, Y.; Jiang, C.; Pei, Y. The use of lactoferrin as a ligand for targeting the
polyamidoamine-based gene delivery system to the brain. Biomaterials 2008, 29, 238–246. [CrossRef]
[PubMed]
Bu, G.; Maksymovitch, E.A.; Nerbonne, J.M.; Schwartz, A.L. Expression and function of the low
density lipoprotein receptor-related protein (LRP) in mammalian central neurons. J. Biol. Chem. 1994,
269, 18521–18528. [PubMed]
Demeule, M.; Currie, J.-C.; Bertrand, Y.; Ché, C.; Nguyen, T.; Régina, A.; Gabathuler, R.; Castaigne, J.-P.;
Béliveau, R. Involvement of the low-density lipoprotein receptor-related protein in the transcytosis of the
brain delivery vector angiopep-2. J. Neurochem. 2008, 106, 1534–1544. [CrossRef] [PubMed]
Ke, W.; Shao, K.; Huang, R.; Han, L.; Liu, Y.; Li, J.; Kuang, Y.; Ye, L.; Lou, J.; Jiang, C. Gene delivery targeted
to the brain using an Angiopep-conjugated polyethyleneglycol-modified polyamidoamine dendrimer.
Biomaterials 2009, 30, 6976–6985. [CrossRef] [PubMed]
Molecules 2018, 23, 2238
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
18 of 18
Auletta, M.; Nielsen, F.C.; Gammeltoft, S. Receptor-mediated endocytosis and degradation of insulin-like
growth factor I and II in neonatal rat astrocytes. J. Neurosci. Res. 1992, 31, 14–20. [CrossRef] [PubMed]
Urayama, A.; Grubb, J.H.; Sly, W.S.; Banks, W.A. Developmentally regulated mannose 6-phosphate
receptor-mediated transport of a lysosomal enzyme across the blood-brain barrier. Proc. Natl. Acad.
Sci. USA 2004, 101, 12658–12663. [CrossRef] [PubMed]
Kumar, P.; Wu, H.; McBride, J.L.; Jung, K.-E.; Kim, M.H.; Davidson, B.L.; Lee, S.K.; Shankar, P.; Manjunath, N.
Transvascular delivery of small interfering RNA to the central nervous system. Nature 2007, 448, 39–43.
[CrossRef] [PubMed]
Liu, Y.; Bryantsev, V.S.; Diallo, M.S.; Goddard, W.A. PAMAM dendrimers undergo pH responsive
conformational changes without swelling. J. Am. Chem. Soc. 2009, 131, 2798–2799. [CrossRef] [PubMed]
Serramía, M.J.; Álvarez, S.; Fuentes-Paniagua, E.; Clemente, M.I.; Sánchez-Nieves, J.; Gómez, R.; de la Mata, J.;
Muñoz-Fernández, M.Á. In vivo delivery of siRNA to the brain by carbosilane dendrimer. J. Control. Release
2015, 200, 60–70. [CrossRef] [PubMed]
Márquez-Miranda, V.; Peñaloza, J.P.; Araya-Durán, I.; Reyes, R.; Vidaurre, S.; Romero, V.; Fuentes, J.; Céric, F.;
Velásquez, L.; González-Nilo, F.D.; Otero, C. Effect of Terminal Groups of Dendrimers in the Complexation
with Antisense Oligonucleotides and Cell Uptake. Nanoscale Res. Lett. 2016, 11, 66. [CrossRef] [PubMed]
Vidal, F.; Vásquez, P.; Díaz, C.; Nova, D.; Alderete, J.; Guzmán, L. Mechanism of PAMAM Dendrimers
Internalization in Hippocampal Neurons. Mol. Pharm. 2016, 13, 3395–3403. [CrossRef] [PubMed]
Kurokawa, Y.; Sone, H.; Win-Shwe, T.-T.; Zeng, Y.; Kimura, H.; Koyama, Y.; Yagi, Y.; Matsui, Y.; Yamazaki, M.;
Hirano, S. Aggregation is a critical cause of poor transfer into the brain tissue of intravenously administered
cationic PAMAM dendrimer nanoparticles. Int. J. Nanomed. 2017, 12, 3967–3975. [CrossRef] [PubMed]
Zhang, F.; Trent Magruder, J.; Lin, Y.-A.; Crawford, T.C.; Grimm, J.C.; Sciortino, C.M.; Wilson, M.A.;
Blue, M.E.; Kannan, S.; Johnston, M.V.; et al. Generation-6 hydroxyl PAMAM dendrimers improve CNS
penetration from intravenous administration in a large animal brain injury model. J. Control. Release 2017,
249, 173–182. [CrossRef] [PubMed]
Thanki, K.; Gangwal, R.P.; Sangamwar, A.T.; Jain, S. Oral delivery of anticancer drugs: Challenges and
opportunities. J. Control. Release 2013, 170, 15–40. [CrossRef] [PubMed]
Ke, W.; Zhao, Y.; Huang, R.; Jiang, C.; Pei, Y. Enhanced oral bioavailability of doxorubicin in a dendrimer
drug delivery system. J. Pharm. Sci. 2008, 97, 2208–2216. [CrossRef] [PubMed]
Sadekar, S.; Ghandehari, H. Transepithelial transport and toxicity of PAMAM dendrimers: Implications for
oral drug delivery. Adv. Drug Deliv. Rev. 2012, 64, 571–588. [CrossRef] [PubMed]
Wiwattanapatapee, R.; Carreño-Gómez, B.; Malik, N.; Duncan, R. Anionic PAMAM dendrimers rapidly
cross adult rat intestine in vitro: A potential oral delivery system? Pharm. Res. 2000, 17, 991–998. [CrossRef]
[PubMed]
Hubbard, D.; Enda, M.; Bond, T.; Moghaddam, S.P.H.; Conarton, J.; Scaife, C.; Volckmann, E.; Ghandehari, H.
Transepithelial Transport of PAMAM Dendrimers Across Isolated Human Intestinal Tissue. Mol. Pharm.
2015, 12, 4099–4107. [CrossRef] [PubMed]
Lu, C.-T.; Zhao, Y.-Z.; Wong, H.L.; Cai, J.; Peng, L.; Tian, X.-Q. Current approaches to enhance CNS delivery
of drugs across the brain barriers. Int. J. Nanomed. 2014, 9, 2241–2257. [CrossRef] [PubMed]
Win-Shwe, T.-T.; Sone, H.; Kurokawa, Y.; Zeng, Y.; Zeng, Q.; Nitta, H.; Hirano, S. Effects of PAMAM
dendrimers in the mouse brain after a single intranasal instillation. Toxicol. Lett. 2014, 228, 207–215.
[CrossRef] [PubMed]
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Planar Microwave Sensor for Theranostic Therapy of Organic Tissue Based on Oval Split Ring Resonators
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Sensors
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cc-by
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Planar Microwave Sensor for Theranostic Therapy of
Organic Tissue Based on Oval Split Ring Resonators Carolin Reimann 1,*, Margarita Puentes 1, Matthias Maasch 2, Frank Hübner 3,
Babak Bazrafshan 3, Thomas J. Vogl 3, Christian Damm 2 and Rolf Jakoby 1 Carolin Reimann 1,*, Margarita Puentes 1, Matthias Maasch 2, Frank Hübner 3,
Babak Bazrafshan 3, Thomas J. Vogl 3, Christian Damm 2 and Rolf Jakoby 1 1
Institute for Microwave Engineering and Photonics, Technische Universität Darmstadt, Merckstr. 25,
Darmstadt 64283, Germany; puentes@imp.tu-darmstadt.de (M.P.); jakoby@imp.tu-darmstadt.de (R.J.)
2
Terahertz Sensors Group, Technische Universität Darmstadt, Merckstr. 25,
Darmstadt 64283, Germany; maasch@imp.tu-darmstadt.de (M.M.); damm@imp.tu-darmstadt.de (C.D.)
3
Institute of Diagnostic and Interventional Radiology, Goethe University, Theodor-Stern-Kai 7,
Frankfurt am Main 60590, Germany; Frank.Huebner@kgu.de (F.H.); Babak.Bazrafshan@kgu.de (B.B.);
Thomas.Vogl@kgu.de (T.J.V.) ,
y;
p
(
);
p
(
)
3
Institute of Diagnostic and Interventional Radiology, Goethe University, Theodor-Stern-Kai 7,
Frankfurt am Main 60590, Germany; Frank.Huebner@kgu.de (F.H.); Babak.Bazrafshan@kgu.de (B.B.);
Thomas.Vogl@kgu.de (T.J.V.) g
g
*
Correspondence: reimann@imp.tu-darmstadt.de; Tel.: +49-6151-16-28462 Academic Editors: Ferran Martín and Jordi Naqui q
Received: 29 July 2016; Accepted: 3 September 2016; Published: 8 September 2016 Abstract: Microwave sensors in medical environments play a significant role due to the contact-less
and non-invasive sensing mechanism to determine dielectric properties of tissue. In this work,
a theranostic sensor based on Split Ring Resonators (SRRs) is presented that provides two operation
modes to detect and treat tumor cells, exemplary in the liver. For the detection mode, resonance
frequency changes due to abnormalities are evaluated, and in the treatment mode, microwave
ablation is performed. The planar sensor structure can be integrated into a needle like a surgery
tool that evokes challenges concerning size limitations and biocompatibility. To meet the size
requirements and provide a reasonable operating frequency, properties of oval shaped SRRs are
investigated. By elongating the radius of the SRR in one direction, the resonance frequency can
be decreased significantly compared to circular SRR by a factor of two below 12 GHz. In order
to validate the detection and treatment characteristics of the sensor, full wave simulations and
measurements are examined. Clear resonance shifts are detected for loading the sensor structures
with phantoms mimicking healthy and malignant tissue. For treatment mode evaluation, ex vivo beef
liver tissue was ablated leading to a lesion zone 1.2 cm × 1 cm × 0.3 cm with a three minute exposure
of maximum 2.1 W. Keywords: theranostics; oval split ring resonator; microwave ablation sensors sensors sensors 1. Introduction The future of medicine is going in the direction of personalized treatments for each individual
patient. A promising approach is the use of theranostic devices or systems that provide a combination
of diagnosis and therapy. The objective of theranostics is to apply a personalized therapy plan for each
patient to improve the individual treatment success. For this purpose, microwave devices are well
suited. Especially in cancer therapy, microwave technology could prove very useful in diagnostics and
for treatment. The main advantages of microwaves are the contact-less and non-destructive sensing
mechanism as well as the good electromagnetic wave penetration properties into human tissue. Nowadays, imaging techniques are widely used for tumor detection with the most established
systems being computed tomography (CT) and magnetic resonance imaging (MRI). However, CT
brings the disadvantage of ionizing radiation to the patient. MRI solves this problem by applying
a gradient in a static magnetic field. However, this imaging process is very time consuming and Sensors 2016, 16, 1450; doi:10.3390/s16091450 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors 2 of 13 Sensors 2016, 16, 1450 not suitable for claustrophobic and adipose people. An emerging technique for tumor detection is
microwave imaging where dielectric properties of tissue are evaluated in order to detect malignancies
that can be seen as a complementary approach to standard imaging techniques [1]. Generally,
malignant and non-malignant tissue can be distinguished by microwaves due to different dielectric
properties. These are strongly dependent on the type of tissue of the tumor. Regarding cancer treatment, current techniques are based on radiotherapy, chemotherapy and
open surgery. However, thermal ablation presents an alternative treatment option with increasing
significance [2]. The provided temperature rise at a specific location is sufficient to eradicate tumor cells
with the advantages of a less invasive therapy compared to open surgery. Additionally, thermal ablation
can be performed very precisely so that difficult accessible tumors and tumors located deep inside the
organs can be treated with a lower risk to damage important blood vessels. All in all, thermal therapy
leads to a shorter recovery time for the patient and less risk for complications. During the ablation
therapy, the surgeon targets the tumor mostly with the help of continuous CT imaging. py
g
g
y
p
g
g
In this work, a theranostic microwave device is proposed that improves current ablation therapy
systems by adding a sensing mechanism to the applicator to detect malignant cells. 1. Introduction With this
theranostic approach, the accurate localization and precise treatment at the right timing and proper
dose becomes possible. Furthermore, monitoring of the treatment progress is enabled. The proposed
applicator is aimed to be integrated in a minimally invasive surgery tool that leads to a strict size
limitation for such a theranostic structure. Therefore, the key elements of the sensor are split ring
resonators (SRRs) with the feature of a physically small size that are coupled to a coplanar waveguide. The SRRs are excited in two different modes: the detection and treatment mode. For the detection
mode, a frequency sweep around the resonance of the SRR with low input power is applied and
relative changes of resonance frequency are evaluated in order to extract permittivity changes due to
tumor cells. In the treatment mode, the input power is increased at the specific resonance frequency
resulting in the generation of heat at the surrounding tissue of the operation tool. The detection of relatively small variations of permittivity values caused by abnormalities is
a challenging factor for the sensor design process. Therefore, a comprehensive study of SRRs as sensing
particles that are integrated in a minimally invasive surgery applicator is examined. Moreover, the
thermal characteristics and size of the ablation zone are analyzed in Section 2. Section 3 introduces the
fabricated prototype and experimental setup for the validation of the detection mode and treatment
mode, respectively. In Section 4, the corresponding measurement results are shown that are further
discussed in Section 5. Finally, conclusions are given in Section 6. 2.1. Oval Split Ring Resonators The general structure of the sensor device is based on pairs of oval SRRs coupled to a coplanar
waveguide as investigated in [5]. In Figure 1, the schematic drawings of a circular and an oval SRR
with corresponding dimensions are shown. From the property of SRRs providing a small electrical
size, a quasi-static equivalent circuit model can be applied that corresponds to an LC resonant circuit. When a SRR is excited by a time varying magnetic field along the z-axis, a current is induced along the
rings. The resonance frequency of circular SRR can be calculated as [6] f0 = 1
2π ·
s
1
LSRRCSRR
,
(2) (2) where LSRR is the total inductance of the SRR, and CSRR describes the total capacitance that is composed
by the capacitance between the rings and the gap capacitance. A detailed electromagnetic analysis
in [7] leads to the consideration that the total capacitance CSRR is determined by two series capacitances
of the two halves of the SRR. For a circular SRR, the total capacitance yields CSRR =
πr0Cp.u.l. 2
,
(3) (3) where Cp.u.l. is the per unit length capacitance between the rings and r0 is the average radius of both
rings of the SRR according to Figure 1a. The inductance LSRR can be computed by the inductance
of a single ring with average radius of both concentric split rings r0 and circular cross section with
diameter equal to the width a of the original rings. The self inductance of a ring with a circular cross
section can be approximated by [8] LSRR = µ0r0
ln 2 · 8r0
a
−1.75
. (4) (4) For the elliptical shaped SRR, both the inductance and capacitance of SRR are modified due to the
varied circumference of an ellipse UEllipse compared to a circle that is given by [9] For the elliptical shaped SRR, both the inductance and capacitance of SRR are modified due to the
varied circumference of an ellipse UEllipse compared to a circle that is given by [9] UEllipse = 4rxE(e, π
2 ),
(5) (5) with E being the complete elliptic integral of second kind with E being the complete elliptic integral of second kind E
e, π
2
=
Z π/2
0
q
1 −e2 sin2 ϕ dϕ. 2. Theoretical Considerations The proposed theranostic microwave sensor is designed for use as a needle-like surgery tool for
minimally invasive operations. From that field of application, a strict size limitation can be derived that
is 2 mm × 2 mm × 200 mm. Consequently, the operating frequency of such a small device is relatively
high. Although SRRs as subwavelength resonators provide a small physical size of about one-tenth
of the free space wavelength, the sensor design with pairs of circular SRR coupled to a coplanar
waveguide for the given size limitation would lead to the operating frequency range above 30 GHz [3]. The cost and effort to perform high power thermal ablation treatment in this frequency range are
not practicable for future scenarios as a surgical tool in a clinical environment. Hence, the resonance
frequency of the SRRs is a critical design parameter that leads to a trade-off between the physical size
of the device and the system complexity for the future application. The prospective compromise is
to design a sensor tool that works in the X-band between 8 GHz and 12 GHz. A further advantage of
decreasing the resonance frequency below 12 GHz is evident by considering the penetration depth of
an electromagnetic field into tissue. Generally, the relative permittivity of biological material has a
complex form that can be written as εr = ε′
r −jε′′
r , while the permeability is close to that of free space. The penetration depth is defined as the depth where the electric field density has decreased to 1/e of 3 of 13 Sensors 2016, 16, 1450 the initial value at the surface of the applicator. Assuming that the condition ε′′
r ≫ε′
r is valid for tissue
due to ion mobility [4], the penetration depth δp can be approximated by the initial value at the surface of the applicator. Assuming that the condition ε′′
r ≫ε′
r is valid for tissue
due to ion mobility [4], the penetration depth δp can be approximated by δp ≈1
ω
s
2
µ0ε0ε′′r
,
(1) (1) where µ0 and ε0 describe the permeability and permittivity of free space, respectively. According to
Equation (1), a larger penetration depth of microwave energy into tissue is achieved for lower
frequencies. All in all, the objective to develop a device with decreased operating frequency that
fulfills the given size requirements is achieved by investigating oval shaped SRR structures as
sensing elements. 2.1. Oval Split Ring Resonators (6) (6) The integral is a function of the eccentricity e =
q
r2x −r2y/rx of the ellipse and can be solved
numerically. Here, the radius rx describes the semi-major axis and ry the semi-minor axis of the ellipse. 4 of 13 Sensors 2016, 16, 1450 By including this geometrical relation into Equations (3) and (4), the following adjusted equations for
the inductance Loval SRR and capacitance Coval SRR can be formulated and inserted in Equation (2) to
compute the resonance frequency of an oval SRR: By including this geometrical relation into Equations (3) and (4), the following adjusted equations for
the inductance Loval SRR and capacitance Coval SRR can be formulated and inserted in Equation (2) to
compute the resonance frequency of an oval SRR: Loval SRR = 2
π µ0rxE(e)
ln 32rxE(e)
πa
−1.75
,
(7)
Coval SRR = 2rxE(e) · Cp.u.l.. (8) (7) (8) rx
ry
s
a
x
(b) z
y
x
x
a
r0
s
(a)
rx
ry
s
a
x
(b)
Figure 1. Drawings of circular (a) and oval Split Ring Resonator (SRR) (b) with corresponding
dimensions. x
a
r0
s
(a) z
y
x (b) (a) Figure 1. Drawings of circular (a) and oval Split Ring Resonator (SRR) (b) with corresponding
dimensions Figure 1. Drawings of circular (a) and oval Split Ring Resonator (SRR) (b) with corresponding
dimensions. In order to verify the accuracy of the introduced derivation for the resonance frequency of the oval
SRR, a comparison between the theoretical determined and simulated resonance frequencies is shown
in Figure 2. Full wave simulations of SRRs surrounded by air and excited by a plane electromagnetic
wave with magnetic components in z-direction and electric field components in x-direction were
performed in CST Microwave Studio with varying values for the semi-major axis rx and the ring
width a. The semi-minor axis ry of the SRRs is set to 0.45 mm. The value corresponds to the maximum
radius in y-direction assuming that a non-intersecting ring pair is coupled to a coplanar waveguide
(CPW) transmission line and does not exceed the given size limitation of 2 mm. From the resulting
plots in Figure 2, the theoretical and simulated results being in good agreement can be observed. Hence, the proposed approach to compute the resonance frequency of oval SRR by considering the
geometrical relation of an ellipse can be used for further investigations in this work. 2.2. Sensitivity Analysis
circumference of the ellips
leads to a decrease of reso The main challenge regarding tumor detection is to determine relatively small permittivity
changes due to abnormalities in tissue. In previous studies, the dielectric properties of ex vivo organic
tissue were determined [10]. Exemplary for liver tissue, a clinical study to measure the dielectric
properties of normal, malignant and cirrhotic liver tissue was carried out in 2007 [11], which came to
the conclusion that the permittivity of ex vivo malignant liver tissue is 19% to 30% larger than that of
normal tissue. The detection of such differences leads to high demands for the sensitivity of the sensor
device. For the evaluation of the sensing mechanism, full wave simulations with CST Microwave
Studio were performed when the sensor device consisting of oval SRR coupled to a CPW is loaded
with material mimicking normal and malignant liver tissue, respectively. The corresponding material
models are based on the dielectric properties for normal and malignant liver tissue given in [11]. decreased, leading to higher resonance frequencies of the oval SRR. 2.2. Sensitivity Analysis
The main challenge regarding tumor detection is to determine relatively small permittivity
changes due to abnormalities in tissue. In previous studies, the dielectric properties of ex vivo organic
tissue were determined [10]. Exemplary for liver tissue, a clinical study to measure the dielectric
properties of normal, malignant and cirrhotic liver tissue was carried out in 2007 [11], which came to
the conclusion that the permittivity of ex vivo malignant liver tissue is 19% to 30% larger than that of
normal tissue. The detection of such differences leads to high demands for the sensitivity of the sensor
device. For the evaluation of the sensing mechanism, full wave simulations with CST Microwave
Studio were performed when the sensor device consisting of oval SRR coupled to a CPW is loaded
with material mimicking normal and malignant liver tissue respectively The corresponding material Generally, resonant methods for sensing mechanisms have higher accuracies and sensitivities than
non resonant methods [12]. However, they are more sensitive against the high losses inherent to the
tissue. Therefore, an isolation layer on top of the SRRs is needed. The equivalent circuit model of SRRs
coupled to a CPW and loaded with an isolation layer and tissue sample is shown in Figure 3a, and the
corresponding schematic drawings of the sensor is shown in Figure 3b. 2.2. Sensitivity Analysis
circumference of the ellips
leads to a decrease of reso The resonance frequency of
this model is extended by the addition of the capacitances caused by the isolation layer CIso and the
tissue CTissue, and can be written as
with material mimicking normal and malignant liver tissue, respectively. The corresponding material
models are based on the dielectric properties for normal and malignant liver tissue given in [11]. Generally, resonant methods for sensing mechanisms have higher accuracies and sensitivities than
non resonant methods [12]. However, they are more sensitive against the high losses inherent to the
tissue. Therefore, an isolation layer on top of the SRRs is needed. The equivalent circuit model of SRRs
coupled to a CPW and loaded with an isolation layer and tissue sample is shown in Figure 3a, and the
corresponding schematic drawings of the sensor is shown in Figure 3b. The resonance frequency of
this model is extended by the addition of the capacitances caused by the isolation layer CIso and the
ti
C
d
b
itt f0 = 1
2π ·
s
1
LSRR(CSRR + CIso + CTissue). (9)
n as
0 = 1
2π ·
s
1
LSRR(CSRR + CIso + CTissue). (9) (9) LCPW
CCPW
M
LSRR
CSRR
CIso
CTissue
(a) oval SRR
CPW
Substrate
Isolation Layer
Isolation Layer
Substrate
CPW
oval SRR
(b)
ti
i
l ti
l
( )
i
l
t i
it
d l LCPW
CCPW
M
LSRR
CSRR
(a)
oval SRR
CPW
Substrate
Isolation Layer
Isolation Layer
Substrate
CPW
oval SRR
(b)
Figure 3. Schematic drawings of sensor with protective isolation layer. (a) equivalent circuit model;
(b) cross section and perspective view. Figure 3. Schematic drawings of sensor with protective isolation layer. (a) equivalent circuit model;
(b) cross section and perspective view. oval SRR
CPW
Isolation Layer
Substrate
CPW
(b) (b) (a) Figure 3. Schematic drawings of sensor with protective isolation layer. (a) equivalent circuit model;
(b) cross section and perspective view. Figure 3. Schematic drawings of sensor with protective isolation layer. (a) equivalent circuit model;
(b) cross section and perspective view. When loading the sensor with different tissue types, in this case with healthy and malignant tissue,
the permittivity change of normal vs. malignant tissue leads to a decreased value for the capacitance
CTissue while the other parameters are not influenced. 2.1. Oval Split Ring Resonators 0.6
0.8
1
1.2
1.4
20
30
40
50
rx/mm
f0/GHz
Theory
Simulation
(a)
60
70
80
90
100
110
120
20
21
22
23
24
a/µm
f0/GHz
Theory
Simulation
(b)
Figure 2. Comparison of theoretical and simulated values for the resonance frequency of oval SRRs (a,b). The distance between the rings is s = 60 µm and the semi-minor axis is ry = 0.45 mm. 0.6
0.8
1
1.2
1.4
20
30
40
50
rx/mm
f0/GHz
Theory
Simulation
(a)
f0/GHz 60
70
80
90
100
110
120
20
21
22
23
24
a/µm
Theory
Simulation
(b) f0/GHz (a) (b) Figure 2. Comparison of theoretical and simulated values for the resonance frequency of oval SRRs (a,b). The distance between the rings is s = 60 µm and the semi-minor axis is ry = 0.45 mm. Regarding the dependencies caused by parameter variation of the ring width a and the semi-major
axis rx of the oval SRR, shown in Figure 2, reasonable relations can be observed. For larger 5 of 13 Sensors 2016, 16, 1450 circumference of the ellipse, the total inductance LSRR and the capacitance CSRR increase, which
leads to a decrease of resonance frequency. By increasing the ring width a, the inductance LSRR is
decreased, leading to higher resonance frequencies of the oval SRR. Sensors 2016, 16, 1450
5 of 13 2.2. Sensitivity Analysis
circumference of the ellips
leads to a decrease of reso The relative frequency shift can be written as ∆f 2
f 2
0
=
f 2
0,H −f 2
0,M
f 2
0,M
=
CM −CH
CH + CSRR + CIso
,
(10) (10) 6 of 13 Sensors 2016, 16, 1450 where CM is the equivalent capacitance when the sensor is loaded with malignant tissue and CH for
healthy tissue. From the relation in Equation (10) can be extracted that the main design parameters to
maximize the relative frequency shift are the equivalent capacitances CSRR and CIso. With the limitation
that the overall resonance frequency of the sensor should not exceed 12 GHz, the capacitance CSRR can
be mainly adjusted by the semi-major axis radius rx with values above 1 mm. In order to influence
CIso, the selected material and thickness of the isolation layer tIso are possible parameters to alter. For the evaluation of corresponding simulation results, a Figure of Merit (FoM) is defined that takes
into account the relative frequency shift ∆f / f0 and the sharpness of the resonance peak presented by
the normalized 1-dB bandwidth B1dB/ f0: FoM = ∆f
B1dB
. (11) (11) For the substrate and isolation layer, thin glass was selected due to its biocompatibility and proper
sterilization properties. Additionally, glass provides a small permittivity value of εr,Glass = 4.6 and
relatively low losses in the X-band, tan δ = 0.006 at 12 GHz, to reduce the influence of CSRR and CIso
on the resonance frequency of the sensor. The proposed thin glass is produced with certain thicknesses,
whereby the relevant values for our purposes range from 200 µm to 400 µm. In Table 1, the trade off between the ellipse radius, the isolation layer thickness and sensitivity
can be clearly identified. For a larger isolation layer, the capacitance CIso decreases; however,
the electromagnetic field penetrates less into the targeted tissue. Regarding the semi-major axis
of the oval SRR, a larger size leads to an increased capacitance CSRR and decreased resonance frequency
that results in a larger value for the normalized resonance shift. According to these results, the
maximum value for the FoM was achieved for an isolation of thickness tIso = 300 µm and a semi-major
radius of rx = 1.2 mm. Table 1. Sensitivity observations for different isolation layer thicknesses and semi-major axis values. 2.2. Sensitivity Analysis
circumference of the ellips
leads to a decrease of reso tIso/µm
rx/mm
f0,M/GHz
∆f/MHz
B1dB/MHz
FoM/%
200
1
11.69
24
745
3.22
1.2
9.77
22.1
443.2
4.98
1.4
8.38
18
292.3
6.16
300
1
11.83
20
430
4.65
1.2
9.88
19
269.7
7.04
1.4
8.47
8
181
4.41
400
1
11.9
6
133
4.51
1.2
9.95
6
183.8
3.26
1.4
8.53
6
257.4
2.33 tIso/µm
rx/mm
f0,M/GHz
∆f/MHz
B1dB/MHz
FoM/% 2.3. Heat Generation 2.3. Heat Generation In the treatment mode of the sensor, microwave ablation is performed. With the information
about relative dielectric property changes detected by the sensing mechanism at the tip of a needle-like
surgery tool, the precise position of the tumor can be defined and subsequently be ablated. For this,
the input power is amplified at the specific resonance frequency of the SRR that detected abnormalities. At microwave frequencies, heat is generated due to molecules such as water in tissues that realign
themselves to the applied field. The resulting kinetic energy is converted into heat. A temperature
increase at the location of the tumor of up to 60 ◦C leads to irreversible damage of the cells [2]. In ablation theory, it is important to find a way to quantify electrical fields and their influence in organic
tissue, especially the heat transfer that becomes more complex due to the presence of continuous blood
flow through a complex network of branching vessels as well as several further biomechnanics in
cells and organs. In addition, the heat effect of ablation does not only depend on the temperature 7 of 13 Sensors 2016, 16, 1450 increase but also on the duration of the thermal exposure. The bioheat equation, firstly introduced by
Pennes [13], describes a model for the heat transfer in tissue that includes the effects of metabolism
and blood perfusion. The equation is given by ρcδT
δt = ∆· k∆T + (ρc)bωb(Tb −T) + ˙Qmet,
(12) (12) with mass density ρ, the specific thermal capacity c, the thermal conductivity k, and the Temperature T
of tissue, respectively. The subscript b denotes these properties for blood and ωb is the blood perfusion
in units of volume of blood flowing per unit time per unit of tissue volume. The metabolic rate is given
by ˙Qmet [14]. In the thermal simulations of the proposed theranostic device, the corresponding
ablation zone can be visualized. For that, CST Microwave Studio provides materials that take
into account the proposed bioheat model. For liver tissue, the thermal parameters are the thermal
capacity per volume ρc/V = 3.6 kJ·K−1·kg−1, thermal conductivity k = 0.469 WK−1·m−1, the blood
perfusion ωb = 68,000 W·K−1·m−3, and the metabolic rate ˙Qmet = 12,000 Wm−3. In Figure 4, the
resulting temperature distribution in liver tissue is presented, when the sensor is excited at a discrete
frequency with an input power of 2.1 W. 2.3. Heat Generation The blood temperature was set to 37 ◦C and the environment
temperature to 21 ◦C, the standard temperature in the measurement lab. The area inside the tissue
with a temperature rise up to 60 ◦C can be measured and equals 0.8 cm × 0.6 cm × 0.3 cm. 0.6 cm
0.8 cm
0.3 cm
Sensor
Isolation
Tissue
Figure 4. Simulated ablation zone with dimension 0.8 cm × 0.6 cm × 0.3 cm. The temperature is given
in ◦C. Figure 4. Simulated ablation zone with dimension 0.8 cm × 0.6 cm × 0.3 cm. The temperature is given
in ◦C 3. Experimental Setup For the evaluation of the sensing and heating functionality of the proposed design, the structures
were fabricated in a photolithographic process. The resulting prototype and relevant dimensions are
given in Figure 5. The lower surface of the structure is colored black due to chrome as adhesive agent
for the gold metallization. In the following, the fabrication process is described more detailed and the
measurement setups for the detection and treatment mode evaluation are presented. (b) (a)
(b)
Figure 5. Prototype with the following dimensions: length = 12 mm, width = 2 mm, semi-major
axis rx = 1.2 mm, semi-minor axis rz = 0.45 mm, ring width a = 0.12 mm, distance between rings
s = 0.06 mm, split size x = 0.1 mm. (a) frontside is structured with CPW line; (b) backside is structured
with oval SRRs. (a) (b) Figure 5. Prototype with the following dimensions: length = 12 mm, width = 2 mm, semi-major
axis rx = 1.2 mm, semi-minor axis rz = 0.45 mm, ring width a = 0.12 mm, distance between rings
s = 0.06 mm, split size x = 0.1 mm. (a) frontside is structured with CPW line; (b) backside is structured
with oval SRRs. Sensors 2016, 16, 1450 8 of 13 3.1. Fabrication For the fabrication of the sensor, both sides of the substrate, the front and back side, were
structured. Therefore, a successive process was developed that includes the continuous protection of
the not-processed side with a layer consisting of photoresist and an adhesive foil. In addition, a precise
alignment is needed to ensure the accurate position of the SRR with the center point in the center of the
gaps of the CPW in order to achieve the highest coupling between line and SRR. As substrate material,
thin glass of height 400 µm with a permittivity of εr= 4.58 is used. A seed layer out of chrome and gold
is vapored on each side followed by electroplating to heighten the gold metallization layer up to 2 µm. The main objective of the design is to obtain an operating frequency below 12 GHz and
simultaneously meet the critical size requirement that limits the width of the sensor to 2 mm. Therefore,
the strip width of the CPW is constructed in a way offering sufficient space for the pair of SRRs to
achieve a maximum possible radius in the y-direction without any intersections of the rings. The gap
width of the CPW is adapted to obtain a 50 Ωline impedance. Towards the edges of the CPW, a linear
taper is designed to enable measurements with on-wafer probes for the connection of the sensor to
external equipment. 3.2. Measurement Setup On wafer probes with a pitch distance of 250 µm are used to connect the CPW of the sensor
structure to the specific measurement equipment that varies for the detection and treatment mode. Since the SRRs are located on the back side of the structure, a holding device was designed that
provides space for the isolation layer and loading of the SRRs. In Figure 6, the measurement setup is
shown when the sensor is loaded with Rohacell that has an εr ≈1 to imitate the unloaded case. Holding Device
Sensor
Rohacell
On Wafer Probes
Figure 6. Sensor device is contacted with on wafer probes for the connection to external
measurement equipment. On Wafer Probes Holding Device
Sensor
Rohacell Figure 6. Sensor device is contacted with on wafer probes for the connection to external
measurement equipment. 3.2.1. Detection Mode First, the measurement setup for the detection mode is prepared. The probes are connected to
a vectorial network analyzer followed by a careful calibration in the frequency range from 5 GHz
to 18 GHz with 6001 measurement points, resulting in a resolution of 2.2 MHz. In order to evaluate
the sensing properties, the container in the holing device is successively filled with phantoms that
mimic dielectric properties of healthy and malignant tissue instead of Rohacell, and the transmission
characteristics are measured. The phantoms are fabricated according to the recipe given in [15]. By changing the ratio of the main ingredients—water and oil—the dielectric properties of the phantoms
can be controlled to approximate the wide band properties of ex vivo healthy and malignant liver
tissues that are examined in [11]. The corresponding permittivity of the normal and malignant
phantoms are given in Figure 7 for the X-band frequency range. 9 of 13 9 of 13 Sensors 2016, 16, 1450 6
7
8
9
10
11
12
13
14
25
30
35
40
45
50
55
Frequency/GHz
ε′
r
Phantom malignant
Phantom normal
Figure 7. Permittvity of phantoms mimicking normal and malignant tissue. Phantom malignant
Phantom normal Figure 7. Permittvity of phantoms mimicking normal and malignant tissue. 3.2.2. Treatment Mode For the validation of the treatment mode of the sensor, the measurement setup is changed to
provide a high input power at one distinct frequency. For that, the vectorial network analyzer is
replaced by a discrete source generating a signal with certain frequency and a power amplifier suitable
for frequencies up to 12 GHz. The measurement setup for thermal ablation experiments was introduced
in more detail in [16]. In this work, the treatment efficiency was firstly validated by ablating ex vivo beef liver tissue. The tissue is placed in the container of the holding device and covered with the isolation layer. In a first step, the resonance frequency of the SRR loaded with the liver tissue was determined using
the measurement setup of the detection mode. Then, the input power is successively increased from
30 mW to 2.1 W. After three minutes, the experiment is completed and the size of the ablated tissue
can be obtained by analyzing the color of the tissue. 4. Measurement Results Measurements were performed for the detection mode and the treatment mode with previously
introduced experimental setups. In the following, the corresponding results are presented and
compared with full wave simulations. 4.1. Detection Mode To prove the concept of the sensing properties, measurements were performed using the proposed
measurement setup for the detection mode. The first test was to check if the actual prototype shows
an expected behavior by comparing the transmission coefficient measurements for an unloaded sensor
with corresponding simulation results. The resulting plot is shown in Figure 8. A frequency offset
of about 2 GHz between the measurements and simulation can be extracted for each measurement. One possible reason for this discrepancy can be the presence of air gaps between the sensor structure
and the isolation layer that leads to an increase of resonance frequency due to the decrease of the
effective surrounding permittivity in the area of the SRRs. Besides this offset, the measurement shows
an expected behavior in terms of shape and depth of the resonance peak. Sensors 2016, 16, 1450 10 of 13 10 of 13 5
6
7
8
9
10
11
12
13
14
15
−8
−6
−4
−2
0
Frequency/GHz
|S21|/dB
Simulation
Measurement
Figure 8. Comparison of simulated and measured transmission coefficient for the unloaded sensor. Figure 8. Comparison of simulated and measured transmission coefficient for the unloaded sensor. The next step is to observe if the resonance frequency changes when the sensor is loaded with
a phantom mimicking normal tissue compared to that mimicking tumorous tissue. The measured
graphs of the two transmission coefficients in comparison with the unloaded case are presented in
Figure 9. A clear resonance shift of 350 MHz that corresponds to a relative frequency shift of 3.2 % is
detected. Regarding the previously introduced sensitiviy analysis, the 1 dB bandwidth B1dB = 330 MHz,
which results in an FoM of 1.06. The frequency shifts, and, consequently, the FoM observed in
the measurements is increased compared to simulations due to the fact that simulated material
models have a smaller difference between permittivity of normal and malignant tissue. According
to Figure 7, the corresponding permittivity change of the different phantoms at around 11 GHz is
∆ε = εhealthy −εtumor ≈10, and for the simulated material models ∆ε = εsim,healthy −εsim,tumor ≈7. A further observation is that the resonance peaks of the loaded sensor are degraded compared to the
unloaded case due to the losses of the phantoms mimicking organic tissue. 4.1. Detection Mode 5
6
7
8
9
10
11
12
13
14
15
−6
−4
−2
0
∆f
Frequency/GHz
|S21|/dB
unloaded
healthy phantom
tumor phantom
Figure 9. Measured transmission coefficient of sensor loaded with phantom mimicking normal and
tumor tissue. The absolute frequency shift ∆f = 350 MHz. Figure 9. Measured transmission coefficient of sensor loaded with phantom mimicking normal and
tumor tissue. The absolute frequency shift ∆f = 350 MHz. 4.2. Treatment Mode The treatment mode validation of the theranostic device is focused on the achievable dimension
of the ablation zone. For this purpose, ex vivo beef liver tissue was ablated by increasing the input
power at specific resonance frequency that was measured prior to the ablation procedure and equals
f0 = 11.48 GHz. Figure 10 shows the liver tissue after three minutes of exposure with successive
increase of input power up to 2.1 W. The progress of input power at the device under test and time
is depicted in Figure 11. Every half a minute, the input power of the discrete signal generated was 11 of 13 Sensors 2016, 16, 1450 increased logarithmically from 0.01 mW to 3.2 mW and amplified. The actual input power at the device
under is determined with the help of a connected power meter. The initial temperature of the liver
sample was 21 ◦C. Version September 1, 2016 submitted to Sensors
11 of 14 (a)
(b)
(c)
Figure 10. Liver tissue after thermal ablation. The ablation zone can be extracted by evaluating the
color of the tissue. (a) ablated liver tissue in the container of the holding device; (b) front side of ablated
liver tissue; (c) cross section of liver tissue to extract penetration depth. (a) Ablated liver tissue in the
container of the holding device
(b) Front side of ablated liver
tissue
(c) Cross Section of liver tissue to
extract penetration depth
Figure 10. Liver tissue after thermal ablation. The ablation zone can be extracted by evaluating the
color of the tissue. 0
0.5
1
1.5
2
2.5
3
0
0.5
1
1.5
2
time/min
Pin/W
Figure 11. Input Power at device under test over exposure time during ablation process. Figure 11. Input Power at device under test over exposure time during ablation process. (a)
(b)
(c)
Figure 10. Liver tissue after thermal ablation. The ablation zone can be extracted by evaluating the
color of the tissue. (a) ablated liver tissue in the container of the holding device; (b) front side of ablated
liver tissue; (c) cross section of liver tissue to extract penetration depth. (a) Ablated liver tissue in the
container of the holding device
(b) Front side of ablated liver
tissue
(c) Cross Section of liver tissue to
extract penetration depth
Figure 10. Liver tissue after thermal ablation. The ablation zone can be extracted by evaluating the
color of the tissue. 4.2. Treatment Mode (b)
e
(b) Front side of ablated liver
tissue (c)
(c) Cross Section of liver tissue to
extract penetration depth (a)
(a) Ablated liver tissue in t
container of the holding device (c)
of live
n depth (b)
ide of (a)
ated l
er of th Figure 10. Liver tissue after thermal ablation. The ablation zone can be extracted by evaluating the
color of the tissue. (a) ablated liver tissue in the container of the holding device; (b) front side of ablated
liver tissue; (c) cross section of liver tissue to extract penetration depth. Figure 10. Liver tissue after thermal ablation. The ablation zone can be extracted by evaluating the
color of the tissue. 0
0.5
1
1.5
2
2.5
3
0
0.5
1
1.5
2
time/min
Pin/W
Figure 11. Input Power at device under test over exposure time during ablation process. Figure 11. Input Power at device under test over exposure time during ablation process. Figure 11. Input Power at device under test over exposure time during ablation process. Figure 11. Input Power at device under test over exposure time during ablation process. 4.2. Treatment Mode
265
The treatment mode validation of the theranostic device is focused on the achievable dimension
266
of the ablation zone. For this purpose, ex vivo beef liver tissue was ablated by increasing the input
267
power at specific resonance frequency that was measured prior to the ablation procedure and equals
268
f0 = 11.48 GHz. Fig. 10 shows the liver tissue after three minutes exposure with successively increase
269
of input power up to 2.1 W. The progress of input power at the device under test and time is depicted
270
in Fig. 11. Every half a minute the input power of the discrete signal generated was increased
271
logarithmically from 0.01 mW to 3.2 mW and amplified. The actual input power at the device under
272
is determined with the help of a connected power meter. The initial temperature of the liver sample
273
The ablation zone was determined by measuring the area where the tissue is discolored from
dark red towards rose. The liver sample was cut to extract the ablation depth. The resulting size
equals 1.2 cm × 1 cm × 0.3 cm. was 21 ◦C.
274
The a
275
5. Discussion dark red towards rose. The liver sample was cut to extract the ablation depth. The resulting size
276
equals 1.2 cm x 1 cm x 0.3 cm. Compared to the thermal simulations that were presented in Fig. 10, the
277
actual measured lesion zone has a slightly larger dimension but with a similar shape.The simulated
278
tissue material models present averaged thermal and dielectric properties that changes even in the
279
same tissue for instance dependent on the presence of larger blood vessels
All in all the thermal
280
The proposed theranostic microwave device with oval SRRs provides two modes of operation
for the detection and treatment of cancerous tissue. In the following, the results concerning the
introduced theoretical model of the oval SRRs as well as the detection and treatment mode properties
are discussed. 4.2. Treatment Mode Compared to the thermal simulations that were presented in Figure 10,
the actual measured lesion zone has a slightly larger dimension but with a similar shape.The simulated
tissue material models present averaged thermal and dielectric properties that changes even in the
same tissue for instance dependent on the presence of larger blood vessels. All in all, the thermal
simulation results can be seen as a good indicator of how well the theranostic tool functions and can
be used for future sensor designs. 5.3. Treatment Properties From the experimental validation of the treatment mode of the sensor, the power efficiency can be
observed. In comparison with commercial microwave ablation systems that work with an input power
up to 200 W, the proposed applicator requires about 2 W of input power, and, therefore, has no need of
a cooling mechanism. Additionally, the size of the ablation zone obtained in the measurements is in the
range of 1 cm3 due to conductive and forced convective heat transfer in tissue. Generally, a reduction
of power consumption is expected since the proposed applicator has a higher operating frequency than
commercial microwave ablation tools working at 915 MHz and 2.45 GHz. With increasing frequency,
the conductivity of tissue increases, leading to a higher heat generation. Considering the ablation zone
size of the proposed device, the commercial competitors are able to ablate much larger areas of up to
3 cm3. However, the theranostic device provides a more targeted way to focus the EM field around the
resonator gaps [18]. Together with the fact that the physical size of the sensor is small, the advantage
of a more controllable ablation zone can be obtained, and therefore, less healthy tissue is damaged
during the thermal treatment. 5.2. Detection Properties The detection mode of the theranostic applicator provides a control system for thermal ablation
systems to monitor the progress of the therapy almost in real-time. Regarding the simulation and
experimental results of the detection mode, relative permittivity changes can be extracted to discern
between healthy and malignant tissue not only prior to the ablation process but also afterwards
without removing the applicator from the patient. This feature is useful to control the progress of the
treatment. However, more information about in vivo dielectric properties and differences between
healthy, tumorous and ablated tissue is needed for further adjustments to the sensitivity. simula
281
used f
282
5.1. Design g
The main challenge concerning the design of a minimally invasive applicator is given by
the size limitation that the sensor can be integrated in a needle-like operation tool. The general
functional principle was previously proven by using circular SRR with a larger dimension in the 12 of 13 Sensors 2016, 16, 1450 range of several cm [17]. By simply decreasing the size of the SRR, the operating frequency increases
significantly. Therefore, the evolution towards oval shaped SRRs was investigated that provides an
appropriate resonance frequency and fulfills the given size requirements. The reason for aiming for a
lowest possible operating frequency is to enable cost efficient, robust, and user friendly measurement
setups that can be installed in a clinical environment. The theoretical model that includes the
geometrical relation between circle and ellipses explains the frequency behavior of oval SRRs. It not
only validates the designed structures but is also suitable for further investigations of similar resonant
particles. References 1. Chandra, R.; Zhou, H.; Balasingham, I.; Narayanan, R.M. On the Opportunities and Challenges in Microwave
Medical Sensing and Imaging. IEEE Trans. Biomed. Eng. 2015, 62, 1667–1682. 2. Brace, C. Thermal tumor ablation in clinical use. Pulse IEEE 2011, 2, 28–38. 3. Reimann, C.; Puentes, M.; Schüßler, M.; Jakoby, R. Design and Realization of a Microwave Applicator
for Diagnosis and Thermal Ablation Treatment of Cancerous Tissue. In Procedings of the 10th German
Microwave Conference, Bochum, Germany, 14–16 March 2016. 4. Schwan, H.; Foster, K. RF - field interactions with biological systems: Electrical properties and biophysical
mechanisms. Proc. IEEE 1980, 68, 104–113. 5. Baena, J.D.; Bonache, J.; Martin, F.; Sillero, R.M.; Falcone, F.; Lopetegi, T.; Laso, M.A.G.; Garcia-Garcia, J.;
Gil, I.; Portillo, M.F.; et al. Equivalent-circuit models for split-ring resonators and complementary split-ring
resonators coupled to planar transmission lines. IEEE Trans. Microw. Theory Tech. 2005, 53, 1451–1461. p
p
y
6. Marqués, R.; Martín, F.; Sorolla, M. Metamaterials with Negative Parameters: Theory, Design and Microwave
Applications; John Wiley & Sons: Hoboken, NJ, USA, 2011; Volume 183. 7. Marqués, R.; Medina, F.; Rafii-El-Idrissi, R. Role of bianisotropy in negative permeability and left-handed
metamaterials. Phys. Rev. B 2002, 65, 144440. 8. Rosa, E.B.; Cohen, L. On the self inductance of circles. Bull. Bur. Stand. 1908, 4, 149–159. 9. Bronstein, I.N.; Semendjajew, K.A.; Musiol, G.; Mühlig, H. Taschenbuch der Mathematik; Verlag Harri Deutsch:
Frankfurt am Main, Germany, 2005. y
10. Gabriel, S.; Lau, R.; Gabriel, C. The dielectric properties of biological tissues: III. Parametric models for the
dielectric spectrum of tissues. Phys. Med. Biol. 1996, 41, 2271–2293. 11. O’Rourke, A.P.; Lazebnik, M.; Bertram, J.M.; Converse, M.C.; Hagness, S.C.; Webster, J.G.; Mahvi, D.M. Dielectric properties of human normal, malignant and cirrhotic liver tissue: in vivo and ex vivo measurements
from 0.5 to 20 GHz using a precision open-ended coaxial probe. Phys. Med. Biol. 2007, 52, 4707–4719. 12. Chen, L.; Ong, C.; Neo, C.; Varadan, V.; Varadan, V. Microwave Electronics Measurement and Materials
Characterization; John Wiley & Sons, Ltd.: Chichester, England, 2004. 13. Pennes, H.H. Analysis of tissue and arterial blood temperatures in the resting human f
J. Appl. Physiol. 1948, 1, 93–122. 14. Moros, E. Physics oF Thermal Therapy: Fundamentals and Clinical Applications; CRC Press: Boca Raton, FL,
USA, 2012. 15. Lazebnik, M.; Madsen, E.L.; Frank, G.R.; Hagness, S.C. Tissue-mimicking phantom materials for narrowband
and ultrawideband microwave applications. Phys. Med. Biol. 2005, 50, 4245–4258. 16. 6. Conclusions In this work, a planar microwave sensor for the theranostic therapy of organic tissue was presented
that proposes a microwave ablation applicator with a feature to detect malignant tissue before and
after the therapy. The key elements of such a device with an adequate size for the integration in
a needle-like operation tool are oval SRRs that work in a frequency range between 8 GHz and 12 GHz. The resonator based approach have beneficial properties to detect small relative permittivity changes
due to malignancies in organic tissue. When the sensor was loaded with phantoms mimicking healthy
compared to tumorous tissue, a frequency shift of 350 MHz was measured. Moreover, thermal ablation
therapy can be performed by increasing the input power at the specific resonance frequency to 2.1 W. An ablation zone of 1.2 cm × 1 cm × 0.3 cm was determined for thermal measurements with a sample
of ex vivo beef liver. Future work will focus on the packaging of the sensor tool and the possibility of
MRI compatibility. Acknowledgments: The authors would like to express their thanks for the financial support of the German
Research Foundation (DFG) in the project JA921/52-1 within the priority program “ESSENCE” SPP 1857. Furthermore, the authors wish to thank CST AG for providing the CST Microwave Studio software package. 13 of 13 Sensors 2016, 16, 1450 Author Contributions: C.R. and M.P. conceived and performed the experiments; C.R. analyzed the data; C.R. and
M.M. studied the theoretical considerations on the design of oval SRRs. The manuscript was written by C.R. and
revised by M.P., F.H., B.B., T.J.V., C.D. and R.J. Author Contributions: C.R. and M.P. conceived and performed the experiments; C.R. analyzed the data; C.R. and
M.M. studied the theoretical considerations on the design of oval SRRs. The manuscript was written by C.R. and
revised by M.P., F.H., B.B., T.J.V., C.D. and R.J. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Author Contributions: C.R. and M.P. conceived and performed the experiments; C.R. analyzed the data; C.R. and
M.M. studied the theoretical considerations on the design of oval SRRs. The manuscript was written by C.R. and
revised by M.P., F.H., B.B., T.J.V., C.D. and R.J. References Puentes, M.; Bashir, F.; Schussler, M.; Jakoby, R. Dual mode microwave tool for dielectric analysis and
thermal ablation treatment of organic tissue. In Proceedings of the 2012 Annual International Conference of
the IEEE Engineering in Medicine and Biology Society (EMBC), San Diego, CA, USA, 28 August–1 September
2012; pp. 4026–4029. 17. Puentes, M.; Schussler, M.; Damm, C.; Jakoby, R. Evolution of a microwave instrument for analysis and
thermal ablation of organic tissue. In Proceedings of the 2014 44th European Microwave Conference (EuMC),
Rome, Italy, 6–9 October 2014; pp. 283–286. y
pp
18. Puentes, M. Planar Metamaterial Based Microwave Sensor Arrays for Biomedical Analysis and Treatment; Springer:
Heidelberg, Germany, 2014; Chapter 6. c⃝2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
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https://europepmc.org/articles/pmc4687427?pdf=render
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English
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Thiopurine methyltransferase and treatment outcome in the <scp>UK</scp> acute lymphoblastic leukaemia trial <scp>ALL</scp>2003
|
British journal of haematology
| 2,015
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cc-by
| 7,630
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Summary The influence of thiopurine methyltransferase (TPMT) genotype on treat-
ment outcome was investigated in the United Kingdom childhood acute
lymphoblastic leukaemia trial ALL2003, a trial in which treatment intensity
was adjusted based on minimal residual disease (MRD). TPMT genotype
was measured in 2387 patients (76% of trial entrants): 2190 were homozy-
gous wild-type, 189 were heterozygous for low activity TPMT alleles (166
TPMT*1/*3A, 19 TPMT*1/*3C, 3 TPMT*1/*2 and 1 TPMT*1/*9) and 8
were TPMT deficient. In contrast to the preceding trial ALL97, there was
no difference in event-free survival (EFS) between the TPMT genotypes. The 5-year EFS for heterozygous TPMT*1/*3A patients was the same in
both trials (88%), but for the homozygous wild-type TPMT*1/*1 patients,
EFS improved from 80% in ALL97% to 88% in ALL2003. Importantly, the
unexplained
worse
outcome
for
heterozygous
TPMT*1/*3C
patients
observed in ALL97 (5-year EFS 53%) was not seen in ALL2003 (5-year EFS
94%). In a multivariate Cox regression analysis the only significant factor
affecting EFS was MRD status (hazard ratio for high-risk MRD patients
422, 95% confidence interval 297–599, P < 00001). In conclusion, refine-
ments in risk stratification and treatment have reduced the influence of
TPMT genotype on treatment outcome in a contemporary protocol. Keywords: thiopurine methyltransferase, mercaptopurine, acute lympho-
blastic leukaemia, minimal residual disease. The thiopurine drug mercaptopurine has been an integral
component of the maintenance chemotherapy within proto-
cols for childhood acute lymphoblastic leukaemia (ALL) for
many decades (Burchenal et al, 1953); disease relapse is sig-
nificantly reduced by long-term maintenance (Richards et al,
1996; Schrappe et al, 2000). The precise mechanism of action
of mercaptopurine in the control and eradication of residual
leukaemia cells is open to debate (Gale & Butturini, 1991),
but the thioguanine nucleotide (TGN) active metabolites can
exert their effects in a number of ways. The TGNs can
induce apoptotic cell death by inhibition of intracellular sig-
nalling pathways (Tiede et al, 2003; Poppe et al, 2006; Bour-
gine
et al,
2011). The
TGNs
can
also
inhibit
DNA
methylation and so promote cytotoxicity (Hogarth et al,
2008) whist cytotoxicity can be triggered by the direct incor-
poration of drug-derived TGN metabolites into DNA (Tidd
& Paterson, 1974; Karran, 2006). The polymorphic enzyme thiopurine methyltransferase
(TPMT) regulates intracellular TGN metabolite production
from the mercaptopurine pro-drug; there is an inverse rela-
tionship between TPMT activity and TGN production (Len-
nard et al, 1990, 2013; Relling et al, 1999a). Received 13 January 2015; accepted for
publication 15 March 2015
Correspondence: Dr Lynne Lennard,
Department of Human Metabolism, Academic
Unit of Clinical Pharmacology, University of
Sheffield, Medical School Floor D, Beech Hill
Road, Sheffield S10 2RX, UK.
E-mail: l.lennard@sheffield.ac.uk research paper research paper Thiopurine methyltransferase and treatment outcome in the
UK acute lymphoblastic leukaemia trial ALL2003 Lynne Lennard,1 Cher S. Cartwright,1
Rachel Wade2 and Ajay Vora3
1Department of Human Metabolism, University
of Sheffield, Sheffield, 2Clinical Trials Service
Unit, Oxford, and 3Department of Paediatric
Haematology, Children’s Hospital, Sheffield, UK Lynne Lennard,1 Cher S. Cartwright,1
Rachel Wade2 and Ajay Vora3
1Department of Human Metabolism, University
of Sheffield, Sheffield, 2Clinical Trials Service
Unit, Oxford, and 3Department of Paediatric
Haematology, Children’s Hospital, Sheffield, UK First published online 5 May 2015
doi: 10.1111/bjh.13469
ª 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.
British Journal of Haematology, 2015, 170, 550–558
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. Lynne Lennard,1 Cher S. Cartwright,1
Rachel Wade2 and Ajay Vora3
1Department of Human Metabolism, University
of Sheffield, Sheffield, 2Clinical Trials Service
Unit, Oxford, and 3Department of Paediatric
Haematology, Children’s Hospital, Sheffield, UK ª 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.
British Journal of Haematology, 2015, 170, 550–558 Thiopurine Methyltransferase and Outcome in ALL2003 Thiopurine Methyltransferase and Outcome in ALL2003 Both North American and European childhood ALL trials
have shown that patients with lower TPMT activities and/or
higher TGN levels have a lower relapse-risk (Lennard & Lil-
leyman, 1989; Schmeigelow et al, 1995; Balis et al, 1998; Rel-
ling
et al,
1999b;
Schmiegelow
et al,
2009);
TPMT
heterozygotes have fewer relapses than those with a wild-type
genotype (Schmiegelow et al, 2009; Lennard et al, 2015). In
the Berlin-Frankfurt-Munster (BFM) 2000 study, patients
heterozygous for TPMT low activity variant alleles had
greater clearance of minimal residual disease (MRD) load,
following the initial course of mercaptopurine (Stanulla et al,
2005). Minimal residual disease is the best measure of early
response to chemotherapy and a sensitive and specific pre-
dictor of relapse risk in children with ALL in remission
(Conter et al, 2010). Within the UK ALL97 and ALL97/99
trials the TPMT*1/*3A heterozygous patients had a better
outcome than TPMT wild-type patients (Lennard et al,
2015). The aim of this study was to re-evaluate the impact of
TPMT on treatment outcome in UKALL 2003, a trial with
significantly improved outcomes compared to ALL97 (Vora
et al, 2013). induction. The treatment intensity randomizations of one or
two delayed intensive blocks (reduced versus standard treat-
ment) for low risk patients and standard treatment versus an
intensive schedule for high-risk patients, have been previ-
ously described along with the complete chemotherapy regi-
mens (Vora et al, 2013). Laboratory measurements Minimal residual disease was measured by a standardized
real-time quantitative polymerase chain reaction method for
immunoglobulin and T-cell receptor antigen gene rearrange-
ments within four UK laboratories participating in a Euro-
pean
quality-assurance
scheme
(Flohr
et al,
2008;
Bruggemann et al, 2010). The quantitative range of the assay
was 104 (001%): 1 leukaemic cell in 10 000 cells. The Thiopurine Study protocol required a blood sample
at disease diagnosis for classification of TPMT genotype
prior to the start of mercaptopurine therapy. An additional
blood sample (5 ml lithium heparin) was requested during
remission maintenance chemotherapy for confirmation of
TPMT
genotype
and
measurement
of
mercaptopurine
metabolites. The metabolite measurement was used as a ref-
erence sample for any future clinical thiopurine metabolism
queries. The mercaptopurine chemotherapy blood sample
was taken immediately before a monthly vincristine injection
and requested at the earliest point in a maintenance cycle
when patients were tolerating mercaptopurine at the stan-
dard protocol, or the maximum tolerated dose, for 2 weeks
or more. If the patient’s mercaptopurine dosage had been
reduced or withdrawn the sample was taken on recovery of
the cell counts during the next maintenance cycle. The pri-
mary thiopurine study was of TPMT genotype and mercap-
topurine metabolite formation, if the chemotherapy blood
sample was taken at least 2 months after the last red cell
transfusion TPMT activity was also measured. Thiopurine
metabolite concentrations, TPMT activities and TPMT geno-
types were measured as previously described (Lennard et al,
2013). Thiopurine metabolite concentrations are measured
as pmol/8 9 108 red blood cells and stated in the text as
pmol. TPMT activity is measured as units/ml packed red
cells and stated in the text as units. The lower limit of
detection and quantitation for the TGN metabolites were 6
and
30 pmol,
respectively,
and
were
15
and
60 pmol,
respectively,
for
the
methyl-mercaptopurine
nucleotide
metabolites (MeMPNs; products of the TPMT reaction). The lower limit of detection and quantitation for TPMT
activity was 075 units (= nil activity, TPMT deficiency). Blood samples were genotyped for TPMT*3A, TPMT*3B
and TPMT*3C by amplification of exons 7 and 10 of the
TPMT gene (TPMT*3A is an exon 7 and 10 double
mutant); TPMT*2 and TPMT*9 were detected by sequencing
exon 5 of the TPMT gene (Lennard et al, 2013). The TPMT
*3 family and TPMT *2 low activity variant alleles account
for ≥95% of variant TPMT alleles. Summary TPMT deficiency
(homozygous for a variant low activity allele, 1 in 300 sub-
jects) is associated with an excess production of TGN metab-
olites and life-threatening bone-marrow toxicity if such
patients are treated with standard doses of thiopurine drugs
(Weinshilboum & Sladek, 1980; Lennard et al, 1989; Evans
et al, 1991; McBride et al, 2000). TPMT heterozygotes (inter-
mediate activity, 11% of subjects) accumulate higher TGN
concentrations than those subjects with a wild-type genotype
and the former are more sensitive to mercaptopurine-
induced myelosupression than the latter (Relling et al, 1999a,b,
2011, 2013; Karas-Kuzelicki et al, 2009 Peregud-Pogorzelski
et al, 2011). First published online 5 May 2015
doi: 10.1111/bjh.13469 Patients The Medical Research Council (MRC) UK ALL 2003 (UK-
ALL 2003) randomized control trial (registration number
ISRCTN07355119) tested whether MRD-based risk stratifi-
cation allows the intensity of therapy to be adapted to the
risk of relapse. The trial had an add-on thiopurine biologi-
cal study. The trial protocol was approved by the Scottish
Multi-Centre Research Ethics Committee. Initially patients
between 1 and 18 years were recruited from 45 centres in
the UK and Ireland, but the upper age limit was gradu-
ally increased to 20 years from February 2006 and finally
to 25 years by August 2007. Patient recruitment, National
Cancer
Institute
(NCI)
risk
stratification
and
clinical
high-risk
groups
have
been
previously
described
(Vora
et al, 2013). Patients classified as clinical high risk (NCI
re-classified cohorts, high-risk cytogenetics or slow mor-
phological
early
response)
were
not
eligible
for
MRD
stratification. The stratification of clinical standard and intermediate risk
groups by bone-marrow MRD has been previously described
(Vora et al, 2013). Briefly, MRD was measured after induc-
tion (day 29) and again after the recovery from consolidation
but prior to the start of interim maintenance. Minimal resid-
ual disease low-risk patients were defined as those with no
detectable disease and those patients who were MRD nega-
tive
prior
to
interim
maintenance. Indeterminate
risk
patients had detectable disease (<001% MRD = <104 leu-
kaemia cells) prior to interim maintenance; this group also
included those patients with no MRD measurement. High-
risk patients had detectable disease (≥001%) at the end of 551 L. Lennard et al overall survival (OS), which was defined as time to death. Kaplan–Meier
curves
were
calculated
and
comparisons
between groups were performed by the log-rank statistic with
stratification by age, gender and white blood cell (WBC)
count at presentation. Cox regression multivariate analysis
was used to test whether the effects of variables were inde-
pendent. Statistical analyses were by SAS, version 92 (SAS
Marlow, Buckinghamshire, UK) or Minitab 16 (Minitab Ltd,
Coventry, Warwickshire, UK). Follow- up was to 31 October
2013, with median follow-up (of those with TPMT geno-
types) of 5 years 10 months, range 3 months to 10 years
1 month. Compliance with oral mercaptopurine chemotherapy Clinicians forwarded additional blood samples for metabolite
monitoring if non-compliance with oral mercaptopurine was
suspected. Patients were suspected of non-compliance if
blood counts remained high when the patient was prescribed
prolonged mercaptopurine at the protocol standard, or
higher, dose. Statistics Within ALL97 the event-free survival (EFS) for the TPMT*1/
*3A heterozygote was far better than for TPMT*1/*3C chil-
dren (P = 0002). For the a priori power calculation we
anticipated TPMT genotypes on 1845 patients over a six-year
ALL2003 trial period. With these patient numbers we would
expect approximately 20 heterozygote TPMT*1/*3C patients
and 142 TPMT*1/*3A. To test the hypothesis that there is
about a four-fold difference in event rates between these
groups, as seen in ALL97, these numbers will give over 95%
power to detect this with similar event rates (55% and 14%
for TPMT*1/*3C and TPMT*1/*3A patients respectively),
using a 2-sided P-value of 005. The EFS in ALL2003 is
higher than for ALL97 (Vora et al, 2006, 2013). There is over
85% power to detect a similar difference but with decreased
event rates of 40% and 10%, and over 80% for 32% and 8%
for TPMT*1/*3C and TPMT*1/*3A patients, respectively. Table I. Patient characteristics. Characteristic
Thiopurine
data
No thiopurine
data
Total
P-value
Sex
Male
1369 (569%)
407 (565%)
1776
09
Female
1037 (431%)
313 (435%)
1350
Age Group
<10 years
1795 (746%)
492 (683%)
2287
00009
≥10 years
611 (254%)
228 (317%)
839
WBC group
<50 9 109/l
1876 (780%)
559 (776%)
2435
09
≥50 9 109/l
530 (220%)
161 (224%)
691
NCI risk group
Standard risk
1428 (594%)
388 (539%)
1816
0009
High risk
978 (406%)
332 (461%)
1310
CNS disease at diagnosis
No
2365 (983%)
708 (983%)
3073
09
Yes
41 (17%)
12 (17%)
53
Immunophenotype
B/N
2102 (876%)
629 (875%)
2731
09
T
298 (124%)
90 (125%)
388
Slow early response
No
2142 (890%)
620 (861%)
2762
003
Yes
264 (110%)
100 (139%)
364
MRD
High
793 (333%)
237 (333%)
1030
00004
Indeterminate
710 (298%)
261 (367%)
971
Low
877 (368%)
213 (300%)
1090
Cytogenetic risk group (BCP ALL only)
Good
1252 (627%)
332 (578%)
1584
004
Intermediate/
Poor/High
745 (373%)
242 (422%)
987
Treatment given
A
1212 (504%)
326 (453%)
1538
0009
B
652 (271%)
194 (269%)
846
C
542 (225%)
200 (278%)
742
WBC, white blood cell count (at diagnosis); NCI, National Cancer
Institute; CNS, central nervous system; MRD, minimal residual dis-
ease; BCP ALL, B-cell precursor acute lymphoblastic leukaemia. ª 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.
British Journal of Haematology, 2015, 170, 550–558 Patients Very low or absent mercaptopurine metabolite
concentrations are strong indications of non-compliance. Thiopurine Methyltransferase and Outcome in ALL2003 Thiopurine Methyltransferase and Outcome in ALL2003 Thiopurine Methyltransferase and Outcome in ALL2003 C skewed the data because these patients did not enter mer-
captopurine maintenance until week 47; Regimen C patients
had their reference sample at a median of week 66 (range
49–162), the median for Regimens A+B remained at week
17. In agreement with previous UK ALL trials (Lennard et al,
1990, 2013, 2015) mercaptopurine metabolite accumulation
differed by genotype with TPMT wild-type patients accumu-
lating lower concentrations of the TGN active metabolites
than TPMT heterozygotes (Table III) or TPMT-deficient
patients. The ALL2003 trial recommendation was for the
TPMT-deficient patient to start mercaptopurine on a much-
reduced dose (10% protocol), and titrate to the protocol tar-
get cell counts. Seven of the 8 TPMT-deficient children,
identified pre-treatment, tolerated mercaptopurine dosages
ranging from 8 to 26 mg/m2 (median 9 mg/m2) and TGN
concentrations ranged from 970 to 2569 pmol (median
1329) in blood samples taken after a median of 8 weeks mer-
captopurine (range 2 to >12 weeks). One child, who lacked a
pre-treatment blood sample, was identified during mainte-
nance chemotherapy with a history of repeated cytopenias
and an inability to tolerate mercaptopurine; TGNs after
50 mg/m2 mercaptopurine for 6 weeks were 2347 pmol. TPMT-deficient patients do not accumulate MeMPNs. Results Thiopurine analysis The patient numbers and samples available for analysis are
summarized in Fig 1. Significantly more blood samples were
received for thiopurine analysis from younger patients and
from less high-risk patients (Table I). Of the 2387 patients
with a TPMT genotype available, 2190 were homozygous
wild-type (TPMT*1/*1), 189 were heterozygous for low activ-
ity TPMT alleles (166 TPMT*1/*3A, 19 TPMT*1/*3C, 3
TPMT*1/*2 and 1 TPMT*1/*9) and 8 were TPMT-deficient
(4 TPMT*3A/*3C, 3 TPMT*3A/*3A and 1 TPMT*2/*3A). Compared to the TPMT*3A allele there was an excess of the
TPMT*3C allele in ethnic minorities (Chi-squared 1057,
P = 0001; Table II). Of those patients categorized as MRD high or low risk
1657 had TPMT genotypes available: 791 high-risk MRD
patients (728 TPMT wild-type and 63 heterozygotes) and
866 low-risk MRD patients (803 TPMT wild-type and 63
heterozygotes). There was no difference in the distribution
of TPMT genotypes between the two MRD risk groups
(Chi-squared 028, P = 0597). Additional blood samples were forwarded from some cli-
nicians when patients were unduly sensitive to mercaptopu-
rine or tolerating mercaptopurine prior to dose escalation. From 1304 patients we received 3514 blood samples taken
during chemotherapy (median 2 samples per patient, range Statistics Comparing the n = 2406 patients who have thiopurine data (TPMT
genotype and/or mercaptopurine metabolites) to the n = 720 with
no data, there is some bias in the thiopurine dataset towards younger
patients and those who are less high risk. British Journal of Haematology published by John Wiley & Sons Ltd Table I. Patient characteristics. The Anderson–Darling test was used to examine the fit of
observations to a normal distribution. Metabolite values are
stated as median and range. Differences between groups were
compared by the Chi-square statistic, or the Mann–Whitney
test. Outcome analysis was of EFS, with an event defined as
time to relapse, secondary tumour or death, relapse-free sur-
vival (RFS), which was defined as time to relapse (excluding
those patients who did not achieve a remission or died dur-
ing initial induction or consolidation chemotherapy) and Fig 1. ALL2003 trial data profile. The numbers of individual patients
providing blood samples for thiopurine analysis. TPMT, thiopurine
methyltransferase; MP, mercaptopurine; MRD, minimal residual dis-
ease. WBC, white blood cell count (at diagnosis); NCI, National Cancer
Institute; CNS, central nervous system; MRD, minimal residual dis-
ease; BCP ALL, B-cell precursor acute lymphoblastic leukaemia. Comparing the n = 2406 patients who have thiopurine data (TPMT
genotype and/or mercaptopurine metabolites) to the n = 720 with
no data, there is some bias in the thiopurine dataset towards younger
patients and those who are less high risk. Fig 1. ALL2003 trial data profile. The numbers of individual patients
providing blood samples for thiopurine analysis. TPMT, thiopurine
methyltransferase; MP, mercaptopurine; MRD, minimal residual dis-
ease. Fig 1. ALL2003 trial data profile. The numbers of individual patients
providing blood samples for thiopurine analysis. TPMT, thiopurine
methyltransferase; MP, mercaptopurine; MRD, minimal residual dis-
ease. 552 ª 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.
British Journal of Haematology 2015 170, 550–558 A comparison of mercaptopurine (MP) metabolite formation in thiopurine methyltransferase (TPMT) wild-type and heterozygous patients. The
MP dose is that tolerated at the time of metabolite measurement. TGNs, thioguanine nucleotides; MeMPNs, methylmercaptopurine nucleotides;
CI, confidence interval. TGN and MeMPN units are pmol/8 9 108 red cells. Values are given as median (range). ª 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.
553
British Journal of Haematology, 2015, 170, 550–558 TPMT genotype-phenotype discordance TPMT activity was available for 1045 patients who were
TPMT wild-type, 92 heterozygotes and eight patients homo-
zygous for variant alleles. The concordance in the homozy-
gous variant allele cohort was 100%, all eight patients lacked
TPMT activity. The break-point of the nadir of the TPMT
‘intermediate’ and ‘high’ frequency distributions was 105
units, a value determined by sensitivity and specificity analy-
sis of the distribution of the TPMT heterozygous genotype
over the TPMT activity range (Lennard et al, 2013). At 105
units the sensitivity for the detection of the TPMT heterozy-
gous variant allele was 95% (specificity 87%). The specificity
of 87% results in 13% of wild-type alleles in the intermediate
activity cohort and a concordance of 39% (i.e. 61% of the
intermediate activity cohort patients had a wild-type geno-
type). Within the intermediate activity cohort both the het-
erozygous variant allele (n = 87) and homozygous wild-type
allele patients (n = 139) had similar TPMT activities ranging
from 53–105 units and 55–105 units respectively (Fig 2). The concordance in the high activity group (activity range
105–264 units) was 995% (906 wild-type alleles and 5 het-
erozygous variant alleles, the highest TPMT activity of a vari-
ant allele heterozygote was 116 units). As observed in ALL97
(Lennard et al, 2013), the median mercaptopurine metabolite
concentrations measured in the intermediate activity TPMT
wild-type genotype patients (317 pmol TGNs, 15 937 pmol
MeMPNs) were similar to the concentrations measured in
the
high
activity
TPMT
wild-type
genotype
patients
(311 pmol TGNs, 14 380 pmol MeMPNs) and significantly
different from the metabolite concentrations recorded for
TPMT
variant
allele
heterozygotes
(747 pmol
TGNs,
3407 pmol MeMPNs); intermediate activity heterozygous vari-
ant allele vs intermediate activity wild-type TPMT genotype
patients, median difference 390 pmol TGNs (95% confidence
interval, CI, 323–469, P < 00001) and 9995 pmol MeMPNs
(95% CI 13446 to 6675, P < 00001). Fig 2. TPMT genotype-phenotype discordance, 61% of the interme-
diate TPMT activity cohort have a TPMT wild-type genotype. The
solid line at TPMT 105 units indicates the nadir of the TPMT high
and intermediate frequency distributions. for TPMT *1/*3C and 100% for all other TPMT genotypes. Five-year EFS was 88% for TPMT*1/*1 (n = 2190, 95% con-
fidence interval, CI, 87–89%) and TPMT *1/*3A (n = 166,
95% CI 82–93%), 94% for TPMT *1/*3C (n = 19, 95% CI
84–100%) and 100% for all other TPMT genotypes (Fig 3). Mercaptopurine metabolites The median week for the reference blood sample was week
17 (range 11–162). Blood samples from patients on Regimen Table II. TPMT genotype by ethnicity. Table II. TPMT genotype by ethnicity. Table II. TPMT genotype by ethnicity. *1/*1
*1/*2
*1/*3A
*1/*3C
*1/*9
*2/*3A
*3A/*3A
*3A/*3C
Unknown
Total
White
1790
2
152
12
1
1
3
4
567
2532
Asian
154
1
5
4
68
232
Black
53
1
1
19
74
Mediterranean
16
2
18
Middle Eastern
14
1
8
23
Mixed
60
2
11
73
Oriental
7
1
2
10
Other
22
6
28
Unknown
74
4
2
56
136
Total
2190
3
166
19
1
1
3
4
739
3126
Including the thiopurine methyltransferase (TPMT)-deficient children in the analysis there were 8 TPMT*3A alleles in ethnic minorities (160 in
white patients) and 5 TPMT*3C alleles in ethnic minorities (16 in white patients), Chi-squared = 1057 P = 0001. Including the thiopurine methyltransferase (TPMT)-deficient children in the analysis there were 8 TPMT*3A alleles in ethnic minorities (160 in
white patients) and 5 TPMT*3C alleles in ethnic minorities (16 in white patients), Chi-squared = 1057 P = 0001. Table III. Thiopurine methyltransferase genotype and metabolite formation. Wild-type TPMT*1/*1
Heterozygous TPMT
Median difference (95%CI)
Patients
1187
109
MP dose mg/m2
75 (7–232)
74 (17–93)
10 (20 to 0001), P = 0046
TGNs pmol
312 (0–1449)
751 (174–2597)
425 (372 to 482), P < 00001
MeMPNs pmol
14808 (0–83904)
4205 (0–37362)
9055 (10824 to 7350), P < 00001
A comparison of mercaptopurine (MP) metabolite formation in thiopurine methyltransferase (TPMT) wild-type and heterozygous patients. The
MP dose is that tolerated at the time of metabolite measurement. TGNs, thioguanine nucleotides; MeMPNs, methylmercaptopurine nucleotides;
CI, confidence interval. TGN and MeMPN units are pmol/8 9 108 red cells. Values are given as median (range). Table III. Thiopurine methyltransferase genotype and metabolite formation. Table III. Thiopurine methyltransferase genotype and metabolite formation. A comparison of mercaptopurine (MP) metabolite formation in thiopurine methyltransferase (TPMT) wild-type and heterozygous patients. The
MP dose is that tolerated at the time of metabolite measurement. TGNs, thioguanine nucleotides; MeMPNs, methylmercaptopurine nucleotides;
CI, confidence interval. TGN and MeMPN units are pmol/8 9 108 red cells. Values are given as median (range). 553 L. Lennard et al Wild-type
Heterozygous
TPMT genotype
ALL 2003 : TPMT genotype and activity
TPMT activity (units / m IRBCs)
Fig 2. ª 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.
British Journal of Haematology, 2015, 170, 550–558 Mercaptopurine metabolites TPMT genotype-phenotype discordance, 61% of the interme-
diate TPMT activity cohort have a TPMT wild-type genotype. The
solid line at TPMT 105 units indicates the nadir of the TPMT high
and intermediate frequency distributions. ALL 2003 : TPMT genotype and activity 1–72). Thirty-nine patients (3% of total cohort) had metabo-
lite levels at the lower limit of detection or lacked measurable
metabolites, six of these patients on multiple occasions. At
the time of nil metabolites the mercaptopurine dosage ran-
ged from 70 to 130 mg/m2 (median 76) for a median of
4 weeks (range 2–15); non-compliance with oral chemother-
apy is the most logical explanation for these findings. There
was no difference between the age range of those children
with compliance problems and those without (median age
49 years, range 11–239). Wild-type
Heterozygous
TPMT genotype
ALL 2003 : TPMT genotype and activity
TPMT activity (units / m IRBCs) TPMT activity (units / m IRBCs) TPMT genotype-phenotype discordance The corresponding five-year EFS estimates for the previous
ALL97 trial were 80% for TPMT*1/*1 (n = 1206, 95% CI
78–82%), 88% for TPMT *1/*3A (n = 99, 95% CI 81–94%)
and 53% for TPMT *1/*3C (n = 17, 95% CI 29–77%) (Len-
nard et al, 2015). For patients with metabolite data there was no difference
in EFS between the nil metabolite, non-compliant, cohort
and the rest of the group. There was no relationship between
the trial reference metabolite concentrations and EFS when
the data was analysed either as continuous variables or split
into quartiles. There was no difference in EFS between the
TPMT phenotype groups. Clinical outcome There was no difference in OS, RFS or EFS between the
TPMT genotypes. Five-year OS was 93% for TPMT*1/*1 and
TPMT *1/*3A and 100% for all other TPMT genotypes. Five-
year RFS was 92% for TPMT*1/*1 and TPMT *1/*3A, 94% Although EFS differed by MRD risk group in ALL2003 there
was no difference in EFS, with respect to TPMT genotype,
within those risk groups (Table IV). In a multivariate Cox
regression analysis the only significant factor affecting EFS in 554 Fig 3. UKALL2003 – Event-free survival by TPMT genotype
Thiopurine Methyltransferase and Outcome in ALL2003 Fig 3. UKALL2003 – Event-free survival by TPMT genotype
Thiopurine Methyltransferase and Outcome in ALL2003 Fig 3. UKALL2003 – Event-free survival by TPMT genotype
Table IV. Event-free survival (EFS) and TPMT genotype within subgroups defined by MRD risk status. TPMT genotype
MRD high-risk
MRD low-risk
Events/Patients
O/E
5-year EFS (95% CI)
Events/Patients
O/E
5-year EFS (95% CI)
*1/*1
140/728
102
805% (776–834%)
37/803
097
954% (938–970%)
*1/*3A, *1/*2, *1/*9
9/58
080
835% (735–935%)
4/56
153
929% (862–996%)
*1/*3C
1/5
119
750% (325–1000%)
0/7
0
100%
P (heterogeneity)
08
06
TPMT, thiopurine methyltransferase; MRD, minimal residual disease; O/E, Observed/Expected; CI, confidence interval. There was no heterogene-
ity with respect to TPMT genotype within the MRD subgroups defined by MRD risk status. Thiopurine Methyltransferase and Outcome in ALL2003 Thiopurine Methyltransferase and Outcome in ALL2003 Fig 3. UKALL2003 – Event-free survival by TPMT genotype Table IV. Event-free survival (EFS) and TPMT genotype within subgroups defined by MRD risk status. Table IV. Event-free survival (EFS) and TPMT genotype within subgroups defined by MRD risk status. TPMT genotype
MRD high-risk
MRD low-risk
Events/Patients
O/E
5-year EFS (95% CI)
Events/Patients
O/E
5-year EFS (95% CI)
*1/*1
140/728
102
805% (776–834%)
37/803
097
954% (938–970%)
*1/*3A, *1/*2, *1/*9
9/58
080
835% (735–935%)
4/56
153
929% (862–996%)
*1/*3C
1/5
119
750% (325–1000%)
0/7
0
100%
P (heterogeneity)
08
06
TPMT, thiopurine methyltransferase; MRD, minimal residual disease; O/E, Observed/Expected; CI, confidence interval. There was no heterogene-
ity with respect to TPMT genotype within the MRD subgroups defined by MRD risk status. TPMT, thiopurine methyltransferase; MRD, minimal residual disease; O/E, Observed/Expected; CI, confidence interval. There was no heterogene-
ity with respect to TPMT genotype within the MRD subgroups defined by MRD risk status. this subset of patients was MRD status (hazard ratio for high-
risk MRD patients = 422, 95% CI 297–599, P < 00001). Clinical outcome The chemotherapy backbone of ALL2003 was identical to
the ALL97/99 phase of the ALL97 trial (Vora et al, 2006,
2013), but in ALL2003 all patients received dexamethasone
instead of prednisolone and pegylated asparaginase instead of
native Escherichia Coli asparaginase. Also, treatment intensity
was stratified by MRD response. MRD low risk patients fared
much better than the high-risk patients (5-year EFS 95% ver-
sus 80%, respectively). Overall there was no difference in sur-
vival, with respect to TPMT genotype, within the two MRD
risk groups. The BFM2000 trial used a two time-point assess-
ment protocol, measuring the disease load before (day 33) and
after (day 78) the initial course of mercaptopurine chemother-
apy (Conter et al, 2010), to study the clearance of disease with
respect to TPMT genotype: patients heterozygous for variant
TPMT alleles had an increased clearance of disease, a lower
rate of MRD positivity (Stanulla et al, 2005). Minimal residual ª 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.
British Journal of Haematology, 2015, 170, 550–558 ª 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.
British Journal of Haematology, 2015, 170, 550–558 Acknowledgements The thiopurine studies within ALL2003 were supported by
Leukaemia and Lymphoma Research. The ALL2003 trial was
supported from grants from both Leukaemia and Lymphoma
Research (UK) and the Medical Research Council (UK). LL,
CSC and AV were supported by Leukaemia and Lymphoma
Research. RW was supported by the Medical Research Coun-
cil. We wish to thank all the clinicians who entered patients
into this trial, the nurses and pharmacists who supported
them, and the participating patients and their families. We
thank Sue Richards (Clinical Trial Service Unit, Oxford) for
her work
on the ALL2003 trial
and Joanna
Chowdry
(Department of Human Metabolism, University of Sheffield)
for her contributions to the TPMT genotyping studies. We
thank the trial co-ordinators and co-investigators (Nick
Goulden, Chris Mitchell, Clare Rowntree, Rachael Hough
and Jeremy Hancock) for supporting the thiopurine studies
within ALL2003 and access to the minimal residual disease
data. In a study of adolescents with ALL, improved compliance
with mercaptopurine was associated with parental supervision
of tablet taking (Malbasa et al, 2007). Evaluation of mercap-
topurine non-compliance in ALL children by mercaptopurine
metabolite monitoring coupled with structured interviews has
indicated that medication non-compliance rates could be as
high as 26% (Hawwa et al, 2009) with a lower non-compli-
ance associated with adverse socioeconomic factors (De
Oliveira et al, 2004). Evaluation of mercaptopurine non-com-
pliance by an event-monitoring cap on the medication bottle
associated non-compliance with ethnicity and an increased
relapse risk; the association with ethnicity was linked to a
lower socioeconomic status (Bhatia et al, 2012). The previ-
ously reported worse outcome for TPMT*1/*3C patients
(Lennard et al, 2015) may have been influenced by the
increased frequency of this TPMT allele in ethnic minorities. This study has also confirmed the previously reported dis-
cordance between TPMT genotype and phenotype, which has
been mainly attributed to the undue influence of the disease
process and chemotherapy on red blood cell TPMT enzyme
activity (Lennard et al, 2013). In this patient group, TPMT
activity should not be used to predict TPMT heterozygosity. An improvement in outcome for TPMT wild-type patients
has closed the EFS gap with TPMT*1/*3A heterozygous
patients. However, the EFS for the TPMT*1/*3A heterozy-
gous cohort has large confidence intervals and so a small dif-
ference
in
outcome
between
the
TPMT
wild-type
and
TPMT*1/*3A patients cannot be excluded. Acknowledgements The improvement
is likely to be due to a combination of better risk stratifica-
tion and use of dexamethasone and pegylated asparagin-
ase throughout treatment. Treatment intensification
can
influence
the
subsequent
response
to
mercaptopurine Author contribution LL, RW and AV contributed to the design of the study. AV
was a trial co-ordinator. LL and CSC were responsible for
the thiopurine studies. LL, CSC and RW were involved in
data collection and data analysis, RW was responsible for
outcome and minimal residual disease analysis. LL, CSC, RW
and AV were involved in data interpretation. LL wrote the
manuscript. All authors were involved in the revision and
editing of the manuscript. All authors approved the final ver-
sion of the manuscript. Discussion A major outcome of the ALL2003 trial was the much improved
survival (5-year EFS 87%, 5-year OS 92%) compared to
its predecessor ALL97 (5-year EFS 80%, 5-year OS 89%) (Vora
et al, 2006, 2013). In contrast to ALL97, there was no differ-
ence in EFS between the TPMT genotypes. In both trials, the
5-year EFS for the TPMT*1/*3A cohort remained the same
(88%), but the 5-year EFS for the TPMT*1/*1 cohort
improved, from 80% in ALL97 (Lennard et al, 2015) to 86%
in ALL2003. Importantly, the unexplained worse outcome for
TPMT*1/*3C patients observed in ALL97 (5-year EFS 53%)
was not observed in ALL2003 (5-year EFS 94%). 555 L. Lennard et al disease was not monitored at two points in UK ALL2003 thus;
the association of TPMT genotype with MRD response follow-
ing thiopurine exposure could not be investigated. maintenance chemotherapy. This was initially reported in
MRC UKALL X (Chessells et al, 1997) and observed subse-
quent to asparaginase therapy in the United States Dana Far-
ber Cancer Institute protocols (Merryman et al, 2012) and
could contribute to the improved outcome for the TPMT
wild-type patients. This study confirms previous observations on non-compli-
ance with oral chemotherapy within the UK ALL protocols
(Lennard et al, 1995, 2015) and illustrates the usefulness of
metabolite monitoring in the identification of the non-com-
pliant patient. A complete lack of mercaptopurine metabo-
lites when taking prolonged high doses, as observed in 3% of
patients in this study, is a strong indication of non-compli-
ance with oral chemotherapy. ª 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.
British Journal of Haematology, 2015, 170, 550–558 The authors have no competing interests. Bourgine, J., Garat, A., Allorge, D., Crunelle-Thi-
baut, A., Lo-Guidice, J.M., Colombel, J.F., Brolly,
F. & Billaut-Laden, I. (2011) Evidence for a func-
tional genetic polymorphism of Rho-GTPase
Rac1. Implication in azathioprine response? Phar-
macogenetics and Genomics, 21, 313–324. Bruggemann, M., Schrauder, A., Raff, T., Pfeifer,
H., Dworzak, M., Ottmann, O.G., Asnafi, V.,
Baruchel, A., Bassan, R., Benoit, Y., Biondi, A.,
Cave, H., Dombret, H., Fielding, A.K., Foa, R.,
Goekbuget, N., Goldstone, A.H., Goulden, N., Bhatia, S., Landier, W., Shangguan, M., Hageman,
L., Schaible, A.N., Carter, A.R., Hanby, C.L.,
Leisenring, W., Yasui, Y., Kornegay, N.M., Mas-
carenhas, L., Ritchey, A.K., Casillas, J.N., Dick-
ens, D.S., Meza, J., Carroll, W.L., Relling, M.V.
& Wong, F.L. (2012) Nonadherence to oral mer-
captopurine and risk of replapse in hispanic and
non-hispanic white children with acute lympho-
blastic leukemia: a report from the Children’s
Oncology Group. Journal of Clinical Oncology,
30, 2094–2101. Bourgine, J., Garat, A., Allorge, D., Crunelle-Thi-
baut, A., Lo-Guidice, J.M., Colombel, J.F., Brolly,
F. & Billaut-Laden, I. (2011) Evidence for a func-
tional genetic polymorphism of Rho-GTPase
Rac1. Implication in azathioprine response? Phar-
macogenetics and Genomics, 21, 313–324. Bhatia, S., Landier, W., Shangguan, M., Hageman,
L., Schaible, A.N., Carter, A.R., Hanby, C.L.,
Leisenring, W., Yasui, Y., Kornegay, N.M., Mas-
carenhas, L., Ritchey, A.K., Casillas, J.N., Dick-
ens, D.S., Meza, J., Carroll, W.L., Relling, M.V.
& Wong, F.L. (2012) Nonadherence to oral mer-
captopurine and risk of replapse in hispanic and
non-hispanic white children with acute lympho-
blastic leukemia: a report from the Children’s
Oncology Group. Journal of Clinical Oncology,
30, 2094–2101. Balis, F.M., Holcenberg, J.S., Poplack, D.G., Ge, J.,
Sather,
H.N.,
Murphy,
R.F.,
Ames,
M.M.,
Waskerwitz, M.J., Tubergen, D.G., Zimm, S.,
Gilchrist, G.S. & Bleyer, W.A. (1998) Pharmaco-
kinetics and pharmacodynamics of oral metho-
trexate and mercaptopurine in children with
lower risk acute lymphoblastic leukaemia: a joint
Children’s Cancer Group and Pediatric Oncology
Branch study. Blood, 92, 3569–3577. Bourgine, J., Garat, A., Allorge, D., Crunelle-Thi-
baut, A., Lo-Guidice, J.M., Colombel, J.F., Brolly,
F. & Billaut-Laden, I. (2011) Evidence for a func-
tional genetic polymorphism of Rho-GTPase
Rac1. Implication in azathioprine response? Phar-
macogenetics and Genomics, 21, 313–324.
Bruggemann, M., Schrauder, A., Raff, T., Pfeifer,
H., Dworzak, M., Ottmann, O.G., Asnafi, V.,
Baruchel, A., Bassan, R., Benoit, Y., Biondi, A.,
Cave, H., Dombret, H., Fielding, A.K., Foa, R.,
Goekbuget, N., Goldstone, A.H., Goulden, N., Bourgine, J., Garat, A., Allorge, D., Crunelle-Thi-
baut, A., Lo-Guidice, J.M., Colombel, J.F., Brolly,
F. & Billaut-Laden, I. (2011) Evidence for a func-
tional genetic polymorphism of Rho-GTPase
Rac1. Implication in azathioprine response? Phar-
macogenetics and Genomics, 21, 313–324.
Bruggemann, M., Schrauder, A., Raff, T., Pfeifer,
H., Dworzak, M., Ottmann, O.G., Asnafi, V.,
Baruchel, A., Bassan, R., Benoit, Y., Biondi, A.,
Cave, H., Dombret, H., Fielding, A.K., Foa, R.,
Goekbuget, N., Goldstone, A.H., Goulden, N., Bruggemann, M., Schrauder, A., Raff, T., Pfeifer,
H., Dworzak, M., Ottmann, O.G., Asnafi, V.,
Baruchel, A., Bassan, R., Benoit, Y., Biondi, A.,
Cave, H., Dombret, H., Fielding, A.K., Foa, R.,
Goekbuget, N., Goldstone, A.H., Goulden, N., References Chessells, J.M., Harrison, G., Lilleyman, J.S., Bai-
ley, C.C. & Richards, S.M. (1997) Continuing
(maintenance) therapy in lymphoblastic leukae-
mia: lessons from MRC UKALL X. British Jour-
nal of Haematology, 98, 945–951. Lennard, L. & Lilleyman, J.S. (1989) Variable 6-
mercaptopurine metabolism and treatment out-
come
in
childhood
lymphoblastic
leukaemia
[published erratum appears in J Clin Oncol. 1990;8:567]. Journal of Clinical Oncology, 7,
1816–1823. Conter, V, Bartram, CR, Valsecchi, MG, Schraud-
er, A, Panzer-Grumayer, R, Moricke, A, Arico,
M, Zimmerman, M, Mann, G, De Rossi, G,
Stanulla, M, Locatelli, F, Basso, G, Niggli, F,
Barisone, E, Henze, G, Ludwig, WD, Haas, OA,
Cazzangigna, G, Koehler, R, Silvestri, D, Brad-
tke, J, Parasole, R, Beier, R, van Dongen, JJM,
Biondi, A & Schrappe, M. (2010) Molecular
response to treatment redefines all prognostic
factors in children and adolescents withB-cell
precurrrsor
acute
lymphoblastic
leukaemia:
results in 3184 patients of the AIEOP-BFM ALL
2000 study. Blood, 115, 3206–3214. Relling, M.V., Hancock, M.L., Rivera, G.K., Sandl-
und, J.T., Ribeiro, R.C., Krynetski, E.Y., Pui, C.-
H. & Evans, W.E. (1999a) Mercaptopurine ther-
apy intolerance and heterozygosity at the thiop-
urine S-methyltransferase gene locus. Journal of
the National Cancer Institute, 91, 2001–2008. Lennard, L., Van Loon, J.A. & Weinshilboum,
R.M. (1989) Pharmacogenetics of acute azathio-
prine toxicity: relationship to thiopurine methyl-
transferase
genetic
polymorphism. Clinical
Pharmacology and Therapeutics, 46, 149–154. Relling, M.V., Hancock, M.L., Boyett, J.M., Pui,
C.-H. & Evans, W.E. (1999b) Prognostic impor-
tance of 6-mercaptopurine dose intensity in
acute lymphoblastic leukemia. Blood, 93, 2817–
2823. Lennard, L., Lilleyman, J.S., Van Loon, J.A. &
Weinshilboum, R.M. (1990) Genetic variation in
response to 6-mercaptopurine for childhood
acute
lymphoblastic
leukaemia. Lancet,
336,
225–229. Relling, M.V., Gardner, E.E., Sandborn, W.J., Pui,
C-H, Stein, C.M., Carrillo, M., Evans, W.E. &
Klein, T.E. (2011) Clinical pharmacogenetics
implementation consortium guidelines for thi-
opurine methyltransferase genotype and thiopu-
rine
dosing. Clinical
Pharmacology
and
Therapeutics, 89, 387–391. De Oliveira, B.M., Viana, M.B., Zani, C.L. & Ro-
manha, A.J. (2004) Clinical and laboratory eval-
uation of compliance in acute lymphoblastic
leukaemia. Archives of Disease in Childhood, 89,
785–788. Lennard, L., Welch, J. & Lilleyman, J.S. (1995)
Intracellular metabolites of 6-mercaptopurine in
children with lymphoblastic leukaemia: a possi-
ble indicator of non-compliance. British Journal
of Cancer, 72, 1004–1006. Evans, W.E., Horner, M., Chu, Y.Q., Kalwinsky, D. & Roberts, W.M. References Balis, F.M., Holcenberg, J.S., Poplack, D.G., Ge, J.,
Sather,
H.N.,
Murphy,
R.F.,
Ames,
M.M.,
Waskerwitz, M.J., Tubergen, D.G., Zimm, S.,
Gilchrist, G.S. & Bleyer, W.A. (1998) Pharmaco-
kinetics and pharmacodynamics of oral metho-
trexate and mercaptopurine in children with
lower risk acute lymphoblastic leukaemia: a joint
Children’s Cancer Group and Pediatric Oncology
Branch study. Blood, 92, 3569–3577. 556 Thiopurine Methyltransferase and Outcome in ALL2003 Thiopurine Methyltransferase and Outcome in ALL2003 Henze, G., Hoelzer, D., Janka-Schaub, G.E.,
Macintyre, E.A., Pieters, R., Rambaldi, A., Riber-
a, J.M., Schmiegelow, K., Spinelli, O., Stary, J.,
von Stackelberg, A., Kneba, M., Schrappe, M. &
Van Dongen, J.J.M. (2010) Standardized MRD
quantification in European ALL trials: proceed-
ings of the Second International Symposium on
MRD Assessment in Kiel, Germany, 18-20 Sep-
tember 2008. Leukaemia, 24, 521–535. lymphoblastic leukaemia – an exploratory study. European Journal of Clinical Pharmacology, 65,
1105–1112. suppression and effects on dosing of other
chemotherapeutic
agents
in
childhood
acute
lymphoblastic leukaemia. Pediatric Blood & Can-
cer, 59, 925–927. Hogarth, L.A., Redfern, C.P.F., Teodoridis, J.M.,
Hall, A.G., Anderson, H., Case, M.C. & Coult-
hard, S.A. (2008) The effect of thiopurine drugs
on DNA methylation in relation to TPMT
expression. Biochemical Pharmacology, 76, 1024–
1035. Peregud-Pogorzelski, J., Tetera-Rudnicka, E., Ku-
rzawski, M., Brodkiewicz, A., Adrianowska, N.,
Mlynarski, W., Januszkiewicz, D. & Drozdzik,
M. (2011)
Thiopurine
S-methyltransferase
(TPMT) polymorphisms in children with acute
lymphoblastic leukemia and the need for reduc-
tion or cessation of 6-mercaptopurinedoses dur-
ing maintenance therapy: the Polish multicentre
analysis. Pediatric Blood and Cancer, 57, 578–
582. Burchenal, J.H., Murphy, M.L., Ellison, R.R., Sy-
kes, M.P., Tan, T.C., Leone, L.A., Karnofsky,
D.A., Craver, L.F., Dargeon, H.W. & Rhoads,
C.P. (1953) Clinical evaluation of a new antime-
tabolite, 6-mercaptopurine, in the treatment of
leukaemia and allied diseases. Blood, 8, 965–999. Karas-Kuzelicki, N., Jazbec, J., Milek, M. & Mlina-
ric-Rascan,
I. (2009)
Heterozygosity
at
the
TPMT gene
locus, augumented by mutated
MTHFR gene, predisposes to 6-MP related tox-
icities in childhood ALL patients. Leukemia, 23,
971–1017. Poppe, D., Tiede, I., Fritz, G., Becker, C., Bartsch,
B., Wirtz, S., Strand, D., Tanaka, S., Galle, P.R.,
Bustelo, X.R. & Neurath, M.F. (2006) Azathio-
prine suppresses Ezrin-Radixin-Moesin-depen-
dent T cell-APC conjugation through inhibition
of Vav guanosine exchange activity on Rac pro-
teins. Journal of Immunology, 176, 640–651. Karran, P. (2006) Thiopurines,
DNA damage,
DNA repair and therapy-related cancer. British
Medical Bulletin, 79 and 80, 153–170. References (1991) Altered mercaptopurine
metabolism, toxic effects, and dosage require-
ment in a thiopurine methyltransferase deficient
child with acute lymphoblastic leukaemia. Jour-
nal of Pediatrics, 119, 985–989. Relling,
M.V.,
Gardner,
E.E.,
Sandborn,
W.J.,
Schmiegelow, K., Pui, C-H, Yee, S.W., Stein,
C.M., Carrillo, M., Evans, W.E., Hicks, J.K.,
Schwab, M. & Klein, T.E. (2013) Clinical phar-
macogenetics implementation consortium guide-
lines for thiopurine methyltransferase genotype
and thiopurine dosing: 2013 update. Clinical
Pharmacology and Therapeutics, 93, 324–325. Lennard, L., Cartwright, C.S., Wade, R., Richards,
S.M. & Vora, A. (2013) Thiopurine methyltrans-
ferase genotype-phenotype discordance, and thi-
opurine
active
metabolite
formation,
in
childhood acute lymphoblastic leukaemia. British
Journal of Clinical Pharmacology, 76, 125–136. Flohr, T., Schrauder, A., Cazzaniga, G., Panzer-
Grumayer, R., van der Velden, V., Fischer, S.,
Stannula, M., Basso, G., Niggli, F.K., Schafer,
B.W., Sutton, R., Koehler, R., Zimmermann, M.,
Valsecchi, M.G., Gadner, H., Masera, G., Schr-
appe, M., van Dogen, J.J.M., Biondi, A. & Bar-
tram, C.R. (2008) Minimal residual disease-
directed risk stratification using real-time quan-
titative PCR analysis of immunoglobulin and T-
cell receptor gene rearrangements in the interna-
tional multicentre trial AIEOP-BFM ALL 2000
for childhood acute lymphoblastic leukaemia. Leukaemia, 22, 771–782. Lennard, L., Cartwright, C.S., Wade, R., Richards,
S. & Vora, A. (2015) Thiopurine dose intensity
and treatment outcome in childhood lympho-
blastic leukaemia: the influence of thiopurine
methyltransferase
pharmacogenetics. British
Journal of Haematology, 169, 228–240. Richards, S., Gray, R., Peto, R., Gaynon, P. & Ma-
sera, G. & The Childhood ALL Collaborative
Group. (1996) Duration and intensity of main-
tenance chemotherapy in acute lymphoblastic
leukaemia: overview of 42 trials involving 12,000
randomised children. Lancet, 347, 1783–1788. Malbasa, T., Kodish, E. & Santacroce, S.J. (2007)
Adolescent adherence to oral therapy for leukae-
mia: a focus group. Journal of Pediatric Oncology
Nursing, 24, 139–151. Schmeigelow, K., Schroder, H., Gustafsson, G.,
Kristinsson,
J.,
Glomstein,
A.,
Salmi,
T. &
Wranne, L. (1995) Risk of relapse in childhood
acute lymphoblastic leukaemia is related to RBC
methotrexate and mercaptopurine metabolites
during maintenance chemotherapy. Journal of
Clinical Oncology, 13, 345–351. McBride, K.L., Gilchrist, G.S., Smithson, W.A.,
Weinshilboum, R.M. & Szumlanski, C.L. (2000)
Severe 6-thioguanine-induced marrow aplasia in
a child with acute lymphoblastic leukaemia and
inherited
thiopurine
methyltransferase
defi-
ciency. Journal of Pediatric Hematology/Oncology,
22, 441–445. Gale, R.P. & Butturini, A. (1991) Maintenance
chemotherapy and cure of childhood acute lym-
phoblastic leukaemia. Lancet, 338, 1315–1318. ª 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.
British Journal of Haematology, 2015, 170, 550–558 References Schmiegelow,
K.,
Forestier,
E.,
Kristinsson,
J.,
Soderhall, S., Vettenranta, K., Weinshilboum, R. & Wesenberg, F. (2009) Thiopurine methyl-
transferase activity is related to the risk of
relapse
of
childhood
acute
lymphoblastic Hawwa, A.F., Millership, J.S., Collier, P.S., McCar-
thy, A., Dempsey, S., Cairns, C. & McElnay, J.C. (2009) The development of an objective meth-
odology to measure medication adherehence to
oral thiopurines in paediatric patients with acute Merryman, R., Stevenson, K.E., Gostic, W.J. II,
Neuberg, D., O’Brien, J., Sallan, S.E. & Silver-
man, L.B. (2012) Asparaginase-associated myelo- 557 ª 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd. British Journal of Haematology, 2015, 170, 550–558 L. Lennard et al leukaemia: results from the NOPHO ALL-92
study. Leukaemia, 23, 557–564. leukaemia: results from the NOPHO ALL-92
study. Leukaemia, 23, 557–564. compared with mercaptopurine in childhood
lymphoblastic
leukaemia:
results
of
the
UK
Medical Research Council Randomised Trial
ALL97. Lancet, 368, 1339–1348. compared with mercaptopurine in childhood
lymphoblastic
leukaemia:
results
of
the
UK
Medical Research Council Randomised Trial
ALL97. Lancet, 368, 1339–1348. Tidd, D.M. & Paterson, A.R.P. (1974) A biochemi-
cal mechanism for the delayed cytotoxic reac-
tions of 6–mercaptopurine. Cancer Research, 34,
738–746. Schrappe, M., Reiter, A., Zimmmerman, M., Har-
bott, J., Ludwig, W.-D., Henze, G., Gadner, H.,
Odenwald, E. & Riehm, H. (2000) Long-term
results of four consecutive trials in childhood
ALL performed by the ALL-BFM study group
from 1981 to 1995. Leukaemia, 14, 2205–2222. Tiede, I., Fritz, G., Strand, S., Poppe, D., Dvor-
sky, R., Strand, D., Lehr, H.A., Wirtz, S., Bec-
ker, C., Atreya, R., Mudter, J., Hildner, K.,
Bartsch, B., Holtman, M., Blumberg, R., Walc-
zak, H., Iven, H., Galle, P.R., Ahmadian, M.R. & Neurath, M.F. (2003) CD28-dependent Rac1
activation is the molecular target of azathio-
prine in primary human CD4 +
T lympho-
cytes. The Journal of Clinical Investigation, 111,
1133–1145. Vora, A., Goulden, N., Wade, R., Mitchell, C.,
Hancock,
J.,
Hough,
R.,
Rowntree,
C. &
Richards, S. (2013) Treatment reduction for
children and young adults with low-risk acute
lymphoblastic leukaemia defined by minimal
residual
disease
(UKALL
2003):
a
rando-
mised
controlled
trial. Lancet
Oncology,
14,
199–209. Stanulla, M., Schaeffler, E., Flohr, T., Cario, G.,
Schrauder, A., Zimmerman, M., Welte, K., Lud-
wig, W.-D., Bartram, C.R., Zanger, U.M., Ei-
chelbaum, M., Schrappe, M. & Schwab, M. (2005) Thiopurine methyltransferase genotype
and early treatment response to mercaptopurine
in childhood acute lymphoblastic leukaemia. ª 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.
British Journal of Haematology, 2015, 170, 550–558 References Journal of the American Medical Association, 293,
1485–1489. Weinshilboum, R.M. & Sladek, S.L. (1980) Mer-
captopurine
pharmacogenetics:
monogenic
inheritance of erythrocyte thiopurine methyl-
transferase activity. American Journal of Human
Genetics, 32, 651–662. Vora, A.J., Mitchell, C.D., Lennard, L., Eden,
T.O.B., Kinsey, S.E., Lilleyman, J.S. & Richards,
S. (2006) Toxicity and efficacy of thioguanine 558
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Shifting peripheries: Dhaka's rickshaw garages and mess dormitories as spaces of work and movement
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Article EPD: Society and Space
2023, Vol. 41(2) 231–252
! The Author(s) 2023
Article reuse guidelines:
sagepub.com/journals-permissions
DOI: 10.1177/02637758231168869
journals.sagepub.com/home/epd EPD: Society and Space
2023, Vol. 41(2) 231–252
! The Author(s) 2023 Corresponding author:
Annemiek Prins, Department of Cultural Anthropology and Development Studies, Radboud University, Maria Montessori
Building, Room 02.018, P.O. Box 9104, 6500 HE Nijmegen, The Netherlands.
Email: annemiek.prins@ru.nl Shifting peripheries: Dhaka’s
rickshaw garages and mess
dormitories as spaces of
work and movement 2023, Vol. 41(2) 231–252
! The Author(s) 2023
Article reuse guidelines:
sagepub.com/journals-permissions
DOI: 10.1177/02637758231168869
journals.sagepub.com/home/epd Article reuse guidelines:
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DOI: 10.1177/02637758231168869
journals.sagepub.com/home/epd Annemiek Prins
Radboud University, The Netherlands Shreyashi Dasgupta
University of Cambridge, UK Abstract This article considers how urban peripheries are made and unmade by forms of “shifting”. We
examine these shifts from the perspective of rickshaw garages and mess dormitories in Dhaka,
Bangladesh, which both offer makeshift working and living arrangements to rural–urban migrants. By explicitly situating these spaces as part of the movements and crisscrossing trajectories that
animate urban peripheries, we challenge the tendency in urban scholarship to analyze peripheral
and marginalized spaces primarily through the lens of habitation. Breaking with residentialist and
sedentarist approaches to urban space, we present rickshaw garages and mess dormitories as
spaces that are enabling and undergoing various forms of shifting, as their occupants move and
alternate between different places, neighborhoods, and spatial arrangements to establish a con-
tinuity of work and income. We argue that these forms of manoeuvring are made possible by a
degree of spatial malleability that reflects the territorial impermanence of the periphery itself,
which is continuously pushed sideways through tandem processes of precariousness and
improvement. By directing attention to the “shifting” in “makeshift”, we contribute to a less
static understanding of how labor migrants try to hold their place in the city amidst wider
processes of exclusion, expansion, and densification. p
g
Annemiek Prins, Department of Cultural Anthropology and Development Studies, Radboud University, Maria Montessori
Building, Room 02.018, P.O. Box 9104, 6500 HE Nijmegen, The Netherlands.
Email: annemiek.prins@ru.nl Introduction “In the last ten years, I have moved accommodations eight times,” Ishrat1 explained. She first
arrived in Dhaka 10 years ago, when she and her husband migrated to the capital city of
Bangladesh from their home village in Tangail. Tragically, her husband had died in an accident
a few years later, making Ishrat the sole ricewinner of the family. She currently shared a bed with
her three children at a “mess dormitory” in Rayerbazar, a neighborhood located along the
western fringes of Dhaka city, close to the Buriganga river. Every morning Ishrat rearranged
their small room into a modest workshop from where she taught embroidery work to women in
the neighborhood. It was just one of the many jobs, ranging from domestic work to working at a
fish market, that she had tried out in Dhaka. In her never-ending search for work and income,
Ishrat had moved from one mess accommodation to the other. She explained that one of the
main reasons for moving was the increase in rent: “Neighborhoods that were affordable to rent
in the past have now become expensive. I am forever on the lookout for a new neighborhood
with cheap rent”. Mustafa, who also stayed and worked in Rayerbazar, was on the lookout for empty land as
well as cheap rent. He ran one of the many cycle-rickshaw garages that can be found along
the peripheral edges of Dhaka city, where storage space is relatively easily available. Mustafa had accessed his current storage space – an empty piece of wasteland – only six
months ago, by entering a lease agreement with a powerful landowner. He had relocated
from a more established part of Rayerbazar after the owner of his previous garage site had
decided to use the land for the construction of a high-rise building. Mustafa’s colourful cycle-
rickshaws, which he rented out to individual drivers on a daily basis, were stored in the open
air. While most garages offered some sort of provisional, free-of-cost accommodation to
rickshaw drivers, Mustafa had not been able to invest in such a sleeping place. In two
months or so, he would have to temporarily park his 23 rickshaws at the roadside, because
of the annual mela (“fair”) that would be hosted at the site of his garage. Keywords y
Dhaka, mess dormitories, rickshaw garages, rural–urban movements, shifting, urban p 232 EPD: Society and Space 41(2) Introduction Such practices speak to a
degree of malleability and manoeuvrability that is often central to people’s efforts to carve
out a living for themselves amidst wider processes of urban exclusion, expansion, and
densification. shifting. The example of Mustafa speaks to the fact that rickshaw garages are not just
located in the peripheral areas of Dhaka, but actually move with the periphery as informal
land uses are pushed towards the edges of the city by processes of urban development and
speculative real-estate investments. Moreover, both mess accommodations and rickshaw
garages bear testimony to everyday forms of shifting, such as the rearranging of furniture
to transform a sleeping place into an embroidery workshop or the temporary relocation of a
rickshaw garage to a nearby street to accommodate a cultural fair. Such practices speak to a
degree of malleability and manoeuvrability that is often central to people’s efforts to carve
out a living for themselves amidst wider processes of urban exclusion, expansion, and
densification. By analyzing how rickshaw garages and mess dormitories emerge as spaces “in” and “of”
movement, we seek to contribute to a deeper understanding of the lateral ways in which
urban peripheries are produced “over time and across space” (Caldeira, 2017: 6). In doing
so, we build on Simone’s (2018: 264) conceptualization of “make þ shift life”, which cap-
tures how people “literally ‘make’ and then ‘shift’ to adapt in rapidly changing circum-
stances” (Bhan, 2017: 593). By focusing explicitly on dimensions of shifting, our article
complements the existing literature on peripheral and marginalized urban neighborhoods,
which tends to emphasize forms of “making” by highlighting processes of auto-construction
(Caldeira, 2017), upgrading (Echanove and Srivastava, 2016), repair (Bhan, 2019), squatting
(Vasudevan, 2015), and incremental adaptation (Kamalipour and Dovey, 2020). We offer
that shifting, just like making or building, represents a crucial way in which people try to
“hold their place in the city” (Simone, 2010: 45). Moreover, we argue that whereas making
or building a home entails a negotiation “over the terms of establishing permanency”
(Makhulu, 2015: 11), shifting ultimately relates to the struggle of establishing a continuity
of livelihoods. Our aim in foregrounding dimensions of work and movement is to break with the ten-
dency to interpret and problematize peripheral and marginalized urban spaces solely
through the lens of habitation. Introduction Moreover, the
landowner also had plans to eventually develop the plot for real-estate purposes, so soon he
would be packing up his business again. The prospect of relocating did not particularly bother
him: “I am not worried, it will be easy to find a different spot to run my business from”. ,
y
p
y
Peripheral urban neighborhoods like Rayerbazar are filled with people like Ishrat and
Mustafa who need to continuously shift and alternate between different places and spatial
arrangements to make a living for themselves. In this article, we examine such forms of
shifting, which play out across different scales, from the ethnographic vantage point of mess
dormitories and rickshaw garages. Both of these spaces, each in their own way, highlight the
various movements that animate and help constitute urban peripheries. Both mess accom-
modations and rickshaw garages can be understood as places where “tentative, residual, or
nascent links to both city and rural areas are maintained and intersect” (Simone, 2010: 51). Rickshaw garage spaces provide an important source of labor and shelter to temporary,
seasonal, and long-term rural–urban migrant workers. They thus play a crucial role in
enabling the tidal waves of laborers moving in and out of the city. Mess accommodations
similarly attract rural–urban migrant workers but, as Ishrat’s example highlights, also func-
tion as entry and exit points amidst intra-urban flows, in the sense that people pass through
these spaces in their restless search for cheap rental accommodation in the city. Neither rickshaw garages nor mess accommodations should be understood as a mere
stopover on wider trajectories of movement. Far from representing a stable or static spatial
configuration, these spaces are themselves continuously made and unmade by forms of 233 Prins and Dasgupta shifting. The example of Mustafa speaks to the fact that rickshaw garages are not just
located in the peripheral areas of Dhaka, but actually move with the periphery as informal
land uses are pushed towards the edges of the city by processes of urban development and
speculative real-estate investments. Moreover, both mess accommodations and rickshaw
garages bear testimony to everyday forms of shifting, such as the rearranging of furniture
to transform a sleeping place into an embroidery workshop or the temporary relocation of a
rickshaw garage to a nearby street to accommodate a cultural fair. Introduction Indeed, the tension between precariousness and improve-
ment that fuels the lateral production of urban peripheries (Caldeira, 2017: 6) is often
intuitively framed as a contrast between improvised and permanent dwellings – between
tin sheds and high-rises. This preoccupation with habitation and the “generally substandard
settlements in which large segments of the urban poor live and work” (Weinstein, 2014) has
been encouraged by a myopic concentration on slums as the dominant frame for making
sense of Southern urbanism (see Arabindoo, 2011; Banerjee, 2022; Roy, 2011). Shot through
with allusions to shelter and habitation, the category of “slum” fails to capture how people’s
lives and livelihoods are part of an “intricate set of connections and flows stretching across
multiple physical spaces” (Arabindoo, 2011: 643). What gets lost amidst residentialist and
sedentarist approaches to urban precariousness is that for many people residence in a par-
ticular place hardly constitutes an adequate measure of their presence, engagement, improv-
isations, struggles, and negotiations with/in the city. Indeed, for labor migrants like Ishrat
the city represents an avenue for making a living rather than a place to live. In this article, we present shifting as a set of strategic practices that people engage in as
they try to make a living amidst processes of urban exclusion, expansion, and densification. First, we examine what it means – theoretically – to move away from habitation and outline
our own approach to studying peripheries as shifting configurations. Next, we contextualize
rickshaw garages and mess accommodations as part of wider trajectories of urbanization in
Bangladesh, paying specific attention to the ways in which circulations of capital and labor
shape the formation and shifting of urban peripheries in Dhaka. Our empirical section
highlights the specific forms of shifting that rickshaw garages and mess dormitories are 234 EPD: Society and Space 41(2) enabling and undergoing. The first section focuses on rickshaw garages and shows how
these spaces accommodate rural–urban flows, and move with the periphery as Dhaka con-
tinues to extend outwards. The second section on mess accommodations directs attention to
intra-urban movements and sheds light on the everyday forms of shifting that play out as
people rearrange their furnishings to combine functions of living and working. We argue
that both rickshaw garages and mess dormitories indicate a degree of malleability and
manoeuvrability that is reflective of the tentative nature of peripheries. Introduction In our discussion
section, we further reflect on what this malleability tells us about the tension between pre-
cariousness and improvement that shapes the shifting of urban peripheries. enabling and undergoing. The first section focuses on rickshaw garages and shows how
these spaces accommodate rural–urban flows, and move with the periphery as Dhaka con-
tinues to extend outwards. The second section on mess accommodations directs attention to
intra-urban movements and sheds light on the everyday forms of shifting that play out as
people rearrange their furnishings to combine functions of living and working. We argue
that both rickshaw garages and mess dormitories indicate a degree of malleability and
manoeuvrability that is reflective of the tentative nature of peripheries. In our discussion
section, we further reflect on what this malleability tells us about the tension between pre-
cariousness and improvement that shapes the shifting of urban peripheries. The material presented in this article is based on extensive ethnographic fieldwork in
various neighborhoods along the southwestern and eastern fringes of Dhaka city (Figure 1). Annemiek carried out 11 months of fieldwork in Dhaka between 2015 and 2018. Her
research focused on the mobilities and labor trajectories of cycle-rickshaw drivers. Throughout her fieldwork, Annemiek visited approximately 75 rickshaw garages, located
in peripheral areas like Rayerbazar, Kamrangirchar, Shonir Akhra, and Badda, where she
conducted semi-structured group discussions and individual interviews with both rickshaw
drivers and garage owners. Shreyashi conducted six months of field research in Dhaka
between 2016 and 2018, focusing predominantly on mess accommodations (dormitories)
for low-income workers in Mirpur and Rayerbazar. She conducted participant observation,
carried out semi-structured and unstructured interviews, and shadowed and accompanied
multiple state- (planning officials, administrative staff) and non-state actors (workers, real-
estate builders, caretakers, landowners). While the section on “rickshaw garages” reflects the
ethnographic work of Annemiek, the section on “mess dormitories” builds on the research
findings by Shreyashi. Beyond habitation: Deterritorializing urban peripheries In our attempt to move beyond categories and epistemologies of habitation, we take inspi-
ration from authors who have criticized the centrality of slums in making sense of Southern
urbanism (Arabindoo, 2011; Banerjee, 2022; Gilbert, 2007; Rao, 2006; Roy, 2011). In many
ways, slums have become the predominant frame through which cities in the global South
“are perceived, understood, mapped, and created” (Arabindoo, 2011: 639). Gilbert (2007:
698) points out that the term slum has helped to stir fears about mass-urbanization and
rural–urban migration, thereby essentially morphing the complexities of urban inequality
and precariousness into a recognizable image and distinct habitat. This has impeded the
“recognition of varied forms of dwelling practices” (Banerjee, 2022), while also restricting
the scope of urban analysis. Indeed, Rao (2006: 227) observes a gradual slippage from slum
as population and terrain to “slum as theory”. This conflation of territorial and epistemo-
logical categories implicitly reifies habitation as the main mode of engaging with the city, by
suggesting that urban precarity in the global South is best understood as an extension of a
particular settlement type. What such a residentialist and sedentarist2 approach to urban life fails to address is that
for many people the city primarily constitutes “a place for making a living”. Martinotti
(1994) has pointed out that the experiences of non-residents, especially workers, are too
often side-lined in analyses of cities. Despite the increased intertwinement of rural and urban
realities and the fact that “many people reside in one [place] but work in the other”
(Champion and Hugo, 2004: 3), urban research has long revolved around “residential
units of observation” (Martinotti, 1994). This static approach does not fit the South
Asian context where “movement, both within rural areas and between villages, towns and 235 Prins and Dasgupta igure 1. Map of Dhaka city, highlighting the main fieldwork areas of this research. Informal settlements a
dicated by brown shadings. Map source: World Bank (2017); informal settlement locations taken from
Gruebner et al. (2014). Figure 1. Map of Dhaka city, highlighting the main fieldwork areas of this research. Informal settlements are
indicated by brown shadings. Map source: World Bank (2017); informal settlement locations taken from
Gruebner et al. (2014). cities has always been, and continues to be, a central feature of life” (Gardner and Osella,
2003: vi). Conceptualizing shifting urban peripheries The fact that these processes are inher-
ently diffuse makes it difficult to definitively map and locate the edges of this dynamic. Consequently, the periphery cannot be simply understood as a “zone at the edge of the city”
(Foot, 2000: 8). Instead, Simone (2010: 40) has defined the periphery as a space that has
never been “brought fully under the auspices of the logic and development trajectories that
characterize a center, and therefore embodies an instability that is always potentially desta-
bilising of that center.” This instability coincides with a degree of territorial impermanence,
marking peripheral urbanization as a “process that is always being displaced, reproduced
somewhere else where land is cheaper because it is more precarious or difficult to access”
(Caldeira, 2017: 6). The fact that urban peripheries are often continuously pushed sideways, through pro-
cesses of urban expansion and densification, suggests a tension between urban access and
exclusion. On the one hand, the periphery functions as a repository for inward migrants
searching for work and economic opportunities. Yet, at the same time, urban peripheries are
also “attractive to investors seeking to capture gains from rapidly rising land value”
(Gururani and Kennedy, 2021: 1). Such forms of capitalist expansion, which become man-
ifest through the construction of mega-projects, land and real-estate speculation, and
increasing urban living costs, make it so that urban newcomers are often on the brink of
being “out” again. It is this degree of uncertainty and ambivalence that marks urban periph-
eries as spaces of “aspirational compromise” (Archambault, 2021). Navigating these shifting
configurations requires a constant (re)attunement of “one’s aspirations with one’s circum-
stances” (Archambault, 2021: 303). Such compromises and trade-offs are characteristic of what Simone (2018) has described
as “make þ shift life”. Various authors have explored what it means to “make” and establish
a place for oneself in the urban periphery by focusing on auto-construction, squatting, and
repair (Bhan, 2017, 2019; Caldeira, 2017; Vasudevan, 2015). Less attention has been paid to
shifting as a spatial tactic, with exceptions such as Solomon’s (2022) work on the movement
and adjustment of patients in a Mumbai hospital. While based on a very different context,
his work helpfully unpacks shifting as a form of praxis that implicates bodies, space, and
time in trajectories of survival (Solomon, 2022: 57). Conceptualizing shifting urban peripheries Periphery, in contrast to the notion of slum, facilitates an explicit focus on movement. Although often employed to make sense of informal or marginalized neighborhoods, the
term periphery provides a less stigmatizing, static, and topological alternative to the notion
of “slum”. Roy (2011: 231–231) contends that the concept is more suitable to capture the
“inevitable heterogeneity of Southern urbanism” due to its “ability to transcend territorial
location”. With its origins in Marxist theorizations of economic and political dependency,
the notion of periphery is also more closely connected to circulations of labor and capital. Specifically, the concept has been used to explain how the uneven development of capitalism
permeates city-suburb and city-hinterland relations (Harvey, 1985; Smith, 1984; Soja, 1989). Periphery thus signals the political-economic hierarchies that shape how urban centers
burst apart and dissolve into bordering territories. The fact that these processes are inher-
ently diffuse makes it difficult to definitively map and locate the edges of this dynamic. Consequently, the periphery cannot be simply understood as a “zone at the edge of the city”
(Foot, 2000: 8). Instead, Simone (2010: 40) has defined the periphery as a space that has
never been “brought fully under the auspices of the logic and development trajectories that
characterize a center, and therefore embodies an instability that is always potentially desta-
bilising of that center.” This instability coincides with a degree of territorial impermanence,
marking peripheral urbanization as a “process that is always being displaced, reproduced
somewhere else where land is cheaper because it is more precarious or difficult to access”
(Caldeira, 2017: 6). Periphery, in contrast to the notion of slum, facilitates an explicit focus on movement. Although often employed to make sense of informal or marginalized neighborhoods, the
term periphery provides a less stigmatizing, static, and topological alternative to the notion
of “slum”. Roy (2011: 231–231) contends that the concept is more suitable to capture the
“inevitable heterogeneity of Southern urbanism” due to its “ability to transcend territorial
location”. With its origins in Marxist theorizations of economic and political dependency,
the notion of periphery is also more closely connected to circulations of labor and capital. Specifically, the concept has been used to explain how the uneven development of capitalism
permeates city-suburb and city-hinterland relations (Harvey, 1985; Smith, 1984; Soja, 1989). Periphery thus signals the political-economic hierarchies that shape how urban centers
burst apart and dissolve into bordering territories. Beyond habitation: Deterritorializing urban peripheries The normalcy of such mobilities and translocal relations requires a different way
of looking at “the relationship between the ephemeral and the durable” (Dharia, 2022: 4). Indeed, as can be expected, fostering a sense of continuity in such a context does not always
center around residence or habitation, nor is it necessarily aimed at establishing permanency
in a certain place. For instance, Jackman (2017: 251) has shown that (temporary) labor
migrants in Dhaka often deliberately divest resources away from residence and rent in the 236 EPD: Society and Space 41(2) city in order to advance projects elsewhere, including “supporting family elsewhere, starting
businesses, paying dowries, or purchasing land”. Our article similarly puts emphasis on the
translocal and mobile nature of the “hustle” for work and income that gains shape as people
navigate precarious urban conditions (Thieme, 2018). We turn to the notion of periphery as
a space/function to think about these issues rather than slums or habitation. city in order to advance projects elsewhere, including “supporting family elsewhere, starting
businesses, paying dowries, or purchasing land”. Our article similarly puts emphasis on the
translocal and mobile nature of the “hustle” for work and income that gains shape as people
navigate precarious urban conditions (Thieme, 2018). We turn to the notion of periphery as
a space/function to think about these issues rather than slums or habitation. Situating garage and mess spaces as part of shifting peripheries As we have seen, urban peripheries are ambivalent places – situated in the interstices
between rural and urban – where the expansionist tendencies of capital meet the restless
search for work and opportunities that animate the movements and trajectories of rural–
urban migrants. Garages and mess dormitories can be viewed as nodes where these different
tendencies intersect, while also highlighting how urban peripheries themselves are continu-
ously shifting amidst processes of urbanization and land speculation. y
g
p
p
Urbanization in Bangladesh is characterized by intertwining processes of expansion,
densification, and rural–urban migration. The capital city Dhaka counts as one of the
fastest-growing and most densely populated megacities in the world and has an estimated
population of 22 million (World Population Review, 2022). This reality of urbanization is
intimately tied to rural and agrarian transformations (Dasgupta, 2019: 289), as trajectories
of rural–urban migration are fuelled by varying degrees of landlessness, rural displacement,
and ecological vulnerabilities such as river bank erosion (Hossain, 2013: 370). Despite this
constant influx of new people, which is intensified by realities of climate change, Dhaka does
not merely constitute a space of arrival. In fact, as Etzold (2016: 171–173) has pointed out,
urban newcomers do not always settle in Dhaka, but often remain situated in a “translocal
social field” as they “organise their livelihoods dynamically across different places”. Both rickshaw garages and mess accommodations speak to a longstanding history of
tentative, seasonal, and tidal movements into the city. Historically, messbari or mess dor-
mitories emerged as an affordable boarding option for people migrating from rural areas to
cities in Bengal during the colonial period. Basu (2019) highlights the cultural and social
significance of these spaces for those who pursued educational and job opportunities in
Kolkata. Dhaka had similar boarding houses and mess dormitories, catering to government
officers and students. In the 1970s, the need for low-income housing in the city intensified, as
Bangladesh shifted its economic focus toward export-oriented industrialization and the
number of readymade garment units in Dhaka started to increase (Ghafur, 2010: 8). With
official planning practices focusing exclusively on higher and middle-income groups
(Ghafur, 2010; Kabir and Parolin, 2012), the formal housing market largely failed the
influx of new workers. To this day, private rental dormitories play an important role in
meeting this demand for affordable lodging, by offering a variety of rental arrangements
ranging from one-day to weekly, monthly, and yearly options. Conceptualizing shifting urban peripheries This article focuses on trajectories of
economic survival, by honing in on the different practices of “shifting” that play out as 237 Prins and Dasgupta people move and alternate between different places, neighbourhoods, and spatial arrange-
ments in order to establish a continuity of work and income. Such a focus on shifting allows
for a further examination of the malleable and indeterminate nature of urban peripheries,
which are often caught between processes of concentration and dispersal; arrival and exclu-
sion; and precariousness and possibilities. Situating garage and mess spaces as part of shifting peripheries The (temporary) occupants
of dormitories work as domestic workers, construction laborers, garment factory workers,
rickshaw- and car drivers, small-scale entrepreneurs, street food vendors, and local restau-
rant workers. Whereas contemporary mess dormitories represent a broad segment of the rural–urban
migrant population and cater to men, women, and families, rickshaw garages focus almost
exclusively on men who work as cycle-rickshaw drivers. With an estimated 1.1 million cycle-
rickshaws operating in Dhaka (Karim and Salam, 2019), the rickshaw industry constitutes
an important source of (seasonal) labor for rural–urban migrants. The fact that rickshaws
can be rented on a per diem basis, makes for a relatively accessible avenue of work that
requires no investment capital and enables workers to come and go according to their needs 238 EPD: Society and Space 41(2) and wishes (Prins, 2021). The only real requirement for prospective rickshaw pullers is that
they have a neighbor or family member, usually someone from their home village, who can
vouch for them with the so-called rickshaw malik (owner). The vast majority of drivers rent
their vehicles from rickshaw owners, who are also responsible for running and maintaining
the rickshaw garage. The fact that only a small percentage3 of workers actually own their
rickshaw is a reflection of the translocal lives that most of them lead. Over 80 percent of
rickshaw pullers spend time at their rural home every six months, and only 45 percent of
them rent a room in the city with their family (Karim and Salam, 2019: 38). The rickshaw
garage provides a form of improvised shelter to those who do not rent a room. and wishes (Prins, 2021). The only real requirement for prospective rickshaw pullers is that
they have a neighbor or family member, usually someone from their home village, who can
vouch for them with the so-called rickshaw malik (owner). The vast majority of drivers rent
their vehicles from rickshaw owners, who are also responsible for running and maintaining
the rickshaw garage. The fact that only a small percentage3 of workers actually own their
rickshaw is a reflection of the translocal lives that most of them lead. Over 80 percent of
rickshaw pullers spend time at their rural home every six months, and only 45 percent of
them rent a room in the city with their family (Karim and Salam, 2019: 38). Situating garage and mess spaces as part of shifting peripheries The rickshaw
garage provides a form of improvised shelter to those who do not rent a room. Their crucial role as urban entry and exit points amidst rural–urban flows marks mess
accommodations and rickshaw garages as peripheral spaces. They are not only part of wider
“territories of transition and connection” (Simone, 2010: 51), but also implicated in the
distinct hierarchies that shape the relation between urban centers and their hinterlands. These hierarchies, which relate to both class and geographical dimensions, become clear
from the way in which Dhaka’s built-up area has expanded and densified over time. Closed
off by the Buriganga river along its southwestern fringe, Dhaka initially expanded north-
wards from what is now called Old Dhaka. The higher grounds along the city’s south–north
axis were singled out to establish planned residential projects for middle- and higher-income
groups (Kabir and Parolin, 2012: 12). The low-lying floodplains on the western and eastern
flanks of the city, on the other hand, have long attracted rural–urban migrant workers,
informal settlements, and low-cost housing (Hafiz, 2007: 61). This peri-urban belt is con-
tinuously pushed sideways, as the rapid increase in land-prices encourages real-estate devel-
opers to fill up the water bodies that can be found along these low-lying edges (Alam, 2018). Such forms of encroachment and extension, fueled by the expansionist tendencies of
capital, make it so that inhabiting the urban periphery often requires “moving with-” the
periphery as informal land-uses are pushed away from the center. At the same time, these
shifting edges also retain a degree of fluidity (Dasgupta and Prins, 2023), open-endedness,
and possibility, as “the city continues to host migrants as they come” (Sowgat and Roy,
2022: 425). In what follows, we further unpack this malleability, and the forms of shifting
that correspond with it, from the explicit perspective of rickshaw garages and mess
dormitories. Rickshaw garages The above example shows how processes of densification, specifically the mushrooming
of high-rise buildings, push rickshaw garages away from more central areas of Dhaka. This
movement is fuelled by the constant search for open stretches of wasteland, which results in
a shift to peripheral lowlands near water bodies. One example of such an area is Rayerbazar
(Figure 2), which is located along the western fringe of the city and inextricably entangled
with the floodplains of the Buriganga river. With the construction of an embankment road
in the 1990s, the low-lying swamplands along the western edge were gradually made inhab-
itable, as ponds were filled up with mud and rubbish to accommodate informal dwellings,
workshops, and rickshaw garages. Today, these informal land uses are increasingly under
pressure from an influx of private and public investments toward real-estate development
and infrastructural upgrading. The fact that Rayerbazar is located directly next to the
upscale residential area of Dhanmondi has further stimulated these speculative processes
(The Dhaka Tribune, 2020). Areas that used to be largely self-built are increasingly co-opted
and transformed into housing blocks for the (higher) middle classes, thereby highlighting
Simone’s (2018: 264) observation that “whatever is made then shifts in terms of its avail-
ability to specific uses and users”. Rickshaw garages are very much affected by such forms of expansion and co-optation,
due to their reliance on land that is owned by others and destined for other purposes. Mustafa, the rickshaw malik who was mentioned in the introduction, knew very well that
he would have to vacate his newly found spot in Rayerbazar as soon as the land-owner
decided to strike a deal with a real-estate company to develop the plot into a high-rise
building. Most rickshaw owners, including Mustafa, relied on temporary lease constructions
with private landowners or local political leaders to obtain access to stretches of land wait-
ing to be converted for different purposes. The inherent temporariness of such lease con-
structions, which function as a bridge between current and future land values, explains why
rickshaw owners often had to move. In Rayerbazar, such shifts followed existing geograph-
ical and socio-economic fault lines, as garage owners would relocate away from the more
established areas near Dhanmondi towards the lowlands near the embankment – often
literally referred to as nama or niche (“down”, “below”). Rickshaw garages Garage relocations: Moving with- the periphery. From the previous section, it becomes clear that
the urban peripheries of Dhaka are far from static. Land scarcity and population growth
trigger processes of expansion and densification that continuously transform the urban
landscape and the city’s edges. These processes also affect the way in which rickshaw
garages are situated. Historically, the rickshaw and cycle industry has had its mainstay in
Bangshal, which is located in Old Dhaka (Figure 1), the oldest and most congested part of
the city. The area used to be a hub for the different categories of workers involved in the
industry. Central to this article are rickshaw wallahs (drivers) and rickshaw maliks (owners),
but equally important are the manufacturers, mechanics, and artists who make, repair, and
decorate the rickshaw. While Bangshal is still home to a variety of rickshaw art studios and
rickshaw assembly workshops (Islam, 2015), rickshaw garages are increasingly absent from
this bustling part of town. One of the few rickshaw owners who still operated his business from Bangshal was Iqbal,
who had lived in Old Dhaka his whole life. Iqbal did not only rent out his rickshaws; he also
made and designed them. He mentioned that up until 15 to 20 years ago, Bangshal had been 239 Prins and Dasgupta host to many rickshaw garages. Yet, today his garage was only one of the two or three
garages still left in the area. He explained the decline as follows: host to many rickshaw garages. Yet, today his garage was only one of the two or three
garages still left in the area. He explained the decline as follows: To build a garage you need empty (khali) space, but that is difficult to find in Old Dhaka. It is impossible to stay somewhere permanently. If you find an open space, you can make a
garage, but these spaces are also needed for constructing high-rises. build a garage you need empty (khali) space, but that is difficult to find in Old Dhaka. Iqbal himself had experienced this increasing scarcity of space first-hand. The garage where
we first met was demolished a month after our visit in order to make way for the construc-
tion of a high-rise building. When we met again, one and a half years later, he was running a
small workshop from the ground floor of a building, where he could only store six
rickshaws. Rickshaw garages One local rickshaw owner, who
had his current garage almost directly next to the embankment, detailed how he had moved
three times in 30 years, shifting a couple of hundred meters westwards with each relocation
(Figure 2). Literally moving with- the periphery is one way in which rickshaw maliks maintain a
sense of continuity amidst exclusionary processes of expansion and densification. In addi-
tion, garage owners also sometimes resort to more ad-hoc or seasonal forms of shifting in
order to “hold their place” (Simone, 2010: 45) in a certain part of the city. Mustafa, for
instance, mentioned that he would have to temporarily move his rickshaws because of the 240 EPD: Society and Space 41(2) Figure 2. Map Rayerbazar with the approximate locations of one rickshaw garage within 30 years
(1987–2017). Source: World Bank (2017). Figure 2. Map Rayerbazar with the approximate locations of one rickshaw garage within 30 years
(1987–2017). Source: World Bank (2017). annual cultural fair that would be staged at the large playing field from where he operated
his garage. To accommodate this move, the landowner had arranged for Mustafa to store
his vehicles at the roadside. Such forms of spatial overlap between rickshaw garages and
other urban functions – a playing field, a cultural fair, the road – were particularly common
in more densely populated areas. For example, one of the few garage owners who had
managed to hold his place in a bustling market area of Old Dhaka, near the city’s main
boat terminal, had only been able to do so because he was willing to alternate between two
different locations. His rickshaws were usually parked at an open, sandy patch of land
between the higher-up road and the immediate riverside. However, the owner explained
that he could only stay there during the dry season. As soon as the summer rains would
arrive, the Buriganga river would overflow, and he would have to move his 80 rickshaws
higher up, to one of the already crowded roads on the other side of the market. annual cultural fair that would be staged at the large playing field from where he operated
his garage. To accommodate this move, the landowner had arranged for Mustafa to store
his vehicles at the roadside. Rickshaw garages Such forms of spatial overlap between rickshaw garages and
other urban functions – a playing field, a cultural fair, the road – were particularly common
in more densely populated areas. For example, one of the few garage owners who had
managed to hold his place in a bustling market area of Old Dhaka, near the city’s main
boat terminal, had only been able to do so because he was willing to alternate between two
different locations. His rickshaws were usually parked at an open, sandy patch of land
between the higher-up road and the immediate riverside. However, the owner explained
that he could only stay there during the dry season. As soon as the summer rains would
arrive, the Buriganga river would overflow, and he would have to move his 80 rickshaws
higher up, to one of the already crowded roads on the other side of the market. Hence, in the context of the rickshaw garage, it is not so much the investment in a
permanent place, as the willingness to move that allows owners to establish a continuity
of livelihoods. Their reliance on spaces that are inherently peripheral – namely, unused but 241 Prins and Dasgupta soon-to-be exploited stretches of wasteland – leads to a privileging of practices of shifting
over incremental forms of upgrading. To some extent, these shifts are indicative of distinct
forms of marginalization that characterize peripheries. The forms of expansion and densi-
fication that push garages towards precarious, flood-prone, and low-lying spaces are fuelled
by a land-hungry real-estate sector that only caters to middle- and higher-income groups. However, the spatial and temporal indeterminacy that characterizes spaces that have not yet
been co-opted by hegemonic logics of city-making also marks these peripheral wastelands as
potentially generative spaces that allow for a degree of malleability and manoeuvrability
that is often central to people’s efforts to carve out a living for themselves. Malleable spaces: Moving in and out of the city. The rickshaw garage is not just a movable space;
it is also a malleable space where different functions overlap and coalesce. Rickshaw garages
do not always mark a distinct space to begin with and vary significantly in size and shape:
from cramped tin shed storage spaces with a handful of rickshaws, to large open fields with
up to 200 vehicles. Rickshaw garages Whereas most rickshaw garages are built as large, half-open structures,
made out of bamboo and corrugated iron (Figure 3), some garages simply consist of a
collection of rickshaws being stored in the open air, be it on a playing field, under an
elevated expressway, or at the side of the road. This rudimentary shape foreshadows the
forms of shifting that garage owners have to engage in, while also highlighting the multi-
plicity of possibilities that these spaces open up to different groups of people. Indeed, the
rickshaw garage often functions as an office, storage space, repair shop, and dormitory all at
once. This malleability also plays an important role in accommodating the frequent, irreg-
ular, or seasonal movements of cycle-rickshaw drivers. Whereas rickshaw owners had usually settled in Dhaka city more-or-less permanently,
the majority of rickshaw wallahs did not rent a room in the city with their families (Karim
and Salam, 2019: 38). Most of them maintained translocal networks and livelihoods, and
even for drivers who had moved to Dhaka with their wives and children, and rented infor-
mal housing in slums, residence did not necessarily equal settlement. Indeed, it would be
quite common for rickshaw pullers to send back their families to the countryside in times of
economic hardship or increasing living costs to save up on rent. The rickshaw garage, by
offering free-of-cost accommodation, provided drivers with something to fall back on when
they had to resort to such “aspirational compromises” (Archambault, 2021). The fact that
most garages doubled as improvised dormitories made the rickshaw industry accessible to
workers who only came to Dhaka occasionally or seasonally to complement other sources of
income. The extent to which rickshaw garages are able to accommodate such tidal move-
ments relies not only on the availability of space but also on a malleability of form. Figure 3. Picture of a rickshaw garage, photograph taken by Annemiek Prins. Figure 3. Picture of a rickshaw garage, photograph taken by Annemiek Prins. 242 EPD: Society and Space 41(2) Just how malleable these spaces are, becomes clear from the example of a rickshaw
garage that was located under one of the large bridges that stretch across the Buriganga
river. The garage consisted of 200 rickshaws and rickshaw vans (flat-backed carts) that were
stored between the large, cement pillars of the bridge, with the four-lane road essentially
doubling as a roof. Rickshaw garages There seemed to be no place where rickshaw drivers could potentially
sleep, but the rickshaw malik testified that there were 15 rickshaw drivers who frequently
slept at his garage. One of them was Enamul, who had worked as a rickshaw puller in
Dhaka for over 35 years. Enamul had moved to Dhaka by himself, leaving his family behind
in the countryside. He travelled back and forth between the city and his rural home every six
weeks. During his time in Dhaka, he would spend his nights at the garage. He bought his
meals from one of the many roadside restaurants and used the public washroom nearby to
freshen up after work. At night, he slept on top of one of the flat-backed rickshaw-vans that
were parked in the garage, with the bridge functioning as a roof of sorts. When asked
whether sleeping like this was a problem, Enamul explained: “No, when it gets hot in the
summer, we just attach a fan to a bamboo pole to keep cool”. Such signs of (temporary) habitation could also be difficult to recognize when visiting
more established rickshaw garages, consisting of an indoor or partly enclosed storage space. There were often subtle clues in the form of a pillow stacked between two bamboo poles, a
rolled-up mosquito net dangling from the ceiling, or a line of duffle bags leading up to an
attic. The makeshift attic where most rickshaw drivers spend their nights, although clearly
demarcated as a place of sleeping, would often seem rather unfurnished at first sight. During
one garage visit, a rickshaw driver by the name of Jalal offered to show us the attic where he
spent his nights in the city. After wriggling our way through the dimly lit storage space, full
of rickshaws, we climbed a bamboo ladder to access the platform. Jalal first pointed to a
small cabin that served as a bathroom, and then to the right corner of the attic, where a few
pots and some large water jars had been arranged on a plastic piece of sheet: “This is where
we cook our meals”. Apart from the kitchen space and a small television set, the floor of the
attic was mostly empty. Checkered lungis4 had been hung out to dry on a clothesline that
crisscrossed through the room. Mess accommodations In the previous section, we have seen that the malleability of the rickshaw garage foreshad-
ows different shifts and movements. It allows garage owners to move with- the periphery in
their restless search for empty wasteland, while also accommodating the rural–urban com-
ings and goings that enable rickshaw drivers to piece together translocal livelihoods. While
mess dormitories similarly help to facilitate such rural–urban shifts, their nodal function
should also be understood in relation to people’s hustle for money and opportunities within
the city. These forms of “hustling” (Thieme, 2018), although shaped by the same processes
of densification and expansion that push rickshaw garages sideways, do not always follow a
clear pattern or direction. Instead, the functioning of mess accommodations helps to draw
attention to the ad-hoc and haphazard forms of shifting that gain shape as people “make
their moves” and “move on” (Simone, 2018: 271) based on where certain relations, vulner-
abilities, and opportunities might become manifest. Due to their role as part of wider trajectories of work, mess dormitories cannot simply be
viewed as a static point of habitation. While for some workers, mess accommodations act as
a node or springboard for finding work and establishing networks in the city, for others the
mess itself functions as a place of work or entrepreneurship. Dormitories attract workers
from various industries, including garments and construction, and are often located near
small- and medium-scale industries, as is the case in both Rayerbazar and Mirpur. At the
same time, mess accommodations also tend to co-exist side by side with affluent residential
neighborhoods like Gulshan, Banani, and Dhanmondi. Such hierarchical relations of prox-
imity become visible, among other things, through a difference in density. Whereas these
planned residential neighborhoods offer spacious apartments to higher-income groups, mess
dormitories are low-income spaces where multiple people live in close proximity to one
another. These living conditions signal a wider trend towards densification. Roy and
Sowgat’s (2020) study stipulates that about 40% of the residential communities in Dhaka city have a density of over 99,000 people per
km2. Since 1991, population densities in the outskirts of Dhaka have increased 28-fold, whereas
the rise within the city boundary has been twofold. The fact that mess dormitories offer high-density and low-income housing means that they
are often categorized and framed as part of slums (Islam, 1996: 379). Rickshaw garages At night, most drivers would simply roll out a blanket to
sleep on or wrap themselves in their lungis. While this absence of beds and possessions can easily be viewed as a condition of lack, it
also speaks to the malleability of the space, which accommodates varying numbers of rick-
shaw drivers throughout the year. The lack of permanent furniture highlights that for many
rickshaw drivers the city does not represent a permanent place to live. Jackman (2017: 251)
alludes to a similar point by showing that, for rural–urban laborers, accepting very basic
living conditions can be a way of divesting resources away from rent towards other homes,
places, and purposes, including “supporting family elsewhere, starting businesses, paying
dowries, or purchasing land, livestock or new homes” (Jackman, 2017: 251). This also
resonates with Enamul’s situation, who planned to move back to his village to start a
business as soon as his son or daughter was old enough to contribute sufficiently to the
family’s earnings. In the meantime, he used his mobile phone to wire back money to his wife
and children every day. Jalal’s presence in Dhaka was even less permanent, as he only
intended to stay in the city for the duration of the rainy season. Jalal ran a fish farm in
Bangladesh’s coastal area, but this year his fish stock has been washed away by heavy
flooding. To make up for his losses, he had temporarily moved to Dhaka to earn some
extra cash as a rickshaw driver. The examples of Enamul and Jalal thus show the limitations of interpreting the rickshaw
garage and its makeshift form solely in terms of unfinished and unfurnished living condi-
tions. Instead, these malleable spaces and their territorial impermanence bear testimony to 243 Prins and Dasgupta the tidal movements of people moving in and out of the city as they try to establish a sense
of continuity, not by investing in one particular place, but by shifting between different
spatial arrangements, places, and livelihoods. the tidal movements of people moving in and out of the city as they try to establish a sense
of continuity, not by investing in one particular place, but by shifting between different
spatial arrangements, places, and livelihoods. Mess accommodations Mess accommodations,
however, are not always located in slums, nor do they fit the popular binary between tin-
sheds and high-rises that is typically invoked to make sense of relations of inequality in the
city. For example, a local newspaper in Dhaka attributed the “underdevelopment” of
Rayerbazar to the fact that “only about 44% of the buildings in the area are three or
more storied” (Dhaka Tribune, 2020). Mess dormitories complicate this static dichotomy,
as becomes clear from the fact that they can be both horizontal (two-storey) and vertical
(four to five-storey) in nature. Moreover, although usually built with permanent materials,
the spatial organization of messes is characterized by a high degree of fluidity and mallea-
bility. Like rickshaw garages, mess dormitories could seem unfurnished at first sight as the
most common type of living arrangement is on the floor. There are no beds provided in the
dormitories for men. In accommodations for women, on the other hand, there are 244 EPD: Society and Space 41(2) sometimes makeshift partitioned walls, that had been built using materials like cardboard to
subdivide the space into separate spaces of sleeping. Some rooms have big windows, whereas
others have blocked windows due to storage or an area demarcated for windows with no
space for actual ventilation. The everyday functioning of these spaces is characterized by a
similar variety, as dormitories tend to blur residential, labor, and entrepreneurial functions. A closer reading of the mess landscape allows us to view these spaces through all kinds of
adjustments, trade-offs, and forms of shifting that gain shape as people negotiate density
and try to carve out a living for themselves. This hustle for work, as we will see, also often
involves shifting between different mess accommodations. Moving through mess dormitories. The majority of the residents encountered during fieldwork
frequently moved from one mess accommodation to the other as they routinely changed
between jobs. This becomes clear from the example of embroidery worker and teacher,
Ishrat, who was mentioned in the introduction and moved accommodation eight times
until she found a place with affordable rent in Rayerbazar. Her hustle for cheap rent was
intertwined with a restless search for work. Ishrat explained: “I have changed five jobs
before this. I can’t seem to sustain any workplace. Mess accommodations I quit the job of domestic help, and
then worked in a grocery shop, then fish market, and now embroidery.” She added that
home-based embroidery work allowed her to spend time with her kids. Rokib, another
respondent, had also changed mess accommodations several times. He worked at a garment
factory in Mirpur, and lived at a two-storey mess accommodation that was managed by a
tea stall owner who operated his shop from the ground floor of the building. Rokib
explained how he had ended up at this particular dormitory: I arrived in Dhaka to look for work six years ago from a village in Barisal district. Initially,
I lived in a room that had a tin shed roof shared by five people. It was a nice neighborhood but
then we had a fire and lost several important things including money. After that, I decided to
shift but it was a bad experience as the landlord cheated us with deposit money and pressurized
for higher rent. Rokib’s decision to move to the city was similarly shaped by financial vulnerabilities, as
returning to his family was not an option because his father had a huge debt in the village. Rokib’s decision to move to the city was similarly shaped by financial vulnerabilities, as
returning to his family was not an option because his father had a huge debt in the village g
y
p
g
g
Ishrat and Rokib’s movements and aspirations not only reflect wider regimes of capital
and the struggle for work but also strategic efforts through which people try to “hold their
places” in the city (Simone, 2010: 45). For Ishrat, this strategic effort involved
“recalibrating” her choice of work based on how much she will earn, how it will improve
her conditions of living in certain neighborhoods, and whether she can do the work from
home. Rokib, on the other hand, found ways of acknowledging these peripheral spaces as an
opportunity. His constant movements between mess accommodations, being in multiple
jobs, and converting his sleeping space into a storage space for work were not a last
resort. Rather he turned these prospective threats into opportunities. Rokib explained
that he was rather happy with his current living and working situation: One of my relatives from the village moved to this neighborhood. Hence, I thought of living
close by. Mess accommodations I assist my relative in his business on a part-time basis where I store and deliver some
raw materials for garment production. This part-time work allows me to earn extra money and
save for my college education. It is my dream to study and secure a stable job. But before that,
I need to recover all the money I lost in the last few years. 245 Prins and Dasgupta Rokib wrapped up his story by emphasizing that every time he had changed a job or shifted
to a new rental place, he had learned so many things about how to survive in the city. “I keep looking for opportunities to do small tasks and earn money. For instance, I make
extra money when I work all night and allow a friend or acquaintance to sleep inside my
room”. Shifting here, thus, attains a double meaning as it also relates to Rokib’s work shifts
at the factory. Solomon (2022: 243) echoes this temporal dimension, observing that shift can
also “refer to a duration of assigned work, as in an evening shift or a morning shift”. Rokib’s narrative helps us to reflect on the movements that mess accommodations make
possible, as their occupants shift between rooms, neighborhoods, jobs, and time-slots in
order to make a living for themselves. The focus on shifting highlights a continuity of
livelihoods across different neighborhoods where those navigating it can never be sure
about whether they are moving towards new opportunities or are actually on the brink of
being pushed out of the city. These shifts register on different scales, ranging from rural–
urban comings and goings to relocations within- or between neighborhoods, to the constant
rearrangement of rooms, furnishings, and materials. Rearranging rooms, and shifting furnitures. While mess accommodations, at least from the out-
side, seem to represent a relatively static structure, this does not take away from the fact that
the spatial arrangements within dormitories are characterized by a degree of malleability,
which gains shape through practices of shifting. Whereas rickshaw drivers use their work-
space as a sleeping space (to save up on rent), the residents of dormitories use their sleeping
space as a space of work. Both are examples of strategically optimizing space, money, and
time and show how shifting is also a way of making “aspirational compromises”
(Archambault, 2021). Mess accommodations Inside Rokib’s accommodation (Figure 4), for instance, I observed
how the storage boxes in his room were moved around. Sometimes they were piled up
systematically, whereas at other times they were arranged in a haphazard manner. Consequently, there was barely any floor space visible. The small room was full of boxes
with garments: on the floor, on the sides, on his bed. In the other corner of this room, he
kept a few pots and pans, containers of spices, and other cooking-related essentials on the
floor. Rokib explained his situation: I need to make space in order to store all my boxes. These boxes have raw materials/garments
that I need for work. I cannot remove these storage boxes. So I need to stack them up system-
atically everywhere in the room. Figure 4. Picture of Rokib’s room, photograph taken by Shreyashi Dasgupta. Figure 4. Picture of Rokib’s room, photograph taken by Shreyashi Dasgupta. 246 EPD: Society and Space 41(2) His room was organized differently at night. By then, he would shift the boxes again and pile
them up haphazardly. He elaborated: “Even if I feel tired in the night, I need to diligently
put things in order. Only then do I get some space to sleep. I need to use the room as much
as I can for work”. The conversation with Rokib thus illustrates dimensions of movement
and shows that everyday forms of shifting straddle the line between stability and relocations. Perceiving of the mess only as a place for housing does not do justice to these flows and
practices of “multipurposing”. What is missing from such an interpretation is the way in
which the constant shifting of storage boxes, pots and pans, floor mattresses, and pillows
indicates specific ways of investing in the continuity of livelihoods. The above example shows how the dormitory can be read as a malleable space, charac-
terized by fluid boundaries between day and night, working and living, and multiple other
activities. This also becomes clear from the room next to Rokib’s, which had a different
sleeping and working arrangement. Arif and his roommates were embroidery workers, as
was evident from the large, rectangular wooden plank in the middle of the room. On top of
the wooden plank, the cloth for embroidery was laid out so that all four residents of the
room could sit down and work on their designated portions. Mess accommodations Although the room had no
windows, there were five bright tube lights. Arif mentioned: We need these lights for our embroidery work. It is easier for us to see the threads clearly, focus
on the task and work better. We switch off the majority of the lights when we are not working. Now we will cook our lunch, so we just need one tubelight not all of them. Within the small space of Arif’s room, apart from his possessions, there were multiple
aspects of malleability to be recognized. Even switching the lights on and off meant a
shift in arrangements. With all the lights on, it is difficult to understand whether it is day
or night outside, as there are no windows to let in daylight. The bright lights also make the
mess accommodation recognizable as a factory line. Yet, with the lights off and the mat-
tresses out, the space is transformed into a place for sleeping. While his roommates were busy preparing their meals in the corner of the room, Arif
elaborated on how they organized their days: “We spend long hours working on the fabric
every day. But we also take lunch and dinner breaks. We need to cook our own meals.” It
was observed how some of the residents folded the scattered cloth materials from the floor
and put it to the side in order to create some space to sit and eat together. Arif said: This space is like a workshop, it is easier for us to work like this. The most important thing here
is the wooden plank, as that works as a base for our work. The rest of our belongings are stored
in the corners. We have our individual mattresses as well to sleep on during the night. He continued: “In fact, I did not buy my mattress, the previous worker who used to work
here left his room and work. When I moved in, I started using his mattress.” His remark
resonates with Simone’s (2020: 227) observation about the “uncertainty as to what exactly a
person possesses” when they move from one temporary accommodation to another. Arif’s
example shows that this is not about conditions of lack; instead, residents prioritize certain
furnishings based on their immediate needs and are often unsure what they will take along
during their next move. Discussion: Between precariousness and improvement Throughout our analysis, we have used “shifting” as a lens for understanding the reloca-
tions, movements, and forms of malleability that shape the functioning of rickshaw garages
and mess dormitories. The example of the rickshaw garage highlights the importance of
these spaces in facilitating the tidal waves of laborers moving in and out of the city. The
everyday functioning of mess accommodations, on the other hand, speaks to the intra-urban
movements that materialize as people shift between neighborhoods and accommodations in
their hustle for work, income, and cheap rent. Such shifts do not always follow a clear
direction but may take shape in an ad-hoc manner based on where certain relations, vulner-
abilities, and possibilities become manifest. The relocations that rickshaw owners engage in,
on the other hand, do show a clear spatial pattern, as businesses are moved from more
established parts of the city towards the low-lying edges near waterbodies. In both cases, this
restless search for opportunities is intensified by rising land prices, real-estate speculation,
and hegemonic forms of urban development that push peripheries sideways. Practices of
shifting can be viewed as a way of negotiating such forms of urban extension and exclusion. While shifting between rural and urban areas can be a way to ensure a continuation of
livelihoods in the face of both agrarian and urban transformations and enclosures, shifting
between neighborhoods or accommodations can represent efforts to “stay with the city”
amidst exclusionary forms of expansion. Moreover, the shifting of materials, furniture, and
possessions can be a means of negotiating processes of densification. These multi-scalar and multi-directional forms of shifting ultimately direct attention
towards the ambivalence of urban peripheries, where the crisscrossing of trajectories
makes it almost impossible “to clearly identify just what is ‘coming and going’” (Simone,
2010: 54). Peripheries are transient places that highlight tensions between the ephemeral and
the durable (Dharia, 2022: 4). As we have seen, it is difficult to appreciate whether people
are arriving in the city, ready to embrace new opportunities, or whether they are on the
brink of being pushed out and cut off from the city. The question that arises is how to
recognize and make sense of precariousness and improvement in such an ambivalent con-
text. Mess accommodations This malleability of space thus offers scope for experimentation,
while also highlighting the precarious trade-offs that stem from the need to always be pre-
pared to move. Viewing mess accommodations solely through the lens of habitation thus limits our
understanding of the “make þ shift” nature of these places. Both Arif and Rokib’s rooms 247 Prins and Dasgupta highlight practices of multipurposing and strategies of shifting. These movements show that
peripheral space is not fixed, as functions are not neatly tied to one particular space. These
unfinished and unfurnished forms of living do not merely signify a condition of lack but
bear testimony to ways of manoeuvring various opportunities and exclusions. Conclusion In this article, we have focused on the “make þ shift dynamics” (Simone, 2018) that shape
urban peripheries. Referring to the ways in which people “literally ‘make’ and then ‘shift’ to
adapt in rapidly changing circumstances” (Bhan, 2017: 593), this notion helpfully addresses
attention away from static configurations of habitation, to the inherent dynamism and
mobility of urban peripheries and the life projects of those who stay and work there. We
have argued that to gain a better understanding of such moving configurations it is impor-
tant to not just look at processes of auto-construction, squatting, and gradual upgrading,
but to also pay attention to spatial practices that are shaped by a willingness to move and
relocate. Specifically, we have sought to demonstrate that the improvised nature of these
peripheral spaces should not be read as a sign of the apparent lack of more permanent
structures or furnishings, but as a “generative space” that allows for strategic forms of
shifting. Throughout this article, we have shown that shifting constitutes a crucial way
through which people try to “hold their place in the city” (Simone, 2010: 45). Taking shifting seriously as both a spatial tactic and an economic strategy not only paves
the way for a less sedentarist and residentialist approach to urban space but also invites a
reconsideration of some of the taken-for-granted notions that are used to make sense of
urban struggles. Too often, the struggles and improvisations of those living precarious
urban lives are only interpreted as efforts to establish permanence. In contrast, mobility
practices, including migration, moving, and shifting, are typically associated with conditions
of transience and the inability to forge lasting connections with the city. We offer that it is
also possible to interpret such practices differently, namely as efforts to establish a conti-
nuity of work and income. Shifting thus helps us to appreciate that there are “always
invisible durabilities within the ephemeral” (Dharia, 2022: 22), while also addressing
“attention to practices and negotiations within the city that are spatially distinct from
those of slums” (Banerjee, 2022: 4). It highlights how the improvisations that characterize
precarious urban life in South Asia (Dharia, 2022; Kamalipour and Dovey, 2020;
Vasudevan, 2015) are not always aimed at establishing a permanence of place and living. While our analysis of shifting is firmly situated within South Asian urbanism, the appli-
cation of this notion carries relevance beyond this specific context. Discussion: Between precariousness and improvement A focus on shifting has allowed us to examine how
people try to foster a sense of continuity amidst processes of densification and expansion,
and the trade-offs they end up making in the process. The fact that peripheries are shifting
thus requires an understanding of precariousness and improvement that pays attention to
what is either cut off or enabled in terms of movements, trajectories, and relations. Discussion: Between precariousness and improvement Caldeira (2017: 6) has argued that in order to capture “the simultaneous processes of
improvement and the reproduction of inequality and precariousness” it is important to
focus on the “lateral reproduction of peripheries”. The relocations of rickshaw garages
and the way in which workers move between dormitories expose this lateral reproduction
of peripheries. However, a focus on shifting not only makes visible how peripheries are
pushed sideways but also poses a challenge to sedentarist and residentialist notions of what
precariousness and improvement might look like. Too often, this tension is envisioned in
terms of habitation and simply understood as the intuitive contrast between improvised and
permanent dwellings. What is overlooked in such accounts is that for many rural–urban workers the city
represents an avenue for making a living rather than a place to live. This is true for
Ishrat, who professed that she did not mind sleeping on the floor as long as her room
has sufficient electricity to continue her work after dark. And for Rokib, who approached
his sleeping space as an opportunity for making money by renting out his bed to friends and
offering up his room as a storage space. It is also true for rickshaw drivers like Enamul and 248 EPD: Society and Space 41(2) Jalal, who have their families living in the countryside and are merely looking for a tem-
porary sleeping place in the city. What is at stake for them is not so much the (im)possibility
of acquiring and negotiating a permanent place to stay in the city, but the (im)possibility of
ensuring a continuation of livelihoods. A focus on shifting has allowed us to examine how
people try to foster a sense of continuity amidst processes of densification and expansion,
and the trade-offs they end up making in the process. The fact that peripheries are shifting
thus requires an understanding of precariousness and improvement that pays attention to
what is either cut off or enabled in terms of movements, trajectories, and relations. Jalal, who have their families living in the countryside and are merely looking for a tem-
porary sleeping place in the city. What is at stake for them is not so much the (im)possibility
of acquiring and negotiating a permanent place to stay in the city, but the (im)possibility of
ensuring a continuation of livelihoods. Conclusion First, shifting allows us
to understand how migrants and newcomers try to implant themselves into the urban
system, without reducing such efforts to “finding and negotiating a literal place in the
city”. Secondly, our conceptualization of shifting offers an analytical point of departure
for studying other spaces that are inherently bound with the ever-shifting edges of the city,
such as waste plants, transportation hubs, manufacturing workshops, and wholesale mar-
kets. Lastly, shifting enables us to interrogate the changing nature of urban peripheries, by
highlighting the processes through which these tentative edges are constantly made and
remade. 249 Prins and Dasgupta Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or
publication of this article: Shreyashi Dasgupta received funding for the completion of writing this
article through the Economic and Social Research Council ESRC – Cambridge Postdoctoral
Fellowship Grant ES/W006391/1. The research conducted by Annemiek Prins was completed with
the support of a grant from the Rosanna Fund for Women, as well as an Elphinstone Scholarship from
the University of Aberdeen. Notes 1. We use pseudonyms throughout this article. 1. We use pseudonyms throughout this article. 2. See the work of Malkki (1992) for an extensive analysis of the way in which the assumption of
sedentarism has shaped scholarly perspectives on identity, belonging, and culture. 3. Begum and Sen (2005) found that only 13 percent of the rickshaw drivers they surveyed owned their
own vehicle. The Bangladesh Institute of Labour Studies (BILS) reported an even lower percentage
of rickshaw ownership and detailed that 96 percent of their interlocuters rented their vehicle (Karim
and Salam, 2019: 51). 3. Begum and Sen (2005) found that only 13 percent of the rickshaw drivers they surveyed owned their
own vehicle. The Bangladesh Institute of Labour Studies (BILS) reported an even lower percentage
of rickshaw ownership and detailed that 96 percent of their interlocuters rented their vehicle (Karim
and Salam, 2019: 51). 4. A lungi is a tube-shaped sarong, which is worn by men and somewhat characteristic for cycle-
rickshaw drivers. 4. A lungi is a tube-shaped sarong, which is worn by men and somewhat characteristic for cycle-
rickshaw drivers. ORCID iD Annemiek Prins
https://orcid.org/0000-0001-9182-4889 Acknowledgments We wish to thank the editor and anonymous reviewers for their helpful comments and feedback. In
addition, Annemiek would like to extend her gratitude to Yasin and Aslam, both of whom offered
invaluable support as research assistants. She also wishes to thank Work for Better Bangladesh
(WBB), who hosted her throughout her fieldwork, and the many rickshaw drivers who generously
shared their time and stories. Shreyashi would like to thank all her interlocuters who supported her
during her research. Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/
or publication of this article. References Alam MJ (2018) Rapid urbanization and changing land values in mega cities: Implications for housing
development projects in Dhaka, Bangladesh. Bandung 5(1): 1–19. Arabindoo P (2011) Rhetoric of the slum: Rethinking urban poverty. City 15(6): 636–646 Archambault JS (2021) Urban precarity and aspirational compromise: Feeling otherwise in a
Mozambican suburb. City & Society 33(2): 303–323. Banerjee P (2022) Abandon the slum? Toward an alternative recognition of urban informal dwelling. Journal of Urban History. Advance online publication. https://doi.org/10.1177/00961442221127311
(accessed 7 April 2023). Basu B (2019) The mess bari project: Documenting Kolkata’s diverse boarding houses. The Mess Bari
Project 8 January. Available at: https://messbariproject.wordpress.com/2019/01/08/the-mess-bari-
project-documenting-kolkatas-diverse-boarding-houses/ (accessed 7 April 2023). Begum S and Sen B (2005) Pulling rickshaws in the city of Dhaka: A way out of poverty? Environment
and Urbanization 17(2): 11–25. Bhan G (2017) From the basti to the ‘house’: Socio-spatial readings of housing policy in India. Current
Sociology 65(4): 587–602. 250 EPD: Society and Space 41(2) Bhan G (2019) Notes on a southern urban practice. Environment and Urbanization 31(2): 639–654. Caldeira TPR (2017) Peripheral urbanization: Autoconstruction, transversal logics, and politics in
cities of the Global South. Environment and Planning D: Society and Space 35(1): 3–20. Champion T and Hugo G (2004) New Forms of Urbanization Beyond the Urban-Rural Dichotomy. London: Routledge. Dasgupta R (2019) On the edge of Dhaka: The agrarian urbanization of South Asia. Pathways for
Changing Rural Landscape 49(1–2): 287–302. Dasgupta S and Prins A (2023) ‘Worlding’ Bangladeshi urbanism through water. Urban Geography. Advance online publication: https://doi.org/10.1080/02723638.2023.2184583 (accessed 10 April
2023). Dhaka Tribune (2020) The roar of Rayerbazar. The Dhaka Tribune 5 October. Available at: www.dh
akatribune.com/business/real-estate/2020/10/05/the-roar-of-rayer-bazar (accessed 7 April 2023). Dharia NV (2022) The Industrial Ephemeral: Labor and Love in Indian Architecture and Construction. Oakland: University of California Press. Echanove M and Srivastava R (2016) This is not a slum: What the world can learn from Dharavi. World Policy Journal 33(2): 19–24. Etzold B (2016) Migration, informal labour and (trans)local productions of urban space – The case of
Dhaka’s street food vendors. Population, Space and Place 22(2): 170–184. Foot J (2000) The urban periphery, myth and reality: Milan, 1950–1990. City 4(1): 7–26. Gardner K and Osella F (2003) Migration, modernity and social transformation in South
overview. Contributions to Indian Sociology 37(1–2): v–xxviii. Ghafur S (2010) Imprints of the changing doctrines on housing in Dhaka. References In: Hafiz R and Rabbani
AKMG (eds) 400 Years of Capital Dhaka and Beyond, Vol 3: Urbanization and Urban Development. Dhaka: Asiatic Society of Bangladesh, pp.153–168. Gilbert A (2007) The return of the slum: Does language matter? International Journal of Urban and
Regional Research 31(4): 697–713. Gruebner O, Sachs J, Nockert A, et al. (2014) Mapping the slums of Dhaka from 2006 to 2010. Dataset Papers in Science (172182): 1–7. Gururani S and Kennedy L (2021) The co-production of space, politics, and subjectivities in India’s
urban peripheries. South Asia Multidisciplinary Academic Journal 26. Available at: http://journals. openedition.org/samaj/7365; DOI: https://doi.org/10.4000/samaj.7365 (accessed 7 April 2023). Gururani S and Kennedy L (2021) The co-production of space, politics, and subjectivities in India’s
urban peripheries. South Asia Multidisciplinary Academic Journal 26. Available at: http://journals. Hafiz R (2007) The urban frontiers of Dhaka: Creating space above the water. In: Jahan S and Zaman
M (eds) Urbanization in Bangladesh: Emerging Challenges. Dhaka: Bangladesh Institute of
Planners, pp.55–67. Harvey D (1985) The Urbanization of Capital: Studies in the History and Theory of Capitalist urban-
ization. Baltimore (Maryland): Johns Hopkins University Press. Hossain S (2013) Migration, urbanization and poverty in Dhaka. Bangladesh. Journal of t
Society of Bangladesh 58(2): 369–382. Hossain S (2013) Migration, urbanization and poverty in Dhaka. Bangladesh. Journal of the Asiatic
Society of Bangladesh 58(2): 369–382. Islam N (2015) A study on material culture in Dhaka: Rickshaw a motion craft. MA Dissertation, Islam N (2015) A study on material culture in Dhaka: Rickshaw a motion craft. MA Di
Department of Architecture, McGill University. Islam N (1996) Sustainability issues in urban housing in a low-income country: Bangladesh. Habitat
International 20(3): 377–388. Jackman D (2017) Are ‘the destitute’ destitute? Understanding micro-inequalities through the concept
of defiled surpluses. Environment and Urbanization 29(1): 251–266. Kabir A and Parolin B (2012) Planning and development of Dhaka – A story of 400 years. In: Proceedings of the 15th international planning history society conference, S~ao Paulo, Brazil,
15–18 July. Kamalipour H and Dovey K (2020) Incremental production of urban space: A typology of informal
design. Habitat International 98: 102133. Karim RM and Salam KA (2019) Organizing the informal economy workers: A study of rickshaw pullers
in Dhaka City. Prepared for the Bangladesh Institute of Labour Studies, BILS. Available at: http://
bilsbd.org/wp-content/uploads/2019/06/A-Study-of-Rickshaw-Pullers-in-Dhaka-City.pdf (accessed
7 April 2023). 251 Prins and Dasgupta Makhulu A (2015) Making Freedom: Apartheid, Squatter Politics, and the Struggle for Home. References Durham:
Duke University Press. Makhulu A (2015) Making Freedom: Apartheid, Squatter Politics, and the Struggle for Home. Durham:
Duke University Press. Malkki L (1992) National geographic: The rooting of peoples and the territorialization of national Malkki L (1992) National geographic: The rooting of peoples and the territorialization of
identity among scholars and refugees. Cultural Anthropology 7(1): 24–44. Martinotti G (1994) The new social morphology of cities. In: Proceedings of the UNESCO/MOST
conference, Vienna, Austria, 10–12 February. Prins A (2021) Earning money as the wheels turn around: Cycle-rickshaw drivers and experiences of
wageless work in Dhaka. In: Monteith W, Vicol DO and Williams P (eds) Beyond the Wage:
Ordinary Work in Diverse Economies. Bristol: Bristol University Press, pp.187–210. Rao V (2006) Slum as theory: The South/Asian city and globalization. International Journal of Urban
and Regional Research 30(1): 225–232. Roy A (2011) Slumdog cities: Rethinking subaltern urbanism. International Journal of Urban And
Regional Research 35(2): 223–238. Roy S and Sowgat T (2020) Dhaka: Diverse, Dense, and Damaged Neighbourhoods and the Impacts of
Unplanned Urbanisation (SHLC Research Summary 8). Bangladesh: GCRF Centre for Sustainable,
Healthy and Learning Cities and Neighbourhoods, Khulna University. Roy S and Sowgat T (2020) Dhaka: Diverse, Dense, and Damaged Neighbourhoods and the Impacts of
Unplanned Urbanisation (SHLC Research Summary 8). Bangladesh: GCRF Centre for Sustainable, Healthy and Learning Cities and Neighbourhoods, Khulna University. Roy S, Sowgat T and Mondal J (2019) City profile: Dhaka, Bangladesh. Environment and Urbanization
ASIA 10(2): 216–232. Simone AM (2010) City Life from Jakarta to Dakar: Movements at the Crossroads. London:
Routledge. Simone AM (2018) Living as logistics: Tenuous struggles in the remaking of collective urban life. In:
Watson V, Bhan G and Srinivas S (eds) Routledge Companion to Planning in the Global South. London: Routledge, pp.264–274. Simone AM (2020) Securing “standby” and urban space making in Jakarta: Intensities in search of
forms. In: Ghertner DA, McFann H and Goldstein DM (eds) Futureproof: Security Aesthetics and
the Management of Life. London: Duke University Press, pp.225–244. Smith N (1984) Uneven Development: Nature, Capital, and the Production of Space. Oxford: Blackwell. Soja EW (1989) Postmodern Geographies: The Reassertion of Space in Critical Social Theory. London:
Verso. 1984) Uneven Development: Nature, Capital, and the Production of Space. Oxford: Blackwell. References 1989) P
t
d
G
hi
Th R
ti
f S
i C iti
l S
i l Th
L
d Smith N (1984) Uneven Development: Nature, Capital, and the Production of Space. Oxford Soja EW (1989) Postmodern Geographies: The Reassertion of Space in Critical Social Theory. London:
Verso. Solomon H (2022) Lifelines: The Traffic of Trauma. Durham: Duke University Press. Sowgat T and Roy S (2022) Throwntogetherness in Dhaka: Rethinking urban planning: A visual
essay. City 26(2–3): 422–432. y
y
(
)
Thieme TA (2018) The hustle economy: Informality, uncertainty and the geographies of getting by. Progress in Human Geography 42 (4): 529–548. van A (2015) The makeshift city: Towards a global geography of squatting. Progress in Hum
graphy 39(3): 338–359. Weinstein L (2014) The Durable Slum: Dharavi and the Right to Stay Put in Globalizin
Minneapolis: Univeristy of Minnesota Press. World Bank (2017) Dhaka (Bangladesh) – Land use/land cover maps (ESA EO4SD-Urban). Available
at: https://datacatalog.worldbank.org/search/dataset/0039604 (accessed 9 August 2022). World Population Review (2022) Dhaka population 2022. Available at: https://worldpopulationre
view.com/world-cities/dhaka-population (accessed 29 August 2022). Annemiek Prins works as a postdoctoral researcher in the Department of Anthropology and
Development Studies at the Radboud University of Nijmegen. She obtained her PhD from
the University of Aberdeen, focusing on the contestation of cycle-rickshaw mobilities and
labor in Bangladesh. Her doctoral dissertation, “Unfixing the City: Rickshaw Mobilities,
Modernities and Urban Change in Dhaka”, explores different aspects of contemporary
urbanism, including mobility, informal labor, peripheral urbanization, and expectations
of modernity. Her current research project, which is also based in Bangladesh, tries to
better understand and situate realities of urbanization as part of the dynamic context of 252 EPD: Society and Space 41(2) 1(2) Bengal delta and looks at the interplay between infrastructure, sand extraction, and envi-
ronmental change in the wake of Padma Bridge construction. Shreyashi Dasgupta is an Economic and Social Research Council (ESRC) Postdoctoral
Fellow at the Department of Geography, University of Cambridge, and a Research
Associate at Homerton College. Her work focuses on housing, infrastructure, and everyday
politics in South Asia. Shreyashi’s doctoral dissertation, “The Accommodation City”,
explores the emerging forms and processes of temporary accommodation (dormitories,
mess housing, work-residence rentals, and room sharing) for low-income workers in
Dhaka, Bangladesh, and Mumbai, India. She is interested in studying how accommodation
matters “in” and “for” cities. References Shreyashi completed her PhD and MPhil in Development
Studies at the University of Cambridge and Girton College.
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https://link.springer.com/content/pdf/10.1140/epjc/s10052-020-08813-y.pdf
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English
| null |
On leptonic width of X(4260)
| null | 2,021
|
cc-by
| 7,028
|
On leptonic width of X(4260) Qin-Fang Cao1,a
, Hong-Rong Qi2,b
, Guang-Yi Tang3, Yun-Feng Xue1, Han-Qing Zheng1,4
1 Department of Physics and State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing 100871,
People’s Republic of China
2 Department of Engineering Physics, Tsinghua University, Beijing 100084, People’s Republic of China
3 Institute of High Energy Physics, Beijing 100049, People’s Republic of China
4 Collaborative Innovation Center of Quantum Matter, Beijing 100871, People’s Republic of China Qin-Fang Cao1,a
, Hong-Rong Qi2,b
, Guang-Yi Tang3, Yun-Feng Xue1, Han-Qing Zheng1,4
1 Department of Physics and State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing 100871,
People’s Republic of China
2 Department of Engineering Physics, Tsinghua University, Beijing 100084, People’s Republic of China
3 Institute of High Energy Physics, Beijing 100049, People’s Republic of China Qin-Fang Cao1,a
, Hong-Rong Qi2,b
, Guang-Yi Tang3, Yun-Feng Xue1, Han-Qing Zheng1,4
1 Department of Physics and State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing 100871,
People’s Republic of China
2 Department of Engineering Physics, Tsinghua University, Beijing 100084, People’s Republic of China
Institute of High Energy Physics, Beijing 100049, People’s Republic of China g
gy
y
j g
p
p
Collaborative Innovation Center of Quantum Matter, Beijing 100871, People’s Republic of China Received: 28 October 2020 / Accepted: 28 December 2020 / Published online: 26 January 2021
© The Author(s) 2021 Received: 28 October 2020 / Accepted: 28 December 2020 / Published online: 26 January 2021
© The Author(s) 2021 Received: 28 October 2020 / Accepted: 28 December 2020 / Published online: 26 January 2021
© The Author(s) 2021 quark model predictions [6]. The only such 1−−states
expected up to 4.4 GeV are generally 1S, 2S, 1D, 3S, 2D and
4S, and they seem to be well established [7] – the situation
is depicted in Fig. 1. Abstract
New measurements on cross sections in e+e−→
J/ψπ+π−, hcπ+π−, D0D∗−π+ + c.c., ψ(2S)π+π−,
ωχc0 and J/ψη channels have been carried out by BESIII,
Belle and BABAR experiments, as well as in the D∗+
s
D∗−
s
channel. We perform extensive numerical analyses by com-
bining all these data available, together with those in
D+D∗−+ c.c. and D∗+D∗−channels. Though the latter
show no evident peak around √s = 4.230 GeV, the missing
X(4260) is explained as that it is concealed by the inter-
ference effects of the well established charmonia ψ(4040),
ψ(4160) and ψ(4415). 1 Here we apologize for only able to provide an incomplete list of
references. On leptonic width of X(4260) Our analyses reveal that the leptonic
decay width of X(4260) ranges from O(102) eV to O(1) keV,
and hence it is probably explained in the conventional quark
model picture. That is, the X(4260) may well be interpreted
as a mixture of 43S1 and 33D1 (23D1) states. It is noticed that above D ¯D threshold the number of
1−−states given by quark model prediction is inconsistent
with that given by experiments. It is considered that the dis-
crepancy between the naive quark model prediction and the
observed spectrum is ascribed, at least partially, to the exis-
tence of many open charm thresholds, since the latter will
distort the spectrum (see related discussions in, for example,
Refs. [8,9]). The situation is depicted in Fig. 2. Because of the situation as described above many studies
have been devoted to the investigation of X(4260).1 The sug-
gestions given by these studies may be classified into three
types: hadronic molecule [10–15]; c¯c state [16–20]; hybrid
state [21,22]; or non-resonant enhancement [23]. The open charm channels such as D ¯D, D ¯D∗, D∗¯D∗
do not seem to be found in the final states of X(4260)
decays [24–26]. If this is indeed the case, then it would make
X(4260) even more mysterious, since the J/ψππ channel
would become a very important, if not the dominant one. Hence the leptonic width e+e−of X(4260) would become
very small, making it even harder to be understood as a con-
ventional 1−−¯cc state, since the nearby 43S1 state is expected
to have a leptonic width at the order ∼103 eV [19]. b e-mail: qihongrong@tsinghua.edu.cn (corresponding author) Regular Article - Theoretical Physics Regular Article - Theoretical Physics a e-mail: caoqf@pku.edu.cn Eur. Phys. J. C (2021) 81:83
https://doi.org/10.1140/epjc/s10052-020-08813-y 2 We do not consider a term ∝DD1 here, as it is found vanishing in
Ref. [27]. Also the experimental data in Zcπ channel is absent. Since
the Zc(3900) state is identified as a DD∗molecule [40,41], the exper-
imental branching ratio is naturally expected to be small. 1 Introduction The X(4260) resonance established by BABAR Collab-
oration in initial-state radiation (ISR) process e+e−→
γI SR J/ψπ+π−in year 2005 [1] (subsequently confirmed
by CLEO [2] and Belle [3]), has attracted much attention
since then. The mass and width of this resonance are given
with M = 4230±8 MeV and = 55±19 MeV [4], respec-
tively, and ee ×Br(J/ψππ) = 9.7±1.1 eV [3] or 9.2±1.5
eV [5]. In a previous publication, we have also studied the
X(4260) issue and suggested that there could be a siz-
able ωχc0 coupling [27], later confirmed by experimental
researches [28]. At the same time, a very small e+e−was
found ∼25eV. However, many new experimental results
have appeared since then, measured by BESIII, Belle and
BABARexperiments,suchase+e−→J/ψπ+π−[3,5,29], The property of X(4260) becomes a very interesting
topic since its discovery, because it is generally thought that
there are not enough unassigned vector states in charmo-
nium spectrum (taking into account the recently reported
X(4360), X(4630)/X(4660) states), according to the naive 12 3 83
Page 2 of 10 Eur. Phys. J. C (2021) 81 :83 Fig. 1 X(4260) and nearby
resonances from naive quark
model calculation [6]
X(4660)
Ψ(4415)
X(4360)
X(4260)
Ψ(4160)
Ψ(4040)
Ψ(3770)
Ψ(2S)
Ψ(1S)
33D1(4.52)
43S1(4.45)
23D1(4.19)
33S1(4.10)
13D1(3.82)
23S1(3.68)
13S1(3.1)
Ψ f0(980)
Λ+
c Λ−
c
D∗+
s D∗−
s
D∗¯D∗
D+
s D−
s
D ¯D∗
D ¯D
DD1(2420)
1−−particle
quark model
threshold
Fig. 2 Location of X(4260)
and nearby thresholds
X(4260)
D∗+D∗−(4020)
χc0ω(4197)
χc0ρ0(4185)
D∗+
s D∗−
s (4224)
χc1ω(4293)
J/ψ ¯KK(4086)
D1 ¯D(4289)
Λc¯Λc(4572) Fig. 1 X(4260) and nearby
resonances from naive quark
model calculation [6] Fig. 1 X(4260) and nearby
resonances from naive quark
model calculation [6] Fig. 2 Location of X(4260)
and nearby thresholds Fig. 2 Location of X(4260)
and nearby thresholds hcπ+π−[30], D0D∗−π++c.c. [31], ψ(2S)π+π−[32–34],
ωχc0 [35,36], J/ψη [37,38] and D∗+
s
D∗−
s
[39]. Hence the
analyses of Ref. [27] urgently need to be upgraded. Among
all it is worthwhile mentioning the D∗+
s
D∗−
s
data near the
X(4260) region [39], which indicates a strong enhance-
ment of events above the D∗+
s
D∗−
s
threshold. If this is true,
our analyses show that it decisively changes our previous
understandings on X(4260) resonance: It could probably be
described by the conventional 43S1 state heavily renormal-
ized by the D∗+
s
D∗−
s
continuum (maybe a small mixing with
the 33D1 state as well). 1 Introduction If the D∗+
s
D∗−
s
data are excluded
from the fit, however, the final result on e+e−can still be
at least O(102) eV, i.e., much larger comparing with that of
Ref. [27], owing to other new data available as mentioned
above. As a consequence, the X(4260) resonance may still
be considered as a mixture of 33D1 and 43S1 states, i.e., a
conventional ¯cc resonance. 2 Theoretical discussions To begin with, the X(4260) propagator is written in the fol-
lowing form: 1
DX(s) =
1
s −M2
X + i√stot(s),
(1) (1) (1) where MX represents the mass of X(4260) and tot(s) is the
total momentum dependent width comprising of all partial
ones2: tot(s) = J/ψππ(s) + hcππ(s) + D ¯D∗π(s)
+ψ(2S)ππ(s) + ωχc0(s) + J/ψη(s)
+D∗+
s
D∗−
s (s) + D ¯D(s)
+D ¯D∗(s) + D∗¯D∗(s) + 0. (2) (2) The paper is organized as follows: this Sect. 1 is the intro-
duction. A detailed description of hadronic cross sections of
e+e−annihilation will be given in Sect. 2. In Sect. 3, com-
bined fits to the hidden charm and open charm decay channels
are performed, with two scenarios: one includes the D∗+
s
D∗−
s
cross section data and the other does not. We leave physical
discussions and conclusions in Sect. 4. Consideringtheisospinsymmetry,itisnoticedthatJ/ψππ(s) =
3
2J/ψπ+π−(s), hcππ(s) =
3
2hcπ+π−(s), D ¯D∗π(s) = 123 123 123 Eur. Phys. J. C (2021) 81 :83 Page 3 of 10
83 3D0D∗−π++c.c.(s),ψ(2S)ππ(s) = 3
2ψ(2S)π+π−(s),D ¯D =
2D+D−, D ¯D∗= 2D+D∗−+c.c. and D∗¯D∗= 2D∗+D∗−.3 −
In above kωχc0, kJ/ψη, kD∗+
s
D∗−
s , kD+D−, kD+D∗−and
kD∗+D∗−are three momentums of ωχc0, J/ψη, D∗+
s
D∗−
s ,
D+D−, D+D∗−and D∗+D∗−in X(4260) rest frame,
respectively. Further, except for the channels just discussed,
there could be other channels with lower thresholds, for these
channels we use a constant width 0 to describe the overall
effects. Asforthe J/ψπ+π−,hcπ+π−, D0D∗−π+ andψ(2S)π+π
channels, the three body partial decay width takes the stan-
dard form [4],4 d f =
1
(2π)5
1
16M2
X
|M|2|p∗
1||p3|dm12d ∗
1d 3,
(3) (3) Because the quantum number of X(4260) is J PC = 1−−,
the interaction between X(4260) and photon can be written
as as where pi j = pi + p j, m2
i j = p2
i j, |p∗
1|, ∗
1 are the momen-
tum and angle of particle 1 in the rest frame of the system
of particle 1 and 2, respectively; 3 is the angle of parti-
cle 3 in the rest frame of particle MX; and |p∗
1|, |p3| are
defined as Lγ X = g0
MX
Xμν Fμν,
(7) (7) where Xμν = ∂μVν −∂νVμ and V μ represents the X(4260)
field, Fμν = ∂μAν −∂ν Aμ describes the photon field. g0
is the coefficient between the photon and X(4260). So the
decay width of X(4260) →e+e−reads |p∗
1| = [(m2
12 −(m1 + m2)2)(m2
12 −(m1 −m2)2)]1/2
2m12
, (4)
|p3| = [(M2
X −(m12 + m3)2)(M2
X −(m12 −m3)2)]1/2
2MX
. (5) e+e−= 4α
3
g2
0
MX
. (8) e+e−= 4α
3
g2
0
MX
. (8) (5) In hcπ+π−, D0D∗−π+, ψ(2S)π+π−, ωχc0, J/ψη,
D∗+
s
D∗−
s , D+D∗−and D∗+D∗−channels, using narrow
width approximation, the cross section formulae take the
form With respect to J/ψπ+π−channel, the amplitude in Eq. (3)
is calculated using the effective Lagrangian as Eq. (6) in
Ref. [27] to describe the X(4260) →J/ψπ+π−process
and the interaction coefficients hi(i = 1, 2, 3) are listed in
Tables 1 and 2. Since the production ratio R = (σ(e+e−→
Zc(4020)∓π± →hcπ+π−)/σ(e+e−→hcπ+π−)) <
20% [42], ghcπ+π−can be used to describe the invariant
amplitude in Eq. (3) when ignoring the resonance structure
in the X(4260) decay. 123 Analogously, gD0D∗−π+, gψ(2S)π+π−
can also be introduced as the invariant amplitude in Eq. (3)
for D0D∗−π+ and ψ(2S)π+π−channels for simplicity. Besides, in the analyses below, it is noticed that the decay
ratios of these channels account for only a tiny share. Hence,
it is feasible to employ ghcπ+π−, gD0D∗−π+ and gψ(2S)π+π−
for simplicity. With respect to J/ψπ+π−channel, the amplitude in Eq. (3) σe+e−→X(4260)→f = 3π
k2 |
see f
s −M2
X + i√stot(s)
+
i
cieiφi
s −M2
i + i√si
+ ˜c |2,
(9) (9) where k is the 3-momentum of incoming electron in c.m. frame, ee follows Eq. (8) and f takes the form of Eqs. (3)
and (6). The term parameterized as a resonance propagator
with a mass Mi and width i and the complex constant ˜c
play the role of a background in each decay channel here, as
will be declared in details in the forthcoming Sect. 3. where k is the 3-momentum of incoming electron in c.m. frame, ee follows Eq. (8) and f takes the form of Eqs. (3)
and (6). The term parameterized as a resonance propagator
with a mass Mi and width i and the complex constant ˜c
play the role of a background in each decay channel here, as
will be declared in details in the forthcoming Sect. 3. In addition, the two body decay widths take the following
simple forms: 3 Hereafter,thenotation D0D∗−π+ and D+D∗−indicate D0D∗−π++
c.c. and D+D∗−+ c.c. for simplicity, respectively. 3 Hereafter,thenotation D0D∗−π+ and D+D∗−indicate D0D∗−π++
c.c. and D+D∗−+ c.c. for simplicity, respectively.
4 Here we use the subscript “ f ” to represent various three body decay
final states. 4 Here we use the subscript “ f ” to represent various three body decay
final states. and the fit results are illustrated as Fig. 3f. and the fit results are illustrated as Fig. 3f. 3.1.1 The J/ψπ+π−process 3.1.1 The J/ψπ+π−process 3.1.6 The D+D∗−and D∗+D∗−processes 3.1.6 The D+D∗−and D∗+D∗−processes For the D+D∗−channel, we take the Belle data [25], √s ∈
[3.93, 4.37] GeV, with 23 data points, as shown in Fig. 3i. Two additional Breit–Wigner resonances and a complex con-
stant are imposed in the fit to simulate the contribution of
interference backgrounds: For the D+D∗−channel, we take the Belle data [25], √s ∈
[3.93, 4.37] GeV, with 23 data points, as shown in Fig. 3i. σi = 3π
k2 |
see f
s −M2
X + i√stot
+
ci1eiφi1
s −M2
4415 + i√s4415
|2 ,
(11) Two additional Breit–Wigner resonances and a complex con-
stant are imposed in the fit to simulate the contribution of
interference backgrounds: σD+D∗−+c.c. = 3π
k2 |
√seeD+D∗−
s −M2
X + i√stot
+
c71eiφ71
s −M2
4040 + i√s4040
+
c72eiφ72
s −M2
4160 + i√s4160
+c73 + ic74|2,
(15) σD+D∗−+c.c. = 3π
k2 |
√seeD+D∗−
s −M2
X + i√stot
+
c71eiφ71
s −M2
4040 + i√s4040
+
c72eiφ72
s −M2
4160 + i√s4160
+c73 + ic74|2, where i = 2, 3, 4 represents the D0D∗−π+, hcπ+π−and
ψ(2S)π+π−channels, respectively, as shown in Fig. 3c–e. where i = 2, 3, 4 represents the D0D∗−π+, hcπ+π−and
ψ(2S)π+π−channels, respectively, as shown in Fig. 3c–e. 3 Numerical analyses and discussions ωχc0(s) = gωχc0kωχc0,
J/ψη(s) = gJ/ψηk3
J/ψη,
D∗+
s
D∗−
s (s) = gD∗+
s
D∗−
s k3
D∗+
s
D∗−
s ,
D+D−(s) = gD+D−k3
D+D−,
D+D∗−(s) = gD+D∗−k3
D+D∗−,
D∗+D∗−(s) = gD∗+D∗−k3
D∗+D∗−. ωχc0(s) = gωχc0kωχc0,
J/ψη(s) = gJ/ψηk3
J/ψη,
D∗+
s
D∗−
s (s) = gD∗+
s
D∗−
s k3
D∗+
s
D∗−
s ,
D+D−(s) = gD+D−k3
D+D−,
D+D∗−(s) = gD+D∗−k3
D+D∗−,
D∗+D∗−(s) = gD∗+D∗−k3
D∗+D∗−. In Sect. 3.1, we will perform comprehensive fits to relevant
data available in the vicinity of X(4260), which include
the J/ψπ+π−[3,5,29], hcπ+π−[30], D0D∗−π+ [31],
ψ(2S)π+π−[32–34], ωχc0 [35,36], J/ψη [37,38], D∗+
s
D∗−
s
[39], together with the previous D+D∗−and D∗+D∗−
data in Ref. [25]. To be cautious, for the reason as already
mentioned previously, the fit without D∗+
s
D∗−
s
cross section
data is also performed in Sect. 3.2. Different results are care-
fully compared and discussed, and we believe that a clearer
understanding on the nature of X(4260) emerges. (6) 12 3 83
Page 4 of 10 Eur. Phys. J. C (2021) 81 :83 3.1.4 The J/ψη process For the J/ψπ+π−channel, the experimental data sets
come from: BESIII [29], √s ∈[3.81, 4.60] GeV, 121 data
points; BABAR [5], √s ∈[4.15, 4.47] GeV, 17 data points;
Belle [3], √s ∈[4.15, 4.47] GeV, 17 data points. For the fit to
theabovedata, weadopt theamplitudeas Eq. (14) inRef. [27]
to describe the e+e−→γ ∗→X(4260) →J/ψπ+π−
process and the propagator
1
DX(s)
5 of that equation is rewrit-
ten as the following form: Concerning the J/ψη channel, 8 data points ranging from
√s ∈[4.09, 4.31] GeV measured by BESIII [37] and 13 data
points from √s ∈[4.07, 4.31] GeV measured by Belle [38]
are adopted simultaneously. On account of the influence of
ψ(4160), which was taken into account in the fit by Belle in
Ref. [38], the cross section is written as the following: σJ/ψη = 3π
k2 |
seeJ/ψη
s −M2
X + i√stot
+
c51eiφ51
s −M2
4160+i√s4160
|2. (13) 1
DX(s) ⇒
1
s −M2
X + i√stot
+
c11eiφ11
s −M2
4415+i√s4415
+c12eiφ12e−c13(√s−mth) ,
(10) (13) (10) Besides, M4160 and 4160 are introduced to represent the
mass and width of ψ(4160), respectively. Besides, M4160 and 4160 are introduced to represent the
mass and width of ψ(4160), respectively. where mth is the threshold of J/ψπ+π−, M4415 and 4415
are introduced to represent the mass and width of ψ(4415),
respectively; φ11 and φ12 are interference phases; c11, c12,
and c13 are free constants (see Fig. 3a and b for fit results). 3.1.5 The D∗+
s
D∗−
s
process In D∗+
s
D∗−
s
channel we take the data from Ref. [39], √s ∈
[4.23, 4.36] GeV, 5 data points. Then the cross section reads 3.1.2 The D0D∗−π+, hcπ+π−and ψ(2S)π+π−
processes 3.1.2 The D0D∗−π+, hcπ+π−and ψ(2S)π+π−
processes σD∗+
s
D∗−
s
= 3π
k2 |
seeD∗+
s
D∗−
s
s −M2
X + i√stot
+
c61eiφ61
s −M2
4415 + i√s4415
|2. (14) The BESIII data for e+e−→D0D∗−π+ [31] ranging from
√s ∈[4.05, 4.45] GeV are chosen with 51 points. In hcππ
channel, the experimental data in √s ∈[4.09, 4.32] GeV
region with 45 data points are also chosen from BESIII Col-
laboration [30]. For the ψ(2S)π+π−process, the experi-
mental data come from: BESIII [32], √s ∈[4.085, 4.308]
GeV, 8 data points; Belle [33], √s ∈[4.11, 4.31] GeV, 11
data points; BABAR [34], √s ∈[4.13, 4.32] GeV, 5 data
points. The fit formula is (14) See Fig. 3h for fit results. 3.1.4 The J/ψη process See Fig. 3h for fit results. 5 Here we use s = q2. 3.1.3 The ωχc0 process The ωχc0 data comes from Ref. [35], √s ∈[4.21, 4.39] GeV,
8 data points and Ref. [36], √s ∈[4.199, 4.278] GeV, 7 data
points. The fit formula can be parameterized as (15) σωχc0 = 3π
k2 |
seeωχc0
s −M2
X + i√stot
|2 ,
(12) in which M4040 (4040) is used to describe the mass (width)
of ψ(4040). (12) Additionally, 18 data points released by Belle [25] from
√s ∈[4.11, 4.45] are adopted in the D∗+D∗−process and Eur. Phys. J. C (2021) 81 :83 Page 5 of 10
83 Page 5 of 10
83 Eur. Phys. J. C (2021) 81 :83
Page 5 of 10
83
(a)
(b)
(c)
(d)
(e)
(g)
(h)
(f)
(i)
(j)
Fig. 3 The results of the fit with D∗+
s
D∗−
s
cross section data. The solid
curves in all subgrpaphs are the projections from the best fit: a and b
fit to the cross section of J/ψπ+π−; c fit to D0D∗−π+ data, where
the dashed black one represents X(4260) components; d fit to hcπ+π−
data, where the dashed black one indicates X(4260) components; e fit
to ψ(2S)π+π−data, where the dashed black one describes X(4260)
components; f fit to ωχc0 cross section data, and we do not fit the two
data points (orange squres) on the left side since they are below the
threshold; g fit to J/ψ η data; h fit to D∗+
s
D∗−
s
data; i fit to D+D∗−
cross section, where the dashed curves show the individual components
of ψ(4040), ψ(4160), X(4260), respectively; j fit to D∗+D∗−cross
section, where the dashed curves show the individual components of
ψ(4040), ψ(4160), X(4260) and ψ(4415), respectively (b) (a) (c) (a) (b) (f) (f) (f) (j) Fig. 3 The results of the fit with D∗+
s
D∗−
s
cross section data. 3.1.3 The ωχc0 process The solid
curves in all subgrpaphs are the projections from the best fit: a and b
fit to the cross section of J/ψπ+π−; c fit to D0D∗−π+ data, where
the dashed black one represents X(4260) components; d fit to hcπ+π−
data, where the dashed black one indicates X(4260) components; e fit
to ψ(2S)π+π−data, where the dashed black one describes X(4260)
components; f fit to ωχc0 cross section data, and we do not fit the two data points (orange squres) on the left side since they are below the
threshold; g fit to J/ψ η data; h fit to D∗+
s
D∗−
s
data; i fit to D+D∗−
cross section, where the dashed curves show the individual components
of ψ(4040), ψ(4160), X(4260), respectively; j fit to D∗+D∗−cross
section, where the dashed curves show the individual components of
ψ(4040), ψ(4160), X(4260) and ψ(4415), respectively 123 123 123 Eur. Phys. J. C (2021) 81 :83 83
Page 6 of 10 Table 1 Summary of numerical results associated with the D∗+
s
D∗−
s
data
Parameters
Value
g0 (MeV)
23.865 ± 0.602
MX (GeV)
4.219 ± 0.001
0 (GeV)
0.005 ± 0.002
h1
−0.0003 ± 0.0005
h2
−0.062 ± 0.005
h3
0.016 ± 0.001
gD0 D∗−π+
234.750 ± 34.787
ghcπ+π−
68.248 ± 22.801
gψ(2S)π+π−
12.538 ± 8.588
gωχc0
0.0007 ± 0.0001
gJ/ψη (GeV−2)
0.0002 ± 0.00006
gD∗+
s
D∗−
s
(GeV−2)
1.101 ± 0.004
gD+ D−(GeV−2)
0.004 ± 0.0003
4040 (GeV)
0.090 ± 0.016
4160 (GeV)
0.080 ± 0.020
4415 (GeV)
0.082 ± 0.002 Table 1 Summary of numerical results associated with the D∗+
s
D∗−
s
data
Parameters
Value
g0 (MeV)
23.865 ± 0.602
MX (GeV)
4.219 ± 0.001
0 (GeV)
0.005 ± 0.002
h1
−0.0003 ± 0.0005
h2
−0.062 ± 0.005
h3
0.016 ± 0.001
gD0 D∗−π+
234.750 ± 34.787
ghcπ+π−
68.248 ± 22.801
gψ(2S)π+π−
12.538 ± 8.588
gωχc0
0.0007 ± 0.0001
gJ/ψη (GeV−2)
0.0002 ± 0.00006
gD∗+
s
D∗−
s
(GeV−2)
1.101 ± 0.004
gD+ D−(GeV−2)
0.004 ± 0.0003
4040 (GeV)
0.090 ± 0.016
4160 (GeV)
0.080 ± 0.020
4415 (GeV)
0.082 ± 0.002 lized in our fit. Therefore, there is only one parameter in need
of describing the coupling coefficient in these three channels. The goodness of the fit is χ2/d.o. f. = 292.84/(357−43) =
0.93. 3.2 Discussions on the fit without D∗+
s
D∗−
s
data Since the D∗+
s
D∗−
s
cross section data from BESIII [39] are
preliminary, the program without fitting the D∗+
s
D∗−
s
has
also been carried out. In this subsection, the total width of
the X(4260) propagator is also Eq. (2), which includes the
D∗+
s
D∗−
s
decay width, even though the data are not fitted. It is noticed that the branching ratios of each decay chan-
nel remain similar whether the program includes fitting the
D∗+
s
D∗−
s
cross section data or not. Likewise, the fit formula
for the J/ψπ+π−, D0D∗−π+, hcπ+π−, ψ(2S)π+π−,
ωχc0, J/ψη, D+D∗−and D∗+D∗−processes are used as
the forms in the Sect. 3.1 respectively. The fit results are
displayed in Fig. 4. (16) The fit projection is presented in Fig. 3j. The fit projection is presented in Fig. 3j. 3.1.3 The ωχc0 process The mass (MX) is determined to be 4.219±0.001 GeV,
and the sum of all partial wave widths excluding the coupling
to D∗+
s
D∗−
s
is 51.44 ± 3.92 MeV at √s = MX. s
s
The value of g0 corresponds to the leptonic decay width
e+e−= 1.314 ± 0.066 keV, which gives a strong support
for X(4260) to be a 43S1 vector charmonium [19]. The above conclusions are rather stable against variations
of background parameterizations. For example, the complex-
constant coherent background can be employed in Eq. (16)
for the D∗+D∗−channel, and the expression is σD∗+D∗−= 3π
k2 |
√seeD∗+D∗−
s −M2
X + i√stot
+
c81eiφ81
s −M2
4040 + i√s4040
+
c82eiφ82
s −M2
4160 + i√s4160
+
c83eiφ83
s −M2
4415 + i√s4415
+ c84 + ic85|2. (17) the cross section is written as the cross section is written as
σD∗+D∗−= 3π
k2 |
√seeD∗+D∗−
s −M2
X + i√stot
+
c81eiφ81
s −M2
4040 + i√s4040
+
c82eiφ82
s −M2
4160 + i√s4160
+
c83eiφ83
s −M2
4415 + i√s4415
|2. (16)
The fit projection is presented in Fig. 3j. the cross section is written as
σD∗+D∗−= 3π
k2 |
√seeD∗+D∗−
s −M2
X + i√stot
+
c81eiφ81
s −M2
4040 + i√s4040
+
c82eiφ82
s −M2
4160 + i√s4160
+
c83eiφ83
s −M2
4415 + i√s4415
|2. (16) It turns out that the fit quality is χ2/d.o. f. = 282.38/(357−
45) = 0.91 and the fit results for each decay channel make
practically little difference. 3.2 Discussions on the fit without D∗+
s
D∗−
s
data 3.1.7 The fit results We have attempted to fit the experimental data with three well
established charmonia, ψ(4040), ψ(4160) and ψ(4415),
together with other coherent background contributions in the
above decay channels. Since X(4260) is our only interest
here, the mass of ψ(4040), ψ(4160) and ψ(4415) is fixed
whereas the widths are left free in this research. The param-
eters related to backgrounds in each process are mentioned
above, and the widths of ψ(4040), ψ(4160) and ψ(4415) are
listed in Table 1, which are found in reasonable agreement
with the widths given by Particle Data Group [4]. The cou-
pling coefficients between X(4260) and different final states
are presented in the Table 1 as well. Especially, with heavy
quark spin symmetry considered, the relationship between
gD+D−, gD+D∗−+c.c. and gD∗+D∗−can be calculated [43] to
be gD+D−: gD+D∗−+c.c. : gD∗+D∗−= 1 : 4 : 7, which is uti- The widths of ψ(4040), ψ(4160) and ψ(4415) as well as
thecouplingcoefficients between X(4260) anddifferent final
states are presented in Table 2. The fit quality is χ2/d.o. f. =
291.86/(352 −41) = 0.94. The value of g0 corresponds to
a leptonic decay width e+e−= 0.385 ± 0.060 keV, which
may imply that X(4260) is a mixture of 43S1 and 33D1 c¯c
charmonium state [19]. In addition, another solution to the fit without D∗+
s
D∗−
s
cross section data is found, with the fit quality to be
χ2/d.o. f. = 288.41/(352 −41) = 0.93, where g0 =
21.212 ± 1.604 MeV. The leptonic width is changed to 12 123 123 Eur. Phys. J. C (2021) 81 :83 Page 7 of 10
83 Page 7 of 10
83 (a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
Fig. 4 The results of the fit without D∗+
s
D∗−
s
cross section data, where the descriptions of the components in subgraphs are similar to those of
Fig. 3 (b) (a) (c) (d) (e)
(h) (i) (g) (g) Fig. 4 The results of the fit without D∗+
s
D∗−
s
cross section data, where the descriptions of the components in subgraphs are similar to those of
Fig. 3 thout D∗+
s
D∗−
s
cross section data, where the descriptions of the components in subgraphs are similar to those of 1.038±0.157 keV, which is different from the previous solu-
tion but is similar to the fit with D∗+
s
D∗−
s
cross section data. 3.1.7 The fit results We find that, unfortunately, the destructive interference in
the open charm channels is rather unstable, which makes it
impossible to distinguish the physical one between the two
solutions with the similar fit quality, and we can not figure
out another solution better than the two. So only the range
of the leptonic width rather than a definitive value may be
trustworthy. Even so, the leptonic width of over hundreds eV
still favor X(4260) as a conventional charmonium. 1.038±0.157 keV, which is different from the previous solu-
tion but is similar to the fit with D∗+
s
D∗−
s
cross section data. We find that, unfortunately, the destructive interference in
the open charm channels is rather unstable, which makes it
impossible to distinguish the physical one between the two
solutions with the similar fit quality, and we can not figure
out another solution better than the two. So only the range
of the leptonic width rather than a definitive value may be
trustworthy. Even so, the leptonic width of over hundreds eV
still favor X(4260) as a conventional charmonium. and 5: the interference between different resonances are done
in rather different manner. The leptonic width behaves quite
differently, with a value of e+e−= 0.317±0.064 keV, com-
paring with the result of D∗+D∗−channel without constant
background. Moreover, we also find another new solution to the fit
with a complex coherent background in D∗+D∗−channel. The fit quality is χ2/d.o. f. = 277.20/(352 −43) = 0.90
and e+e−= 1.077 ± 0.164 keV, which is compatible with
the second solution to the fit without a complex coherent
background in D∗+D∗−channel. The physical solution is
not able to be picked out from the two solutions. Owing to the instability brought by the open charm
channels, then we add a complex coherent background in
D∗+D∗−channel following the strategy of Sect. 3.1.7 to
test the stability of outputs against the variation of back-
grounds. The fit is plotted in Fig. 5. The fit quality is
χ2/d.o. f. = 275.67/(352 −43) = 0.89. It is found, how-
ever, unlike the result of Sect. 3.1.7, the fit is not quite stable
here. The difference is clearly seen when comparing Figs. 3j 3.3 Summary and discussions on numerical fits To compare with the fits discussed above and to further
test the stability of the whole fit program, here we also test
the fit without including the D+D∗−and D∗+D∗−cross 123 123 Eur. Phys. J. C (2021) 81 :83 83
Page 8 of 10 Table 2 Summary of relevant parameters without the D∗+
s
D∗−
s
data
Parameters
Value
g0 (MeV)
12.928 ± 1.000
MX (GeV)
4.221 ± 0.001
0 (GeV)
0.000 ± 0.0002
h1
0.0004 ± 0.0008
h2
0.103 ± 0.008
h3
−0.026 ± 0.002
gD0 D∗−π+
603.980 ± 122.580
ghcπ+π−
197.150 ± 71.981
gψ(2S)π+π−
31.475 ± 21.230
gωχc0
0.002 ± 0.0003
gJ/ψη (GeV−2)
0.0003 ± 0.0001
gD∗+
s
D∗−
s
(GeV−2)
1.112 ± 0.002
gD+ D−(GeV−2)
0.004 ± 0.0002
4040 (GeV)
0.090 ± 0.014
4160 (GeV)
0.080 ± 0.014
4415 (GeV)
0.082 ± 0.004
Fig. 5 Given the complex coherent background in D∗+D∗−process,
the fit result of the D∗+D∗−cross section. The blue dots come from
Belle results [25]. The solid curve is the projection from the best fit. The
dashed curves show the individual components of ψ(4040), ψ(4160),
X(4260) and ψ(4415), respectively. The contribution of the complex
background is not presented Table 2 Summary of relevant parameters without the D∗+
s
D∗−
s
data
Parameters
Value
g0 (MeV)
12.928 ± 1.000
MX (GeV)
4.221 ± 0.001
0 (GeV)
0.000 ± 0.0002
h1
0.0004 ± 0.0008
h2
0.103 ± 0.008
h3
−0.026 ± 0.002
gD0 D∗−π+
603.980 ± 122.580
ghcπ+π−
197.150 ± 71.981
gψ(2S)π+π−
31.475 ± 21.230
gωχc0
0.002 ± 0.0003
gJ/ψη (GeV−2)
0.0003 ± 0.0001
gD∗+
s
D∗−
s
(GeV−2)
1.112 ± 0.002
gD+ D−(GeV−2)
0.004 ± 0.0002
4040 (GeV)
0.090 ± 0.014
4160 (GeV)
0.080 ± 0.014
4415 (GeV)
0.082 ± 0.004 Ref. [27]. However, we believe this scenario does not have
much chance to be physically correct, since there is no rea-
son a priori to exclude the couplings between X(4260)
and these states. We may conclude, in the most conserva-
tive situation, one still get a leptonic width well above 102
eV, which is compatible with the upper limit 580 eV [44]
obtained by reanalyzing BESII R-scan data [45,46]. If tak-
ing the D∗+
s
D∗−
s
data into account, the leptonic width will
easily exceed 1 keV. Inaddition,thebranchingratioof X(4260) →J/ψπ+π−
isdeterminedtobeO(10−4),sothattheproduct(X(4260) →
e+e−) × B(X(4260) →J/ψπ+π−) is about O(10−1) eV,
which is smaller than the old data [1] measured by BABAR
Collaboration in 2005. 3.3 Summary and discussions on numerical fits Yet the product (X(4260) →
e+e−) × B(X(4260) →J/ψπ+π−) is the same order of
magnitude as those of other charmonia [4] (such as ψ(4040)
and ψ(4160)) to J/ψπ+π−near 4 GeV. It may further imply
that X(4260) serves as a conventional charmonium state. The pole location of X(4260) propagator with D∗+
s
D∗−
s
coupling is also searched for in order to achieve a better
understanding on the nature of X(4260). Since D ¯D, D ¯D∗
and D∗¯D∗channels play a vital role and the threshold of
D∗+
s
D∗−
s
channel is close to the location of X(4260), the
complicated multi-sheets structure of the complex plane is
simplified as 4 sheets defined in Table 3. It is noticed that
there are two poles on sheet II and III as shown in Table 4. The pole width is a bit smaller than the line shape width
excluding the coupling to D∗+
s
D∗−
s . We think this is well
understood and be a typical situation in p waves, when the
pole lies below the second threshold. Since one partial width
is ∝k3 where k is the 2nd channel momentum, below the
second threshold the k2 factor provides an additional minus
sign. However, it should be emphasized that the appearance
of two poles, is not a manifestation of the “elementariness” of Fig. 5 Given the complex coherent background in D∗+D∗−process,
the fit result of the D∗+D∗−cross section. The blue dots come from
Belle results [25]. The solid curve is the projection from the best fit. The
dashed curves show the individual components of ψ(4040), ψ(4160),
X(4260) and ψ(4415), respectively. The contribution of the complex
background is not presented Table 3 Definition of the four Riemann sheets with D∗+
s
D∗−
s
channel
and the D ¯D, D ¯D∗and D∗¯D∗channels as a whole
Sheet I
Sheet II
Sheet III
Sheet IV
D ¯D + D ¯D∗+ D∗¯D∗
+
−
−
+
D∗+
s
D∗−
s
+
+
−
−
Table 4 Pole positions of X(4260). References 1. B. Aubert et al., (BABAR Collaboration). Phys. Rev. Lett. 95,
142001 (2005) 2. T.E.Coanetal.,(CLEOCollaboration).Phys.Rev.Lett. 96,162003
(2006) 3. C.Z. Yuan et al., (Belle Collaboration). Phys. Rev. Lett. 99, 182004
(2007) 4. M. Tanabashi et al., (Particle Data Group). Phys. Rev. D 98, 030001
(2018) 5. J.P. Lees et al., (BABAR Collaboration). Phys. Rev. D 86, 051102
(2012) 6. S. Godfrey, N. Isgur, Phys. Rev. D 32, 189 (1985) 7. E.S. Swanson, Phys. Rep. 429, 243 (2006) 8. Z.Y. Zhou, Z.G. Xiao, Eur. Phys. J. A 50, 165 (2014) 9. Z.Y. Zhou, Z.G. Xiao, Phys. Rev. D 84, 034023 (2011) 10. S.R. Xue, H.J. Jing, F.K. Guo, Q. Zhao, Phys. Lett. B 779, 402–408
(2018) 11. W. Qin, S.R. Xue, Q. Zhao, Phys. Rev. D 94, 054035 (2016) 12. G.J. Ding, Phys. Rev. D 79, 014001 (2009) 13. F. Close, C. Downum, Phys. Rev. Lett. 102, 242003 (2009) 14. M.T. Li, W.L. Wang, Y.B. Dong, Z.Y. Zhang, arXiv:1303.4140 15. Y.D. Chen, C.F. Qiao, P.N. Shen, Z.Q. Zeng, Phys. Rev. D 88,
114007 (2013) 16. J.Z. Wang, D.Y. Chen, X. Liu, T. Matsuki, Phys. Rev. D 99, 114003
(2019) 17. Q. Huang, D.Y. Chen, X. Liu, T. Matsuki, Eur. Phys. J. C 79, 613
(2019) 18. D.Y. Chen, X. Liu, T. Matsuki, Eur. Phys. J. C 78, 136 (2018) 19. B.Q. Li, K.T. Chao, Phys. Rev. D 79, 094004 (2009) 20. F.J. Llanes-Estrada, Phys. Rev. D 72, 031503 (2005) Acknowledgements We are grateful to illuminating discussion with
Kuang-ta Chao, Ce Meng and Chang-Zheng Yuan at early stage of this
work. This work is supported in part by National Natural Science Foun-
dations of China (NSFC) under Contract nos. 10925522, 11021092;
and China Postdoctoral Science Foundation under Contract Number
2020M680500. 21. E. Kou, O. Pene, Phys. Lett. B 631, 164–169 (2005) 22. S.L. Zhu, Phys. Lett. B 625, 212 (2005) 23. S. Coito, F. Giacosa, Acta Phys. Pol. B. 51(8), 1713–1737 (2020) 23. S. Coito, F. Giacosa, Acta Phys. Pol. B. 51(8 24. G. Pakhlova et al., (Belle Collaboration). Phys. Rev. D 77, 011103
(2008) 25. G. Pakhlova et al., (Belle Collaboration). Phys. Rev. Lett. 98,
092001 (2007) Data Availability Statement This manuscript has no associated data
or the data will not be deposited. [Authors’ comment: All of data in our
article are from publications, i.e. no extra data need to be deposited.] 26. B. Aubert et al., (BABAR Collaboration). Phys. Rev. 6 The pole counting criteria is originally proposed in Ref. [47], and has
been applied to the discussions of the X, Y, Z states in, for example,
Refs. [40,48–50]. 4 Conclusions g
y
Funded by SCOAP3. Studieson X(4260)resonanceplayanimportantroleindeep-
ening our understandings on exotic particles and strong inter-
actions. Ref. [27] pointed out that X(4260) is a confining
state with a very small leptonic decay width which is hard to
be understood by a simple quark model calculation. Thanks
to the new experimental data available, a correct understand-
ing gradually emerges, as we believe: a combined fit with
the “old” D+D∗−and D∗+D∗−data – even though there is
no apparent X(4260) peak showing up in these channels –
reveals that the X(4260) can have a sizable leptonic width up
to at least O(102)eV. Further the fit including the D∗+
s
D∗−
s
data can raise the value up to 1 keV. It is worth mentioning
that Ref. [51] gives the muonic width to be from 1.09 to 1.53
keV in the range from 4212.8 to 4219.4 MeV, which provides
a strong support to our results.7 In Ref. [19], which uses a
screening potential instead of a linear confining potential to
calculate the spectrum, it is estimated that a 43S1 state has a
leptonic width ∼1 keV, whereas a 33D1 state has a leptonic
width ∼50 eV. Hence the smaller e+e−(∼300 eV) obtained
in this paper may be provided by a 33D1 state (maybe a small
portion of 23D1 state as well) mixed with certain portion of
43S1 state, and the larger value estimated in this paper may
corresponds to a 43S1 state, and is probably largely renor-
malized by the D∗+
s
D∗−
s
continuum. To further determine
the accurate portion of these mixing is still an open question
awaiting more fine studies both theoretically and experimen-
tally. 3.3 Summary and discussions on numerical fits The value of √s ≡Mpole −
ipole/2 is given in unit of GeV
Sheet I
Sheet II
Sheet III
Sheet IV
−
4.218 −i0.015
4.218 −i0.009
− Table 3 Definition of the four Riemann sheets with D∗+
s
D∗−
s
channel
and the D ¯D, D ¯D∗and D∗¯D∗channels as a whole
Sheet I
Sheet II
Sheet III
Sheet IV
D ¯D + D ¯D∗+ D∗¯D∗
+
−
−
+
D∗+
s
D∗−
s
+
+
−
−
Table 4 Pole positions of X(4260). The value of √s ≡Mpole −
ipole/2 is given in unit of GeV
Sheet I
Sheet II
Sheet III
Sheet IV
−
4.218 −i0.015
4.218 −i0.009
− Table 3 Definition of the four Riemann sheets with D∗+
s
D∗−
s
channel
and the D ¯D, D ¯D∗and D∗¯D∗channels as a whole section, but with the D∗+
s
D∗−
s
data included. The fit qual-
ity is χ2/d.o. f. = 266.82/(316 −29) = 0.93, with the
leptonic width 1.119 ± 0.081 keV. Besides, the constant
width 0 describing other decay channels is raised up to
48.95 MeV, which indicates that the dominant decays are out
of J/ψπ+π−, hcπ+π−, D0D∗−π+, ψ(2S)π+π−, ωχc0,
J/ψη and D∗+
s
D∗−
s . As is mentioned above, the decay
widths of D+D−, D+D∗−and D∗+D∗−are indeed about 40
MeV, which implies that the fit programs are self-consistent. Forthefitexcludingboth D+D∗−, D∗+D∗−and D∗+
s
D∗−
s
cross sections, the parameter g0 is 4.58 MeV with e+e−=
0.048 ± 0.018 keV, which approaches the result given in 123 Page 9 of 10
83 Eur. Phys. J. C (2021) 81 :83 X(4260), since the pole counting criteria only applies when
couplings are in s waves.6 are included in the article’s Creative Commons licence, unless indi-
cated 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 permit-
ted use, you will need to obtain permission directly from the copy-
right holder. To view a copy of this licence, visit http://creativecomm
ons.org/licenses/by/4.0/. 7 We point out that the first version of the present manuscript was put
on arXiv 4 months before Ref. [51]. References D 79, 092001
(2009) 27. L.Y. Dai, M. Shi, G.Y. Tang, H.Q. Zheng, Phys. Rev. D 92, 014020
(2015) Open Access This article is licensed under a Creative Commons Attri-
bution 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, pro-
vide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article 28. M. Ablikim et al., (BESIII Collaboration). Phys. Rev. Lett. 114,
092003 (2015) 29. M. Ablikim et al., (BESIII Collaboration). Phys. Rev. Lett. 118,
092001 (2017) 30. M. Ablikim et al., (BESIII Collaboration). Phys. Rev. Lett. 118,
092002 (2017) 31. M. Ablikim et al., (BESIII Collaboration) Phys. Rev. Lett. 122,
102002 (2019) 32. M. Ablikim et al., (BESIII Collaboration) Phys. Rev. Lett. 96,
032004 (2017) 33. X.L. Wang et al., (Belle Collaboration). Phys. Rev. D 91, 112007
(2015) 7 We point out that the first version of the present manuscript was put
on arXiv 4 months before Ref. [51]. 12 3 3 83
Page 10 of 10 Eur. Phys. J. C (2021) 81 :83 34. J.P. Lees et al., (BABAR Collaboration). Phys. Rev. D 89, 111103
(2014) 42. M. Ablikim et al., (BESIII Collaboration). Phys. Rev. Lett. 111,
242001 (2013) 35. M. Ablikim et al., (BESIII Collaboration). Phys. Rev. D 93, 011102
(2016) 43. M.B. Voloshin, Phys. Rev. D 85, 034024 (2012) 44. X.H. Mo, G. Li, C.Z. Yuan, K.L. He, H.M. Hu, J.H. Hu, P. Wang,
Z.Y. Wang, Phys. Lett. B 640, 182 (2006) (
)
36. M. Ablikim et al., (BESIII Collaboration). Phys. Rev. D 99, 091103
(2019) 45. J.Z. Bai et al., (BESII Collaboration). Phys. Rev. Lett. 84, 594
(2000) 37. M. Ablikim et al., (BESIII Collaboration). Phys. Rev. D 91, 112005
(2015) 46. J.Z. Bai et al., (BES Collaboration). Phys. Rev. Lett. 88, 101802
(2002) 38. X.L. Wang, X.L. Han, C.Z. Yuan, C.P. Shen, P. Wang, Phys. Rev. D 87, 051101 (2013) 47. D. Morgan, Nucl. Phys. A 543, 632 (1992) 48. Q.F. Cao, H.R. Qi, Y.F. Wang, H.Q. Zheng, Phys. Rev. D 100,
054040 (2019) 39. W.M. Song,
Ph.D
thesis,
“Study
of
the
production
and
decay of the charmed hadron at BESIII”, Beijing IHEP,
CAS, 2015. http://www.wanfangdata.com.cn/details/detail.do?_
type=degree&id=Y2957455 49. C. Meng et al., Phys. Rev. References D 92, 034020 (2015) 50. O. Zhang, C. Meng, H.Q. Zheng, Phys. Lett. B 680, 453 (2009) 40. Q.R. Gong et al., Phys. Rev. D 94, 114019 (2016) 51. M. Ablikim et al. (BESIII Collaboration), arXiv:2007.12872 41. Q.R. Gong, J.L. Pang, Y.F. Wang, H.Q. Zheng, Eur. Phys. J. C 78,
276 (2018) 123 123
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2-Complex Symmetric Composition Operators on H2
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Axioms
| 2,022
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| 5,870
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axioms
Article
2-Complex Symmetric Composition Operators on H 2
Lian Hu, Songxiao Li *
and Rong Yang
Institute of Fundamental and Frontier Sciences, University of Electronic Science and Technology of China,
Chengdu 610054, China; 202111210323@std.uestc.edu.cn (L.H.); 201921210221@std.uestc.edu.cn (R.Y.)
* Correspondence: lisongxiao@uestc.edu.cn or jyulsx@163.com
Abstract: In this paper, we study 2-complex symmetric composition operators with the conjugation
J, defined by J f (z) = ( f (z̄)), on the Hardy space H 2 . More precisely, we obtain the necessary and
sufficient condition for the composition operator Cφ to be 2-complex symmetric with J when φ is an
automorphism of D. We also characterize 2-complex symmetric with J when φ is a linear fractional
self-map of D.
Keywords: composition operator; m-complex symmetric; normal
MSC: 30H10; 47B38
1. Introduction
Throughout this paper, H and B( H ) will always denote a separable complex Hilbert
space and the set of all continuous linear operators on H, respectively.
Definition 1. An operator C : H → H is said to be a conjugation on H if it is
(a)
Citation: Hu, L.; Li, S.; Yang, R.
2-Complex Symmetric Composition
Operators on H 2 . Axioms 2022, 11,
(b)
(c)
anti-linear or conjugate-linear: C (αx + βy) = ᾱC ( x ) + β̄C (y), for all α, β ∈ C and
x, y ∈ H,
isometric: kCx k = k x k, for all x ∈ H,
involutive: C2 = Id , where Id is an identity operator.
358. https://doi.org/10.3390/
axioms11080358
Academic Editor: Palle E. T.
Jorgensen
It is easy to check that ( J f )(z) = f (z̄) is a conjugation on the Hardy space H 2 .
Definition 2. For a conjugation C on H, an operator T ∈ B( H ) is said to be complex symmetric
(complex symmetric with C or a C-symmetric operator) if T = CT ∗ C.
Received: 24 June 2022
Accepted: 21 July 2022
Published: 23 July 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affiliations.
The class of complex symmetric operators includes all normal operators, binormal
operators, Hankel operators, compressed Toeplitz operators and Volterra integration operators. The study of complex symmetric operators was initiated by Garcia, Putinar, and
Wogen in [1–4]. See [5–14] for more results on complex symmetric operators.
Definition 3. Let m be a positive integer and T ∈ B( H ). T is said to be a m-complex symmetric
operator (m-complex symmetric with C) if there exists a conjugation C such that
m
∑ (−1)
Copyright: © 2022 by the authors.
j =0
m− j
m
j
T ∗ j CT m− j C = 0.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
The above definition was introduced by Chō, Ko, and Lee in [15]. When m = 2,
we obtain
CT 2 C − 2T ∗ CTC + T ∗ 2 = 0,
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
which is equivalent to
4.0/).
Axioms 2022, 11, 358. https://doi.org/10.3390/axioms11080358
CT 2 − 2T ∗ CT + T ∗ 2 C = 0.
https://www.mdpi.com/journal/axioms
Axioms 2022, 11, 358
2 of 12
It is clear that 1-complex symmetric operator is just the complex symmetric operator.
From [15], we see that all complex symmetric operators are 2-complex symmetric operators.
Thus, the set of all 2-complex symmetric operators is larger than the set of all complex
symmetric operators. In [16], the authors studied m-complex symmetric weighted shifts on
Cn . We refer the reader to [15–18] for more results about m-complex symmetric operators.
Inspired by these papers, in this paper, we study 2-complex symmetric composition
operators, induced by linear fractional self-maps of D, with J on the Hardy space H 2 . When
the symbol φ is an automorphism of D, we show that the composition operator Cφ is
2-complex symmetric with J if and only if Cφ is normal. Furthermore, we also characterize
2-complex symmetric composition operators with J on H 2 when the induced maps are
linear fractional self-maps of D.
2. Preliminaries
Let D and ∂D be the open unit disk C and the unit circle in the complex plane,
respectively. Let H (D) be the set of all analytic functions on D. The Hardy space H 2 (D) is
the space of all f ∈ H (D) such that
1
0≤r <1 2π
k f k2 = sup
Z 2π
0
| f (reiθ )|2 dθ < ∞.
The space H 2 (D) is a reproducing kernel Hilbert space, that is, for each w ∈ D and
f ∈ H 2 (D), there is a unique function Kw such that
h f , K w i = f ( w ),
where Kw (z) = 1−1wz is said to be the reproducing kernel at w. For g ∈ L∞ (∂D), the Toeplitz
operator Tg is defined as Tg f = P( f g) for f ∈ H 2 (D), where P is the orthogonal projection
of L2 onto H 2 (D). Recall that
Tg∗ Kw = f (w)Kw
for each w ∈ D and f ∈ H ∞ .
Let φ be an analytic self-map of D. Recall that the composition operator Cφ is defined by
Cφ f (z) = f (φ(z)),
f ∈ H (D), z ∈ D.
It is easy to see that Cφ∗ Kw = Kφ(w) for each w ∈ D.
3. Main Results
We begin this section with Cowen’s formula for the adjoint of a linear fractional selfaz+b
map. For a linear fractional self-map φ(z) = cz
+d , Cowen in [19] obtained the following
important formula:
Cφ∗ = Tg Cσ Th∗ ,
where a, b, c, d ∈ C, ad − bc 6= 0 and
σ(z) =
1
āz − c̄
, g(z) =
, h(z) = cz + d.
¯
−b̄z + d
−b̄z + d¯
Next, we state some lemmas which will be used in our mian results.
Lemma 1 ([20]). Let φ be an analytic self-map of D. Then, Cφ is normal if and only if φ(z) = az
with | a| ≤ 1.
az+b
Lemma 2. Let φ(z) = cz
+d be a linear fractional self-map of D. If w ∈ D satisfies āw 6 = c̄, then
the following statements hold:
(a)
JCφ2 Kw (z) = Kw̄ (φ2 (z̄)),
Axioms 2022, 11, 358
3 of 12
g(w)
(b)
Cφ∗ JCφ Kw (z) = −c̄ σ(w) Kφ(0) (z) + h
(c)
Cφ∗ 2 JKw (z) = Kφ2 (w̄) (z),
(d)
JCφ JCφ∗ JKw (z) = Kφ(w̄) (φ(z̄)),
where σ (z) =
āz−c̄
,
−b̄z+d¯
g(z) =
1
−b̄z+d¯
1
σ(w)
g(w)Kφ(σ(w)) (z),
and h(z) = cz + d.
Proof. Since J f (z) = f (z̄) and f (w) = h f , Kw i for any w ∈ D and f ∈ H 2 , we see that ( a)
and (c) hold obviously. Now, we only verify (b) and (d). For any w ∈ D with āw 6= c̄,
we obtain
Cφ∗ JCφ Kw (z) =Cφ∗ Kw̄ (φ(z̄))
c̄z + d¯
(c̄ − āw)z + (d¯ − b̄w)
!
¯ − b̄w)
c̄
(
d
c̄
1
1
=Cφ∗
+ d¯ −
− c̄
c̄ − āw
c̄ − āw
d¯ − b̄w 1 − dāw
¯ − b̄w z
c̄
c̄(d¯ − b̄w)
1
∗
¯
(z)
=
C K0 ( z ) + d −
C∗ K
c̄ − āw φ
c̄ − āw
d¯ − b̄w φ σ(w)
!
g(w)
1
= − c̄
(z) + h
K
g(w)Kφ(σ(w)) (z).
σ ( w ) φ (0)
σ(w)
=Cφ∗
Clearly,
JCφ JCφ∗ JKw (z) = JCφ JCφ∗ Kw̄ (z) = JCφ JKφ(w̄) (z)
= JCφ Kφ(w̄) (z) = JKφ(w̄) (φ(z))
= Kφ(w̄) (φ(z̄)).
The proof is complete.
Lemma 3. Let φ be an analytic self-map of D. If Cφ is normal, then Cφ∗ and Cφ is 2-complex
symmetric with J.
Proof. Assume that Cφ is normal. From Corollary 3.10 in [21], we see that Cφ is complex
symmetric with J. Hence, Cφ is 2-complex symmetric with J. Write φ(z) = az with | a| ≤ 1
from Lemma 1. We notice that T ∗ is 2-complex symmetric with J if and only if
T ∗ 2 J − 2JT JT ∗ J + JT 2 = 0.
In view of Lemma 2, set σ (z) = āz, g(z) = h(z) = 1, we have
Cφ∗ 2 JKw (z) = Kφ2 (w̄) (z) =
1
,
1 − ā2 wz
JCφ JCφ∗ JKw (z) = Kφ(w̄) (φ(z̄)) =
and
JCφ2 Kw (z) = Kw̄ (φ2 (z̄)) =
1
1 − ā2 wz
1
1 − wφ2 (z̄)
=
1
.
1 − ā2 wz
Thus, we obtain that Cφ∗ 2 J − 2JCφ JCφ∗ J + JCφ2 = 0, which means that Cφ∗ is 2-complex
symmetric with J.
Axioms 2022, 11, 358
4 of 12
Lemma 4. Let φ be an analytic self-map of D. If Cφ is 2-complex symmetric with J, then
2φ2 (0) − 4φ(0) + φ(0)φ2 (0) = 0.
Proof. Since f (w) = h f , Kw i for any w ∈ D and f ∈ H 2 , we have
h JCφ2 K0 , K 1 i = h JK0 , K 1 i = 1,
2
2
hCφ∗ JCφ K0 , K 1 i = hCφ∗ K0 , K 1 i = hKφ(0) , K 1 i =
2
2
2
2
2 − φ (0)
and
hCφ∗ 2 JK0 , K 1 i = hCφ∗ 2 K0 , K 1 i = hKφ2 (0) , K 1 i =
2
2
2
2
2 − φ2 (0)
.
By the assumption that Cφ is 2-complex symmetric with J, we obtain that
1−
4
2 − φ (0)
+
2
2 − φ2 (0)
= 0.
By a simple calculation, we obtain the desired result.
The following result gives a necessary and sufficient condition for Cφ to be 2-complex
symmetric with J when φ is an automorphism of D.
Theorem 1. Let φ be an automorphism of D. Then, Cφ is 2-complex symmetric with J if and only
if Cφ is normal.
Proof. Assume that Cφ is normal. From Lemma 3, we see that Cφ is 2-complex symmetric
with J.
z
Now, suppose that φ(z) = λ 1a−−āz
with |λ| = 1, a ∈ D and Cφ is 2-complex symmetric
with J. Then, σ (z) =
−λ̄z+ a ,
−λaz+1
g(z) =
φ2 (z) =
1
−λaz+1
and h(z) = − āz + 1. Noting that
λ(λz − λa) − λa( āz − 1)
,
ā(λz − λa) − ( āz − 1)
then, for λ̄w 6= a, Lemma 2 gives that
JCφ2 Kw (z) = Kw̄ (φ2 (z̄)) =
Cφ∗ JCφ Kw (z)
a(λ̄z − λa) − ( az − 1)
,
a(λ̄z − λa) − ( az − 1) − w[λ̄(λ̄z − λa) − λa( az − 1)]
(1)
!
g(w)
1
=a
K
(z) + h
g(w)Kφ(σ(w)) (z)
σ ( w ) φ (0)
σ(w)
=
a
λ̄w(| a|2 − 1)
+
(−λ̄w + a)(1 − λaz) (−λ̄w + a)(−λaw + 1)
·
(2)
−λaw + 1 + aλ̄w − a2
−λaw + 1 + aλ̄w − a2 − (λ̄2 w − λ̄2 ā2 w − λ̄a + λa)z
and
Cφ∗ 2 JKw (z) = Kφ2 (w̄) (z) =
a(λ̄w − λa) − ( aw − 1)
a(λ̄w − λa) − ( aw − 1) − [λ̄(λ̄w − λa) − λa( aw − 1)]z
for any w, z ∈ D. Taking w = 0 in (1)–(3), we have
JCφ2 K0 (z) = 1,
Cφ∗ JCφ K0 (z) =
1
,
1 − λaz
(3)
Axioms 2022, 11, 358
5 of 12
and
Cφ∗ 2 JK0 (z) =
−λ̄| a|2 + 1
−λ̄| a|2 + 1 + (λ̄2 ā − λa)z
for any z ∈ D. Since Cφ is 2-complex symmetric with J, we obtain
1 − λ̄| a|2
1 + λaz
=
.
1 − λaz
−λ̄| a|2 + 1 + (λ̄2 ā − λa)z
(4)
Calculating and noting that the coefficient of z2 must be 0, we obtain that λ2 a2 (λ − 1) = 0.
Since λ and a are non-zero, then λ = 1. Hence, (4) becomes
1 + āz
= 1,
1 − āz
which implies that āz = 0 for all z ∈ D. Thus, a = 0. Hence, φ(z) = −λz with |λ| = 1.
Lemma 1 gives that Cφ is normal. The proof is complete.
4. Linear Fractional Self-Maps
In this section, we first consider 2-complex symmetric composition operators with J
which are induced linear fractional self-maps with φ(0) = 0. In the following, we obtain a
sufficient and necessary condition for this case.
az+b
∗
Theorem 2. Let φ(z) = cz
+d be a linear fractional self-map of D and φ (0) = 0. Then, Cφ (Cφ ) is
2-complex symmetric with J if and only if Cφ is normal.
Proof. Assume first that Cφ is normal. Lemma 3 gives that Cφ (Cφ∗ ) is 2-complex symmetric
with J.
Conversely, suppose that Cφ is 2-complex symmetric with J. Since a 6= 0 and φ(0) = 0,
set φ(z) = szz+t , where s = ac and t = da satisfy |t| ≥ |s| + 1. Then, σ (z) = z−t̄ s̄ , g(z) = 1t̄
and h(z) = sz + t. If s = 0, then φ(z) = 1t z. Therefore, Lemma 1 gives that Cφ is normal.
Now, we suppose that s 6= 0. After a calculation, we have that
φ2 (z) =
z
(s + st)z + t2
and
z̄ − s
1
φ(σ(z)) =
, h
sz̄ − s2 + t2
σ(z)
!
=
st
+ t.
z̄ − s
For any w, z ∈ D with w 6= s̄, employing Lemma 2, we obtain that
JCφ2 Kw (z) = Kw̄ (φ2 (z̄)) =
Cφ∗ JCφ Kw (z)
1
1−
zw
s̄z + stz + t̄2
=
(s̄ + st)z + t̄2
,
(s̄ + st)z + t̄2 − wz
!
g(w)
1
= − s̄
K
(z) + h
g(w)Kφ(σ(w)) (z)
σ ( w ) φ (0)
σ(w)
−s̄
st
1
1
=
+
+ t̄ ·
s̄
w − s̄
w − s̄
t̄ 1 − s̄w−ws̄−
2 + t̄2 z
=
(5)
(6)
s̄
wt̄
s̄w − s̄2 + t̄2
+
,
s̄ − w
(w − s̄)t̄ s̄w − s̄2 + t̄2 − (w − s̄)z
and
Cφ∗ 2 JKw (z) = Kφ2 (w̄) (z) =
1
1−
zw
s̄w + stw + t̄2
=
(s̄ + st)w + t̄2
.
(s̄ + st)w + t̄2 − wz
(7)
Axioms 2022, 11, 358
6 of 12
Since Cφ is 2-complex symmetric with J, we obtain from (5)–(7) that
(s̄ + st)z + t̄2
(s̄ + st)w + t̄2
+
2
(s̄ + st − w)z + t̄
(s̄ + st)w + t̄2 − wz
=
2s̄
2wt̄
s̄w − s̄2 + t̄2
+
s̄ − w
(w − s̄)t̄ s̄w − s̄2 + t̄2 − (w − s̄)z
for any w, z ∈ D with w 6= s̄, which gives that
[(s̄ + st)z + t̄2 ][(s̄ + st)w + t̄2 − wz] + [(s̄ + st)w + t̄2 ][(s̄ + st − w)z + t̄2 ]
[(s̄ + st − w)z + t̄2 ][(s̄ + st)w + t̄2 − wz]
2st(w − s̄)[s̄w − s̄2 + t̄2 − (w − s̄)z] + 2wt̄(s̄ − w)(s̄w − s̄2 + t̄2 )
=
−(w − s̄)2 t̄[s̄w − s̄2 + t̄2 − (w − s̄)z]
(8)
for any w, z ∈ D with w 6= s̄. Taking w = 0 in (8), then
t̄2 [(s̄ − st)z + t̄2 ] + t̄2 [(s̄ + st)z + t̄2 ]
2s̄2 t̄(−s̄2 + t̄2 + s̄z)
= 2.
=
s̄2 t̄(−s̄2 + t̄2 + s̄z)
t̄2 [(s̄ + st)z + t̄2 ]
Therefore, (s̄ + st)z + t̄2 = (s̄ − st)z + t̄2 for all z ∈ D. Since s 6= 0, then 1 + t = 1 − t.
Hence, t = 0, a contradiction. Thus, the hypothesis is not true that is s = 0.
Now, assume that Cφ∗ is 2-complex symmetric with J. We also assume that s 6= 0. By
Lemma 2, we have
JCφ JCφ∗ JKw (z) = Kφ(w̄) (φ(z̄)) =
1
1−
w
s̄w+t̄
·
z
s̄z+t̄
(s̄w + t̄)(s̄z + t̄)
=
(s̄w + t̄)(s̄z + t̄) − wz
(9)
for any w, z ∈ D. Since Cφ∗ is 2-complex symmetric with J, we obtain from (5), (7),
and (9) that
[(s̄ + st)z + t̄2 ][(s̄ + st)w + t̄2 − wz] + [(s̄ + st)w + t̄2 ][(s̄ + st − w)z + t̄2 ]
[(s̄ + st − w)z + t̄2 ][(s̄ + st)w + t̄2 − wz]
2(s̄w + t̄)(s̄z + t̄)
=
(s̄w + t̄)(s̄z + t̄) − wz
(10)
for any w, z ∈ D with w 6= s̄. Taking w = 0 in (10), we also have that
t̄2 [(s̄ − st)z + t̄2 ] + t̄2 [(s̄ + st)z + t̄2 ]
= 2.
t̄2 [(s̄ + st)z + t̄2 ]
The other arguments are similar to the case of Cφ . Then, we obtain the desired result.
The proof is complete.
We now consider 2-complex symmetric composition operators with J which are
induced linear fractional self-maps with c = 0.
Lemma 5 ([22]). Let φ(z) =
Cφ =
Cξ∗ Tτ∗ ,
where ξ (z) =
az+b
cz+d
āz
−b̄z+d¯
be a linear fractional self-mapof D such that c = 0. Then,
and τ (z) =
d¯
.
−b̄z+d¯
In the next two results, we give the necessary and sufficient conditions for Cφ and its
adjoint operator Cφ∗ to be 2-complex symmetric with J when c = 0.
Axioms 2022, 11, 358
7 of 12
az+b
Theorem 3. Let φ(z) = cz
+d be a linear fractional self-map of D such that c = 0. Then, Cφ is
2-complex symmetric with J if and only if Cφ is normal.
Proof. Assume first that Cφ is normal. Lemma 3 gives that Cφ is 2-complex symmetric
with J.
Conversely, suppose that Cφ is 2-complex symmetric with J. Since c = 0, then
m̄z
φ(z) = mz + n, where m = da and n = db satisfy |m| + |n| ≤ 1. Let ξ (z) = −n̄z
+1 and
1
τ (z) = −n̄z+1 . Note that
φ2 (z) = m2 z + mn + n
and
ξ 2 (z) =
m̄2 z
m2 z̄ − n2 z̄ + n
−n̄z + 1
, φ(ξ (z)) =
, τ (ξ (z)) =
.
−mnz − n̄z + 1
−nz̄ + 1
−mnz − n̄z + 1
Lemmas 2 and 5 give that
JCφ2 Kw (z) = JCξ∗ Tτ∗ Cξ∗ Tτ∗ Kw (z) = Jτ (w)Cξ∗ Tτ∗ Kξ (w) (z)
= Jτ (w)τ (ξ (w))Kξ 2 (w) (z) = τ (w)τ (ξ (w))Kξ
1
−n̄w + 1
·
−n̄w + 1 −mnw − n̄w + 1 1 −
1
,
=
−mnw − n̄w + 1 − m̄2 wz
=
2 (w)
(z)
1
m̄2 w
−mnw − n̄w + 1 z
(11)
Cφ∗ JCφ Kw (z) = Cφ∗ JCξ∗ Tτ∗ Kw (z) = Cφ∗ Jτ (w)Kξ (w) (z) = Cφ∗ τ (w)Kξ (w) (z̄)
= Cφ∗ τ (w)Kξ (w) (z) = τ (w)Kφ(ξ (w)) (z)
1
1
·
−n̄w + 1 1 − m̄2 w − n̄2 w + n̄ z
−n̄w + 1
1
=
−n̄w + 1 − (m̄2 w − n̄2 w + n̄)z
(12)
=
and
Cφ∗ 2 JKw (z) = Kφ2 (w̄) (z) =
1
1 − (m̄2 w + mn + n̄)z
(13)
for any w, z ∈ D. Since Cφ is 2-complex symmetric with J, then we obtain from (11)–(13) that
1
1
+
2
2
−mnw − n̄w + 1 − m̄ wz 1 − (m̄ w + mn + n̄)z
2
=
2
−n̄w + 1 − (m̄ w − n̄2 w + n̄)z
(14)
for any w, z ∈ D. Taking w = 0 in (14), we have that
1+
1
2
=
1 − (mn + n̄)z
1 − n̄z
for any z ∈ D. Then,
2 − (mn + n̄)z
2
=
.
1 − (mn + n̄)z
1 − n̄z
Noting that the coefficient of z2 must be 0, then n2 (m + 1) = 0, which means that
n = 0 or m = −1. Similarly, noting that the coefficient of z must be also 0, then n = mn,
which means that n = 0 or m = 1. Therefore, n = 0. This implies that φ(z) = mz with
|m| ≤ 1. Lemma 1 gives that Cφ is normal.
Axioms 2022, 11, 358
8 of 12
az + b
∗
Theorem 4. Let φ(z) = cz
+ d be a linear fractional self-map of D such that c = 0. Then, Cφ is
2-complex symmetric with J if and only if Cφ is normal.
Proof. Since c = 0, we can set φ(z) = mz + n where m =
1
m̄z
Let ξ (z) = −n̄z
+1 and τ ( z ) = −n̄z+1 . Noting that
a
d
and n =
b
d
satisfy |m| + |n| ≤ 1.
φ2 (z) = m2 z + mn + n,
and
τ (φ(z̄)) =
1
m̄(m̄z + n̄)
, ξ (φ(z̄)) =
.
−n̄(m̄z + n̄) + 1
−n̄(m̄z + n̄) + 1
Using Lemma 5 and Theorem 3, we obtain that
JCφ2 Kw (z) = JCξ∗ Tτ∗ Cξ∗ Tτ∗ Kw (z) =
1
,
−mnw − n̄w + 1 − m̄2 wz
(15)
JCφ JCφ∗ JKw (z) = JCξ∗ Tτ∗ Kφ(w̄) (z)
= Jτ (φ(w̄))Kξ (φ(w̄)) (z) = τ (φ(w̄))K
=
1
·
−n̄(m̄w + n̄) + 1 1 −
=
1
1 − (m̄w + n̄)(m̄z + n̄)
ξ (φ(w̄))
(z)
1
(16)
m̄(m̄w+n̄)z
−n̄(m̄w+n̄)+1
and
Cφ∗ 2 JKw (z) = Kφ2 (w̄) (z) =
1
1 − (m̄2 w + mn + n̄)z
(17)
for any w, z ∈ D. Since Cφ∗ is 2-complex symmetric with J, then we obtain from (15)–(17) that
1
1
2
+
=
1 − (m̄w + n̄)(m̄z + n̄)
−mnw − n̄w + 1 − m̄2 wz 1 − (m̄2 w + mn + n̄)z
for any w, z ∈ D. Taking w = 0, we get
2 − (mn + n̄)z
2
=
.
1 − (mn + n̄)z
1 − n̄(m̄z + n̄)
Noting that the coefficient of constant term must be 0, then n̄2 = 0, which means that
n = 0. Thus, φ(z) = mz with |m| ≤ 1. Lemma 1 assures that Cφ is normal.
Conversely, assume that Cφ is normal. Lemma 3 gives that Cφ∗ is 2-complex symmetric
with J.
Furthermore, we prove that there is no 2-complex symmetric composition operators
with J which are induced linear fractional self-maps with a = 0 and φ(0) 6= 0.
Lemma 6. Let φ : D → D be a constant function. Then, Cφ (Cφ∗ ) is 2-complex symmetric with J
if and only if φ(z) ≡ 0.
Proof. The sufficiency is obvious.
Now, we assume that φ(z) ≡ c for some c ∈ D and Cφ is 2-complex symmetric with J.
Then, we obtain that
JCφ2 Kw (z) = JKw (φ2 (z)) = J
1
1
=
,
1 − w̄c
1 − wc̄
Axioms 2022, 11, 358
9 of 12
Cφ∗ JCφ Kw (z) = Cφ∗ JKw (φ(z)) =
1
1
C ∗ K0 ( z ) =
,
1 − wc̄ φ
(1 − wc̄)(1 − c̄z)
Cφ∗ 2 JKw (z) = Kφ2 (w̄) (z) =
1
1 − c̄z
and
JCφ JCφ∗ JKw (z) = Kφ(w̄) (φ(z̄)) =
1
1 − c̄2
for any w, z ∈ D. Since Cφ is 2-complex symmetric with J, we obtain
1
2
1
−
+
=0
1 − wc̄ (1 − wc̄)(1 − c̄z) 1 − c̄z
for any w, z ∈ D. By a simple calculation, we see that c = 0.
Assume that Cφ∗ is 2-complex symmetric with J. Similarly, we have
2
1
1
−
+
=0
2
1 − wc̄ 1 − c̄
1 − c̄z
for any w, z ∈ D. Therefore, c = 0. The proof is complete.
az+b
Theorem 5. Let φ(z) = cz
+d be a linear fractional self-map of D such that a = 0 and φ (0) 6 = 0.
Then, Cφ is not 2-complex symmetric with J.
Proof. Since a = 0 and φ(0) 6= 0, we set φ(z) = mz1+n , where m = bc and n = db . Then,
m̄
1
1
σ (z) = −−z+
n̄ , g ( z ) = −z+n̄ and h ( z ) = mz + n. When m = 0, φ ( z ) = n 6 = 0, then Lemma 6
gives that Cφ is not 2-complex symmetric with J. Now, we assume that m 6= 0, n 6= 0 and
Cφ is 2-complex symmetric with J. Note that
mz + n
1
φ2 (z) =
, h
m + mnz + n2
σ(z)
!
= z̄, φ(σ(z)) =
−z̄ + n
.
−m2 − nz̄ + n2
Lemma 2 gives that
JCφ2 Kw (z) = Kw̄ (φ2 (z̄)) =
Cφ∗ JCφ Kw (z)
1
+n̄
1 − w m̄+m̄z
mnz+n̄2
=
m̄ + mnz + n̄2
,
m̄ + mnz + n̄2 − (m̄z + n̄)w
!
g(w)
1
K
(z) + h
= − m̄
g(w)Kφ(σ(w)) (z)
σ ( w ) φ (0)
σ(w)
w
= K φ (0) ( z ) +
K
(z)
−w + n̄ φ(σ(w))
1
1
w
=
·
+
w+n̄
1 − n̄z
−w + n̄ 1 − −m̄−
2 − n̄w + n̄2 z
=
(18)
(19)
n̄
w
−m̄2 − n̄w + n̄2
+
·
n̄ − z −w + n̄ −m̄2 − n̄w + n̄2 − (−w + n̄)z
and
Cφ∗ 2 JKw (z) = Kφ2 (w̄) (z) =
1
+n
1 − z m̄ +m̄w
mnw + n̄2
=
m̄ + mnw + n̄2
m̄ + mnw + n̄2 − (m̄w + n̄)z
(20)
for any w, z ∈ D. Since Cφ is 2-complex symmetric with J, we obtain from (18)–(20) that
Axioms 2022, 11, 358
10 of 12
m̄ + mnw + n̄2
m̄ + mnz + n̄2
+
2
m̄ + mnz + n̄ − (m̄z + n̄)w m̄ + mnw + n̄2 − (m̄w + n)z
n̄
w
−m̄2 − n̄w + n̄2
=2
+
·
n̄ − z −w + n̄ −m̄2 − n̄w + n̄2 − (−w + n̄)z
(21)
for any w, z ∈ D. Taking w = 0 in (21), we have that
(−n̄z + m̄ + n̄2 ) + (m̄ + n̄2 )
2n̄
=
2
n̄ − kz
−n̄z + m̄ + n̄
for any z ∈ D. Thus, the coefficient of z2 must be 0. This implies that n = 0, which is a
contradiction. The proof is complete.
In the remainder of this paper, we consider 2-complex symmetric composition operators with J which are induced linear fractional self-maps with a 6= 0, c 6= 0 and φ(0) 6= 0
and obtain that all composition operators are not 2-complex symmetric with J.
az+b
Theorem 6. Let φ(z) = cz
+d be a linear fractional self-map of D such that a 6 = 0, c 6 = 0 and
φ(0) 6= 0. Then, Cφ is not 2-complex symmetric with J.
Proof. We prove it by contradiction. Assume that Cφ is 2-complex symmetric with J. Since
+n
a
b
c
a 6= 0, c 6= 0 and φ(0) 6= 0, set φ(z) = mz
sz+1 , where m = d , n = d and s = d . Then,
1
−s̄
σ (z) = −m̄z
n̄z+1 , g ( z ) = −n̄z+1 and h ( z ) = sz + 1. After a calculation, we obtain
(m2 + ns)z + mn + n
φ2 (z) =
,
(ms + s)z + ns + 1
and
φ(σ (z)) =
h
1
!
σ(z)
=
(m − ns)z̄
mz̄ − s
(m2 − n2 )z̄ + n − ms
.
(ms − n)z̄ + 1 − s2
Lemma 2 gives that
JCφ2 Kw (z) = Kw̄ (φ2 (z̄)) =
1
(m̄2 + ns)z + mn + n̄
1 − w (ms + s̄)z + ns + 1
(22)
(ms + s̄)z + ns + 1
=
,
(ms + s̄)z + ns + 1 − [(m̄2 + ns)z + mn + n̄]w
Cφ∗ JCφ Kw (z)
!
g(w)
1
= − s̄
K
(z) + h
g(w)Kφ(σ(w)) (z)
σ ( w ) φ (0)
σ(w)
=
=
−s̄
1
1
(m̄ − ns)w
+
·
m̄w − s̄ 1 − n̄z
m̄w − s̄ −n̄w + 1 1 −
1
(m̄2 −n̄2 )w−ms+n̄
z
(ms−n̄)w+1−s̄2
(23)
−s̄
(m̄ − ns)w
+
(m̄w − s̄)(1 − n̄z) (m̄w − s̄)(−n̄w + 1)
(ms − n̄)w + 1 − s̄2
·
(ms − n̄)w + 1 − s̄2 − [(m̄2 − n̄2 )w − ms + n̄]z
and
Cφ∗ 2 JKw (z) = Kφ2 (w̄) (z) =
1
(m̄2 + ns)w + mn + n̄
1 − z (ms + s̄)w + ns + 1
(ms + s̄)w + ns + 1
=
(ms + s̄)w + ns + 1 − [(m̄2 + ns)w + mn + n̄]z
(24)
Axioms 2022, 11, 358
11 of 12
for any w, z ∈ D. Taking w = 0, then we obtain from (22)–(24) that
JCφ2 K0 (z) = 1, Cφ∗ JCφ K0 (z) =
and
Cφ∗ 2 JK0 (z) =
1
1 − n̄z
ns + 1
ns + 1 − (mn + n̄)z
for any z ∈ D. Since Cφ is 2-complex symmetric with J, we obtain that
1+
ns + 1
2
=
ns + 1 − (mn + n̄)z
1 − n̄z
for any z ∈ D, which implies that
2ns + 2 − (mn + n̄)z
2
=
ns + 1 − (mn + n̄)z
1 − n̄z
for any z ∈ D. Noting that the coefficient of z2 must be 0, then we have that n̄2 (m + 1) = 0.
Since φ(0) 6= 0, then m = −1.
Noting that φ2 (z) =
(m2 + ns)z + mn + n
,
(ms + s)z + ns + 1
2φ2 (0) − 4φ(0) + φ(0)φ2 (0) =
then Lemma 4 gives that
2(mn + n)
n(mn + n)
− 4n +
= 0.
ns + 1
ns + 1
By a simple calculation, we see that 2m − 4ns + mn + n = 2. Since m = −1, we obtain
that ns = −1. Therefore, m − ns = 0, which means that φ is not a linear fractional self-map
of D, a contradiction. The proof is complete.
5. Conclusions
In this paper, we obtained some characterizations for composition operators Cφ to
be 2-complex symmetric with respect to the conjugation J on the Hardy space H 2 . To
be specific, in Theorem 1, we obtain the necessary and sufficient condition for Cφ to be
2-complex symmetric with J when φ is an automorphism of D. Next, we discuss 2-complex
symmetric composition operators Cφ which were induced by linear fractional self-maps of
D with four different forms. The necessary and sufficient conditions for Cφ to be 2-complex
symmetric with J in the first two cases are given in Theorems 2 and 3, respectively. In
Theorems 5 and 6, we obtain that there is no 2-complex symmetric composition operators
with J in the latter two cases.
Author Contributions: Conceptualization, S.L.; investigation, S.L., L.H. and R.Y.; writing—original
draft preparation, L.H. and R.Y.; writing—review and editing, S.L. and L.H.; project administration,
S.L. 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: No data were used to support this study.
Conflicts of Interest: The authors declare that they have no conflict of interest.
References
1.
2.
3.
4.
Garcia, S.; Putinar, M. Complex symmetric operators and applications. Trans. Am. Math. Soc. 2006, 358, 1285–1315. [CrossRef]
Garcia, S.; Putinar, M. Complex symmetric operators and applications, II. Trans. Am. Math. Soc. 2007, 359, 3913–3931. [CrossRef]
Garcia, S.; Wogen, W. Complex symmetric partial isometries. J. Funct. Anal. 2009, 257, 1251–1260. [CrossRef]
Garcia, S.; Wogen, W. Some new classes of complex symmetric operators. Trans. Am. Math. Soc. 2010, 362, 6065–6077. [CrossRef]
Axioms 2022, 11, 358
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
12 of 12
Fatehi, M. Complex symmetric weighted composition operators. Complex Var. Elliptic Equ. 2019, 64, 710–720. [CrossRef]
Gupta, A.; Malhotra, A. Complex symmetric weighted composition operators on the space H12 (D). Complex Var. Elliptic Equ. 2020,
65, 1488–1500. [CrossRef]
Gao, Y.; Zhou, Z. Complex symmetric composition operators induced by linear fractional maps. Indiana Univ. Math. J. 2020, 69,
367–384. [CrossRef]
Han, K.; Wang, M. Weighted composition-differentiation operators on the Hardy space. Banach J. Math. Anal. 2021, 15, 44.
[CrossRef]
Jung, S.; Kim, Y.; Ko, E.; Lee, J. Complex symmetric weighted composition operators on H 2 (D). J. Funct. Anal. 2014, 267, 323–351.
[CrossRef]
Lim, R.; Khoi, L. Complex symmetric weighted composition operators on Hγ (D). J. Math. Anal. Appl. 2018, 464, 101–118.
[CrossRef]
Narayan, S.; Sievewright, D.; Thompson, D. Complex symmetric composition operators on H 2 . J. Math. Anal. Appl. 2016, 443,
625–630. [CrossRef]
Narayan, S.; Sievewright, D.; Tjani, M. Complex symmetric composition operators on weighted Hardy spaces. Proc. Am. Math.
Soc. 2020, 148, 2117–2127. [CrossRef]
Thompson, D.; McClatchey, T.; Holleman, C. Binormal, complex symmetric operators. Linear Mult. Algebra 2021, 69, 1705–1715.
[CrossRef]
Yao, X. Complex symmetric composition operators on a Hilbert space of Dirichlet series. J. Math. Anal. Appl. 2017, 452, 1413–1419.
[CrossRef]
Chō, M.; Ko, E.; Lee, J. On m-complex symmetric operators. Mediterr. J. Math. 2016, 13, 2025–2038. [CrossRef]
Exner, G.; Jin, J.; Jung, I.; Lee, J. On m-complex symmetric weighted shift operators on Cn . Linear Algebra Appl. 2020, 603, 130–153.
[CrossRef]
Chō, M.; Ko, E.; Lee, J. On m-complex symmetric operators, II. Mediterr. J. Math. 2016, 13, 3255–3264. [CrossRef]
Jeridi, N.; Rabaoui, R. On ( A, m)-symmetric operators in a Hilbert space. Results Math. 2019, 74, 124. [CrossRef]
Cowen, C. Composition operators on H 2 . J. Oper. Theory 1983, 9, 77–106.
Cowen, C.; MacCluer, B. Composition Operators on Spaces of Analytic Functions; Studies in Advanced Mathematics; CRC Press:
Boca Raton, FL, USA, 1995; 388p, ISBN 0-8493-8492-3.
Garcia, S.; Hammond, C. which weighted composition operators are complex symmetric? Oper. Theory Adv. Appl. 2014, 236,
171–179.
Jung, S.; Kim, Y.; Ko, E. Composition operators for which Cφ∗ Cφ and Cφ + Cφ∗ commute. Complex Var. Elliptic Equ. 2014, 59,
1608–1625. [CrossRef]
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English
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Boosting with AIDSVAX B/E Enhances Env Constant Region 1 and 2 Antibody-Dependent Cellular Cytotoxicity Breadth and Potency
|
Journal of virology
| 2,020
|
cc-by
| 16,870
|
Boosting with AIDSVAX B/E Enhances Env Constant Region 1
and 2 Antibody-Dependent Cellular Cytotoxicity Breadth and
Potency David Easterhoff,a Justin Pollara,a Kan Luo,a William D. Tolbert,l Brianna Young,k Dieter Mielke,a Shalini Jha,a
Robert J. O’Connell,c
Sandhya Vasan,b,c,j Jerome Kim,b* Nelson L. Michael,b,j Jean-Louis Excler,b,j* Merlin L. Robb,b,j
Supachai Rerks-Ngarm,e Jaranit Kaewkungwal,f Punnee Pitisuttithum,f Sorachai Nitayaphan,g Faruk Sinangil,h
James Tartaglia,i Sanjay Phogat,i* Thomas B. Kepler,d S. Munir Alam,a Kevin Wiehe,a Kevin O. Saunders,a
David C. Montefiori,a Georgia D. Tomaras,a M. Anthony Moody,a Marzena Pazgier,l Barton F. Haynes,a Guido Ferraria aDuke University, Durham, North Carolina, USA
bU.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA
cU.S. Army Medical Directorate, AFRIMS, Bangkok, Thailand
dBoston University, Boston, Massachusetts, USA
eThai Ministry of Public Health, Nonthaburi, Thailand
fMahidol University, Bangkok, Thailand
gRoyal Thai Army Component, AFRIMS, Bangkok, Thailand
hGlobal Solutions of Infectious Diseases, South San Francisco, California, USA
iSanofiPasteur, Swiftwater, Pennsylvania, USA
jThe Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, Maryland, USA
kDepartment of Biochemistry and Molecular Biology, University of Maryland, Baltimore, Maryland, USA
lInfectious Diseases Division, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA aDuke University, Durham, North Carolina, USA
bU.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA
cU.S. Army Medical Directorate, AFRIMS, Bangkok, Thailand
dBoston University, Boston, Massachusetts, USA
eThai Ministry of Public Health, Nonthaburi, Thailand
fMahidol University, Bangkok, Thailand
gRoyal Thai Army Component, AFRIMS, Bangkok, Thailand
hGlobal Solutions of Infectious Diseases, South San Francisco, California, USA
iSanofiPasteur, Swiftwater, Pennsylvania, USA
jThe Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, Maryland, USA
kDepartment of Biochemistry and Molecular Biology, University of Maryland, Baltimore, Maryland, USA
lInfectious Diseases Division, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA lInfectious Diseases Division, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA ABSTRACT
Induction of protective antibodies is a critical goal of HIV-1 vaccine de-
velopment. One strategy is to induce nonneutralizing antibodies (NNAbs) that kill
virus-infected cells, as these antibody specificities have been implicated in slowing
HIV-1 disease progression and in protection. HIV-1 Env constant region 1 and 2
(C1C2) monoclonal antibodies (MAbs) frequently mediate potent antibody-dependent
cellular cytotoxicity (ADCC), making them an important vaccine target. Here, we ex-
plore the effect of delayed and repetitive boosting of RV144 vaccine recipients with
AIDSVAX B/E on the C1C2-specific MAb repertoire. It was found that boosting in-
creased clonal lineage-specific ADCC breadth and potency. VACCINES AND ANTIVIRAL AGENTS VACCINES AND ANTIVIRAL AGENTS crossm VACCINES AND ANTIVIRAL AGENTS Boosting with AIDSVAX B/E Enhances Env Constant Region 1
and 2 Antibody-Dependent Cellular Cytotoxicity Breadth and
Potency A ligand crystal structure
of a vaccine-induced broad and potent ADCC-mediating C1C2-specific MAb showed
that it bound a highly conserved Env gp120 epitope. Thus, boosting to affinity ma-
ture these types of IgG C1C2-specific antibody responses may be one method by
which to make an improved HIV vaccine with higher efficacy than that seen in the
RV144 trial. Citation Easterhoff D, Pollara J, Luo K, Tolbert
WD, Young B, Mielke D, Jha S, O’Connell RJ,
Vasan S, Kim J, Michael NL, Excler J-L, Robb ML,
Rerks-Ngarm S, Kaewkungwal J, Pitisuttithum P,
Nitayaphan S, Sinangil F, Tartaglia J, Phogat S,
Kepler TB, Alam SM, Wiehe K, Saunders KO,
Montefiori DC, Tomaras GD, Moody MA,
Pazgier M, Haynes BF, Ferrari G. 2020. Boosting
with AIDSVAX B/E enhances Env constant
region 1 and 2 antibody-dependent cellular
cytotoxicity breadth and potency. J Virol
94:e01120-19. https://doi.org/10.1128/JVI
.01120-19. Editor Viviana Simon, Icahn School of
Medicine at Mount Sinai Editor Viviana Simon, Icahn School of
Medicine at Mount Sinai Copyright © 2020 Easterhoff et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license. IMPORTANCE Over one million people become infected with HIV-1 each year, mak-
ing the development of an efficacious HIV-1 vaccine an important unmet medical
need. The RV144 human HIV-1 vaccine regimen is the only HIV-1 clinical trial to date
to demonstrate vaccine efficacy. An area of focus has been on identifying ways by
which to improve upon RV144 vaccine efficacy. The RV305 HIV-1 vaccine regimen
was a follow-up boost of RV144 vaccine recipients that occurred 6 to 8 years after
the conclusion of RV144. Our study focused on the effect of delayed boosting in hu-
mans on the vaccine-induced Env constant region 1 and 2 (C1C2)-specific antibody
repertoire. It was found that boosting with an HIV-1 Env vaccine increased C1C2-
specific antibody-dependent cellular cytotoxicity potency and breadth. KEYWORDS HIV vaccine, antibody function
February 2020
Volume 94
Issue 4
e01120-19 KEYWORDS HIV vaccine, antibody function
February 2020
Volume 94
Issue 4
e01120-19
Journal of V KEYWORDS HIV vaccine, antibody function
February 2020
Volume 94
Issue 4
e01120-19 Journal of Virology jvi.asm.org
1 Easterhoff et al. Easterhoff et al. Journal of Virology C
D4-inducible (CD4i) epitopes within HIV-1 envelope (Env) constant regions 1 and 2
(C1C2) are targets for antibodies that mediate antibody-dependent cellular cyto-
toxicity (ADCC) (1). Boosting with AIDSVAX B/E Enhances Env Constant Region 1
and 2 Antibody-Dependent Cellular Cytotoxicity Breadth and
Potency C1C2-specific antibody epitopes have been termed cluster A (1) and
defined by two Env-targeted monoclonal antibodies (MAbs), A32 (2) and C11 (1). Structural analyses of antigen complexes formed by A32, A32-like (3–5), and C11-like (6)
MAbs indicate that these MAbs bind distinct Env epitopes. The A32 epitope involves a
discontinuous sequence within Env layers 1 and 2 of the inner domain (4, 5), while the
C11 epitope maps to the inner domain eight-stranded sandwich (6). Importantly,
both MAbs are nonneutralizing for tier 2 HIV strains but are capable of broad and
potent ADCC (1, 2). The secondary analysis of HIV-1 infection risk in RV144 (ClinicalTrials registration no. NCT00223080) indicated that ADCC in the presence of low anti-Env IgA responses
correlated with decreased HIV-1 acquisition (7). While antibodies representative of the
Env variable region 2 (V2) response inversely correlated with HIV-1 acquisition (7),
we previously demonstrated that synergy between A32-blockable C1C2-specific
MAbs and V2-specific MAbs increased ADCC potency of the V2 MAbs induced in the
RV144 trial (8). Here, we studied the effect of late boosting of RV144 vaccinees in the RV305 HIV-1
vaccine trial (ClinicalTrials registration no. NCT01435135), focusing specifically on C1C2-
specific MAb affinity maturation, ADCC potency, and ADCC breadth. We found that the
RV144 ALVAC/AIDSVAX B/E immunization regimen induced durable C1C2-specific
memory B cells and that boosting with AIDSVAX B/E could increase C1C2-specific MAb
variable heavy and variable light (VH VL) chain gene mutation frequency along with
increasing ADCC breadth and potency. (This article was submitted to an online preprint archive [9]). February 2020
Volume 94
Issue 4
e01120-19 RESULTS An A32-specific mutant protein was designed (AE.A244g120Δ11 F53S H72L V75A E106K D107H S110A Q114L) to identify A32-like MAb
responses. 19B was used as a positive control and CH65 as a negative control. (B) RV305 nonneutralizing MAbs were assayed for A32 blocking by ELISA. (C)
RV305 nonneutralizing A32-blockable MAb heavy- and light-chain gene sequence mutation frequencies were analyzed by Cloanalyst (13) and compared to
previously published RV144 heavy- and light-chain gene sequence mutation frequencies (percent nucleotide) (12). Statistical significance was determined using
a Wilcoxon rank sum test. Red bar represents the means. (D) RV305 nonneutralizing A32-blockable MAbs were assayed by ELISA for binding to AE.A244g120Δ11
and AE.A244g120Δ11 F53S H72L V75A E106K D107H S110A Q114L. Data are expressed as percent binding of the mutant protein relative to the wild type. Shown
are the means with standard deviations from two independent experiments. FIG 1 Identification of RV305 C1C2-specific MAbs. (A) The C1C2-specific MAbs A32 and C11 were assayed by ELISA for reactivity with full-length AE.A244gp120
or AE.A244g120Δ11. An A32-specific mutant protein was designed (AE.A244g120Δ11 F53S H72L V75A E106K D107H S110A Q114L) to identify A32-like MAb
responses. 19B was used as a positive control and CH65 as a negative control. (B) RV305 nonneutralizing MAbs were assayed for A32 blocking by ELISA. (C)
RV305 nonneutralizing A32-blockable MAb heavy- and light-chain gene sequence mutation frequencies were analyzed by Cloanalyst (13) and compared to
previously published RV144 heavy- and light-chain gene sequence mutation frequencies (percent nucleotide) (12). Statistical significance was determined using
Wil
k
R d b
h
(D) RV30
li i
A32 bl
k bl MAb
d b ELISA f
bi di
AE A2
20Δ FIG 1 Identification of RV305 C1C2-specific MAbs. (A) The C1C2-specific MAbs A32 and C11 were assayed by ELISA for reactivity with full-length AE.A244gp120
or AE.A244g120Δ11. An A32-specific mutant protein was designed (AE.A244g120Δ11 F53S H72L V75A E106K D107H S110A Q114L) to identify A32-like MAb
responses. 19B was used as a positive control and CH65 as a negative control. (B) RV305 nonneutralizing MAbs were assayed for A32 blocking by ELISA. (C)
RV305 nonneutralizing A32-blockable MAb heavy- and light-chain gene sequence mutation frequencies were analyzed by Cloanalyst (13) and compared to
previously published RV144 heavy- and light-chain gene sequence mutation frequencies (percent nucleotide) (12). Statistical significance was determined using
a Wilcoxon rank sum test. Red bar represents the means. RESULTS AIDSVAX B/E N-terminal deletion alters C1C2-specific antibody responses. The
AIDSVAX B/E protein used in the RV144 and RV305 HIV-1 vaccine trials had an
eleven-amino-acid (aa) N-terminal deletion (10) that removed a majority of the C11-like
MAb epitope (6), whereas the CRF_01 AE gp140 Env 92TH023 in ALVAC (vCP1521)
retained the gp120 N-terminal 11 amino acids (11). To determine if C11 could bind to
gp120 proteins with an 11-aa N-terminal deletion, we assayed A32 and C11 MAbs for
binding to full-length AE.A244gp120 or to AE.A244gp120Δ11 (N-terminal 11 aa de-
leted). A32 bound to full-length AE.A244gp120, and binding was enhanced on
AE.A244gp120Δ11 (Fig. 1A) (10). In contrast, C11 only bound to the full-length
AE.A244gp120 (Fig. 1A). From these data we concluded that C11-like antibody re-
sponses were unlikely to be boosted by AIDSVAX B/E. Peripheral blood mononuclear cells (PBMCs) collected from four vaccine recipients
2 weeks after the second RV305 boost with AIDSVAX B/E (RV305 group II) were used
for AE.A244gp120-specific single B cell sorting and antibody-variable region reverse
transcription-PCR (RT-PCR). A total of 19 RV305-derived nonneutralizing antibodies
(NNAbs) were identified that blocked the C1C2 MAb A32 binding to AE.A244gp120Δ11
(Fig. 1B and Table 1). Compared to previously published RV144 C1C2-specific MAbs (12),
the RV305 C1C2-specific MAbs had significantly more VH and VL chain gene mutations
(P 0.0001 by Wilcoxon rank sum test) (Fig. 1C), suggesting that RV305 boosting
induced additional somatic mutations in C1C2-specific antibodies. To determine if the RV305-boosted A32 blockable MAbs contained a binding
epitope similar to that of A32, we used the A32 ligand crystal structure (5) to identify
critical A32 antibody contact residues and then designed an AE.A244gp120Δ11 mutant
protein (AE.A244gp120Δ11 F53S, H72L, V75A, E106K, D107H, S110A, Q114L) to elimi-
nate A32 MAb binding (Fig. 1A). In an enzyme-linked immunosorbent assay (ELISA), the
RV305 antibody, DH838, was the only MAb with binding eliminated by mutating the
A32 epitope (Fig. 1D). Likewise, DH838 was the only MAb that used a VH3 family gene,
while all other ALVAC/AIDSVAX B/E-induced C1C2-specific MAbs used VH1 genes (Table
1). Thus, as in RV144, AIDSVAX B/E boosting preferentially expanded C1C2-specific jvi.asm.org
2 February 2020
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e01120-19 Boosting to Increase ADCC Breadth and Potency Journal of Virology FIG 1 Identification of RV305 C1C2-specific MAbs. (A) The C1C2-specific MAbs A32 and C11 were assayed by ELISA for reactivity with full-length AE.A244gp120
or AE.A244g120Δ11. February 2020
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e01120-19 RESULTS (D) RV305 nonneutralizing A32-blockable MAbs were assayed by ELISA for binding to AE.A244g120Δ11
and AE.A244g120Δ11 F53S H72L V75A E106K D107H S110A Q114L. Data are expressed as percent binding of the mutant protein relative to the wild type. Shown
are the means with standard deviations from two independent experiments. antibodies that used VH1 family genes (12). A majority of the MAbs assayed in this
study bound epitopes distinct from A32 but in close enough proximity to be sterically
cross-blocked by A32 (Fig. 1B). Boosting increased C1C2-specific ADCC breadth and potency. RV305 C1C2-
specific MAbs and a subset of RV144 C1C2-specific MAbs were next assessed for ADCC
against CEM.NKRCCR5 cells infected with one of seven HIV-1 infectious molecular clones
(IMCs), representing three different clades (HIV-1 AE.CM235, B.WITO, C.TV-1. C.MW965,
C.1086C, C.DU151, and C.DU422) (Table 2). These IMCs were chosen because they
represent clusters of IMCs with different sensitivity to ADCC (unpublished data). To
ascertain ADCC breadth and potency, MAbs were ranked using an ADCC score (see
Materials and Methods) analogous to calculating MAb neutralization breadth. Apart
from the RV144-derived A32 blockable MAb CH38, which was naturally an IgA MAb but jvi.asm.org
3 February 2020
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e01120-19 Easterhoff et al. February 2020
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Issue 4
e01120-19 RESULTS Journal of Virology TABLE 1 Immunogenetics of nonneutralizing A32-blocking RV305 C1C2-specific MAbsa
Study
Visit
PTID
DH
Parameters for:
Heavy chains
Light chains
Variable
segment
Joining
segment
CDR3
length (aa)
% mut
(nt)
Ig isotype/
subclass
Variable
segment
Joining
segment
CDR3
length (aa)
% mut
(nt)
RV144
8
RV144_140
DH677.1
18
1
16
1.04
IgG1
127
5
9
0.857
RV305
5
RV305_094
DH677.3
18
1
16
3.47
IgG1
127
5
9
2.87
RV305
5
RV305_082
DH697
146
6
24
7.99
IgG1
144
3
11
4.87
RV305
5
RV305_094
DH677.2
18
1
16
2.43
IgG1
127
5
9
2.86
RV305
5
RV305_082
DH838
323
4
12
4.17
IgG1
861
3
10
2.22
RV305
5
RV305_094
DH677.4
18
1
16
4.51
IgG1
127
5
9
2.57
RV305
5
RV305_082
DH695
12
6
22
5.56
IgG1
139
3
9
5.30
RV305
5
RV305_082
DH694
146
6
22
10.42
IgG1
139
5
9
8.71
RV305
5
RV305_094
DH688.1
146
6
23
6.97
IgG3
139
4
9
6.00
RV305
5
RV305_094
DH705.5
146
6
23
5.77
IgG3
320
2
10
4.49
RV305
5
RV305_094
DH705.4
146
6
23
6.01
IgG1
139
5
9
3.43
RV305
5
RV305_3119
DH886
12
4
12
8.38
IgG3
139
4
9
4.92
RV305
5
RV305_031
DH690
146
6
24
6.92
IgG1
144
2
11
3.62
RV305
5
RV305_094
DH692
146
6
25
1.66
IgG1
139
4
9
2.57
RV305
5
RV305_094
DH693
146
4
26
5.18
IgG1
19
5
9
2.86
RV305
5
RV305_094
DH836
146
6
16
5.32
IgG1
112
4
9
2.86
RV305
5
RV305_094
DH688.2
146
6
23
7.69
IgG3
139
4
9
6.57
RV305
5
RV305_031
DH689.1
146
6
22
10.17
IgG3
139
2
9
9.71
RV305
5
RV305_031
DH689.2
146
6
22
10.41
IgG3
139
1
8
8.55
RV305
5
RV305_031
DH687.2
146
4
23
5.77
IgG1
320
3
10
1.97
aPBMCs used for MAb isolation were collected 2 weeks after the final boost in RV144 or 2 weeks after the second boost in RV305. All four RV305 vaccinees studied
were from group II, boosted 2 with AIDSVAX B/E. RT-PCR-amplified variable heavy- and light-chain genes were Sanger sequenced (Genewiz) and analyzed with
Cloanalyst (13). PTID, patient identifier; DH, antibody identifier. TABLE 1 Immunogenetics of nonneutralizing A32-blocking RV305 C1C2-specific MAbsa aPBMCs used for MAb isolation were collected 2 weeks after the final boost in RV144 or 2 weeks after the second boost in RV305. RESULTS All four RV305 vaccinees studied
were from group II, boosted 2 with AIDSVAX B/E. RT-PCR-amplified variable heavy- and light-chain genes were Sanger sequenced (Genewiz) and analyzed with
Cloanalyst (13). PTID, patient identifier; DH, antibody identifier. tested here as a recombinant IgG1 MAb, ADCC scores for 16/19 RV305 MAbs ranked
higher than those of the RV144 MAbs (Tables 3 and 4). Boosting of RV144 vaccine recipients with AIDSVAX B/E in the RV305 trial
increased ADCC breadth and potency of the RV144-derived C1C2-specific, DH677
clonal lineage. The C1C2-specific DH677 memory B cell clonal lineage next was used
to study affinity maturation and ontogeny of AIDSVAX B/E-induced ADCC responses. B
cell clonal lineage member DH677.1 was isolated from PBMCs collected from a vaccine
recipient 2 weeks after the last boost in the original RV144 trial (ALVAC plus AIDSVAX
B/E). DH677.2, DH677.3, and DH677.4 clonal lineage members were isolated from
PBMCs collected from the same vaccine recipient 2 weeks after the second boost with
AIDSVAX B/E alone given in the RV305 clinical trial (RV305 group II). Thus, this B cell
clonal lineage belongs to a long-lived memory B cell pool started by the RV144 vaccine
regimen and boosted many years later with the RV305 vaccine regimen (Fig. 2). RESULTS DH677.1, DH677.2, DH677.3, and DH677.4 MAb sequences were used to infer with TABLE 2 Properties and subtypes of viruses used in luciferase and infected cell
elimination assays
Virus name
Subtype
IMC type
Assay used
1086c
C
Luciferase Env-IMC
Luciferase
CM235
AE
Luciferase Env-IMC
Luciferase
Du151
C
Luciferase Env-IMC
Luciferase
Du422
C
Luciferase Env-IMC
Luciferase
MW96.5
C
Luciferase Env-IMC
Luciferase
TV-1
C
Luciferase Env-IMC
Luciferase
WITO
B
Luciferase Env-IMC
Luciferase
CH77
B
Full-length IMC
Infected cell elimination assay
CH264
C
Full-length IMC
Infected cell elimination assay
CH0470
B
Full-length IMC
Infected cell elimination assay
CH042
C
Full-length IMC
Infected cell elimination assay
CH185
C
Full-length IMC
Infected cell elimination assay
CH162
C
Full-length IMC
Infected cell elimination assay
CH236
C
Full-length IMC
Infected cell elimination assay TABLE 2 Properties and subtypes of viruses used in luciferase and infected cell
elimination assays jvi.asm.org
4 Boosting to Increase ADCC Breadth and Potency Journal of Virology TABLE 3 C1C2-specific ADCC of infectious molecular clone-infected cells
Antibody
Study
IMC-infected cell ADCC endpoint concn (g/ml)
AE.CM235
B.WITO
C.TV-1
C.MW96.5
C1086.C
C.DU151
C.DU422
A32
0.0006104
0.002097
0.0006104
0.009766
0.0009997
0.001628
0.009766
CH38
RV144
0.0006104
0.00675
0.001343
0.01723
0.008663
0.0333
40
CH57
RV144
0.008608
40
5.368
40
9.307
0.1427
7.142
CH90
RV144
0.06479
23
1.281
40
40
0.4033
4.474
CH54
RV144
0.08913
1.079
0.1192
40
1.944
0.1169
40
DH677.1
RV144
0.008816
1.01
40
40
40
40
40
DH677.3
RV305
0.0007527
0.006146
0.002376
6.151
0.01439
0.02068
0.02528
DH697
RV305
0.004967
0.03909
0.02134
0.0657
0.08982
0.1096
1.047
DH677.2
RV305
0.002912
0.02705
0.01351
40
0.03581
0.5741
40
DH838
RV305
0.0006104
0.008903
0.03242
40
0.005042
40
40
DH677.4
RV305
0.00182
0.008885
0.03447
6.947
0.02838
0.3027
40
DH695
RV305
0.005664
0.5867
0.07879
1.325
0.3076
0.3342
12.15
DH694
RV305
0.0091
0.1159
0.1249
40
0.2651
1.854
40
DH688.1
RV305
0.03942
0.09033
0.1375
8.079
0.3554
1.268
40
DH705.5
RV305
0.0122
0.07738
0.09824
40
0.08681
0.3366
40
DH705.4
RV305
0.03119
0.2424
0.1735
2.813
0.1999
1.8
40
DH886
RV305
0.01321
0.1425
0.1367
40
1.583
0.1721
40
DH690
RV305
0.09713
0.1758
0.3679
7.525
0.6221
2.862
4.34
DH692
RV305
0.07932
0.1438
0.3851
5.228
0.1444
6.399
40
DH693
RV305
0.01995
2.168
0.05216
0.8583
1.361
2.194
6.363
DH836
RV305
0.02868
0.6244
0.1854
40
0.1397
12.02
40
DH688.2
RV305
0.009277
0.1436
0.1338
40
0.5658
4.409
40
DH691
RV305
0.03596
0.9201
0.465
8.113
0.6149
2.376
40
DH689.1
RV305
0.352
1.629
1.69
40
4.817
9.348
30.49
DH689.2
RV305
0.3429
2.225
0.4746
40
5.814
6.567
1.094
DH687.2
RV305
40
40
20.61
40
2.033
40
40 TABLE 3 C1C2-specific ADCC of infectious molecular clone-infected cells Cloanalyst (13) three intermediate ancestors (IA), IA1, IA2, and IA3, and an unmutated
common ancestor (UCA) for the DH677 clonal lineage. February 2020
Volume 94
Issue 4
e01120-19 RESULTS The DH677 clonal lineage then
was assayed by surface plasmon resonance for binding to the AIDSVAX B/E proteins,
AE.A244gp120Δ11 and B.MNgp120Δ11, as well as full-length AE.A244gp120. The
DH677 UCA did not bind to B.MNgp120Δ11 and had minimal binding to the full-length
AE.A244gp120, and this binding was enhanced with AE.A244gp120Δ11 (Fig. 2). The
RV305 boosts more than doubled the VH chain gene mutation frequency from 1.04%
(DH677.1; RV144) up to 4.51% (DH677.4; RV305), which resulted in a 100-fold increase
in apparent affinity (Kd) for the AIDSVAX B/E proteins (DH677.1 AE.A244gp120Δ11, Kd
45.2 nM; B.MNgp120Δ11, Kd 219 nM; DH677.4 AE.A244gp120Δ11, Kd 0.49 nM;
B.MNgp120Δ11, Kd 2.86 nM) and also improved binding to full-length AE.A244gp120
(DH677.1 AE.A244gp120 full length, Kd 152 nM; DH677.4, Kd 2.29 nM) (Fig. 2). The ontogeny of vaccine-induced ADCC was studied by assaying the DH677 clonal
lineage against a panel of IMC-infected CEM.NKRCCR5 cells (AE.CM235, B.WITO, C.TV-1,
C.MW965, C.1086C, C.DU151, and C.DU422) (Table 2). The RV144 prime-boost immu-
nization regimen minimally increased ADCC breadth and potency (DH677 UCA ADCC
score, 2.32; DH677.1 ADCC score, 2.20 [see Materials and Methods]). Conversely,
RV305 boosting substantially increased ADCC breadth and potency (DH677.3 ADCC
score, 4.56) (Fig. 2). These data indicate that the RV144 prime-boost regimen was
insufficient to fully affinity mature this C1C2-specific B cell clonal lineage. Rather, RV305
trial boosting of this particular RV144 vaccine recipient profoundly enhanced DH677
lineage ADCC breadth and potency. Crystal structure of the potent ADCC-mediating MAb DH677.3. We next deter-
mined the crystal structure of the antigen binding fragment (Fab) of the highest-
ranking RV305 ADCC MAb, DH677.3 (Table 4), alone and in complex with clade AE
gp12093TH057 coree plus the CD4-mimetic M48-U1 (Fig. 3 and Table 5). DH677.3
Fab-gp12093TH057 coree-M48U1 complex (Fig. 4) showed that, similar to other cluster A
MAbs, DH677.3 approaches gp120 at the face that is buried in the native Env trimer
(3–5) and binds the C1C2 region exclusively within the gp120 inner domain. The gp120 jvi.asm.org
5 February 2020
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e01120-19 Journal of Virology Easterhoff et al. TABLE 4 Ranking C1C2-specific MAbs by ADCC breadth and potencya TABLE 4 Ranking C1C2-specific MAbs by ADCC breadth and potencya
Rank
Antibody
Study
Score
No. RESULTS of strains
recognized
1
A32
6.62
7
2
CH38
RV144
6.28
6
3
DH677.3
RV305
4.56
7
4
DH697
RV305
2.70
7
5
DH677.2
RV305
1.72
5
6
DH838
RV305
1.62
4
7
DH677.4
RV305
1.30
6
8
DH695
RV305
0.86
7
9
DH688.1
RV305
0.66
6
10
DH694
RV305
0.58
5
11
DH705.5
RV305
0.52
5
12
DH705.4
RV305
0.46
6
13
DH690
RV305
0.36
7
14
DH692
RV305
0.08
6
15
DH693
RV305
0.06
7
16
DH886
RV305
0.08
5
17
DH688.2
RV305
0.30
5
18
DH836
RV305
0.32
5
19
CH57
RV144
0.48
5
20
CH90
RV144
1.06
5
21
CH54
RV144
1.20
5
22
DH689.2
RV305
1.42
6
23
DH689.1
RV305
1.68
6
24
DH677.1
RV144
2.20
2
25
DH687.2
RV305
2.70
2
aRV305 and RV144 C1C2-specific MAbs were assayed for antibody-dependent cellular cytotoxicity against
AE.CM235, B.WITO, C.TV-1, C.MW965, C.1086C, C.DU151, and C.DU422 infectious molecular clone-infected
CEM.NKRCCR5 cells. MAbs were ranked using an ADCC score that accounts for breadth and potency (see
Materials and Methods). Number of strains recognized was determined by ADCC endpoint concentration. aRV305 and RV144 C1C2-specific MAbs were assayed for antibody-dependent cellular cytotoxicity against
AE.CM235, B.WITO, C.TV-1, C.MW965, C.1086C, C.DU151, and C.DU422 infectious molecular clone-infected
CEM.NKRCCR5 cells. MAbs were ranked using an ADCC score that accounts for breadth and potency (see
Materials and Methods). Number of strains recognized was determined by ADCC endpoint concentration. residues involved in DH677.3 binding map to the base of the 7-stranded -sandwich
(residues 82, 84, 86 to 87, 222 to 224, 244 to 246, and 491 to 492) and extend into the
mobile layers 1 (residues 53, 60, and 70 to 80) and 2 (residues 218 to 221). By docking
at the layer 1/2/-sandwich junction, the Fab buried surface area (BSA) utilizes 248 Å2
of the -sandwich, 542 Å of layer 1, and 135 Å2 of layer 2 (Fig. 4 and Table 5). The
majority of contacts providing specificity involve a network of hydrogen bonds and a
salt bridge (Fig. 4, inset) contributed by the antibody heavy-chain and gp120 side-chain
atoms of layer 1 ( turn connecting the 1-0 strands, D78 and N80) and the 7-stranded
-sandwich (strand 7, Q246). The contacts provided by the light chain are less specific
and consist of hydrogen bonds to the gp120 main-chain atoms and hydrophobic
contacts within a hydrophobic cleft formed at the layer 1/2/-sandwich junction (Fig. 4B and C). February 2020
Volume 94
Issue 4
e01120-19 aRV305 and RV144 C1C2-specific MAbs were assayed for antibody-dependent cellular cytotoxicity against
AE.CM235, B.WITO, C.TV-1, C.MW965, C.1086C, C.DU151, and C.DU422 infectious molecular clone-infected
CEM.NKRCCR5 cells. MAbs were ranked using an ADCC score that accounts for breadth and potency (see
Materials and Methods). Number of strains recognized was determined by ADCC endpoint concentration. RESULTS Overall, DH677.3 utilizes all six of its complementary determining regions
(CDRs) and relies approximately equally on both heavy chain and light chain with a
total BSA of 973 Å2, 498 Å2 for the light chain, and 475 Å2 for the heavy chain (Fig. 5
and Table 5). Interestingly, 25 of 29 gp120 contact residues are conserved in 80% of
sequences in the HIV Sequence Database Compendium (https://www.hiv.lanl.gov/
content/sequence/HIV/COMPENDIUM/compendium.html), with 15 of 29 being effec-
tively invariant ( 99% conserved) (Fig. 4B). Comparison of the DH677.3 mode of binding and epitope footprint to cluster
A prototype MAbs. Antigen complex structures of A32 and N12-i3 (C11-like) (3, 6),
MAbs isolated from HIV-1-infected individuals, confirm that DH677.3 recognized a
unique epitope between the A32 and C11 antibody-binding sites involving Env epitope
elements of both (Fig. 6). While the A32 MAb epitope consists exclusively of gp120
mobile layers 1 and 2 (76% and 24% of gp120 BSA, respectively; Fig. 6 and Table 5),
DH677.3 relies less on layers 1 and 2 (53% and 14% of gp120 BSA, respectively) and
effectively utilizes the gp120 7-stranded -sandwich (24% of gp120 BSA) (Fig. 6 and
Table 5). The ability to recognize the 7-stranded -sandwich renders DH677.3 similar to
the C11-like antibody N12-i3, which almost exclusively depends on the -sandwich for jvi.asm.org
6 February 2020
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e01120-19 Boosting to Increase ADCC Breadth and Potency Journal of Virology FIG 2 RV305 boosting increased the apparent affinity and antibody-dependent cellular cytotoxicity breadth and potency of the C1C2-specific RV144-derived
DH677 memory B cell clonal lineage. DH677.1 was isolated by AE.A244gp120Δ11-specific single-cell sorting of PBMCs collected from a vaccinee 2 weeks after
the final boost in the RV144 vaccine trial. DH677.2, DH677.3, and DH677.4 were isolated by AE.A244gp120Δ11-specific single-cell sorting of PBMCs collected
from the same vaccinee after the second AIDSVAX B/E (RV305 group II) boost given in RV305 (7 years later). The intermediate ancestor one (IA1), intermediate
ancestor two (IA2), intermediate ancestor three (IA3), and unmutated common ancestor (UCA) were inferred using Cloanalyst (13). The MAbs were
recombinantly expressed and assayed by surface plasmon resonance for binding to the AIDSVAX B/E proteins AE.A244g120 full length, AE.A244g120Δ11, and
B.MNg120Δ11. Shown are the antibody apparent affinity measurements (Kd), expressed in nanomolars. NB, no detectable binding. MAbs were also assayed for
ADCC against AE.C235-, B.WITO-, C.TV-1-, C.MW965-, C.1086C-, C.DU151-, and C.DU422-infected CEM.NKRCCR5 cells. RESULTS An ADCC score (see Materials and Methods)
was used to account for ADCC breadth and potency. FIG 2 RV305 boosting increased the apparent affinity and antibody-dependent cellular cytotoxicity breadth and potency of the C1C2-specific RV144-derived
DH677 memory B cell clonal lineage. DH677.1 was isolated by AE.A244gp120Δ11-specific single-cell sorting of PBMCs collected from a vaccinee 2 weeks after
the final boost in the RV144 vaccine trial. DH677.2, DH677.3, and DH677.4 were isolated by AE.A244gp120Δ11-specific single-cell sorting of PBMCs collected
from the same vaccinee after the second AIDSVAX B/E (RV305 group II) boost given in RV305 (7 years later). The intermediate ancestor one (IA1), intermediate
ancestor two (IA2), intermediate ancestor three (IA3), and unmutated common ancestor (UCA) were inferred using Cloanalyst (13). The MAbs were
recombinantly expressed and assayed by surface plasmon resonance for binding to the AIDSVAX B/E proteins AE.A244g120 full length, AE.A244g120Δ11, and
B.MNg120Δ11. Shown are the antibody apparent affinity measurements (Kd), expressed in nanomolars. NB, no detectable binding. MAbs were also assayed for
ADCC against AE.C235-, B.WITO-, C.TV-1-, C.MW965-, C.1086C-, C.DU151-, and C.DU422-infected CEM.NKRCCR5 cells. An ADCC score (see Materials and Methods)
was used to account for ADCC breadth and potency. binding (94% of its total gp120 BSA; Fig. 6 and Table 5). Interestingly, N12-i3 and other
C11-like MAbs require the N terminus of gp120 for binding and recognize a unique
gp120 conformation formed by docking of the gp120 N terminus as an 8th strand to
the -sandwich to form an 8-stranded--sandwich structure (6). The DH677.3 complex
crystals were obtained with gp12093TH057 coree, which lacks the N terminus (11-aa
deletion), and therefore a direct judgment, based on structure, of whether or not the
8th strand is involved in binding was not possible. However, we were able to model the
N/C termini-gp12093TH057 coree from the N12-i3 Fab complex structure (PDB code
5W4L) to the DH677.3 Fab-gp12093TH057 coree-M48U1 complex without any steric
clashes (Fig. 6A, inset). Both the conformation and orientation of CDR H1 and H2 of
DH677.3 allowed easy access to the 8-stranded -sandwich structure and enabled
contacts to the 8th strand. These data indicated that DH677.3 is capable of accommo-
dating both the 7- and 8-stranded--sandwich conformations of gp120 with effective
contacts to the 8th strand. Thus, the vaccine-induced C1C2-specific MAb DH677.3 has
a unique binding angle to the C1C2 region compared to the infection-induced C1C2-
specific MAbs C11 and A32. binding (94% of its total gp120 BSA; Fig. RESULTS The -carbon backbone diagram
of superposition of the structures of DH677.3 Fab alone (dark cyan, heavy chain; light cyan, light chain) and N5-i5 Fab bound to CD4-triggered gp120 (dark
brown, heavy chain; light brown, light chain). The average RMSD between free and bound Fabs is 0.818 Å for main-chain residues. FIG 3 Comparison of the two copies of the DH677.3 Fab-gp12093TH057 coree-M48U1 complex and the two Fab copies in the apo Fab structure from the
asymmetric unit of crystals. (A) The root mean square deviation (RMSD) between complex copies is 0.946 Å for main-chain residues. (B) The RMSD between the
Fab copies in the apo Fab structure is 0.540 Å for main-chain residues. (C) Comparison of the free and bound DH677.3 Fab. The -carbon backbone diagram
of superposition of the structures of DH677.3 Fab alone (dark cyan, heavy chain; light cyan, light chain) and N5-i5 Fab bound to CD4-triggered gp120 (dark
brown, heavy chain; light brown, light chain). The average RMSD between free and bound Fabs is 0.818 Å for main-chain residues. FIG 3 Comparison of the two copies of the DH677.3 Fab-gp12093TH057 coree-M48U1 complex and the two Fab copies in the apo Fab structure from the
asymmetric unit of crystals. (A) The root mean square deviation (RMSD) between complex copies is 0.946 Å for main-chain residues. (B) The RMSD between the
Fab copies in the apo Fab structure is 0.540 Å for main-chain residues. (C) Comparison of the free and bound DH677.3 Fab. The -carbon backbone diagram
of superposition of the structures of DH677.3 Fab alone (dark cyan, heavy chain; light cyan, light chain) and N5-i5 Fab bound to CD4-triggered gp120 (dark
brown, heavy chain; light brown, light chain). The average RMSD between free and bound Fabs is 0.818 Å for main-chain residues. infected with IMCs containing the Renilla luciferase (LucR) reporter gene, which restricts
Nef expression, leading to incomplete CD4 downregulation (17). Nevertheless, Vpu
expression can compensate for Nef function and induce CD4 downregulation during
the 72-h incubation of the target cells before assays were performed. To exclude any
possible impact of this technical aspect of IMCs with LucR on our ADCC results,
full-length IMCs (n 7) that do not contain a report gene were used to evaluate ADCC
of the affinity-matured RV305 C1C2-specific MAbs DH677.3 and DH677.4 (Fig. 2) and
A32 (2). RESULTS 6 and Table 5). Interestingly, N12-i3 and other
C11-like MAbs require the N terminus of gp120 for binding and recognize a unique
gp120 conformation formed by docking of the gp120 N terminus as an 8th strand to
the -sandwich to form an 8-stranded--sandwich structure (6). The DH677.3 complex
crystals were obtained with gp12093TH057 coree, which lacks the N terminus (11-aa
deletion), and therefore a direct judgment, based on structure, of whether or not the
8th strand is involved in binding was not possible. However, we were able to model the
N/C termini-gp12093TH057 coree from the N12-i3 Fab complex structure (PDB code
5W4L) to the DH677.3 Fab-gp12093TH057 coree-M48U1 complex without any steric
clashes (Fig. 6A, inset). Both the conformation and orientation of CDR H1 and H2 of
DH677.3 allowed easy access to the 8-stranded -sandwich structure and enabled
contacts to the 8th strand. These data indicated that DH677.3 is capable of accommo-
dating both the 7- and 8-stranded--sandwich conformations of gp120 with effective
contacts to the 8th strand. Thus, the vaccine-induced C1C2-specific MAb DH677.3 has
a unique binding angle to the C1C2 region compared to the infection-induced C1C2-
specific MAbs C11 and A32. DH677 lineage MAbs mediate ADCC against CD4-downmodulated HIV-1-
infected cells. During natural infection the HIV-1 accessory protein Nef downregulates
CD4 expression on the surface of virus-infected cells (14, 15). Cell surface-expressed
CD4 facilitates the exposure of CD4i Env epitopes, like C1C2, by binding to coexpressed
cell surface Env (16). The analyses of ADCC breadth was performed using target cells DH677 lineage MAbs mediate ADCC against CD4-downmodulated HIV-1-
infected cells. During natural infection the HIV-1 accessory protein Nef downregulates
CD4 expression on the surface of virus-infected cells (14, 15). Cell surface-expressed
CD4 facilitates the exposure of CD4i Env epitopes, like C1C2, by binding to coexpressed
cell surface Env (16). The analyses of ADCC breadth was performed using target cells jvi.asm.org
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e01120-19 Journal of Virology Easterhoff et al. FIG 3 Comparison of the two copies of the DH677.3 Fab-gp12093TH057 coree-M48U1 complex and the two Fab copies in the apo Fab structure from the
asymmetric unit of crystals. (A) The root mean square deviation (RMSD) between complex copies is 0.946 Å for main-chain residues. (B) The RMSD between the
Fab copies in the apo Fab structure is 0.540 Å for main-chain residues. (C) Comparison of the free and bound DH677.3 Fab. February 2020
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e01120-19 RESULTS The two copies in the asymmetric unit of the DH677.3, A32, and N12-i3 complexes are averaged. than that of A32. This is likely related to the unique DH677 clonal lineage epitope,
which may be more frequently exposed on Env conformations on the surface of
IMC-infected cells even in the context of CD4 downmodulation. February 2020
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e01120-19 RESULTS Since clade CRF01_AE possesses a histidine at Env HXB2 position 375 that
influences sensitivity to CD4i antibody binding and ADCC (18, 19), only clade B and
clade C HIV-1 IMCs were used (Table 2). As these full-length IMCs did not contain a reporter gene, we used an infected cell
elimination assay, which measures the reduction of live p24, p24 CD4, and/or p24
CD4 cell populations in the presence of effector cells (Fig. 7). When evaluating
elimination of total p24 cells, no significant difference (P 0.05 by Wilcoxon rank sum
test) in specific killing was noted among the three MAbs (Fig. 8A). However, when
infected cells were separated into p24 CD4 (Fig. 8B) and p24 CD4 (Fig. 8C), it was
found that the RV305-boosted DH677.3 MAb mediated ADCC against 4 out of 7 HIV-1
IMCs, whereas DH677.4 and A32 MAb mediated ADCC against two or none of these
IMCs, respectively. Although DH677.3 percent specific killing against these IMCs was
low (mean, 6%; range, 0 to 24%), the level was significantly higher (P 0.03 by
Wilcoxon rank sum test) for mediating ADCC against p24 CD4-infected cells (Fig. 8C) jvi.asm.org
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e01120-19 Boosting to Increase ADCC Breadth and Potency Journal of Virology TABLE 5 Details of the DH677.3, A32, and N12-i3 interfaces
Region
BSA (Å2) fora:
DH677.3 fab-gp12093TH057
coree-M48U1
A32 Fab- ID293TH057
(4YC2)
N12-i3 fab-gp12093TH057
coreeN/C-M48U1 (5W4L)
gp120 total
925
850
803
7/8-stranded -sheet
248
0
754
Layer 1
542
645
49
Layer 2
135
205
0
Layer 3
0
0
0
Heavy chain total
475
614
711
FWR
0
17
16
CDR H1
16
103
39
CDR H2
84
83
440
CDR H3
375
411
216
Light chain total
498
234
142
FWR
95
0
0
CDR L1
238
126
10
CDR L2
27
0
0
CDR L3
138
108
132
Heavy- and light-chain total
973
848
853
aDetails are based on the DH677.3-gp12093TH057coree -M48U1, A32 Fab-ID293TH057, and N12-i3 Fab-gp12093TH057coreeN/C-M48U1 structures, as calculated by the EBI
PISA server (http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). The two copies in the asymmetric unit of the DH677.3, A32, and N12-i3 complexes are averaged. TABLE 5 Details of the DH677.3, A32, and N12-i3 interfaces of the DH677.3, A32, and N12-i3 interfaces aDetails are based on the DH677.3-gp12093TH057coree -M48U1, A32 Fab-ID293TH057, and N12-i3 Fab-gp12093TH057coreeN/C-M48U1 structures, as calculated by the EBI
PISA server (http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). DISCUSSION In this study, it was found that late boosting of RV144 vaccinees increased C1C2-
specific MAb VH VL chain gene mutation frequency and increased clonal lineage-
specific ADCC breadth and potency (Fig. 1 and 2 and Table 4). We analyzed the somatic
hypermutation frequency of MAbs obtained after boosting and did not observe a clear
correlation between somatic hypermutation frequency and ADCC breadth or potency. Boosting likely affinity matures antibody clonal lineages that are capable of acquiring
broad and potent ADCC, as well as antibody clonal lineages incapable of acquiring
broad and potent ADCC. For example, DH689 clonal lineage members DH689.1 and
DH689.2 were both 10% mutated (Table 1), but ADCC breadth and potency was
worse than that of most of the RV144 MAbs assayed (Table 4). Conversely, boosting
increased somatic hypermutation along with ADCC breadth and potency within the
DH677 clonal lineage (Fig. 2). Thus, clonal lineages with fine epitope specificities
capable of being matured to increased ADCC breadth and potency greatly benefited
from the two AIDSVAX B/E boosts given in the RV305 clinical trial. It should be noted
that in VAX003 and VAX004 clinical trials ADCC responses peaked at 3 to 4 immuni-
zations and declined after 5 to 7 immunizations (20). Collectively these data indicate
that while the RV144 clinical trial was underboosted, repetitive subsequent boosting
beyond RV305 does not necessarily lead to continuously better functional antibody
outcomes. Improving vaccine-induced NNAb effector function will also require more detailed
immunological studies on the timing and frequency of boosting. In the VAX003
(ClinicalTrials registration no. NCT00002441) and VAX004 (ClinicalTrials registration no. NCT00002441) trials, frequent protein immunizations skewed Env-specific antibody
subclass usage from the highly functional IgG3 to IgG4 (21–23). The RV305 boosts that
were studied here occurred several years (6 to 8 years) after the final RV144 boost,
unlike previous HIV-1 vaccine trials. Whether the boosting interval can be shortened
without skewing antibody subclass usage is not known, but it is possible that boosting
with long rest intervals (1 to 2 years) will be necessary. The AIDSVAX B/E protein used for boosting in the RV144 and RV305 HIV-1 vaccine jvi.asm.org
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e01120-19 Journal of Virology Easterhoff et al. FIG 4 Crystal structure of the DH677.3 Fab-gp12093TH057 coree-M48U1 complex. DISCUSSION Complementary determining regions (CDRs) are in the following colors: CDR H1, light blue; CDR H2, dark green; CDR H3, black; CRL1, light green;
(Continued on next page) February 2020
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10 Boosting to Increase ADCC Breadth and Potency Journal of Virology FIG 5 DH677.3 heavy- and light-chain contact residues. MAb side-chain () and main-chain () contact
residues, colored green for hydrophobic, blue for hydrophilic, and black for both, as determined by a 5-Å
cutoff value over the corresponding sequence. CDRs are colored as described for Fig. 1, and buried
surface residues as determined by PISA are shaded. FIG 5 DH677.3 heavy- and light-chain contact residues. MAb side-chain () and main-chain () contact
residues, colored green for hydrophobic, blue for hydrophilic, and black for both, as determined by a 5-Å
cutoff value over the corresponding sequence. CDRs are colored as described for Fig. 1, and buried
surface residues as determined by PISA are shaded. trial contained an N-terminal 11-amino-acid deletion. Previously it was shown that this
modification enhanced exposure of the C1C2 region and V2 loop (10). Here, we show
that this modification disrupts C11-like MAb binding (Fig. 1) but does create a germ
line-targeting immunogen for DH677-like B cell lineages (Fig. 2). Ligand crystal struc-
ture analysis found that DH677.3 recognized a unique C1C2 epitope that involves
epitope footprints of cluster A MAb A32 and N12-i3 (C11-like), as well as new elements
of the inner domain layer 1 and the 7-stranded -sandwich (Fig. 6). The DH677.3
epitope is positioned midway between the A32 and N12-i3 binding sites, with most
residues being highly conserved. Interestingly, DH677.3 binds at the edge of the gp120
inner domain 7-stranded -sandwich and with layers 1 and 2. This binding mode allows
it to bind a gp120 conformation emblematic of the late stages of HIV entry recognized
by C11 and C11-like MAbs (6). Most likely this allows DH677.3 to recognize a broader
range of Env targets, emerging in both early (when the A32 epitope becomes available)
and late (when the C11 epitope becomes available) stages of the viral entry process. Identification of stage 2A of the HIV-1 Env expressed on the surface of infected cells
in the presence of the CD4 molecule or CD4 mimetics reiterate the importance of
targeting these epitopes by vaccine-induced responses as detected in our assays (24). FIG 4 Legend (Continued)
CDR L2, brown; CDRL3, blue. A blow-up view shows the network of hydrogen (H) bonds formed at the Fab-gp120 interface. H bonds contributed by side-chain
and main-chain atoms of gp120 residues are colored magenta and blue, respectively. (B) Fab buried surface area (BSA) and gp120 residues forming DH677.3
epitope are shaded in blue according to BSA (antibody) and percent conservation of gp120 residues (Env). gp120 main-chain (blue) and side-chain (red)
hydrogen bonds (H) and salt bridges (S) are shown above the residue. (C) The DH677.3 Fab-gp12093TH057 coree interface. CDRs are shown as ribbons (left) and
balls and sticks of residues contributing the binding (right) over the gp120 core. The molecular surface of gp120 is colored as described for panel A (left) and
by electrostatic potential (right). DISCUSSION (A) The overall structure of the complex is shown as a ribbon diagram (left)
with the molecular surface displayed over the Fab molecule (middle), colored based on electrostatic charge: red, negative; blue, positive. The gp120 outer
domain is gray and the inner domain colored to indicate inner domain mobile layers 1 (yellow), 2 (cyan), and 3 (light orange) and the 7-stranded -sandwich
(magenta). Complementary determining regions (CDRs) are in the following colors: CDR H1, light blue; CDR H2, dark green; CDR H3, black; CRL1, light green;
Easterhoff et al. Journal of Virology FIG 4 Crystal structure of the DH677.3 Fab-gp12093TH057 coree-M48U1 complex. (A) The overall structure of the complex is shown as a ribbon diagram (left
with the molecular surface displayed over the Fab molecule (middle), colored based on electrostatic charge: red, negative; blue, positive. The gp120 oute
domain is gray and the inner domain colored to indicate inner domain mobile layers 1 (yellow), 2 (cyan), and 3 (light orange) and the 7-stranded -sandwich
(magenta). Complementary determining regions (CDRs) are in the following colors: CDR H1, light blue; CDR H2, dark green; CDR H3, black; CRL1, light green
(C
i
d FIG 4 Crystal structure of the DH677.3 Fab-gp12093TH057 coree-M48U1 complex. (A) The overall structure of the complex is shown as a ribbon diagram (left)
with the molecular surface displayed over the Fab molecule (middle), colored based on electrostatic charge: red, negative; blue, positive. The gp120 outer
domain is gray and the inner domain colored to indicate inner domain mobile layers 1 (yellow), 2 (cyan), and 3 (light orange) and the 7-stranded -sandwich
(magenta). Complementary determining regions (CDRs) are in the following colors: CDR H1, light blue; CDR H2, dark green; CDR H3, black; CRL1, light green;
(Continued on next page) FIG 4 Crystal structure of the DH677.3 Fab-gp12093TH057 coree-M48U1 complex. (A) The overall structure of the complex is shown as a ribbon diagram (left)
with the molecular surface displayed over the Fab molecule (middle), colored based on electrostatic charge: red, negative; blue, positive. The gp120 outer
domain is gray and the inner domain colored to indicate inner domain mobile layers 1 (yellow), 2 (cyan), and 3 (light orange) and the 7-stranded -sandwich
(magenta). February 2020
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e01120-19 DISCUSSION In addition, a model of DH677.3 in complex with gp120 antigen bound to CD4 of a
target/infected cell confirms that the recognition site and angle of approach position
the DH677.3 IgG for easy access for effector cell recognition and Fc-effector complex
formation (Fig. 6A). Interestingly, we recently characterized JR4, a MAb isolated from a
nonhuman primate that, like DH677.3, recognizes an epitope that includes elements of
both the A32 and C11 binding sites (5). JR4 uses its CDR H1 to contact layer 2 residues
of the A32 epitope region and its CDR H3 to reach the residues of the 7-stranded
-sandwich. However, in contrast to DH677.3, which largely relies on recognizing the
7-stranded -sandwich, access of JR4 to this region is limited and involves only a few
residue contacts. In this regard, JR4 is more like A32, with main anchoring contacts to
layer 2 that limit its reach to the 7-stranded -sandwich. DH677.3 misses these A32
layer 2 residues and shifts its epitope footprint more toward the 7-stranded
-sandwich, placing it midway between the two epitope regions. ADCC-mediating antibodies have been shown to reduce mother-to-child HIV-1 FIG 4 Legend (Continued) February 2020
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e01120-19 jvi.asm.org
11 Journal of Virology Easterhoff et al. FIG 6 Recognition of HIV-1 Env by DH677.3 and other cluster A MAbs. (A) The overlay of DH677.3 and cluster A MAbs A32 and N12-i3 (C11-like) bound to the
gp120 core. Crystal structures of the gp120 antigen in complex with the Fab of DH677.3, A32 (PDB code 4YC2), and N12-i3 (PDB code 5W4L), superimposed
based on gp120. The d1 and d2 domains of the target cell receptor CD4 were added to replace peptide mimetic M48U1 of the DH677.3 Fab-gp12093TH057
coree-M48U1 complex. Molecular surfaces are displayed over Fab molecules and colored in lighter and darker shades of brown, blue, and green for the heavy
and light chains of DH677.3, A32, and N12-i3, respectively. A blow-up view shows details of the DH677.3 interaction with the 8-stranded -sandwich of the
gp120 inner domain. The 8th strand (colored in blue) formed by the 11 N-terminal residues of gp120 in the N12-i3 bound conformation (PDB entry 5W4L) was
modeled into the DH677.3 Fab-gp12093TH057 coree-M48U1 complex. CDR H1 and H2 of DH677.3 are colored light blue and dark green, respectively. (B and C)
Comparison of DH677.3, A32, and N12-i3 epitope footprints. (B) The DH677.3 epitope footprint (shown in red) is plotted on the gp120 surface with layers
colored, as described for Fig. 1, with the A32 and N12-i2 epitope footprints shown in black. (C) DH677.3, A32, and N12-i3 gp120 contact residues are mapped
onto the gp120 sequence. Side-chain () and main-chain () contact residues are colored green for hydrophobic, blue for hydrophilic, and black for both, as
determined by a 5-Å cutoff value over the corresponding sequence. Buried surface residues, as determined by PISA, are shaded. The DH677.3 epitope footprint
overlays with the epitopes of both A32 and N12-i3. FIG 6 Recognition of HIV-1 Env by DH677.3 and other cluster A MAbs. (A) The overlay of DH677.3 and cluster A MAbs A32 and N12-i3 (C11-like) bound to the
gp120 core. Crystal structures of the gp120 antigen in complex with the Fab of DH677.3, A32 (PDB code 4YC2), and N12-i3 (PDB code 5W4L), superimposed
based on gp120. The d1 and d2 domains of the target cell receptor CD4 were added to replace peptide mimetic M48U1 of the DH677.3 Fab-gp12093TH057
coree-M48U1 complex. FIG 4 Legend (Continued) Molecular surfaces are displayed over Fab molecules and colored in lighter and darker shades of brown, blue, and green for the heavy
and light chains of DH677.3, A32, and N12-i3, respectively. A blow-up view shows details of the DH677.3 interaction with the 8-stranded -sandwich of the
gp120 inner domain. The 8th strand (colored in blue) formed by the 11 N-terminal residues of gp120 in the N12-i3 bound conformation (PDB entry 5W4L) was
modeled into the DH677.3 Fab-gp12093TH057 coree-M48U1 complex. CDR H1 and H2 of DH677.3 are colored light blue and dark green, respectively. (B and C)
Comparison of DH677.3, A32, and N12-i3 epitope footprints. (B) The DH677.3 epitope footprint (shown in red) is plotted on the gp120 surface with layers
colored, as described for Fig. 1, with the A32 and N12-i2 epitope footprints shown in black. (C) DH677.3, A32, and N12-i3 gp120 contact residues are mapped
onto the gp120 sequence. Side-chain () and main-chain () contact residues are colored green for hydrophobic, blue for hydrophilic, and black for both, as
determined by a 5-Å cutoff value over the corresponding sequence. Buried surface residues, as determined by PISA, are shaded. The DH677.3 epitope footprint
overlays with the epitopes of both A32 and N12-i3. transmission (25–27) and slow virus disease progression (27–29), and in RV144 they
were correlated with reduced risk of infection in vaccine recipients with lower anti-Env
plasma IgA responses (7). Synergy between the RV144 C1C2 and V2 MAbs suggest a
role for the C1C2 plasma responses that could not be directly identified by the
correlates of the protection study. That DH677.3 was better than A32 at mediating
ADCC against HIV-1 clade B and C CD4 downmodulated cells (Fig. 8) makes this MAb
an attractive candidate for targeting HIV-1-infected cells in vivo in the setting of HIV-1
infection. We have previously shown that the C1C2 MAb A32, when formulated as a
bispecific antibody, can potently opsonize and kill HIV-1-infected CD4 T cells (30). jvi.asm.org
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e01120-19 Boosting to Increase ADCC Breadth and Potency Journal of Virology FIG 7 Gating strategy for infected cell elimination assay. Cells are gated on forward scatter and side scatter (FSC and SSC, respectively), followed b
distinguishing viable cells using NFL-1 (a dead cell marker) and TFL-4 (target cell marker). Target cells are then gated on CD4 and p24. February 2020
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e01120-19 FIG 4 Legend (Continued) Mock-infected cells ar
used to determine positioning of the gate for p24. The proportion of p24, p24 CD4, and p24 CD4 target cells in the presence of effectors only, effector
plus negative control, or effectors plus sample was determined. FIG 7 Gating strategy for infected cell elimination assay. Cells are gated on forward scatter and side scatter (FSC and SSC, respectively), followed by
distinguishing viable cells using NFL-1 (a dead cell marker) and TFL-4 (target cell marker). Target cells are then gated on CD4 and p24. Mock-infected cells are
used to determine positioning of the gate for p24. The proportion of p24, p24 CD4, and p24 CD4 target cells in the presence of effectors only, effectors
plus negative control, or effectors plus sample was determined. February 2020
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e01120-19 jvi.asm.org
13 Journal of Virology Easterhoff et al. FIG 8 RV305-derived C1C2-specific MAb DH677.3 is significantly better than A32 at mediating antibody-dependent
cellular cytotoxicity against CD4 downmodulated infectious molecular clone (IMC)-infected cells. Cells were
infected with clade B and clade C full-length IMC that do not contain a reporter gene. Surface CD4 expression was
analyzed by flow cytometry, and p24 expression was measured in live/viable, all p24 (A), p24 CD4 (B), and p24
CD4 (C) IMC-infected cell populations. Data are shown with the means and standard deviations. FIG 8 RV305-derived C1C2-specific MAb DH677.3 is significantly better than A32 at mediating antibody-dependent
cellular cytotoxicity against CD4 downmodulated infectious molecular clone (IMC)-infected cells. Cells were
infected with clade B and clade C full-length IMC that do not contain a reporter gene. Surface CD4 expression was
analyzed by flow cytometry, and p24 expression was measured in live/viable, all p24 (A), p24 CD4 (B), and p24
CD4 (C) IMC-infected cell populations. Data are shown with the means and standard deviations. Whether the DH677.3-type MAbs are superior to A32 for targeting virus-infected cells
remains to be determined. In summary, our data demonstrate that if the RV144 vaccine trial had been boosted,
ADCC-mediating C1C2-specific antibodies would have undergone affinity maturation
for both ADCC potency and breadth of recognition of HIV-1-infected CD4 T cells. February 2020
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e01120-19 MATERIALS AND METHODS Ethics statement. The RV305 clinical trial (ClinicalTrials registration no. NCT01435135) was a boost-
ing of 162 RV144 clinical trial participants (NCT00223080) six to eight years after the conclusion of RV144
(31). Donors used in this study were from groups boosted twice with either AIDSVAX B/E plus ALVAC-HIV
(vCP1521) (group I) or AIDSVAX B/E alone (group II). PBMCs were only collected 2 weeks after the second
boost. The RV305 clinical trial (NCT01435135) received approvals from the Walter Reed Army Institute of
Research, Thai Ministry of Public Health, Royal Thai Army Medical Department, Faculty of Tropical
Medicine, Mahidol University, Chulalongkorn University Faculty of Medicine, and Siriraj Hospital. Written
informed consent was obtained from all clinical trial participants. The Duke University Health System
Institutional Review Board approved all human specimen handling. Antigen-specific single-cell sorting. All PBMCs used in this study were collected 2 weeks after
the second boost in RV305. A total of 1 107 PBMC per vaccine recipient were stained with
AE.A244gp120Δ11 fluorescently labeled proteins and a human B cell flow cytometry panel. Viable
antigen-specific B cells (AqVd CD14 CD16 CD3 CD19 IgD) were single-cell sorted with a BD
FACSAria II SORP (BD Biosciences, Mountain View, CA) into 96-well PCR plates and stored at –80°C for
RT-PCR. Single-cell reverse transcriptase PCR. Single B cell cDNA was generated with random hexamers
using SSIII. The antibody heavy- and light-chain variable regions were PCR amplified using AmpliTaq360
master mix (Applied Biosystems). PCR products were purified (Qiagen, Valencia, CA) and sequenced by
Genewiz. Gene rearrangements, clonal relatedness, unmutated common ancestors, and intermediate
ancestor inferences were made using Cloanalyst (13). The DH677 clonal lineage tree was generated using
FigTree. MAb production. PCR-amplified heavy- and light-chain gene sequences were transiently expressed
as previously described (32). Ig-containing cell culture supernatants were used for ELISA binding assays. For large-scale expression, VH and VL chain genes were synthesized (VH chain in the IgG1 4A backbone)
and transformed into DH5 cells (GeneScript, Piscataway, NJ). Plasmids were expressed in Luria broth
and purified (Qiagen, Valencia, CA), and Expi293 cells were transfected using ExpiFectamine (Life
Technologies, Carlsbad, CA) by following the manufacturer’s protocol. After 5 days of incubation at 37°C
5% CO2, the Ig-containing medium was concentrated and purified with protein A beads, and the
antibody buffer was exchanged into PBS. MAb production. PCR-amplified heavy- and light-chain gene sequences were transiently expressed
as previously described (32). MATERIALS AND METHODS Ig-containing cell culture supernatants were used for ELISA binding assays. For large-scale expression, VH and VL chain genes were synthesized (VH chain in the IgG1 4A backbone)
and transformed into DH5 cells (GeneScript, Piscataway, NJ). Plasmids were expressed in Luria broth
and purified (Qiagen, Valencia, CA), and Expi293 cells were transfected using ExpiFectamine (Life
Technologies, Carlsbad, CA) by following the manufacturer’s protocol. After 5 days of incubation at 37°C
5% CO2, the Ig-containing medium was concentrated and purified with protein A beads, and the
antibody buffer was exchanged into PBS. Antibody binding and blocking assays. Direct ELISAs were performed as previously described (32). Briefly, 384-well microplates were coated overnight with 30 ng/well of protein. Antibodies were diluted
and added for 1 h. Binding was detected with anti-IgG-horseradish peroxidase (HRP) (Rockland) and
developed with SureBlue reserve TMB one component (KPL). Plates were read on a plate reader
(Molecular Devices) at 450 nm. A32-blocking assays were performed by adding the RV305 antibodies
followed by biotinylated A32 and detection with streptavidin HRP. Antibody binding and blocking assays. Direct ELISAs were performed as previously described (32). Briefly, 384-well microplates were coated overnight with 30 ng/well of protein. Antibodies were diluted
and added for 1 h. Binding was detected with anti-IgG-horseradish peroxidase (HRP) (Rockland) and
developed with SureBlue reserve TMB one component (KPL). Plates were read on a plate reader
(Molecular Devices) at 450 nm. A32-blocking assays were performed by adding the RV305 antibodies
followed by biotinylated A32 and detection with streptavidin HRP. Neutralization assays. TZM-bl neutralization assays were performed as previously described (33). No
neutralization was detected for the MAbs assayed in this study. Neutralization assays. TZM-bl neutralization assays were performed as previously described (33). No
neutralization was detected for the MAbs assayed in this study. IMCs. The HIV-1 reporter viruses used were replication-competent IMCs designed to encode the env
genes of CM235 (subtype A/E; GenBank accession no. AF259954.1), WITO (subtype B; GenBank no. IMCs. The HIV-1 reporter viruses used were replication-competent IMCs designed to encode the env
genes of CM235 (subtype A/E; GenBank accession no. AF259954.1), WITO (subtype B; GenBank no. February 2020
Volume 94
Issue 4
e01120-19 MATERIALS AND METHODS IMC infections were performed by incubation of the optimal dilution of virus with CEM. NKRCCR5 cells for 0.5 h at 37°C and 5% CO2 in the presence of DEAE-dextran (7.5 g/ml). The cells were
subsequently resuspended at 0.5 106/ml and cultured for 48 to 72 h in complete medium containing
7.5 g/ml DEAE-dextran. For each ADCC assay, we monitored the frequency of infected target cells by
intracellular p24 staining. Assays performed using infected target cells were considered reliable if cell
viability was 60% and the percentage of viable p24 target cells on assay day was 20%. Infection of CEM.NKRCCR5 cell line with HIV-1 IMCs. CEM.NKRCCR5 cells were infected with HIV-1
IMCs as previously described (36). Briefly, IMCs were titrated in order to achieve maximum expression
within 48 to 72 h postinfection, as determined by detection of luciferase activity and intracellular p24
expression. IMC infections were performed by incubation of the optimal dilution of virus with CEM. NKRCCR5 cells for 0.5 h at 37°C and 5% CO2 in the presence of DEAE-dextran (7.5 g/ml). The cells were
subsequently resuspended at 0.5 106/ml and cultured for 48 to 72 h in complete medium containing
7.5 g/ml DEAE-dextran. For each ADCC assay, we monitored the frequency of infected target cells by
intracellular p24 staining. Assays performed using infected target cells were considered reliable if cell
viability was 60% and the percentage of viable p24 target cells on assay day was 20%. Luciferase ADCC assay. ADCC activity was determined by a luciferase-based assay as previously
described (8, 37). Briefly, CEM.NKRCCR5 cells (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH,
from Alexandra Trkola) (38) were used as targets after infection with the HIV-1 IMCs. PBMCs obtained
from an HIV-seronegative donor with the heterozygous 158F/V and 131H/R genotypes for FcR3A and
FcR2A (39, 40), respectively, were used as a source of effector cells and were used at an effector-to-
target ratio of 30:1. Recombinant MAbs were tested across a range of concentrations using 5-fold serial
dilutions starting at 50 g/ml. The effector cells, target cells, and Ab dilutions were plated in opaque
96-well half area plates and were incubated for 6 h at 37°C in 5% CO2. MATERIALS AND METHODS Eigenvalue is a measure of variance in the data along that principal component (PC), and
a larger value would mean that corresponding PC explains a larger amount of variance in the data. The
rationale behind this methodology of calculating ADCC score is that MAbs will not target all seven HIV-1
IMCs equally, hence, calculation of breadth score for MAbs needs to account for this variation. In this study, a panel of 7 HIV-1 IMCs was tested, which implies that our data set has seven
dimensions. ADCC activity was measured as AUC. In our analysis, PC1 and PC2 have eigenvalues above
1 and together account for 80.57% variance (Table 6). Scores obtained from the first principal component ADCC score. Antibodies were tested across a range of concentrations using 5-fold serial dilutions
starting at 50 g/ml. Since the dilution curves are not monotonic due to prozone effect of MAbs,
nonparametric area under the curve (AUC) was calculated using the trapezoidal rule, with activity of less
than 15% set to 0%. In this study, we used principal component analysis (PCA) to compute an ADCC score
that explains both potency and breadth of the MAbs. This method of using PCA to calculate breadth
score for neutralizing antibodies has been used in a previous study (41). Similarly, in 2011 a study
published in Atherosclerosis aimed to study the effect of various risk factors on increase in carotid and
femoral intima-media thickness used PCA to calculate a cumulative risk score (42). PCA is the most commonly used method to reduce the dimensionality of the data set (43). It uses
eigenvector decomposition of the correlation matrix of the variables, where each variable is represented
by an HIV-1 IMC in our study. Most of the shared variance of the correlations of ADCC AUC of HIV-1 IMCs
is explained by the first principal component (PC1) (41). Ideally, one would want to explain 70% of the
variance, but this should not be at the expense of adding principal components with an eigenvalue of
less than 1 (44). Eigenvalue is a measure of variance in the data along that principal component (PC), and
a larger value would mean that corresponding PC explains a larger amount of variance in the data. MATERIALS AND METHODS jvi.asm.org
14 Boosting to Increase ADCC Breadth and Potency Journal of Virology TABLE 6 Eigenvalues and variance explained by principle componentsa
PC
Eigenvalue
Difference
Proportion of variance
Cumulative variance
1
5.42464828
4.40337817
0.6781
0.6781
2
1.02127012
0.28980217
0.1277
0.8057
3
0.73146795
0.32157054
0.0914
0.8972
4
0.40989741
0.19988330
0.0512
0.9484
5
0.21001411
0.0263
0.9747
aShown are eigenvalues of the correlation matrix. Eigenvalue is a measure of variance in the data along a
particular principal component (PC). Proportion of variance describes the percentage of variability explained
by every principal component. As seen in the table, the first two PCs have eigenvalues of more than 1, and
they account for 80.57% of variance. ADCC scores are derived from PC1 scores, which have an eigenvalue
of 5.424 and cumulative variance of 67.81%. TABLE 6 Eigenvalues and variance explained by principle componentsa
PC
Eigenvalue
Difference
Proportion of variance
Cumulative variance TABLE 6 Eigenvalues and variance explained by principle componentsa aShown are eigenvalues of the correlation matrix. Eigenvalue is a measure of variance in the data along a
particular principal component (PC). Proportion of variance describes the percentage of variability explained
by every principal component. As seen in the table, the first two PCs have eigenvalues of more than 1, and
they account for 80.57% of variance. ADCC scores are derived from PC1 scores, which have an eigenvalue
of 5.424 and cumulative variance of 67.81%. JN944948), 1086.c (subtype C; GenBank no. FJ444395), TV-1 (subtype C; GenBank no. HM215437),
MW96.5 (subtype C), DU151 (subtype C; GenBank no. DQ411851), and DU422 (subtype C; GenBank no. DQ411854) in cis within an Nef-deficient isogenic backbone that expresses the Renilla luciferase reporter
gene (34). The subtype AE Env-IMC-LucR viruses used were the NL-LucR.T2A-AE.CM235-ecto (IMCCM235)
(plasmid provided by Jerome Kim, US Military HIV Research Program) and clinical env IMCs from the
RV144 trial that were built on the 40061-LucR virus backbone. All other IMCs were built using the original
NL-LucR.T2A-ENV.ecto backbone as originally described (35). Reporter virus stocks were generated by
transfecting 293T cells with proviral IMC plasmid DNA, and virus titer was determined on TZM-bl cells for
quality control (35). y
Infection of CEM.NKRCCR5 cell line with HIV-1 IMCs. CEM.NKRCCR5 cells were infected with HIV-1
IMCs as previously described (36). Briefly, IMCs were titrated in order to achieve maximum expression
within 48 to 72 h postinfection, as determined by detection of luciferase activity and intracellular p24
expression. MATERIALS AND METHODS The final read-out was the
luminescence intensity (relative light units, or RLU) generated by the presence of residual intact target
cells that have not been lysed by the effector population in the presence of ADCC-mediating MAb
(ViviRen substrate; Promega, Madison, WI). The percentage of specific killing was calculated using the
formula percent specific killing [(number of RLU of target and effector well number of RLU of test
well)/number of RLU of target and effector well] 100. In this analysis, the RLU of the target plus effector
wells represents spontaneous lysis in the absence of any source of Ab. The ADCC endpoint concentration
(EC), defined as the lowest concentration of MAb capable of mediating ADCC in our in vitro assay, was
calculated by interpolation of the MAb concentration that intersected the positive cutoff of 15% specific
killing. The RSV-specific MAb palivizumab was used as a negative control. g
p
p
g
ADCC score. Antibodies were tested across a range of concentrations using 5-fold serial dilutions
starting at 50 g/ml. Since the dilution curves are not monotonic due to prozone effect of MAbs,
nonparametric area under the curve (AUC) was calculated using the trapezoidal rule, with activity of less
than 15% set to 0%. In this study, we used principal component analysis (PCA) to compute an ADCC score
that explains both potency and breadth of the MAbs. This method of using PCA to calculate breadth
score for neutralizing antibodies has been used in a previous study (41). Similarly, in 2011 a study
published in Atherosclerosis aimed to study the effect of various risk factors on increase in carotid and
femoral intima-media thickness used PCA to calculate a cumulative risk score (42). PCA is the most commonly used method to reduce the dimensionality of the data set (43). It uses
eigenvector decomposition of the correlation matrix of the variables, where each variable is represented
by an HIV-1 IMC in our study. Most of the shared variance of the correlations of ADCC AUC of HIV-1 IMCs
is explained by the first principal component (PC1) (41). Ideally, one would want to explain 70% of the
variance, but this should not be at the expense of adding principal components with an eigenvalue of
less than 1 (44). MATERIALS AND METHODS The
rationale behind this methodology of calculating ADCC score is that MAbs will not target all seven HIV-1
IMCs equally, hence, calculation of breadth score for MAbs needs to account for this variation. In this study, a panel of 7 HIV-1 IMCs was tested, which implies that our data set has seven
dimensions. ADCC activity was measured as AUC. In our analysis, PC1 and PC2 have eigenvalues above
1 and together account for 80.57% variance (Table 6). Scores obtained from the first principal component jvi.asm.org
15 Journal of Virology Easterhoff et al. can be interpreted as a weighted average of the 7 HIV-1 IMCs that would account for both potency as
well as breadth of the MAbs (44). Higher PC1 score would mean that MAb has higher breadth as well as
potency for ADCC activity. To calculate the ADCC score, the standardized AUC value for each MAb is first
calculated for each HIV-1 IMC and then multiplied by factor loading of the corresponding HIV-1 IMCs. Lastly, these products are added together. Factor loadings are the correlation coefficients between PCs
and HIV-1 IMCs (42). The ADCC score obtained from PC1 is a weighted average of the standardized AUC,
where factor loadings obtained from PC1 are used as weights to calculate the weighted average. Standardized AUC values imply zero mean and unit standard deviation. The AUC values below the value
of mean AUC will result in negative PC1 scores. Infection of primary cells with HIV-1 IMCs. IMCs encoding the full-length transmitted/founder
sequence of seven individuals infected with either subtype B or C viruses from the CHAVI acute infection
cohort (CH77, CH264, CH0470, CH042, CH185, CH162, and CH236) were constructed as previously
described (45, 46) and used to infect primary CD4 cells. To infect cells, cryopreserved PBMCs were
thawed and stimulated in R20 medium (RPMI medium [Invitrogen] with 20% fetal bovine serum [Gemini
Bioproducts], 2 mM L-glutamine [Invitrogen], 50 U/ml penicillin [Invitrogen], and 50 g/ml gentamicin
[Invitrogen]) supplemented with interleukin-2 (IL-2) (30 U/ml; Proleukin), anti-CD3 (25 ng/ml clone OKT-3;
Invitrogen), and anti-CD28 (25 ng/ml; BD Biosciences) antibodies for 72 h at 37°C in 5% CO2. CD8 cells
were depleted from the PBMCs using CD8 microbeads (Miltenyi Biotec, Germany) according to the
manufacturer’s instructions, and 1.5 106 cells were infected using 1 ml virus supernatant by spinocu-
lation (1,125 g) for 2 h at 20°C. MATERIALS AND METHODS After spinoculation, 2 ml of R20 supplemented with IL-2 was added to
each infection, and infections were left for 72 h. Infected cells were used if viability was 70% and more
than 5% of cells were p24. Infected cell elimination assay. HIV-1-infected or mock-infected CD8-depleted PBMCs were used as
targets, and autologous cryopreserved PBMCs rested overnight in R10 supplemented with 10 ng/ml IL-15
(Miltenyi Biotec) were used as a source of effector cells. Infected and uninfected target cells were labeled
with a fluorescent target cell marker (TFL4; OncoImmunin) and a viability marker (NFL1; OncoImmunin)
for 15 min at 37°C, as specified by the manufacturer. The labeling of the target cells with these two
markers allowed us to clearly identify only the live viable cells in our gating strategy and exclude artifacts
related to the presence of dead cell staining. Cells were washed in R10 and adjusted to a concentration
of 0.2 106 cells/ml. PBMCs were then added to target cells at an effector/target ratio of 30:1 (6 106
cells/ml). The target-effector cell suspension was plated in V-bottom 96-well plates and cocultured with
10 g/ml of each MAb. Cocultures were incubated for 6 h at 37°C in 5% CO2. After the incubation period,
cells were washed and stained with anti-CD4-peridinin chlorophyll protein-Cy5.5 (clone OKT4; eBiosci-
ence) at a final dilution of 1:40 in the dark for 20 min at room temperature. Cells were then washed,
resuspended in 100 l/well Cytofix/Cytoperm (BD Biosciences), incubated in the dark for 20 min at 4°C,
washed in 1 Cytoperm wash solution (BD Biosciences), coincubated with anti-p24 antibody (clone
KC57-RD1; Beckman Coulter) to a final dilution of 1:100, and incubated in the dark for 25 min at 4°C. Cells
were washed three times with Cytoperm wash solution and resuspended in 125 l PBS–1% paraformal-
dehyde. The samples were acquired within 24 h using a BD Fortessa cytometer. The appropriate
compensation beads were used to compensate for the spillover signal for the four fluorophores. Data
analysis was performed using FlowJo 9.6.6 software (TreeStar). Mock-infected cells were used to
appropriately position live cell p24/ and CD4/ gates. Specific killing was determined by the reduction in percentage of viable p24 cells in the presence
of MAbs after taking into consideration nonspecific killing and was calculated as [% p24(target effector
cells) % p24(targets effectors MAb or plasma)]/[% p24(target effector cells)]. February 2020
Volume 94
Issue 4
e01120-19 MATERIALS AND METHODS CH65 (an anti-influenza monoclonal antibody, kindly provided by M. A. Moody) was used as a
negative control. To remove background signal, the highest value of percent specific killing induced by
CH65 was subtracted from the calculated reduction in percentage of p24 cells, and then negative values
were rounded to 0%. As the data were background subtracted, no positivity criteria were applied to the
data. SPR. The binding and kinetic rate measurements of gp120 proteins against RV305 antibodies were
obtained by surface plasmon resonance (SPR) using the Biacore 3000 instrument (GE Healthcare). SPR
measurements were performed using a CM5 sensor chip with anti-human IgG Fc antibody directly
immobilized to a level of 9,000 to 11,000 RU (response units). Antibodies then were captured at 5 l/min
for 60 s to a level of 100 to 300 RU. For binding analyses, the gp120 proteins were diluted to
approximately 1,000 nM in PBS and injected over the captured antibodies for 3 min at 30 l/min. For
kinetics measurements, the gp120 proteins were diluted from 5 to 750 nM and injected using a
high-performance kinetics injection for 5 min at 50 l/min. This was followed by a dissociation period of
600 s and surface regeneration with glycine, pH 2.0, for 20 s. Results were analyzed using Biacore
BiaEvaluation software (GE Healthcare). Negative-control antibody (Ab82) and blank buffer binding were
used for double reference subtraction to account for nonspecific protein binding and signal drift. Subsequent curve-fitting analysis was performed using a 1:1 Langmuir model with a local Rmax, and the
reported rate constants are representative of two measurements. Protein preparation and complex crystallization. DH677.3 Fab alone was grown and crystallized
at a concentration of 10 mg/ml. The structure was solved by molecular replacement with PDB entry
3QEG in space group P21 to a resolution of 2.6 Å. Clade A/E 93TH057 gp120 coree (gp12093TH057 coree,
residues 42 to 492 [Hxbc2 numbering]), lacking the V1, V2, and V3 variable loops and containing an
H375S mutation to allow binding of the CD4 mimetic M48U1 (47), was used to obtain crystals of DH677.3
Fab-antigen complex. gp12093TH057 coree was prepared and purified as described previously (3). MATERIALS AND METHODS Degly-
cosylated gp12093TH057 coree was first mixed with CD4 mimetic peptide M48U1 at a molar ratio of 1:1.5 jvi.asm.org
16 Boosting to Increase ADCC Breadth and Potency Journal of Virology TABLE 7 DH677.3 structural data collection and refinement statistics
Parameter
Value(s) forh:
DH677.3 fab
DH677.3 fab-gp12093TH057-M48U1
Data collection
Wavelength, Å
0.979
0.979
Space group
P21
P1
Cell parameters
a, b, c (Å)
81.7, 70.5, 84.3
63.5, 80.1, 88.7
, , (°)
90, 98.3, 90
84.8, 82.3, 82.2
Complexes/AU
2
2
Resolution (Å)
50–2.62 (2.67–2.62)
50–3.0 (3.05–3.0)
No. of reflections
Total
96,040
57,891
Unique
28,247
30,469
Rmerge,a %
15.0 (39.1)
9.2 (44.2)
Rpim,b %
9.6 (25.2)
9.2 (44.2)
CC1/2c
0.99 (0.84)
0.99 (0.75)
I/
13.0 (2.6)
10.2 (1.2)
Completeness, %
98.0 (98.0
89.1 (90.7)
Redundancy
3.4 (3.2)
1.9 (1.9)
Refinement statistics
Resolution, Å
50.0–2.62
50.0–3.0
R,d %
19.9
21.4
Rfree,e %
26.1
27.4
No. of atoms
Protein
6,569
12,278
Water
140
Ligand/Ion
28
426
Overall B value (Å2)
Protein
38
102
Water
32
Ligand/ion
58
112
RMSDf
Bond lengths, Å
0.004
0.004
Bond angles, °
1.3
1.3
Ramachandrang (%)
Favored
96.0
86.7
Allowed
3.8
10.3
Outliers
0.2
3.0
PDB code
6MFJ
6MFP
aRmerge |I |/I, where I is the observed intensity and the average intensity obtained from multiple
observations of symmetry-related reflections after rejections. bRpim, defined in reference 1. TABLE 7 DH677.3 structural data collection and refinement statistics aRmerge |I |/I, where I is the observed intensity and the average intensity obtained from multiple
observations of symmetry-related reflections after rejections. bRpim, defined in reference 1. cCC1/2, defined by Karplus and Diederichs (56). dR Fo| | Fc/|Fo |, where Fo and Fc are the observed and calculated structure factors, respectively. eRfree, as defined by Brünger (57). fRMSD Root mean square deviation
gCalculated with MolProbity. hValues in parentheses are for the highest-resolution shell. and purified through gel filtration chromatography using a Superdex 200 16/60 column (GE Healthcare,
Piscataway, NJ). After concentration, the gp12093TH057 coree-M48U1 complex was mixed with a 20%
molar excess of DH677.3 Fab and again passed through the gel filtration column equilibrated with 5 mM
Tris-HCl buffer, pH 7.2, and 100 mM ammonium acetate. The purified complex was concentrated to
10 mg/ml for crystallization experiments. The structure was solved by molecular replacement using the
DH677.3 Fab and PDB entry 3TGT as the searching models in space group P1 to a resolution 3.0 Å. February 2020
Volume 94
Issue 4
e01120-19 MATERIALS AND METHODS The
final Rfactor/Rfree (%) for the Fab structure is 19.9/26.1, and the final Rfactor/Rfree for the complex is 21.4/27.4
(Table 7). The PDB codes for the deposited structures are 6MFJ and 6MFP, respectively. In each case the
asymmetric unit of the crystal contained two almost identical copies of Fab or the Fab-gp12093TH057 coree
complex (Fig. 3). Crystallization and data collection. Initial crystal screens were done in vapor-diffusion hanging-
drop trials using commercially available sparse matrix crystallization screens from Hampton Research
(Index), Emerald BioSystems (Precipitant Wizard Screen), and Molecular Dimensions (Proplex and Mac-
rosol Screens). The screens were monitored periodically for protein crystals. Conditions that produced
crystals were then further optimized to produce crystals suitable for data collection. DH677.3 Fab crystals
were grown from 20% polyethylene glycol (PEG) 3000, 100 mM HEPES, pH 7.5, and 200 mM sodium
chloride. DH677.3 complex crystals were grown from 25% PEG 4000 and 100 mM morpholineethane- jvi.asm.org
17 Journal of Virology Easterhoff et al. sulfonic acid, pH 5.5. Crystals were briefly soaked in crystallization solution plus 20% 2-methyl-2,4-
pentanediol (MPD) before being flash frozen in liquid nitrogen prior to data collection. sulfonic acid, pH 5.5. Crystals were briefly soaked in crystallization solution plus 20% 2-methyl-2,4-
pentanediol (MPD) before being flash frozen in liquid nitrogen prior to data collection. Data collection and structure solution. Diffraction data were collected at the Stanford Synchrotron
Radiation Light Source (SSRL) at beam line BL12-2, equipped with a Dectris Pilatus area detector. All data
were processed and reduced with HKL2000 (48). Structures were solved by molecular replacement with
Phaser (49) from the CCP4 suite (50). The DH677.3 Fab structure was solved based on the coordinates of
the N12-i2 Fab (PDB entry 3QEG), and the DH677.3 complex was then solved with coordinates from the
DH677.3 Fab model, gp120 (PDB entry 3TGT), and M48U1 (PDB entry 4JZW). Refinement was carried out
with Refmac (51) and/or Phenix (52). Refinement was coupled with manual refitting and rebuilding with
COOT (53). Data collection and refinement statistics are shown in Table 7. Structure validation and analysis. The quality of the final refined models was monitored using the
program MolProbity (54). Structural alignments were performed using the program lsqkab from the CCP4
suite (50). The PISA (55) webserver was used to determine contact surfaces and residues. All illustrations
were prepared with the PyMol Molecular Graphic suite (http://pymol.org) (DeLano Scientific, San Carlos,
CA, USA). MATERIALS AND METHODS Conservation of the DH677.3 epitope was calculated using the HIV Sequence Database
Compendium (https://www.hiv.lanl.gov/content/sequence/HIV/COMPENDIUM/compendium.html), com-
paring gp120 residues to those of clade B Hxbc2. Only unique sequences in the database having an
equivalent residue at each position were included in the calculated percentage, representing approxi-
mately 32,000 sequences on average. Statistical methods. For luciferase-based ADCC assay, background correction was performed by
subtracting the highest value of percent specific killing induced by CH65 and then rounding off the
negative values to zero. In Fig. 5, positivity cutoff criteria were not applied but background correction
was performed. In order to assess if two groups have different responses, pairwise comparisons between groups was
conducted using Wilcoxon rank sum test. Statistical analysis was performed using SAS software (SAS
Institute, Inc., Cary, NC). In order to assess if two groups have different responses, pairwise comparisons between groups was
conducted using Wilcoxon rank sum test. Statistical analysis was performed using SAS software (SAS
Institute, Inc., Cary, NC). Data availability. The structures determined here were deposited under PDB codes 6MFJ and 6MFP. Data availability. The structures determined here were deposited under PDB codes 6MFJ and 6MFP. REFERENCES Acharya P, Tolbert WD, Gohain N, Wu X, Yu L, Liu T, Huang W, Huang CC,
Kwon YD, Louder RK, Luongo TS, McLellan JS, Pancera M, Yang Y, Zhang
B, Flinko R, Foulke JS, Jr, Sajadi MM, Kamin-Lewis R, Robinson JE, Martin
L, Kwong PD, Guan Y, DeVico AL, Lewis GK, Pazgier M. 2014. Structural
definition of an antibody-dependent cellular cytotoxicity response im-
plicated in reduced risk for HIV-1 infection. J Virol 88:12895–12906. https://doi.org/10.1128/JVI.02194-14. 3. Acharya P, Tolbert WD, Gohain N, Wu X, Yu L, Liu T, Huang W, Huang CC,
Kwon YD, Louder RK, Luongo TS, McLellan JS, Pancera M, Yang Y, Zhang
B, Flinko R, Foulke JS, Jr, Sajadi MM, Kamin-Lewis R, Robinson JE, Martin
L, Kwong PD, Guan Y, DeVico AL, Lewis GK, Pazgier M. 2014. Structural
definition of an antibody-dependent cellular cytotoxicity response im-
plicated in reduced risk for HIV-1 infection. J Virol 88:12895–12906. https://doi.org/10.1128/JVI.02194-14. 11. Pitisuttithum P, Rerks-Ngarm S, Bussaratid V, Dhitavat J, Maekanantawat
W, Pungpak S, Suntharasamai P, Vanijanonta S, Nitayapan S, Kaewkung-
wal J, Benenson M, Morgan P, O’Connell RJ, Berenberg J, Gurunathan S,
Francis DP, Paris R, Chiu J, Stablein D, Michael NL, Excler J-L, Robb ML,
Kim JH. 2011. Safety and reactogenicity of canarypox ALVAC-HIV
(vCP1521) and HIV-1 gp120 AIDSVAX B/E vaccination in an efficacy trial
in Thailand. PLoS One 6:e27837. https://doi.org/10.1371/journal.pone
.0027837. 4. Tolbert WD, Gohain N, Veillette M, Chapleau JP, Orlandi C, Visciano ML,
Ebadi M, DeVico AL, Fouts TR, Finzi A, Lewis GK, Pazgier M. 2016. Paring
down HIV Env: design and crystal structure of a stabilized inner domain
of HIV-1 gp120 displaying a major ADCC target of the A32 region. Structure 24:697–709. https://doi.org/10.1016/j.str.2016.03.005. 4. Tolbert WD, Gohain N, Veillette M, Chapleau JP, Orlandi C, Visciano ML,
Ebadi M, DeVico AL, Fouts TR, Finzi A, Lewis GK, Pazgier M. 2016. Paring
down HIV Env: design and crystal structure of a stabilized inner domain
of HIV-1 gp120 displaying a major ADCC target of the A32 region. Structure 24:697–709. https://doi.org/10.1016/j.str.2016.03.005. 5. Gohain N, Tolbert WD, Acharya P, Yu L, Liu T, Zhao P, Orlandi C, Visciano ML,
Kamin-Lewis R, Sajadi MM, Martin L, Robinson JE, Kwong PD, DeVico AL, Ray
K, Lewis GK, Pazgier M. 2015. Cocrystal structures of antibody N60-i3 and
antibody JR4 in complex with gp120 define more cluster A epitopes in-
volved in effective antibody-dependent effector function against HIV-1. J
Virol 89:8840–8854. https://doi.org/10.1128/JVI.01232-15. 12. REFERENCES 1. Guan Y, Pazgier M, Sajadi MM, Kamin-Lewis R, Al-Darmarki S, Flinko R,
Lovo E, Wu X, Robinson JE, Seaman MS, Fouts TR, Gallo RC, DeVico AL,
Lewis GK. 2013. Diverse specificity and effector function among human
antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4
binding. Proc Natl Acad Sci U S A 110:E69–E78. https://doi.org/10.1073/
pnas.1217609110. synergize for increased antiviral activities. J Virol 88:7715–7726. https://
doi.org/10.1128/JVI.00156-14. synergize for increased antiviral activities. J Virol 88:7715–7726. https://
doi.org/10.1128/JVI.00156-14. 9. Easterhoff D, Pollara J, Luo K, Tolbert WD, Young B, Mielke D, Jha S,
O’Connell RJ, Vasan S, Kim J, Michael NL, Excler J-L, Robb ML, Rerks-
Ngarm S, Kaewkungwal J, Pitisuttithum P, Nitayaphan S, Sinangil F,
Tartaglia J, Phogat S, Kepler TB, Alam SM, Wiehe K, Saunders KO,
Montefiori DC, Tomaras GD, Moody MA, Pazgier M, Haynes BF, Ferrari G. 2019. Boosting with ALVAC-HIV and AIDSVAX B/E enhances Env con-
stant region 1 and 2 antibody-dependent cellular cytotoxicity. bioRxiv
https://doi.org/10.1101/632844. 2. Ferrari G, Pollara J, Kozink D, Harms T, Drinker M, Freel S, Moody MA,
Alam SM, Tomaras GD, Ochsenbauer C, Kappes JC, Shaw GM, Hoxie JA,
Robinson JE, Haynes BF. 2011. An HIV-1 gp120 envelope human mono-
clonal antibody that recognizes a C1 conformational epitope mediates
potent antibody-dependent cellular cytotoxicity (ADCC) activity and
defines a common ADCC epitope in human HIV-1 serum. J Virol 85:
7029–7036. https://doi.org/10.1128/JVI.00171-11. 2. Ferrari G, Pollara J, Kozink D, Harms T, Drinker M, Freel S, Moody MA,
Alam SM, Tomaras GD, Ochsenbauer C, Kappes JC, Shaw GM, Hoxie JA,
Robinson JE, Haynes BF. 2011. An HIV-1 gp120 envelope human mono-
clonal antibody that recognizes a C1 conformational epitope mediates
potent antibody-dependent cellular cytotoxicity (ADCC) activity and
defines a common ADCC epitope in human HIV-1 serum. J Virol 85:
7029–7036. https://doi.org/10.1128/JVI.00171-11. 10. Alam SM, Liao HX, Tomaras GD, Bonsignori M, Tsao CY, Hwang KK, Chen
H, Lloyd KE, Bowman C, Sutherland L, Jeffries TL, Jr, Kozink DM, Stewart
S, Anasti K, Jaeger FH, Parks R, Yates NL, Overman RG, Sinangil F, Berman
PW, Pitisuttithum P, Kaewkungwal J, Nitayaphan S, Karasavva N, Rerks-
Ngarm S, Kim JH, Michael NL, Zolla-Pazner S, Santra S, Letvin NL,
Harrison SC, Haynes BF. 2013. Antigenicity and immunogenicity of
RV144 vaccine AIDSVAX clade E envelope immunogen is enhanced by a
gp120 N-terminal deletion. J Virol 87:1554–1568. https://doi.org/10
.1128/JVI.00718-12. 3. ACKNOWLEDGMENTS Robb, Supachai Rerks-Ngarm, Jaranit jvi.asm.org
18 February 2020
Volume 94
Issue 4
e01120-19 Boosting to Increase ADCC Breadth and Potency Journal of Virology Kaewkungwal, Punnee Pitisuttithum, Sorachai Nitayaphan, Faruk Sinangil, James Tarta-
glia, Sanjay Phogat, Thomas B. Kepler, S. Munir Alam, Kevin Wiehe, Kevin O. Saunders,
David C. Montefiori, Georgia D. Tomaras, M. Anthony Moody, Marzena Pazgier, Barton
F. Haynes, and Guido Ferrari; preparation of figures, David Easterhoff, Justin Pollara,
Shalini Jha, and Guido Ferrari; Supervision, David Easterhoff, Justin Pollara, William D. Tolbert, Kevin O. Saunders, David C. Montefiori, Marzena Pazgier, Barton F. Haynes, and
Guido Ferrari; project administration, David Easterhoff, Marzena Pazgier, Guido Ferrari,
and Barton F. Haynes; funding acquisition, Marzena Pazgier, Georgia D. Tomaras, Barton
F. Haynes, and Guido Ferrari. y
,
B.F.H., G.F., and D.E. have patents submitted on antibodies listed in this paper. The views expressed are those of the authors and should not be construed to
represent the positions of the Uniformed Services University, U.S. Army, Department of
Defense, or the Department of Health and Human Services. The investigators have
adhered to the policies for protection of human subjects as prescribed in AR-70. ACKNOWLEDGMENTS We acknowledge the Duke Human Vaccine Institute Flow Cytometry Facility (Dur-
ham, NC), Duke Human Vaccine Institute Viral Genetic Analysis Facility (Durham, NC),
and the following individuals for their expert technical assistance: for flow cytometry,
Derek Cain, Patrice McDermott, and Dawn Jones Marshall; for conjugated antigens,
Lawrence Armand; for transient transfections, Andrew Foulger, Erika Dunford, and
Kedamawit Tilahun; for ELISA, Rob Parks, Callie Vivian, and Maggie Barr; for antibody
expression, Giovanna Hernandez, Esther Lee, Emily Machiele, and Rachel Reed; for
neutralization assays, Amanda Eaton, Celia C. LeBranche, Peter Gao, Kelli Greene, and
Hongmei Gao; for surface plasmon resonance, Kara Anasti; for project management,
Cynthia Nagle and Kelly Soderberg. We also thank all of the RV144 and RV305 clinical
trial team members and participants. This work was primarily supported by Collaboration for AIDS Vaccine Discovery
grant OPP1114721 from the Bill & Melinda Gates Foundation to B.F.H., NIH grants NIAID
R01 AI116274 and R01 AI129769 to M.P. and NIAID P01 AI120756 to G.D.T., and Henry
M. Jackson Foundation for the Advancement of Military Medicine grant number 829295
to B.F.H. Author contributions were the following: conceptualization, David Easterhoff, Nel-
son L. Michael, Jean-Louis Excler, Merlin L. Robb, Supachai Rerks-Ngarm, Barton F. Haynes, Justin Pollara, Marzena Pazgier, and Guido Ferrari; methodology, David Easter-
hoff, Nelson L. Michael, Jean-Louis Excler, Merlin L. Robb, Supachai Rerks-Ngarm, Barton
F. Haynes, Justin Pollara, and Guido Ferrari; development of antibody sequence analysis
software, Kevin Wiehe and Thomas B. Kepler; data validation, David Easterhoff, Justin
Pollara, Shalini Jha, Marzena Pazgier, and Guido Ferrari; formal analysis, Shalini Jha and
Kevin Wiehe; investigation, David Easterhoff, Justin Pollara, Kan Luo, William D. Tolbert,
Brianna Young, and Dieter Mielke; provision of clinical trial material (PBMCs and serum),
Robert J. O’Connell, Sandhya Vasan, Jerome Kim, Jaranit Kaewkungwal, Punnee Pitisut-
tithum, Sorachai Nitayaphan, Faruk Sinangil, James Tartaglia, and Sanjay Phogat; data
curation, David Easterhoff, Justin Pollara, Kevin Wiehe, Dieter Mielke, Shalini Jha, S. Munir Alam, David C. Montefiori, Marzena Pazgier, and Guido Ferrari; writing (original
draft), David Easterhoff, Justin Pollara, Marzena Pazgier, and Guido Ferrari; writing
(review and editing), David Easterhoff, Justin Pollara, Kan Luo, William D. Tolbert,
Brianna Young, Dieter Mielke, Shalini Jha, Robert J. O’Connell, Sandhya Vasan, Jerome
Kim, Nelson L. Michael, Jean-Louis Excler, Merlin L. REFERENCES Bonsignori M, Pollara J, Moody MA, Alpert MD, Chen X, Hwang KK,
Gilbert PB, Huang Y, Gurley TC, Kozink DM, Marshall DJ, Whitesides JF,
Tsao CY, Kaewkungwal J, Nitayaphan S, Pitisuttithum P, Rerks-Ngarm S,
Kim JH, Michael NL, Tomaras GD, Montefiori DC, Lewis GK, DeVico A,
Evans DT, Ferrari G, Liao HX, Haynes BF. 2012. Antibody-dependent
cellular cytotoxicity-mediating antibodies from an HIV-1 vaccine efficacy
trial target multiple epitopes and preferentially use the VH1 gene family. J Virol 86:11521–11532. https://doi.org/10.1128/JVI.01023-12. 6. Tolbert WD, Gohain N, AlsahafiN, Van V, Orlandi C, Ding S, Martin L, Finzi
A, Lewis GK, Ray K, Pazgier M. 2017. Targeting the late stage of HIV-1
entry for antibody-dependent cellular cytotoxicity: structural basis for
Env epitopes in the C11 region. Structure 25:1719–1731. https://doi.org/
10.1016/j.str.2017.09.009. Virol 86:11521–11532. https://doi.org/10.1128/JVI.01023-12. 13. Kepler TB, Munshaw S, Wiehe K, Zhang R, Yu JS, Woods CW, Denny TN,
Tomaras GD, Alam SM, Moody MA, Kelsoe G, Liao HX, Haynes BF. 2014. Reconstructing a B-cell clonal lineage. II. Mutation, selection, and affinity
maturation. Front Immunol 5:170. https://doi.org/10.3389/fimmu.2014
.00170. 7. Haynes BF, Gilbert PB, McElrath MJ, Zolla-Pazner S, Tomaras GD, Alam
SM, Evans DT, Montefiori DC, Karnasuta C, Sutthent R, Liao HX, DeVico
AL, Lewis GK, Williams C, Pinter A, Fong Y, Janes H, DeCamp A, Huang Y,
Rao M, Billings E, Karasavvas N, Robb ML, Ngauy V, de Souza MS, Paris R,
Ferrari G, Bailer RT, Soderberg KA, Andrews C, Berman PW, Frahm N, De
Rosa SC, Alpert MD, Yates NL, Shen X, Koup RA, Pitisuttithum P,
Kaewkungwal J, Nitayaphan S, Rerks-Ngarm S, Michael NL, Kim JH. 2012. Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N Engl J
Med 366:1275–1286. https://doi.org/10.1056/NEJMoa1113425. 14. Wildum S, Schindler M, Munch J, Kirchhoff F. 2006. Contribution of Vpu,
Env, and Nef to CD4 down-modulation and resistance of human immu-
nodeficiency virus type 1-infected T cells to superinfection. J Virol 80:
8047–8059. https://doi.org/10.1128/JVI.00252-06. 15. Arganaraz ER, Schindler M, Kirchhoff F, Cortes MJ, Lama J. 2003. En-
hanced CD4 down-modulation by late stage HIV-1 nef alleles is associ-
ated with increased Env incorporation and viral replication. J Biol Chem
278:33912–33919. https://doi.org/10.1074/jbc.M303679200. 8. Pollara J, Bonsignori M, Moody MA, Liu P, Alam SM, Hwang K-K, Gurley
TC, Kozink DM, Armand LC, Marshall DJ, Whitesides JF, Kaewkungwal J,
Nitayaphan S, Pitisuttithum P, Rerks-Ngarm S, Robb ML, O’Connell RJ,
Kim JH, Michael NL, Montefiori DC, Tomaras GD, Liao H-X, Haynes BF,
Ferrari G. February 2020
Volume 94
Issue 4
e01120-19 REFERENCES 2014. HIV-1 vaccine-induced C1 and V2 Env-specific antibodies 16. Veillette M, Desormeaux A, Medjahed H, Gharsallah NE, Coutu M, Baalwa
J, Guan Y, Lewis G, Ferrari G, Hahn BH, Haynes BF, Robinson JE, Kauf-
mann DE, Bonsignori M, Sodroski J, Finzi A. 2014. Interaction with February 2020
Volume 94
Issue 4
e01120-19 jvi.asm.org
19 Easterhoff et al. Journal of Virology cellular CD4 exposes HIV-1 envelope epitopes targeted by antibody-
dependent cell-mediated cytotoxicity. J Virol 88:2633–2644. https://doi
.org/10.1128/JVI.03230-13. JF, Saez-Cirion A, Ferrari G. 2013. High antibody-dependent cellular
cytotoxicity responses are correlated with strong CD8 T cell viral sup-
pressive activity but not with B57 status in HIV-1 elite controllers. PLoS
One 8:e74855. https://doi.org/10.1371/journal.pone.0074855. JF, Saez-Cirion A, Ferrari G. 2013. High antibody-dependent cellular
cytotoxicity responses are correlated with strong CD8 T cell viral sup-
pressive activity but not with B57 status in HIV-1 elite controllers. PLoS
One 8:e74855. https://doi.org/10.1371/journal.pone.0074855. 17. Prevost J, Richard J, Medjahed H, Alexander A, Jones J, Kappes JC,
Ochsenbauer C, Finzi A. 2018. Incomplete downregulation of CD4 ex-
pression affects HIV-1 Env conformation and antibody-dependent cel-
lular cytotoxicity responses. J Virol 92:e00484-18. https://doi.org/10
.1128/JVI.00484-18. One 8:e74855. https://doi.org/10.1371/journal.pone.0074855. 30. 30. Sung JA, Pickeral J, Liu L, Stanfield-Oakley SA, Lam CY, Garrido C, Pollara
J, LaBranche C, Bonsignori M, Moody MA, Yang Y, Parks R, Archin N,
Allard B, Kirchherr J, Kuruc JD, Gay CL, Cohen MS, Ochsenbauer C,
Soderberg K, Liao HX, Montefiori D, Moore P, Johnson S, Koenig S,
Haynes BF, Nordstrom JL, Margolis DM, Ferrari G. 2015. Dual-affinity
re-targeting proteins direct T cell-mediated cytolysis of latently HIV-
infected cells. J Clin Investig 125:4077–4090. https://doi.org/10.1172/
JCI82314. 18. Zoubchenok D, Veillette M, Prevost J, Sanders-Buell E, Wagh K, Korber B,
Chenine AL, Finzi A. 2017. Histidine 375 modulates CD4 binding in HIV-1
CRF01_AE envelope glycoproteins. J Virol 91:e02151-16. https://doi.org/
10.1128/JVI.02151-16. 31. Rerks-Ngarm S, Pitisuttithum P, Excler J-L, Nitayaphan S, Kaewkungwal J,
Premsri N, Kunasol P, Karasavvas N, Schuetz A, Ngauy V, Sinangil F,
Dawson P, deCamp AC, Phogat S, Garunathan S, Tartaglia J, DiazGrana-
dos C, Ratto-Kim S, Pegu P, Eller M, Karnasuta C, Montefiori DC, Sawant
S, Vandergrift N, Wills S, Tomaras GD, Robb ML, Michael NL, Kim JH,
Vasan S, O’Connell RJ, RV305 Study Team. 2017. Randomized, double-
blind evaluation of late boost strategies for HIV-uninfected vaccine
recipients in the RV144 HIV vaccine efficacy trial. J Infect Dis 215:
1255–1263. https://doi.org/10.1093/infdis/jix099. 19. .3007736. 22. Karnasuta C, Akapirat S, Madnote S, Savadsuk H, Puangkaew J, Rittiroon-
grad S, Rerks-Ngarm S, Nitayaphan S, Pitisuttithum P, Kaewkungwal J,
Tartaglia J, Sinangil F, Francis DP, Robb ML, de Souza MS, Michael NL,
Excler J-L, Kim JH, O’Connell RJ, Karasavvas N. 2017. comparison of
Antibody Responses Induced by RV144, VAX003, and VAX004 vaccina-
tion regimens. AIDS Res Hum Retroviruses 33:410–423. https://doi.org/
10.1089/AID.2016.0204. 34. Edmonds TG, Ding H, Yuan X, Wei Q, Smith KS, Conway JA, Wieczorek L,
Brown B, Polonis V, West JT, Montefiori DC, Kappes JC, Ochsenbauer C. 2010. Replication competent molecular clones of HIV-1 expressing Re-
nilla luciferase facilitate the analysis of antibody inhibition in PBMC. Virology 408:1–13. https://doi.org/10.1016/j.virol.2010.08.028. 35. Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin
MA. 1986. Production of acquired immunodeficiency syndrome-
associated retrovirus in human and nonhuman cells transfected with an
infectious molecular clone. J Virol 59:284–291. 23. Yates NL, Liao H-X, Fong Y, deCamp A, Vandergrift NA, Williams WT,
Alam SM, Ferrari G, Yang Z-y, Seaton KE, Berman PW, Alpert MD, Evans
DT, O’Connell RJ, Francis D, Sinangil F, Lee C, Nitayaphan S, Rerks-Ngarm
S, Kaewkungwal J, Pitisuttithum P, Tartaglia J, Pinter A, Zolla-Pazner S,
Gilbert PB, Nabel GJ, Michael NL, Kim JH, Montefiori DC, Haynes BF,
Tomaras GD. 2014. Vaccine-induced Env V1-V2 IgG3 correlates with
lower HIV-1 infection risk and declines soon after vaccination. Sci Transl
Med 6:228ra39. https://doi.org/10.1126/scitranslmed.3007730. 36. Pollara J, Hart L, Brewer F, Pickeral J, Packard BZ, Hoxie JA, Komoriya A,
Ochsenbauer C, Kappes JC, Roederer M, Huang Y, Weinhold KJ, Tomaras
GD, Haynes BF, Montefiori DC, Ferrari G. 2011. High-throughput quan-
titative analysis of HIV-1 and SIV-specific ADCC-mediating antibody
responses. Cytometry A 79:603–612. https://doi.org/10.1002/cyto.a
.21084. 24. AlsahafiN, Bakouche N, Kazemi M, Richard J, Ding S, Bhattacharyya S,
Das D, Anand SP, Prevost J, Tolbert WD, Lu H, Medjahed H, Gendron-
Lepage G, Ortega Delgado GG, Kirk S, Melillo B, Mothes W, Sodroski J,
Smith AB, III, Kaufmann DE, Wu X, Pazgier M, Rouiller I, Finzi A, Munro JB. 2019. An asymmetric opening of HIV-1 envelope mediates antibody-
dependent cellular cytotoxicity. Cell Host Microbe 25:578–587. https://
doi.org/10.1016/j.chom.2019.03.002. 37. .3007736. Liao HX, Bonsignori M, Alam SM, McLellan JS, Tomaras GD, Moody MA,
Kozink DM, Hwang KK, Chen X, Tsao CY, Liu P, Lu X, Parks RJ, Montefiori
DC, Ferrari G, Pollara J, Rao M, Peachman KK, Santra S, Letvin NL,
Karasavvas N, Yang ZY, Dai K, Pancera M, Gorman J, Wiehe K, Nicely NI,
Rerks-Ngarm S, Nitayaphan S, Kaewkungwal J, Pitisuttithum P, Tartaglia
J, Sinangil F, Kim JH, Michael NL, Kepler TB, Kwong PD, Mascola JR, Nabel
GJ, Pinter A, Zolla-Pazner S, Haynes BF. 2013. Vaccine induction of
antibodies against a structurally heterogeneous site of immune pressure
within HIV-1 envelope protein variable regions 1 and 2. Immunity
38:176–186. https://doi.org/10.1016/j.immuni.2012.11.011. 25. Ljunggren K, Moschese V, Broliden PA, Giaquinto C, Quinti I, Fenyo EM,
Wahren B, Rossi P, Jondal M. 1990. Antibodies mediating cellular cyto-
toxicity and neutralization correlate with a better clinical stage in chil-
dren born to human immunodeficiency virus-infected mothers. J Infect
Dis 161:198–202. https://doi.org/10.1093/infdis/161.2.198. 38. Trkola A, Matthews J, Gordon C, Ketas T, Moore JP. 1999. A cell line-
based neutralization assay for primary human immunodeficiency virus
type 1 isolates that use either the CCR5 or the CXCR4 coreceptor. J Virol
73:8966–8974. 26. Nag P, Kim J, Sapiega V, Landay AL, Bremer JW, Mestecky J, Reichelderfer
P, Kovacs A, Cohn J, Weiser B, Baum LL. 2004. Women with cervicova-
ginal antibody-dependent cell-mediated cytotoxicity have lower genital
HIV-1 RNA loads. J Infect Dis 190:1970–1978. https://doi.org/10.1086/
425582. 39. Koene HR, Kleijer M, Algra J, Roos D, von Dem Borne AE, de Haas M. 1997. Fc gammaRIIIa-158V/F polymorphism influences the binding of
IgG by natural killer cell Fc gammaRIIIa, independently of the Fc
gammaRIIIa-48L/R/H phenotype. Blood 90:1109–1114. https://doi.org/
10.1182/blood.V90.3.1109. 27. Lambotte O, Ferrari G, Moog C, Yates NL, Liao HX, Parks RJ, Hicks CB,
Owzar K, Tomaras GD, Montefiori DC, Haynes BF, Delfraissy JF. 2009. Heterogeneous neutralizing antibody and antibody-dependent cell cy-
totoxicity responses in HIV-1 elite controllers. AIDS 23:897–906. https://
doi.org/10.1097/QAD.0b013e328329f97d. 40. Bruhns P, Iannascoli B, England P, Mancardi DA, Fernandez N, Jorieux S,
Daeron M. 2009. Specificity and affinity of human Fcgamma receptors
and their polymorphic variants for human IgG subclasses. Blood 113:
3716–3725. https://doi.org/10.1182/blood-2008-09-179754. 28. Baum LL, Cassutt KJ, Knigge K, Khattri R, Margolick J, Rinaldo C,
Kleeberger CA, Nishanian P, Henrard DR, Phair J. 1996. HIV-1 gp120-
specific antibody-dependent cell-mediated cytotoxicity correlates with
rate of disease progression. J Immunol 157:2168–2173. 41. February 2020
Volume 94
Issue 4
e01120-19 REFERENCES Prevost J, Zoubchenok D, Richard J, Veillette M, Pacheco B, Coutu M,
Brassard N, Parsons MS, Ruxrungtham K, Bunupuradah T, Tovanabutra S,
Hwang KK, Moody MA, Haynes BF, Bonsignori M, Sodroski J, Kaufmann
DE, Shaw GM, Chenine AL, Finzi A. 2017. Influence of the envelope
gp120 Phe 43 cavity on HIV-1 sensitivity to antibody-dependent cell-
mediated cytotoxicity responses. J Virol 91:e02452-16. https://doi.org/
10.1128/JVI.02452-16. 20. Balasubramanian P, Williams C, Shapiro MB, Sinangil F, Higgins K, Nadas
A, Totrov M, Kong XP, Fiore-Gartland AJ, Haigwood NL, Zolla-Pazner S,
Hioe CE. 2018. Functional antibody response against V1V2 and V3 of HIV
gp120 in the VAX003 and VAX004 vaccine trials. Sci Rep 8:542. https://
doi.org/10.1038/s41598-017-18863-0. p
g
j
32. Easterhoff D, Moody MA, Fera D, Cheng H, Ackerman M, Wiehe K, Saunders
KO, Pollara J, Vandergrift N, Parks R, Kim J, Michael NL, O’Connell RJ, Excler
J-L, Robb ML, Vasan S, Rerks-Ngarm S, Kaewkungwal J, Pitisuttithum P,
Nitayaphan S, Sinangil F, Tartaglia J, Phogat S, Kepler TB, Alam SM, Liao H-X,
Ferrari G, Seaman MS, Montefiori DC, Tomaras GD, Harrison SC, Haynes BF. 2017. Boosting of HIV envelope CD4 binding site antibodies with long
variable heavy third complementarity determining region in the random-
ized double blind RV305 HIV-1 vaccine trial. PLoS Pathog 13:e1006182. https://doi.org/10.1371/journal.ppat.1006182. 21. Chung AW, Ghebremichael M, Robinson H, Brown E, Choi I, Lane S,
Dugast AS, Schoen MK, Rolland M, Suscovich TJ, Mahan AE, Liao L,
Streeck H, Andrews C, Rerks-Ngarm S, Nitayaphan S, de Souza MS,
Kaewkungwal J, Pitisuttithum P, Francis D, Michael NL, Kim JH, Bailey-
Kellogg C, Ackerman ME, Alter G. 2014. Polyfunctional Fc-effector pro-
files mediated by IgG subclass selection distinguish RV144 and VAX003
vaccines. Sci Transl Med 6:228ra38. https://doi.org/10.1126/scitranslmed
.3007736. 33. Montefiori DC. 2005. Evaluating neutralizing antibodies against HIV, SIV,
and SHIV in luciferase reporter gene assays. Curr Protoc Immunol Chap-
ter 12:Unit 12.11. .3007736. Moody MA, Pedroza-Pacheco I, Vandergrift NA, Chui C, Lloyd KE, Parks R,
Soderberg KA, Ogbe AT, Cohen MS, Liao HX, Gao F, McMichael AJ,
Montefiori DC, Verkoczy L, Kelsoe G, Huang J, Shea PR, Connors M, 29. Lambotte O, Pollara J, Boufassa F, Moog C, Venet A, Haynes BF, Delfraissy jvi.asm.org
20 jvi.asm.org
20 Boosting to Increase ADCC Breadth and Potency Journal of Virology fraction data collected in oscillation mode. Methods Enzymol 276:
307–326. https://doi.org/10.1016/S0076-6879(97)76066-X. Borrow P, Haynes BF. 2016. Immune perturbations in HIV-1-infected
individuals who make broadly neutralizing antibodies. Sci Immunol
1:aag0851. https://doi.org/10.1126/sciimmunol.aag0851. 1:aag0851. https://doi.org/10.1126/sciimmunol.aag0851. 49. McCoy AJ. 2007. Solving structures of protein complexes by molecular
replacement with Phaser. Acta Crystallogr D Biol Crystallogr 63:32–41. https://doi.org/10.1107/S0907444906045975. 42. Berni A, Giuliani A, Tartaglia F, Tromba L, Sgueglia M, Blasi S, Russo G. 2011. Effect of vascular risk factors on increase in carotid and femoral
intima-media thickness. Identification of a risk scale. Atherosclerosis
216:109–114. https://doi.org/10.1016/j.atherosclerosis.2011.01.034. g
50. Collaborative Computational Project, Number 4. 1994. The CCP4 suite:
programs for protein crystallography. Acta Crystallogr D Biol Crystallogr
50:760–763. https://doi.org/10.1107/S0907444994003112. 216:109–114. https://doi.org/10.1016/j.atherosclerosis.2011.01 43. Hotelling H. 1933. Analysis of a complex of statistical variables into
principal components. J Educ Psychol 24:417–441. https://doi.org/10
.1037/h0071325. 51. Murshudov GN, Vagin AA, Dodson EJ. 1997. Refinement of macromolecular
structures by the maximum-likelihood method. Acta Crystallogr D Biol
Crystallogr 53:240–255. https://doi.org/10.1107/S0907444996012255. 44. Jackson JE. 2005. A user’s guide to principal components. John Wiley &
Sons, Hoboken, NJ. 52. Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ,
Moriarty NW, Read RJ, Sacchettini JC, Sauter NK, Terwilliger TC. 2002. PHENIX: building new software for automated crystallographic structure
determination. Acta Crystallogr D Biol Crystallogr 58:1948–1954. https://
doi.org/10.1107/s0907444902016657. 45. Salazar-Gonzalez JF, Salazar MG, Keele BF, Learn GH, Giorgi EE, Li H,
Decker JM, Wang S, Baalwa J, Kraus MH, Parrish NF, Shaw KS, Guffey MB,
Bar KJ, Davis KL, Ochsenbauer-Jambor C, Kappes JC, Saag MS, Cohen MS,
Mulenga J, Derdeyn CA, Allen S, Hunter E, Markowitz M, Hraber P,
Perelson AS, Bhattacharya T, Haynes BF, Korber BT, Hahn BH, Shaw GM. 2009. Genetic identity, biological phenotype, and evolutionary pathways
of transmitted/founder viruses in acute and early HIV-1 infection. J Exp
Med 206:1273–1289. https://doi.org/10.1084/jem.20090378. 53. Emsley P, Cowtan K. 2004. Coot: model-building tools for molecular
graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132. https://doi
.org/10.1107/S0907444904019158. Med 206:1273–1289. https://doi.org/10.1084/jem.20090378. 54. February 2020
Volume 94
Issue 4
e01120-19 .3007736. Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ,
Murray LW, Richardson JS, Richardson DC. 2010. MolProbity: all-atom struc-
ture validation for macromolecular crystallography. Acta Crystallogr D Biol
Crystallogr 66:12–21. https://doi.org/10.1107/S0907444909042073. 46. Ochsenbauer C, Edmonds TG, Ding H, Keele BF, Decker J, Salazar MG,
Salazar-Gonzalez JF, Shattock R, Haynes BF, Shaw GM, Hahn BH, Kappes
JC. 2012. Generation of transmitted/founder HIV-1 infectious molecular
clones and characterization of their replication capacity in CD4 T lym-
phocytes and monocyte-derived macrophages. J Virol 86:2715–2728. https://doi.org/10.1128/JVI.06157-11. 55. Krissinel E, Henrick K. 2007. Inference of macromolecular assemblies
from crystalline state. J Mol Biol 372:774–797. https://doi.org/10.1016/j
.jmb.2007.05.022. 47. Martin L, Stricher F, Misse D, Sironi F, Pugniere M, Barthe P, Prado-Gotor
R, Freulon I, Magne X, Roumestand C, Menez A, Lusso P, Veas F, Vita C. 2003. Rational design of a CD4 mimic that inhibits HIV-1 entry and
exposes cryptic neutralization epitopes. Nat Biotechnol 21:71–76. https://doi.org/10.1038/nbt768. 56. Karplus PA, Diederichs K. 2012. Linking crystallographic model and data
quality. Science 336:1030–1033. https://doi.org/10.1126/science.1218231. 57. Brunger AT. 1997. Free R value: cross-validation in crystallography, p
366-396. In Methods in enzymology, vol. 277. Academic Press, Cam-
bridge, MA. 48. Otwinowski Z, Minor W, Charles WC, Jr. 1997. Processing of X-ray dif- jvi.asm.org
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Academic work from the perspectives of aspiring academics: a study of doctoral candidates and recent graduates
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The Australian Educational Researcher (2024) 51:1559–1577
https://doi.org/10.1007/s13384-023-00653-5 The Australian Educational Researcher (2024) 51:1559–1577
https://doi.org/10.1007/s13384-023-00653-5 * Ai Tam Le
aitamle@deakin.edu.au Ai Tam Le1 Received: 12 July 2022 / Accepted: 31 July 2023 / Published online: 4 September 2023
© The Author(s) 2023 1
Deakin University, Burwood Highway, Melbourne, Australia Abstract Several studies have highlighted the changing nature and forms of work in the
academic profession, but few have examined the perspectives of aspiring academ-
ics who may contribute to shaping the future academic profession. What types of
activities do aspiring academics consider academic work, and what are the implica-
tions for academic training and the future of the academic profession? This paper
addresses these questions by drawing on qualitative interviews with a group of doc-
toral candidates and recent doctoral candidates at a research-intensive university in
Australia. Building on Ernst Boyer’s scholarship typology, the paper proposes a con-
ceptualisation of academic work domains based on how the outcomes of intellectual
inquiry can be communicated. Conceptualising academic work from a communica-
tion perspective allows for a language to speak of and recognise the diversity of aca-
demic work. The paper also raises questions about how doctoral training could pre-
pare aspiring academics for work areas beyond research, particularly engagement,
and how institutional structure could accommodate and recognise this diversity. Keywords Aspiring academics · Doctoral candidates · Academic profession ·
Boyer · Research training · Engagement Keywords Aspiring academics · Doctoral candidates · Academic profession ·
Boyer · Research training · Engagement Academic work from the perspectives of aspiring
academics: a study of doctoral candidates and recent
graduates Ai Tam Le1 Introduction Change has been a constant faced by academic professions around the world. For the
past few decades, various aspects of change in academia have been discussed in the
book series The changing academy—The changing academic profession in interna-
tional comparative perspective. These changes reflect rapidly changing social condi-
tions, such as mass higher education, globalisation, and new technologies such as (0123456789)
1 3 456789)
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3 A. T. Le A. T. Le 1560 Artificial Intelligence; all of which have placed different demands and expectations
on universities and academic professions (Bearman et al., 2022; Huang et al., 2022;
Teichler et al., 2013). Consequently, these changes have raised questions about what
an academic profession is, who academics are, and what academics do—questions
that have become more pertinent in the time of the university environment charac-
terised by ‘supercomplexity’—different internal and external forces interacting all at
one (Clement & Grant, 2010; Krause, 2020). A changing academic profession begs another question: what academic profes-
sion are aspiring academics trained for? Aspiring academics broadly refer to individ-
uals with an interest in pursuing an academic career, who often have a postgraduate
research degree (a masters by research or doctoral degree). In a context where PhD
training is expected to prepare doctoral graduates for various employment destina-
tions (Sarrico, 2022), efforts to improve doctoral graduates’ employability for non-
academic careers have overshadowed concerns about improving academic training
for doctoral candidates who will stay in academia (Edwards et al., 2011; Probert,
2014a, 2014b). Not all doctoral graduates wish to pursue or will end up in academia,
but for those who do, understanding their perspectives would give insights into
improving academic training and how we can support them better. This paper addresses the following questions: what types of activities do aspir-
ing academics consider academic work? And what are the implications for doctoral
training and the future of the academic profession in Australia? It draws from inter-
view data from a qualitative PhD research project exploring the academic profes-
sion from the perspectives of aspiring academics—doctoral candidates and recent
doctoral graduates with an interest in pursuing an academic career—at one research-
intensive Australian university. Introduction The research centred on the premise that a genuine
effort aiming to improve academic training and the academic profession’s attractive-
ness should take into account the perspectives of aspiring academics and reflect on
how the profession could adjust and evolve to accommodate their needs.ii The rest of the paper is divided into five sections. The first section focuses on
issues related to training doctoral candidates for a changing academic career in Aus-
tralia. The second section discusses Boyer’s scholarship typology as the analytical
framework for this paper. The third section details methodological underpinnings,
data collection, and data analysis. Findings are presented and discussed in the fourth
section. The final section concludes with some reflections and implications for aca-
demic training and the attractiveness of the academic profession. Doctoral training for a changing academic profession in Australia The traditional PhD model—independent research based on supervision—originated
from the University of Berlin founded in 1810 by Wilhelm von Humboldt, which has
evolved and been adapted to national contexts over time (Schneijderberg & Teichler,
2018). While differences in the research training systems exist, the core element of a
traditional PhD remains conducting independent research supervised by experienced
academic staff (Shin et al., 2018). According to the Australian Qualification Frame-
work (2013, p. 65), a doctoral candidate is expected to produce ‘significant and origi-
nal research outcomes’. Thus, one of the aims of PhD training is to ensure the supply 1 3 Academic work from the perspectives of aspiring academics:… 1561 of high-quality researchers and specialists of disciplinary knowledge (McGagh et al.,
2016, p. 36). Due to the emphasis on research, traditionally PhD training was expected
to prepare doctoral candidates to work in academia. of high-quality researchers and specialists of disciplinary knowledge (McGagh et al.,
2016, p. 36). Due to the emphasis on research, traditionally PhD training was expected
to prepare doctoral candidates to work in academia. Although the growing complexity of academic work has been acknowledged (Coates
& Goedegebuure, 2012), less attention is paid to preparing doctoral candidates beyond the
traditional tasks of research and, to a lesser extent, teaching (Probert, 2014a). For exam-
ple, a review of Higher Degree by Research (HDR) training by the Australian Council of
Learned Academies noted, ‘Compared with Doctoral education offered in the US or in
Europe, graduates in Australia are not as well prepared for academic teaching’ (McGagh
et al., 2016, p. 39). Findings from a large-scale survey suggested that Australian HDR
students had a limited understanding of the academic tasks, mostly learning through
observations; some of them did not feel adequately prepared for teaching (Edwards et al.,
2011). As a result, some have called for considering the meaning of scholarship in doc-
toral training (Probert, 2014b), integrating teaching training (Edwards et al., 2011; Prob-
ert, 2014a), and re-designing the structure of doctoral programs (Coates et al., 2020). In addition, the changing nature of academic work in Australia means that the bifur-
cation of academic tasks (research versus teaching) fails to reflect the internal diver-
sification of academic roles (Kenny & Fluck, 2018; Probert, 2014a, 2014b). Doctoral training for a changing academic profession in Australia Gard-
ner (2011) argued that a potential ‘collision’ exists between ‘the reality of academic
work in large, complex universities and our construction and understanding of the
core requirements and accountabilities of those academic roles’ (p. 14). For example,
besides research and teaching, a third area of work is service, which is an umbrella
term encompassing different activities, including service roles internal to the univer-
sity, such as involvement in faculty committees, and those external to the university,
such as reviewing journals or serving on professional bodies (Kenny & Fluck, 2018). In
addition, engagement is another domain of academic work that has attracted significant
scholarly work recently (Doberneck et al., 2010; Renwick et al., 2020). These varia-
tions indicate that what constitutes academic work remains open for debate. Having a
realistic understanding of academic work is important for maintaining the profession’s
attractiveness, as Coates et al. (2015) argued: If academic life is to be an attractive future career choice for clever and dedicated
people, then it is necessary to be able to paint them a realistic picture of what
becoming an academic means, coupled with a career structure that meets the real-
ity and expectations of an increasingly diversifying workforce. (p. 299) A study focusing on aspiring academics provides insights into their expectations and
how the academic profession can work to accommodate those. In addition, focusing on
the perspectives of aspiring academics can open new possibilities in thinking about the
academic profession of the future. These are the foundation for the current research. Analytical framework: Ernst Boyer’s scholarship typology This paper used Boyer’s scholarship typology (Boyer, 1990) as an analytical start-
ing point to examine academic work. His typology was first introduced in the 1990
report Scholarship reconsidered: Priorities the Professoriate, in which he aimed to 3 A. T. Le A. T. Le 1562 ‘move beyond the tired old ‘teaching versus research debate’ to acknowledge and
recognise the diversity of academic work in the US at the time’ (Boyer, 1990, pp. 15–16). Boyer broadly defined scholarship as ‘the ability to think, communicate, and
learn’, which has four ‘separate, yet overlapping’ functions (p. 15). The scholarship
of discovery refers to ‘the commitment to knowledge for its own sake, to freedom of
inquiry and to following, in a disciplined fashion, an investigation wherever it may
lead’ (p. 18). The scholarship of integration recognises scholars who ‘give meaning
to isolated facts, putting them in perspective’ (p. 18). The scholarship of application
emphasises the relevance and application of knowledge. It requires the scholar to
ask questions such as ‘How can knowledge be responsibly applied to consequential
problems? How can it be helpful to individuals as well as institutions?’, or ‘Can
social problems themselves define an agenda for scholarly investigation?’ (Boyer,
1990, p. 21). The scholarship of teaching refers to work based on disciplinary and
pedagogical knowledge that ‘both educates and entices future scholars’ (p. 23). In a
later publication, Boyer (1996b) proposed the fifth aspect of scholarship, the schol-
arship of engagement, which calls for strengthening academia-society partnership
‘in the search for answers to our most pressing social, civic, economic, and moral
problems’ (Boyer, 1996b, p. 27).i Since its publication, Boyer’s typology has attracted significant scholarly interest
and critiques both on the conceptual level—the construction of scholarship dimen-
sions—and practical levels—the operationalisation of these dimensions. For exam-
ple, some authors have argued that the meaning of the scholarship of teaching and
learning remains elusive (Kern et al., 2015), making it challenging to operationalise
this concept (Canning & Masika, 2020). Another critique comes from Angela Brew
(2010), who has argued that scholarship manifests itself in the quality of academic
work, such as meticulousness or rigour, rather than the type of work. In other words,
scholarship is determined by how academic work is done rather than what type
of academic work is done. Research approach and data collection This research adopted a qualitative approach that allows for investigating the
nuances in individuals’ lived experiences and meaning-making (Patton, 2002). Data
were collected through semi-structured interviews, which provides the structure nec-
essary to examine the analytical framework, Boyer’s scholarship typology, while
permitting opportunities to probe into emerging issues or insights during the inter-
views. Ethics approval from the author’s institution had been obtained before data
collection took place. The study recruited doctoral candidates who indicated an interest in pursuing
academia after their graduation. Participants were recruited from four disciplines,
representing soft-hard, pure-applied categories (Becher & Trowler, 2001), including
physics (hard-pure), biomedical engineering (hard-applied), history (soft-pure), and
economics and business (soft-applied). Participants were recruited first through PhD
candidates’ emails published on the departments’ websites. Three rounds of recruit-
ment emails were sent in July 2019, October 2019, and February 2020, respec-
tively. The research was also advertised through student societies and departments’
notice boards. These recruitment strategies resulted in 32 participants, most of those
were in the mid to late PhD stages, and some were already doing their postdoctoral
research while still waiting for PhD examination results. A summary is provided
in Table 1, details are provided in the Appendix. The interview questions revolved
around participants’ doctoral experience, involvement in and understanding of aca-
demic work, values attached to this work, and career prospects. This paper only
focuses on the academic work aspect of the findings. i
The research site was a comprehensive research-intensive university in Australia. The university was chosen because of its comprehensive breadth of the PhD pro-
grams, which allowed for recruiting participants from different disciplinary fields. As a member of the Group of Eight, this university was highly ranked internation-
ally for its education and research. The institution offered PhD programs in a range
of subjects, often in collaboration with industry and other sectors. The university
also actively provided professional development activities to facilitate transferable
skills for their graduate researchers. These characteristics might influence how par-
ticipants view academic work, particularly in terms of research focus and engage-
ment outlook. For example, the promotion of transferable skills through professional
development activities might mean that graduate researchers are oriented towards
work that is other than research and more industry- and public-focused. Analytical framework: Ernst Boyer’s scholarship typology Thus, an essentialist approach to scholarship—putting
scholarship into neat boxes—may not adequately reflect how academics perform and
conceptualise their work in practice (Brew, 2001). These critiques are valid and have contributed to developing a more robust
understanding of scholarship. However, they do not undermine the merit of Boyer’s
proposal: to reframe the conversation about academic work to reflect its diversity
(Boyer, 1990, 1996b; Rice, 2002). Boyer’s typology remains relevant in higher edu-
cation debate not only because the topic of his proposal—scholarship—touches the
heart of academia, but also because it allows for scholarly reinterpretation and crea-
tivity (Rice, 2002). As a heuristic device, these scholarship dimensions can serve
as an analytical framework for exploring the perspectives of aspiring academics by
providing general direction while not restricting the possibilities for discovering new
concepts to think about academic work. In a university environment that has been
described as changing and ‘supercomplex’ (Clement & Grant, 2010; Krause, 2020),
Boyer’s typology allows for an ‘ecosystemic perspective on scholarship’ (Krause,
2020) to account for the increasing diversity of academic work. In addition, the
loose articulation of these dimensions allows for interpretations and development,
which is suitable for exploring the perspectives of aspiring academics in this study. 1 3 Academic work from the perspectives of aspiring academics:… 1563 Data analysis With permission from participants, all interviews were recorded and transcribed. The data analysis approach was most closely aligned with analytic induction, a
process moving between open coding (coding of the interviews from the ground
up) and selective coding (looking for specific concepts and salient themes) 3 1564 A. T. Le A. T. Le Table 1 Research participants
Number
Percentage (%)
By discipline
Physics
6
19
Biomedical Engineering
8
25
Economics and Business
10
31
History
8
25
Total
32
100
By gender
Male
18
56
Female
14
44
Total
32
100
By age group
25–29
15
47
30–34
9
28
35–39
3
9
40–44
4
13
> 50
1
3
Total
32
100 (Given, 2008; Patton, 2002). Ideally, the intention was to document interesting
insights emerging from the interviews that were not captured in the pre-existing
analytical framework. In practice, there was a limited extent to which ’interesting
insights’ could emerge since the interview schedule was designed based on exist-
ing analytical concepts. Nevertheless, semi-structured interviews allowed partici-
pants to introduce their own interpretation of these analytical concepts. 1 3 Limitations Even though the study touched upon issues of academic identity and doctoral
experiences—and benefited greatly from this body of literature—its focus was not
on research development or academic identity formation of early career research-
ers or doctoral candidates; the latter would have required a different research
design (for example, see McAlpine & Amundsen, 2018). The analysis did not
consider factors that could contribute to a fine-grained understanding of the par-
ticipants’ perspectives, such as gender, type of study (part-time or full-time), stu-
dent status (domestic or international, with a scholarship or without a scholar-
ship), or personal preferences and attributes. Consideration of these factors would
require a different research design, for example, a quantitative approach and con-
sequently a larger size of research participants. This limitation points to a poten-
tial avenue for future research.il The findings from this study reflected the views of aspiring academics who had
been trained in a research-focused environment. While the findings are not expected 1 3 Academic work from the perspectives of aspiring academics:… 1565 to be generalisable, they can provide initial starting points for other studies of larger
sample sizes. Acquiring this snapshot of perspectives, captured at a particular stage
of the participant’s current situation, can contribute to the ‘horizontal’ knowledge
building—having a range of perspectives on the same issue—in the social sciences
(Pratt et al., 2020). While the detailed analysis and descriptive findings are not
expected to be replicated, a more abstract level analysis linked to existing literature
can make theoretical contributions (Green et al., 2007).i Finally, data collection was finalised in late March 2020, just when the pandem-
ic’s impact started deepening. The timing of this project means the findings reported
in this paper reflected a pre-COVID-19 perspective that set a comparison point for
future research. Domains of academic work from a communication perspective When aspiring academics were asked about the types of academic activities, their
responses came from a combination of experience, observation, and imagination. Some had experience in academia as a casual tutor or a research assistant; some
deliberately observed academic and professional staff; others simply ‘guessed’ or
‘imagined.’ The responses included a range of activities centred around research and
teaching. Other activities were also mentioned, including industry engagement or
collaboration, community engagement, community outreach, public engagement,
public outreach, or policy engagement (see Appendix for a summary). In general,
these activities fell into Boyer’s broad dimensions of scholarship. However, explicit in some responses, and implicit in most, was a distinction
between doing research and the meaningfulness of research; the latter is attached
to the communication aspect of research. One participant insisted on this distinc-
tion: ‘Research is not meaningful if all it does is build up a folder of information;
the meaningfulness comes from the writing or the teaching’ (History 8). Another
participant was more critical, hypothetically asking: ‘What’s the point of discover-
ing some really valuable knowledge if you can’t communicate it? Yeah, that makes it
utterly useless’ (Economics and Business 4).l These comments reflect the importance of research communication widely
acknowledged in the literature (Becher & Trowler, 2001). The communication
aspect was also reflected in Boyer’s thinking and writing when he remarked, ‘[schol-
arship] only takes on life when it is shared’ (Boyer, 1996a, p. 132). Indeed, underly-
ing Boyer’s scholarship typology is the distinction between doing research—schol-
arship of discovery and scholarship of integration—and sharing or communicating
research outcomes—scholarship of teaching, scholarship of application and scholar-
ship of engagement. While all participants converged on the interest in research—the process of intel-
lectual inquiry driven by personal agency—the majority of them did not see research
as an end in itself. Even for those who valued intellectual inquiry without immedi-
ate contribution, such as those in physics, research must be communicated in some
form. The interview data suggested three domains of academic work based on how
research outcome is communicated.i The first domain was academic publishing, which refers to the publishing of
research outcomes in academic written forms, often directed at an academic audi-
ence. The usual outlets included articles, books, or conference papers. For many
participants, academic publishing was important for sharing research results to
facilitate knowledge development. The essence of academia Participants in this study had different degrees of interest in pursuing academia. Some were committed to pursuing an academic job as their only option, others were
more open to seeking a career outside academia. Despite these differences, there
was a unifying thread in their accounts, which is underpinned by four interrelated
themes. First, most participants associated academia with learning and intellectual
inquiry, using phrases such as ‘learning new things,’ ‘seeking answers for ques-
tions,’ ‘figuring out how the world works,’ ‘[doing] dope [excellent] research.’ For
most participants, academia, or the university environment interchangeably, was a
place where they could focus on deep and rigorous thinking on an idea, a theoretical
or a practical problem. Second, most participants emphasised a sense of agency in
this intellectual process. This sense of agency was often expressed in different terms
such as ‘curiosity,’ ‘creativity,’ ‘inquisitive,’ ‘flexibility,’ and ‘freedom.’ Participants
emphasised the importance of thoughts and actions being personally motivated
rather than externally dictated. The third theme was the view that academia allows for an unending process of
intellectual inquiry whose outcome could be built upon. One participant talked about
the ability to ‘keep chasing the questions’ (Physics 1); another referred to academic
research as a ‘never-ending process’ (Biomedical Engineering 7). For some partici-
pants, the continuity of research distinguished research in industry that is externally
dictated and targeted from research in academia that is curiosity-driven, allowing for
the ‘why’ and ‘how’ to be pursued. The fourth theme referred to the view that the
outcome of the intellectual inquiry would make contributions that are expected to be
wide-ranging and long-lasting—whether it be to the pool of knowledge, the trans-
formation of people’s lives, or the betterment of society. These four themes constitute the essence of academia: a sense of personal agency
in pursuing intellectual inquiry that allows a person to make a contribution that
can be built upon. Establishing the essence of academia is important as it under-
pins what was considered academic work, as opposed to non-academic work, by 3 3 A. T. Le A. T. Le 1566 aspiring academics in this study. The essence of academia is associated with the
act of research, which lays the foundation for what will be argued to constitute three
other domains of academic work based on a communication perspective, as will be
discussed next. 1 3 Domains of academic work from a communication perspective Implicit in this domain was the distinction
between research and academic writing; the former aligns with the process of 1 3 1 3 Academic work from the perspectives of aspiring academics:… 1567 thinking and intellectual inquiry while the latter communicates the result of that
inquiry in a written form. thinking and intellectual inquiry while the latter communicates the result of that
inquiry in a written form. The second domain was teaching, which primarily refers to teaching in the
university classroom context. Some participants believed university teaching dif-
fered from teaching at other educational levels due to its dependence on state-
of-the-art knowledge stemming from research. In addition, the communication
skills required for teaching were considered different from the academic publish-
ing domain. Some participants pointed to the differences by giving the example
of an academic who could write but failed as an effective communicator in the
classroom. The third domain was engagement, which includes a range of activities directed
at the broader public audience and stakeholders beyond the university walls. These
activities reflect the application and engagement dimensions, emphasising the uni-
versity’s commitment to society. Some examples of engagement included build-
ing partnerships with industry; outreach activities to share knowledge and attract
younger generations to science; communicating to a larger public audience in lay-
person terms, whether via writing, public lectures, TV interviews or policy advising. The variety of activities reflects the different forms of engagement reported in the
literature (see, for example, Doberneck et al., 2010). Preferences for academic work domains The previous section outlined three domains of academic work based on a commu-
nication perspective, in addition to research. However, individual preferences to be
involved in these domains varied. Overall, participants’ preferences can be catego-
rised into three groups (see Appendix for a summary). The first group, Academic
publishing, includes participants who preferred to focus on research and academic
publication, which they considered central in establishing and maintaining an aca-
demic career (Fig. 1). In contrast, teaching and engagement were considered periph-
eral, if not distracting, to building an academic career. Seven participants belonged
to this group: two from biomedical engineering (2,5), three from economics and
business (7, 8, 9), and two from physics (3, 6). No participant in history fit in this
model, suggesting that a sole focus on academic publishing was not preferred or
common in history. A response from one participant represents the view of this
group: So publishing means getting your knowledge verified by other experts.… So I
believe, yes, getting your work published is the most important.… So whether
or not teaching is important, yes, it’s not. (Economics and Business 8) The second group, Academic publishing and teaching, includes participants who
preferred to focus on both academic publication and teaching domains that were
considered mutually supportive (Fig. 2). The outer third layer is engagement which
encompasses a group of activities considered an optional component in ensuring the
meaningfulness of research or the social responsibility in exchange for the privilege 1 3 1568 A. T. Le of being an academic. The view of this group resonates with Kogan’s (2000) argu-
ment that academics start with the core tasks of ‘teaching, promoting learning and
the creation and testing of knowledge’ before engaging with other groups ‘outside
its walls’ (p. 208). Sixteen participants belonged to this group: three from biomedi-
cal engineering (3, 4, 6, 8), four from economics and business (1,5, 6, 10), four from
history (2, 4, 6, 7) and four from physics (1, 2, 4, 5). One response from a history
participant illustrates the view of this group:
Fig. 1 Academic publishing
group (Model 1) Fig. 1 Academic publishing
group (Model 1) of being an academic. The view of this group resonates with Kogan’s (2000) argu-
ment that academics start with the core tasks of ‘teaching, promoting learning and
the creation and testing of knowledge’ before engaging with other groups ‘outside
its walls’ (p. 208). Fig. 2 Academic publishing and
teaching group (Model 2) Preferences for academic work domains Sixteen participants belonged to this group: three from biomedi-
cal engineering (3, 4, 6, 8), four from economics and business (1,5, 6, 10), four from
history (2, 4, 6, 7) and four from physics (1, 2, 4, 5). One response from a history
participant illustrates the view of this group: of being an academic. The view of this group resonates with Kogan’s (2000) argu-
ment that academics start with the core tasks of ‘teaching, promoting learning and
the creation and testing of knowledge’ before engaging with other groups ‘outside
its walls’ (p. 208). Sixteen participants belonged to this group: three from biomedi-
cal engineering (3, 4, 6, 8), four from economics and business (1,5, 6, 10), four from
history (2, 4, 6, 7) and four from physics (1, 2, 4, 5). One response from a history
participant illustrates the view of this group: 1 3
Fig. 2 Academic publishing and
teaching group (Model 2) Fig. 2 Academic publishing and
teaching group (Model 2) 1 3 Academic work from the perspectives of aspiring academics:… 1569 I think research and teaching are the main things. They can be beneficial
towards each other and can reinforce each other.… Personally, I think academ-
ics do have a social responsibility rather than just sitting there doing research
for themselves.…You want to produce certain output that can benefit the wider
society rather than just the intellectual community. (History 6) The third model, Academic publishing, teaching, and engagement, includes
participants who preferred to be involved in all three domains alongside research
(Fig. 3). They considered the third domain, engagement, an essential part of aca-
demic work. This view reflects the increasingly emphasised engagement area as the
university’s third mission (Doberneck et al., 2010). Nine participants belonged to
this group: two from biomedical engineering (1, 3), three from economics and busi-
ness (2, 3, 4), four in history (2, 3, 5, 8). No participant in physics fit in this group
indicating that doing engagement at the same time with academic publishing and
research was not preferred or common in physics. One participant in economics and
business who belonged to this group described their ideal work arrangements: Workload wise for me, that would mean some research, so at least 50 percent
research perhaps, then some teaching…and then some kind of direct industry
engagement for me would be also part of my ideal mix. Constraints on academic work in a future academic career Participants’ responses indicated a distinction between an ideal vision, what work
domains they personally preferred, and a reality, what they would actually end up
doing. Most participants showed an understanding and expectations that their prefer-
ences would have to be matched with institutional demands, particularly at this stage
of their career. For example, when asked about ideal work arrangements as an aca-
demic, one participant in economics and business immediately pointed to the com-
promises he would have to consider ’being in a low position of power early in my
career, I have to take what’s out there based on the universities that are offering jobs
and what their prerogatives are... that’s up to them’ (Economics and Business 2). In
addition, participants also mentioned that any academic role would typically involve
non-academic tasks, some of which were considered unimportant or inessential. Among them are the ‘paperwork’ component and the management or administration
of a research project or lab. One physics participant captured this sentiment by refer-
ring to ‘the annoying parts of research that no one really wants to talk about’ that
‘get brushed under the carpet a bit’ (Physics 2).i The non-academic tasks, however, could be divided into two categories. The first
one included activities that were considered non-academic yet essential to keep the
work running, such as grant writing. The second group of non-academic activities
was considered inherent in any academic role yet unessential, such as bureaucratic
procedures or committee work. Some participants thought that these activities do
not contribute to the ‘real’ work and should be ‘got rid of’ to make give them more
time to focus on research. In addition, the mismatch between what was considered personally meaningful
and what was rewarded or incentivised by the university was another concern. For
example, one participant in history highlighted the mismatch between the institu-
tional rhetoric of engagement and how it was recognised ‘I don’t think [engagement]
is judged in the same way as a publication in a prestigious journal’ (History 2). The
focus on academic publication, as a measure of career success, was indeed brought
up frequently. However, what participants seemed to question was not an excessive
focus on research but, rather, research without communicating or communicating
through certain channels to limited audiences, often confined to academic circles. Preferences for academic work domains So like maybe 25 per-
cent of working with companies, going into companies, talking to them about Fig. 3 Academic publishing, teaching, and engagement group (Model 3) Fig. 3 Academic publishing, teaching, and engagement group (Model 3) 3 1570 A. T. Le A. T. Le what problems they’re having.… So I would want to have some sort of balance
between all three of those things. (Economics and Business 4) Conclusions Situated against the backdrop of a changing academic profession, this paper exam-
ined academic work from the perspectives of academics and its implications for aca-
demic training and the future of the academic profession. Here, I highlight three
implications based on the findings discussed above.f i
First, the different domains of academic work based on a communication perspec-
tive—academic publishing, teaching, and engagement—reflect the nuances in how
academic work is undertaken and could be recognised accordingly, at least from the
perspectives of aspiring academics. The research contributes to the scholarly effort
in conceptualising the complexity of academic work (Boyer, 1990; Coates & Goede-
gebuure, 2012), particularly in integrating the domains of engagement (Doberneck
et al., 2010; Renwick et al., 2020). Future research could build on this to investigate
whether these academic work domains represent the views of a larger population of
aspiring academics, especially those in different types of institutions. f
Second, aspiring academics perceived academics work to involve more than
research and publication. This understanding reflects the reality of academic work
that is complex and evolving in Australia (Coates & Goedegebuure, 2012; Krause,
2020). This finding has important implications for the academic training system and
policies in Australia if one of its aims is to equip doctoral candidates to work effec-
tively in academia. Specifically, to prepare aspiring academics to become fully func-
tional academics, perhaps options should be given to them to understand, explore
and gain experience in other domains of academic work, such as teaching or engage-
ment. This is easier said than done given the importance and weight of research
in the current training model, for understandable reasons. Yet, without a systematic
rethinking of the academic training model—training for an academic career—doc-
toral candidates in Australia could be disadvantaged in the international academic
job market (Mantai & Marrone, 2023)—and eventually academic job perfor-
mance—compared to their counterparts in countries where opportunities to engage
in these domains are made open and accessible (see Shin et al., 2018).i Third, findings from this study also pointed to an engaged academic profession:
a profession whose members have an interest in engaging with society beyond the
university walls. But a concern raised among participants was whether this domain
of work would be recognised by the institution, thus creating tension between what
is considered personally meaningful and what is recognised in the institutional
reward structure. Constraints on academic work in a future academic career One participant raised her concern about the ‘publication race’ in which the research
is ‘conducted for the sake of publications, not for the sake of actually production of
new interesting knowledge that is relevant and that is useful’ (Economics and Busi-
ness 3). Another participant likewise emphasised industry engagement to trans-
late his work and preferred ‘a moderately successful career that I felt was actually
impactful rather than a career that was just marked by publication success’ (Eco-
nomics and Business 4). These remarks are telling because they suggested aspiring 3 3 Academic work from the perspectives of aspiring academics:… 1571 academics perceived institutional incentive and reward structures commonly found
in contemporary universities as distorted. These structures could discourage the type
of work that is meaningful to academics and society. academics perceived institutional incentive and reward structures commonly found
in contemporary universities as distorted. These structures could discourage the type
of work that is meaningful to academics and society. Conclusions An implication for the future academic profession is to understand
engagement activities in academic work, what forms of engagement activities take
place in different disciplines, and the possibility to account for them in the workload
model and for evaluation and promotion. This suggestion is particularly relevant 3 A. T. Le A. T. Le 1572 to the context of Australia, where engagement has become a focus on national and
institutional policies in recent years (Barker, 2015). Building a stronger civic agenda
will become an important mission for Australian higher education, as highlighted in
the Australian Universities Accord Interim report that outlines a vision for the future
of Australia’s higher education system (Australian Government, 2023). Indeed, if
engagement has become a focus of academic work and an interest in engagement
existed among doctoral candidates, a question that should be asked is how relevant
engagement is to research training for doctoral candidates, especially those who
would like to pursue academia. The attractiveness of working in academia has been in question for some time
(Coates et al., 2009; Fortune et al., 2023; Le, 2023). Findings from this study sug-
gest that improving the academic profession’s attractiveness means designing an
institutional structure that encourages and recognises diverse career aspirations
based on different preferences for and values attached to academic work. This sug-
gestion reflects the ‘creativity contract’ between a scholar and their institution, one
that allows for each person’s strength to flourish and can be recognised appropri-
ately (Boyer, 1990). Aspiring academics in this study demonstrated a strong drive
for intellectual inquiry that could lead to positive contributions. But to harness their
intellectual enthusiasm, creativity and commitment should be put into making aca-
demia a more attractive place to work. Building a healthy, sustainable academic pro-
fession is a critical and necessary step to achieving a high-quality Australian higher
education system. Appendix See Table 2. 1 3 1 3 3 Academic work from the perspectives of aspiring academics:… 1573 1
Table 2 Research participants’ details and preferences for academic work
Coded name Gender Age
PhD stage
Status Academic activities
Model
Biomed 1
M
30–34 3rd year
D/FT
Research, publication, teaching, service to students, industry engagement, grant writing, administra-
tion
3
Biomed 2
M
25–29 3rd year
I/FT
Research, publication, teaching, professional networking and collaboration, grant writing, lab
administration and management, public engagement
1
Biomed 3
F
25–29 4th year
I/FT
Teaching, communicating and translating knowledge, research, publication
2
Biomed 4
M
25–29 Graduated/Postdoc I/FT
Research, publication, teaching, industry engagement
2
Biomed 5
M
25–29 4th year/Postdoc
D/FT
Research, publication, teaching, professional networking and collaboration, community outreach
1
Biomed 6
F
25–29 3rd year
I/FT
Research, publication, teaching, supervising students, professional networking and collaboration, lab
administration and management, community outreach, engagement in public policies
2
Biomed 7
F
30–34 Graduated/Postdoc I/FT
Research, publication, teaching and supervising, mentoring, professional networking and collabora-
tion, grant writing, industry engagement, administration
3
Biomed 8
M
30–34 5th year/Postdoc
I/FT
Research, publication, teaching, professional networking and collaboration, grant writing, industry
engagement
2
E&B 1
F
40–44 4th year
D/FT
Research, publication, teaching, service to the department, industry engagement
2
E&B 2
M
25–29 4th year
D/FT
Research, publication, teaching, community and policy engagement
3
E&B 3
F
30–34 5th year
D/FT
Research, publication, teaching, industry engagement
3
E&B 4
M
40–44 5th year
I/FT
Research, publication, teaching, industry engagement
3
E&B 5
M
35–39 5th year
D/FT
Research, publication, teaching, professional networking, grant writing, industry engagement
2
E&B 6
F
30–34 5th year
I/FT
Research, publication, teaching, service to the department, industry engagement
2
E&B 7
F
25–29 5th year
I/FT
Research, publication, teaching, administration
1
E&B 8
M
35–39 5th year
D/FT
Research, publication, teaching, administration
1
E&B 9
M
25–29 5th year
I/FT
Research, publication, teaching, administration
1
E&B 10
F
25–29 3rd year
I/FT
Research, publication, teaching, professional networking, service to the university
2
History 1
M
25–29 Graduated
D/FT
Research, publication, teaching, policy engagement
2
History 2
F
30–34 Graduated/Postdoc D/FT
Research, publication, teaching, service to the department, community and public engagement
3
History 3
F
40–44 3rd year
D/FT
Research, publication, teaching, supervising students, community and public engagement, administration
3 3 A. T. Appendix Le 1574 (
)
oded name Gender Age
PhD stage
Status Academic activities
Model
istory 4
F
40–44 4th year
D/FT
Research, publication, teaching, professional networking, community and public engagement,
administration
2
istory 5
F
30–34 4th year
D/FT
Research, publication, professional networking, community and public engagement, administration
3
istory 6
F
35–39 4th year
D/FT
Research, publication, teaching, community and policy engagement, administration
2
istory 7
M
30–34 Graduated
D/FT
Research, publication, teaching, professional networking, administration
2
istory 8
M
> 50
Graduated
D/FT
Research, publication, teaching, professional networking, community and public engagement
3
hysics 1
M
25–29 3rd year
D/FT
Research, publication, teaching, supervising students, professional networking, grant writing,
administration
2
hysics 2
M
25–29 3rd year
D/FT
Research, publication, teaching, professional networking, grant writing, public outreach
2
hysics 3
M
30–34 Graduated/Postdoc D/FT
Research, publication, teaching, public outreach
1
hysics 4
M
25–29 4th year
D/FT
Research, publication, teaching, supervising and mentoring students, project management, adminis-
tration
2
hysics 5
M
25–29 1st year
D/FT
Research, publication, teaching, supervising students, grant writing, public outreach
2
hysics 6
F
25–29 4th year
D/FT
Research, publication, teaching, supervising students, grant writing, public outreach, administration
1
iomed: biomedical engineering; E&B: economics and business; D: domestic; I: international; FT: full-time; PT: part-time; ‘service’ generally refers to institutional ser-
ce contributing back to the running of the institution; ‘administration’ generally refers to the bureaucracy of the work Coded name Gender Age
PhD stage
Status Academic activities
Mod
History 4
F
40–44 4th year
D/FT
Research, publication, teaching, professional networking, community and public engagement,
administration
2
History 5
F
30–34 4th year
D/FT
Research, publication, professional networking, community and public engagement, administration
3
History 6
F
35–39 4th year
D/FT
Research, publication, teaching, community and policy engagement, administration
2
History 7
M
30–34 Graduated
D/FT
Research, publication, teaching, professional networking, administration
2
History 8
M
> 50
Graduated
D/FT
Research, publication, teaching, professional networking, community and public engagement
3
Physics 1
M
25–29 3rd year
D/FT
Research, publication, teaching, supervising students, professional networking, grant writing,
administration
2
Physics 2
M
25–29 3rd year
D/FT
Research, publication, teaching, professional networking, grant writing, public outreach
2
Physics 3
M
30–34 Graduated/Postdoc D/FT
Research, publication, teaching, public outreach
1
Physics 4
M
25–29 4th year
D/FT
Research, publication, teaching, supervising and mentoring students, project management, adminis-
tration
2
Physics 5
M
25–29 1st year
D/FT
Research, publication, teaching, supervising students, grant writing, public outreach
2
Physics 6
F
25–29 4th year
D/FT
Research, publication, teaching, supervising students, grant writing, public outreach, administration
1
Biomed: biomedical engineering; E&B: economics and business; D: domestic; I: international; FT: full-time; PT: part-time; ‘service’ generally refers to institutional
vice contributing back to the running of the institution; ‘administration’ generally refers to the bureaucracy of the work Academic work from the perspectives of aspiring academics:… 1575 Acknowledgements This paper draws on a doctoral study supported by the Research Training Program
funded by the Australian Commonwealth Government. Ethics approval This project was provided ethics approval by the University of Melbourne Research Ethics
Committee (ID 1954070.1). Ethics approval This project was provided ethics approval by the University of Melbourne Research Ethics
Committee (ID 1954070.1). 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 permis-
sion directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/
licenses/by/4.0/. Appendix Funding Open Access funding enabled and organized by CAUL and its Member Institutions. This paper
draws on a doctoral study supported by the Research Training Program funded by the Australian Com-
monwealth Government. Declarations Competing interests The author has no relevant financial or non-financial conflict of interest to disclose References Australian Government. (2023). Australian Universities Accord—Interim Report. In Interim report. https://www.education.gov.au/download/16699/australian-universities-accord-interim-report/33821/
document/pdfii Australian Qualifications Framework Council. (2013). Australian Qualifications Framework: Second Edi-
tion January 2013. Australian Qualifications Framework Council. https://www.aqf.edu.au/sites/aqf/
files/aqf-2nd-edition-january-2013.pdf i
Barker, K. (2015). The challenging Australian policy context for university engagement. Journal of
Higher Education Policy and Management, 37(5), 477–489. https://doi.org/10.1080/1360080X.
2015.1079384i Bearman, M., Ryan, J., & Ajjawi, R. (2022). Discourses of artificial intelligence in higher education:
A critical literature review. Higher Education. https://doi.org/10.1007/s10734-022-00937-2 Becher, T., & Trowler, P. (2001). Academic tribes and territories: Intellectual enquiry and the culture
of disciplines (2nd ed.). Open University Press/SRHE. Becher, T., & Trowler, P. (2001). Academic tribes and territo
of disciplines (2nd ed.). Open University Press/SRHE. Boyer, E. L. (1990). Scholarship reconsidered: Priorities of the professoriate. Carnegie Foundation
for the Advancement of Teaching. Boyer, E. L. (1996a). From scholarship reconsidered to scholarship assessed. Quest, 48(2), 129–139. Boyer, E. L. (1990). Scholarship reconsidered: Priorities of the professoriate. Carnegie Foundatio
for the Advancement of Teaching Boyer, E. L. (1990). Scholarship reconsidered: Priorities of the professoriate. Carnegie Foundation
for the Advancement of Teaching. Boyer, E. L. (1996a). From scholarship reconsidered to scholarship assessed. Quest, 48(2), 129–139. https://doi.org/10.1080/00336297.1996.10484184 for the Advancement of Teaching. Boyer, E. L. (1996a). From scholarship reconsidered to scholarship assessed. Quest, 48(2), 129–13 https://doi.org/10.1080/00336297.1996.10484184 Boyer, E. L. (1996b). The scholarship of engagement. Bulletin of the American Academy of Arts and
Sciences, 49(7), 18–33. https://doi.org/10.2307/3824459 Brew, A. (2001). Conceptions of research: A phenomenographic study. Studies in Higher Education,
26(3), 271–285. https://doi.org/10.1080/03075070120076255 Brew, A. (2001). Conceptions of research: A phenomenographic study. Studies in Higher Education,
26(3), 271–285. https://doi.org/10.1080/03075070120076255
Brew, A. (2010). Transforming academic practice through scholarship. International Journal for Aca- Brew, A. (2010). Transforming academic practice through scholarship. International Journal for Ac
demic Development, 15(2), 105–116. https://doi.org/10.1080/13601441003737618 Canning, J., & Masika, R. (2020). The scholarship of teaching and learning (SoTL): The thorn in the
flesh of educational research. Studies in Higher Education. https://doi.org/10.1080/03075079.
2020.1836485 1 3 A. T. Le A. T. Le 1576 Clement, M., & Grant, B. (2010). Scholarship in times of super-complexity: Various accounts of what
it can be and how we need each other to enact it. International Journal for Academic Develop-
ment, 15(2), 103–105. https://doi.org/10.1080/13601441003737527 p
g
Coates, H., Croucher, G., Weerakkody, U., Moore, K., Dollinger, M., Kelly, P., Bexley, E., & Grose-
mans, I. (2020). An education design architecture for the future Australian doctorate. Higher
Education, 79(1), 79–94. https://doi.org/10.1007/s10734-019-00397-1 Coates, H., Dobson, I., Edwards, D., Friedman, T., Goedegebuure, L., & Meek, L. (2009). The attrac-
tiveness of the Australian academic profession: A comparative analysis. LH Martin Institute,
University of Melbourne & Australian Council for Educational Research & Educational Policy
Institute. https://minerva-access.unimelb.edu.au/handle/11343/28921 Institute. https://minerva-access.unimelb.edu.au/handle/11343/28921 Coates, H., & Goedegebuure, L. (2012). Recasting the academic workforce: Why the attractiveness of
the academic profession needs to be increased and eight possible strategies for how to go about
this from an Australian perspective. Higher Education, 64(6), 875–889. https://doi.org/10.1007/
s10734-012-9534-3 Coates, H., Goedegebuure, L., & Meek, V. L. (2015). Increasing the attractiveness of the academic
profession: A challenge for management. In U. Teichler & W. K. Cummings (Eds.), Forming,
recruiting and managing the academic profession (The Changing Academic Profession in Inter-
national Comparative Perspective, vol 14) (pp. 297–315). Cham: Springer. https://doi.org/10.
1007/978-3-319-16080-1_16 Doberneck, D. M., Glass, C. R., & Schweitzer, J. (2010). From rhetoric to reality: A typology of
publically engaged scholarship. Journal of Higher Education Outreach and Engagement, 14(4),
5–35. Edwards, D., Bexley, E., & Richardson, S. (2011). Regenerating the academic workforce: The
careers, intentions and motivations of higher degree research students in Australia: Findings
of the National Research Student Survey (NRSS). Australian Council for Educational Research
(ACER). https://research.acer.edu.au/higher_education/23f Fortune, T., Fyffe, J., & Barradell, S. https://doi.org/10.1080/00336297.1996.10484184 (2023). Reimagining the university of our dreams: heterotopic
havens for wounded academics. Higher Education Research & Development. https://doi.org/10.
1080/07294360.2023.2228213 Gardner, M. (2011). Just a knowledge worker? Academics, universities and industrial relations (The
Foenander Lecture in Industrial Relations). The University of Melbourne. https://fbe.unimelb.
edu.au/__data/assets/pdf_file/0006/2594922/Foenander_26th_2011.pdf pi
p
Given, L. (2008). Codes and Coding. In L. M. Given (Ed.), The SAGE Encyclopedia of Qualitative
Research Methods. Cham: SAGE Publications. https://doi.org/10.4135/9781412963909.n48 Green, J., Willis, K., Hughes, E., Small, R., Welch, N., Gibbs, L., & Daly, J. (2007). Generating best
evidence from qualitative research: The role of data analysis. Australian and New Zealand Jour-
nal of Public Health, 31(6), 545–550. https://doi.org/10.1111/j.1753-6405.2007.00141.x p
g
j
Huang, F., Aarrevaara, T., & Teichler, U. (2022). Teaching and Research in the Knowledge-Based
Society. Springer. Kenny, J., & Fluck, A. E. (2018). Research workloads in Australian universities. Australian Universi-
ties’ Review, 60(2), 25–37. Kern, B., Mettetal, G., Dixson, M. D., & Morgan, R. K. (2015). The role of SoTL in the academy:
Upon the 25th anniversary of boyer’s “Scholarship reconsidered.” Journal of the Scholarship of
Teaching and Learning., 15(3), 1–14. https://doi.org/10.14434/josotl.v15i3.13623 g
g
( )
p
g
j
Kogan, M. (2000). Higher education communities and academic identity. Higher Education Quar-
terly, 54(3), 207–216. https://doi.org/10.1111/1468-2273.00156 Kogan, M. (2000). Higher education communities and academic ide
terly, 54(3), 207–216. https://doi.org/10.1111/1468-2273.00156 Krause, K.-L. (2020). Scholarship and supercomplexity: Policy implications. In L. Ling & P. Ling
(Eds.), Emerging Methods and Paradigms in Scholarship and Education Research (pp. 263–
282). IGI Global. https://doi.org/10.4018/978-1-7998-1001-8.ch015 Le, A. T. (2023). To be or not to be (in academia)?‘Inward calling’and “academic hazards” in aspir-
ing academics’ career prospects in Australia. Higher Education Research & Development, 42(4),
874–887. https://doi.org/10.1080/07294360.2022.2123901 p
g
Mantai, L., & Marrone, M. (2023). Academic career progression from early career researcher to pro-
fessor: what can we learn from job ads. Studies in Higher Education. https://doi.org/10.1080/
03075079.2023.2167974 McAlpine, L., & Amundsen, C. (2018). Identity-trajectories of early career researchers: Unpacking
the post-PhD experience. Palgrave Macmillan. 1 3 3 Academic work from the perspectives of aspiring academics:… 1577 McGagh, J., Marsh, H., Western, M., Thomas, P., Hastings, A., Mihailova, M., & Wenham, M. (2016). Review of Australia’s Research Training System. Report for the Australian Council of
Learned Academies. https://acola.org/wp-content/uploads/2018/08/saf13-review-research-train
ing-system-report.pdf McGagh, J., Marsh, H., Western, M., Thomas, P., Hastings, A., Mihailova, M., & Wenham, M. (2016). Review of Australia’s Research Training System. Report for the Australian Council of
Learned Academies. Ai Tam Le is Researcher Development Coordinator at Deakin University (Australia). Her current research
interests include building communities in academia, cross-countries analysis of research training systems,
and academic training and development for graduate researchers. https://doi.org/10.1080/00336297.1996.10484184 https://acola.org/wp-content/uploads/2018/08/saf13-review-research-train
ing-system-report.pdf g y
p
p
Patton, M. Q. (2002). Qualitative research & evaluation methods (3rd ed.). SAGE Publications. Pratt, M. G., Kaplan, S., & Whittington, R. (2020). Editorial essay: The tumult over transparency:
Decoupling transparency from replication in establishing trustworthy qualitative research. Administrative Science Quarterly, 65(1), 1–19. https://doi.org/10.1177/0001839219887663 Probert, B. (2014a). Becoming a university teacher: The role of the PhD. Report commissioned by
Australian Government Office for Learning and Teaching Discussion. https://ltr.edu.au/resou
rces/Probert_Becoming_uni_teacher_%20discussion%20paper3.pdf Probert, B. (2014b). Why scholarship matters in higher education. Report commissioned by Austral-
ian Government Office for Learning and Teaching Discussion. https://ltr.edu.au/resources/Probe
rt_Scholarship_discussion_paper2.pdf Renwick, K., Selkrig, M., Manathunga, C., & Keamy, R. ‘. (2020). Community engagement is … :
Revisiting Boyer’s model of scholarship. Higher Education Research & Development., 39(6),
1232–1246. https://doi.org/10.1080/07294360.2020.1712680 Rice, R. E. (2002). Beyond scholarship reconsidered: Toward an enlarged vision of the scholarly work
of faculty members. New Directions for Teaching and Learning, 2002(90), 7–18. https://doi.org/
10.1002/tl.51 Sarrico, C. S. (2022). The expansion of doctoral education and the changing nature and purpose of the
doctorate. Higher Education, 84(6), 1299–1315. https://doi.org/10.1007/s10734-022-00946-1 Schneijderberg, C., & Teichler, U. (2018). Doctoral education, training and work in Germany. In J. C. Shin, B. M. Kehm, & G. A. Jones (Eds.), Doctoral Education for the Knowledge Society: Con-
vergence or Divergence in National Approaches? (pp. 13–34). Springer. https://doi.org/10.1007/
978-3-319-89713-4_2 Shin, J. C., Kehm, B. M., & Jones, G. A. (2018). The increasing importance, growth, and evolution
of doctoral education. In J. C. Shin, B. M. Kehm, & G. A. Jones (Eds.), Doctoral education for
the knowledge society: Convergence or divergence in national approaches? (pp. 1–10). Springer. https://doi.org/10.1007/978-3-319-89713-4_1 Teichler, U., Arimoto, A., & Cummings, W. K. (2013). The changing academic profession: Major
findings of a comparative survey (The Changing Academic Profession in International Compara-
tive Perspective) (Vol. 1). Springer. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published
maps and institutional affiliations. Ai Tam Le is Researcher Development Coordinator at Deakin University (Australia). Her current research
interests include building communities in academia, cross-countries analysis of research training systems,
and academic training and development for graduate researchers. 1 3 1 3 1 3
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ANALISIS DAN EVALUASI IMPLEMENTASI
PENGELOLAAN KEPEMILIKAN UMUM DAN NEGARA DI
INDONESIA (PENDEKATAN MADZHAB HAMFARA) Siti Murtiyani1*
Hery Sasono2
Dwi Condro Triono3
Hanifah Zahra4
1,2,3,4STEI Hamfara, Jogyakarta
Email: *1smurtiyani@yahoo.com
2herysasono@ymail.com ABSTRAK - Kajian ini bertujuan untuk menganalisis dan mengevaluasi secara kritis penerapan
pengelolaan kepemilikan umum (Milkiyah 'Ammah) dan kepemilikan negara (Milkiyah Daulah) di
Indonesia. Studi ini menjelaskan secara deskriptif tentang implementasi pengelolaan kedua kepemilikan
tersebut yang sekarang dipraktikkan di Indonesia. Teori yang digunakan dalam penelitian ini adalah
Sistem Ekonomi Islam Madzhab Hamfara (Hadza Min fadzli Rabbi) yang secara kritis mengevaluasi
implementasi pengelolaan kepemilikan umum dan negara di Indonesia. Kajian ini menggunakan data
sekunder yang berasal buku-buku madzhab Hamfara, literatur, artikel jurnal dan informasi lainnya yang
terkait dengan pengelolaan aset di Indonesia. Analisis perbandingan juga dilakukan untuk memahami
perbedaan pengelolaan kepemilikan umum dan kepemilikan negara. Hasil kajian menunjukkan bahwa
pengelolaan kepemilikan, baik kepemilikan umum maupun negara di Indonesia, tidak dijalankan sesuai
dengan sistem ekonomi Islam Madzhab Hamfara. Bukti-bukti menunjukkan bahwa pengelolaan
kepemilikan publik dan negara tidak sepenuhnya dilakukan oleh negara. Fakta juga menunjukkan bahwa
kepemilikan tersebut lebih banyak diserahkan kepada lembaga-lembaga privat bahkan lembaga-
lembaga asing. Akibatnya, kesenjangan pendapatan yang menjadi pemicu kemiskinan terus terjadi. Kajian ini menyarankan agar pemerintah mengimplementasikan sistem ekonomi Islam Madzhab
Hamfara dalam pengelolaan kepemilikan umum dan negara di Indonesia. p
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Kata kunci: Sistem ekonomi Islam Madzhab Hamfara, kepemilikan umum, kepemilikan negara ABSTRACT - This study aims to analyze and critically evaluate the implementation of the management
of public ownership (Milkiyah 'ammah) and State ownership (Milkiyah Daulah) in Indonesia. This study
descriptively describes the implementation of both ownerships that are currently practicing in Indonesia. Theoretical approaches used is the Islamic Economic System Madzhab Hamfara (Hadza Min fadzli
Rabbi) who critically evaluate the implementation of the public wealth management and state wealth in
Indonesia. This research used secondary data that was obtained from Madzhab Hamfara books,
literature, journal and information relating to the Indonesian State assets management system. A
comparative analysis was conducted to understand the difference between the public wealth
management and state wealth management in Indonesia. Findings show that the management of both,
public and state ownership are not in accordance to the ownership management approach of Madzhab
Hamfara Islamic Economic System. The evidence suggests that both public and state ownership are not
fully managed by the Government of Indonesia. Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 83 ANALISIS DAN EVALUASI IMPLEMENTASI
PENGELOLAAN KEPEMILIKAN UMUM DAN NEGARA DI
INDONESIA (PENDEKATAN MADZHAB HAMFARA) The fact shows that public ownership in the form of
water, fire and pastures are managed by individuals and institutions as well as foreign parties who have
fund to privatize the public ownership. Consequently, it has the impact on unequal distribution of income
that cause poverty in Indonesia. This research suggest that government should implement Islamic
Economic System Madzhab Hamfara in managing public and state ownership in Indonesia. Keywords : Islamic Economic System Madzhab Hamfara, Common Wealth, State Property SHARE | Volume 5 | Number 1 | January - June 2016 84 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ LATAR BELAKANG MASALAH Indonesia merupakan negara yang memiliki kekayaan sumber alam yang luar
biasa, mulai dari Minyak Bumi, Gas, Tambang Emas, Batubara, Nikel dan
kekayaan hutan yang membentang di seluruh kepulauan Indonesia. Ini adalah
karunia Allah SWT yang berlimpah sekaligus amanah kepada manusia untuk
mengelola kekayaan dengan benar dan sebaik-baiknya untuk kemaslahatan
masyarakat, khususnya di Indonesia. Oleh karena itu, Allah SWT
mengkaruniakan juga Agama Islam yang memberikan petunjuk bagaimana
mengatur seluruh aspek kehidupan, mulai dari hubungan antara sesama
manusia dan hubungan dengan Allah SWT Sang Pencipta dan Pengatur
kehidupan ini serta hubungan dengan alam dan seisinya. Agar seluruh umat
bisa mencapai apa yang menjadi tujuan hidup manusia adalah kesejahteraan
manusia di dunia dan juga di akhirat. Indonesia merupakan negara yang kaya sumber alam, tetapi kesejahteraan
rakyat belum bisa terwujud, berbagai permasalahan selalu melanda negara
Indonesia. Beberapa permasalahan yang mendasar berkaitan dengan
pengelolaan kekayaan alam yang ada di Indonesia. Kasus kenaikan Bahan
Bakar Minyak (BBM), kasus PT. Freeport McMoran yang tidak membayarkan
dividen, kasus gugatan PT. Newmont tentang pembatasan ekspor konsentrat
tembaga, dan berbagai persoalan persoalan penguasaan kekayaan Indonesia
melalui investasi yang dibuka selebar-lebarnya oleh pemerintah Indonesia. Indonesia merupakan negara yang kaya sumber alam, tetapi kesejahteraan
rakyat belum bisa terwujud, berbagai permasalahan selalu melanda negara
Indonesia. Beberapa permasalahan yang mendasar berkaitan dengan
pengelolaan kekayaan alam yang ada di Indonesia. Kasus kenaikan Bahan
Bakar Minyak (BBM), kasus PT. Freeport McMoran yang tidak membayarkan
dividen, kasus gugatan PT. Newmont tentang pembatasan ekspor konsentrat
tembaga, dan berbagai persoalan persoalan penguasaan kekayaan Indonesia
melalui investasi yang dibuka selebar-lebarnya oleh pemerintah Indonesia. Berdasarkan peta kepemilikan perusahaan asing di Indonesia, menunjukkan
bahwa sebagian besar kekayaan Negara Indonesia dikuasai dan dikelola oleh
negara lain dengan melakukan pembelian terhadap saham-saham BUMN dan
investasi yang besar-besaran di Indonesia, baik oleh pemilik modal dari luar
negeri maupun pemilik modal swasta yang melakukan privatisasi kekayaan
alam di Indonesia (lihat Gambar 1). Dari gambar tersebut menunjukkan bahwa sebagian besar kekayaan Indonesia
sudah dikuasai pihak asing melalui investasi besar-besaran dengan menguasai
lebih dari 50% saham kepemilikan perusahaan-perusahaan yang dikelola oleh
Negara Indonesia. Namun dalam perjalanannya muncul kasus-kasus yang
berkaitan dengan pengelolaan kekayaan umum dan kekayaan negara di
Indonesia. SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 85 Gambar 1. Peta Penguasaan Asing terhadap Kepemilikan Kekayaan Indonesia
Kasus yang sedang terjadi, dimana sudah sejak tahun 2011 PT. LATAR BELAKANG MASALAH Freeport
McMoran tidak membayar dividen sebesar 1,5 triliun per tahun, pada hal
kenaikan penjualan sebesar 6,2 % menjadi US $.4,34 miliar (Rp.49,59 triliun,
kurs 11,428 per USD) pada akhir tahun 2013, dibandingkan pada periode tahun
2012 sebesar USD $.4,09 miliar. Selain kejadian tersebut, sesuai amanat UU
No.4 tahun 2004 tentang Mineral dan Batubara, seharusnya negara menguasai
saham mayoritas (minimal 51%) dalam setiap usaha tambang. Sekarang
Indonesia hanya menguasai sekitar 9,6 %. Dan sudah tiga tahun ini pemerintah
tidak melaksanakan amanah undang-undang tersebut. Freeport tetap berkuasa
di tanah Papua yang merupakan bagian dari Negara Indonesia. Gambar 1. Peta Penguasaan Asing terhadap Kepemilikan Kekayaan Indonesia Kasus yang sedang terjadi, dimana sudah sejak tahun 2011 PT. Freeport
McMoran tidak membayar dividen sebesar 1,5 triliun per tahun, pada hal
kenaikan penjualan sebesar 6,2 % menjadi US $.4,34 miliar (Rp.49,59 triliun,
kurs 11,428 per USD) pada akhir tahun 2013, dibandingkan pada periode tahun
2012 sebesar USD $.4,09 miliar. Selain kejadian tersebut, sesuai amanat UU
No.4 tahun 2004 tentang Mineral dan Batubara, seharusnya negara menguasai
saham mayoritas (minimal 51%) dalam setiap usaha tambang. Sekarang
Indonesia hanya menguasai sekitar 9,6 %. Dan sudah tiga tahun ini pemerintah
tidak melaksanakan amanah undang-undang tersebut. Freeport tetap berkuasa
di tanah Papua yang merupakan bagian dari Negara Indonesia. PT. Newmont Nusa Tenggara (PTNNT) dan saham mayoritasnya, Nusa
Tenggara Partnership B.V (NTPBV) suatu Badan Usaha yang terdaftar di
Belanda mengajukan gugatan Arbitrase kepada The International Center for
The Settlement of Investment Dispute, untuk mengizinkan kembali mengekspor
konsentrat tembaga agar kegiatan tambang Batu Hijau dapat beroperasi
kembali. Dalam kasus ini pemerintah seharusnya tunduk kepada undang-
undang No. 4 tahun 2004 tentang Minerba yang menyatakan bahwa agar tidak
ada eksploitasi besar-besaran di sektor mineral dan menciptakan nilai tambah. Dengan
memberlakukan
undang-undang
tersebut
akan
mendorong
pertumbuhan dan pembangunan ekonomi di Indonesia, sehingga tidak hanya
mendorong
eksploitasi
kekayaan
alam
oleh
pihak
asing. Berbagai
permasalahan yang muncul dalam perekonomian di Indonesia diawali dengan
proses liberalisasi bidang ekonomi yang diawali pada masa orde baru dengan
terbitnya Undang-Undang Nomor 1 tahun 1967 tentang Penanaman Modal
Asing disahkan pada masa pemerintah orde baru. Dengan Undang-undang SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 86 tersebut memungkinkan investasi dari pihak asing dengan menanamkan
sahamnya secara besar-besaran di Indonesia. Pada tahun 1968 Presiden
Soeharto membentuk kabinet Mafia Berkeley sebagai Team penanaman modal
asing dengan Sudjatmoko sebagai Duta Besar Republik Indonesia di
Washington (Ransom: 2006 dalam Aria Susman, 2009). Mafia Berkeley memformat pembangunan Indonesia bertumpu pada Utang
sesuai dengan arahan International Monetary Fund (IMF) dan bank dunia. Pada tahun 1990-an Indonesia berusaha meningkatkan pertumbuhan ekonomi
dengan meningkatkan investasi asing dan pihak swasta. Akibatnya Utang luar
negeri swasta Indonesia meningkat tajam. Hal inilah yang mendorong
dilakukannya privatisasi perusahaan-perusahaan Indonesia dengan menjual
saham di pasar modal kepada para pengusaha Indonesia maupun pengusaha
tingkat Internasional. Dan sampai saat ini, pemerintah Negara Indonesia selalu
membiayai pembangunan Indonesia dengan berbasis pada Utang. Gambar 1. Peta Penguasaan Asing terhadap Kepemilikan Kekayaan Indonesia Utang inilah
membuat pemerintah melakukan privatisasi dengan menjual saham, dan hasil
penjualan saham digunakan untuk membayar Utang luar negeri. Dari penjelasan di atas perlu dikaji lebih luas, bagaimana perlunya mengkaji
kembali dan melakukan analisis untuk memberikan penjelasan kekeliruan dan
dampak kerusakan yang ditimbulkan dari kesalahan dalam pelaksanaan
pengelolaan kepemilikan umum maupun kepemilikan negara di Indonesia. Oleh karena itu tujuan dari penelitian ini untuk melakukan analisis aplikasi
pengelolaan kepemilikan umum dan kepemilikan Negara, memberikan kritik,
serta rekomendasi-rekomendasi yang diperlukan untuk melakukan perubahan
yang mendasar dalam aplikasi pengelolaan kekayaan umum dan kekayaan
Negara secara syariah. Dalam penelitian ini berbasis pada sistem ekonomi
Islam Madzhab Hamfara “Hadza min Fadli Rabbi”. Metode yang digunakan adalah metode induktif dalam menarik hukum syara’
yaitu dengan memahami fakta (fahmul waqi’) problematika yang terjadi dalam
masyarakat, kemudian dilakukan pemahaman terhadap nash (fahmun nushus)
bagaimana problematika dalam masyarakat terhadap aplikasi kepemilikan
umum dan kepemilikan negara dan dampak terhadap kesejahteraan
masyarakat. Selanjutnya dilakukan penarikan hukum (istinbathul ahkam) yaitu
proses penarikan kesimpulan hukum syari’at terhadap status perbuatan
manusia yang hendak dihukumi yaitu pengelolaan kekayaan umum dan
kekayaan negara dalam Negara Indonesia. Apakah penarikan status hukum
syari’at wajib, sunnah, mubah, makruh atau haram. SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 87 PRIVATISASI DALAM SISTEM NEOLIBERALISME Privatisasi atau denasionalisasi merupakan proses pengalihan kepemilikan dari
kepemilikan umum dan kepemilikan negara menjadi kepemilikan pribadi atau
kelompok pribadi yang memiliki modal dengan membeli sebagian besar
kepemilikan saham perusahaan milik negara, Sedangkan lawan kata dari
privatisasi adalah nasionalisasi (wikipedia). Dengan demikian privatisasi
adalah upaya untuk melakukan perubahan terhadap status kepemilikan
perusahaan milik negara maupun milik umum menjadi kepemilikan individu
atau pribadi serta institusi tertentu baik pihak pribadi dalam Negara Indonesia
maupun oleh pihak asing. Dalam pandangan Islam privatisasi terhadap
kekayaan umum tidak diperbolehkan Nabi Saw bersabda: الن َّاسُ شُرَكَاءُ فِيْ ثَلاَث:
ُاَلْمَاء ُوَالْكَلاَءُ وَالن َّار Masyarakat itu berserikat dalam tiga perkara (barang): air, padang
gembalaan dan api. (HR. Bukhari dan Muslim). Telah diriwayatkan dari Abyadl bin Jamal, bahwa dia pernah datang kepada
Rasulullah SAW lalu dia meminta Rasulullah agar memberinya tambang
garam, dan Rasulullah pun memberinya. Ketika Abyadl pergi, seorang sahabat
di majelis berkata kepada Rasulullah, “Wahai Rasulullah, tahukah Anda, apa
yang telah Anda berikan kepadanya? Sesungguhnya Anda telah memberikan
kepadanya sesuatu (yang bagaikan ) air mengalir.”Rasulullah kemudian
menarik kembali pemberian tersebut. Orang tersebut menyerupakan tambang
garam dengan air mengalir, karena banyaknya produksi pada tambang garam
tersebut. Ini mencakup pula setiap tambang dengan produksi dalam kuantitas
yang banyak atau menguntungkan secara ekonomis, seperti tambang minyak,
gas, fosfat, tembaga, dan sebagainya. Privatisasi lahir bersamaan dengan ide neo-liberalisme yang diperkenalkan
pada tahun 1980-an. Pemikiran ini dicetuskan oleh Milton Freedman, penasihat
ekonomi Presiden AS saat itu, Ronald Reagan, dan Frederick High, penasihat
ekonomi PM Inggris, Margaret Thatcher. Latar belakang neo-liberalisme
berawal pada tahun 1975, di Amerika Serikat, Robert Nozick mengeluarkan
sebuah tulisan berjudul “Anarchy, State, and Utopia“, yang menyatakan
kembali posisi kaum ultra minimalis, ultra libertarian sebagai retorika dari
lembaga pengkajian universitas, yang kemudian disebut dengan istilah
“Reaganomics”. Reaganomics atau Reaganisme ini menyebarkan retorika
kebebasan yang dihubungkan dengan pemikiran Locke. Sedangkan di Inggris,
Keith
Joseph
menjadi
arsitek
“Thatcherisme”. Thatcherisme SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 88 menghubungkannya dengan pemikiran liberal klasik Mill dan Smith. Walaupun sedikit berbeda, tetapi kesimpulan akhirnya sama
yaitu
menghilangkan peran negara dalam pengelolaan kekayaan alam (Soemantri,
2011). Gagasan-gagasan ini kemudian tersebar luas ke berbagai negara, khususnya
Amerika Serikat dan Eropa Barat. SHARE | Volume 5 | Number 1 | January - June 2016 PRIVATISASI DALAM SISTEM NEOLIBERALISME Sebagaimana penelitian World Bank pada
tahun 1992, tercatat semenjak tahun 1980 sudah lebih dari 80 negara yang telah
melaksanakan privatisasi dan melibatkan 6.800 badan usaha milik negara yang
terjadi di seluruh dunia. Perusahaan-perusahaan yang sebelumnya dikelola oleh
negara banyak yang dijual dan dikelola oleh perusahaan-perusahaan swasta
dengan memakai kerangka perjanjian-perjanjian tertentu. Oleh karena itu,
proses privatisasi terhadap perusahaan-perusahaan yang dikelola oleh Negara
semakin sukses sesuai dengan amanat undang-undang yang telah mereka
bentuk melalui parlemen. Kebijakan neoliberal di Indonesia semakin tidak terkendali dengan masuknya
IMF yang lahir pada akhir tahun 1997 dalam penataan ekonomi negara. Melalui pengawasan yang sangat ketat, IMF memaksa Indonesia menjalankan
kebijakan neoliberal. Indonesia ditekan untuk melakukan reformasi ekonomi
yang didasarkan pada pemikiran ekonomi neo-liberal. Di antaranya: 1)
Menghilangkan dan mengurangi Intervensi pemerintah dalam mengelola
perusahaan negara; 2) melaksanakan swastanisasi perekonomian Indonesia
seluas-luasnya; 3) melakukan liberalisasi seluruh kegiatan ekonomi dengan
menghilangkan segala bentuk proteksi dan subsidi; 4) memperbesar dan
memperlancar arus masuk modal asing dengan fasilitas yang lebih besar. Dengan demikian IMF merupakan pemeran utama dalam proses liberalisasi
sistem ekonomi di seluruh dunia. Sebagai upaya melancarkan gagasan neo-liberalisme maka kemudian
dibentuklah berbagai produk undang-undang (UU) yang pro neo-liberalisme
sebagai konsekuensi logis dalam menjalankan letter of intent (LoI) dengan
IMF. Produk undang-undang pada sektor strategis di antaranya: UU nomor 41
tahun 1999 tentang kehutanan; UU no. 29 tahun 2000 tentang Perlindungan
Varietas Tanaman; UU nomor 22 tahun 2001 tentang Minyak dan Gas;UU no. 18 tahun 2004 tentang Perkebunan; UU no. 31 tahun 2004 tentang perikanan;
UU nomor 7 tahun 2004 tentang Sumber Daya Air; UU nomor 25 tahun 2007
tentang Penanaman Modal; UU nomor 36 Tahun 2009 tentang kesehatan; UU
nomor 30 tahun 2009 Tentang Ketenagalistrikan; UU No. 41 tahun 2009
tentang Perlindungan Laba Pertanian Pangan Indonesia, semua undang-undang
tersebut semakin memperlancar masuknya investor asing dalam mengambil SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 89 alih kepemilikan umum maupun kepemilikan Negara di Indonesia sampai
sekarang. Kerjasama yang dilakukan oleh para pemimpin yang berkuasa adalah melalui
parlemen
yang mengesahkan produk undang-undang tersebut untuk
melancarkan kerjasama dengan negara-negara dan pihak-pihak pemilik modal
yang menjadi penggerak dalam rangka penguasaan kekayaan Indonesia melalui
pengambilalihan kekayaan dengan penjualan saham pada perusahaan-
perusahaan milik negara maupun perusahaan umum lainnya. Penjualan saham
tersebut dilakukan dalam rangka memenuhi kebutuhan untuk pembayaran
utang kepada IMF yang jumlahnya setiap tahun meningkat. PENDEKATAN TEORI EKONOMI ISLAM MADZHAB HAMFARA Sistem ekonomi Islam Madzhab Hamfara merupakan sistem ekonomi yang
dibangun dan dikembangkan berdasarkan pada ketentuan-ketentuan yang
ditunjukkan dalam kitab Al-Qur’an dan Al-hadits. Kata Hamfara kependekkan
dari Hadza Min Fadli Rabbi yang artinya karunia dari Tuhanku. Jadi
berdasarkan petunjuk dari kitab-kitab itulah teori ekonomi Islam dibangun dan
dikembangkan, untuk memberikan gambaran yang lebih komprehensif tentang
sistem ekonomi Islam yang Rahmatan lil ‘alamin, serta memberikan solusi
alternatif dalam mengelola kepemilikan umum (milkiyah ‘ammah) dan
kepemilikan Negara (milkiyah daulah) di Indonesia, dan mengembalikan
kekayaan Negara yang dikuasai pihak asing dan para pemilik modal dengan
menerapkan sistem ekonomi Islam Madzhab Hamfara. Ada 3 pilar dalam konsep kepemilikan pada sistem ekonomi Islam yaitu, 1)
kepemilikan individu (milkiyah fardiyah), 2) kepemilikan umum (milkiyah
‘ammah), 3) kepemilikan Negara (milkiyah daulah) (lihat Gambar 2). Dari
seluruh harta kekayaan yang dimiliki, masing-masing dikelola dan
dikembangkan
berdasarkan
kepemilikannya,
kepemilikan
individu
dikembangkan melalui mekanisme pasar syariah, sedangkan pengelolaan
kekayaan umum dan kekayaan Negara dikelola dan dikembangkan oleh Negara
untuk kesejahteraan rakyatnya. Kepemilikan individu didefinisikan sebagai hukum syariat yang berlaku bagi
zat atau manfaat tertentu, yang memungkinkan bagi yang memperolehnya
untuk memanfaatkannya secara langsung atau mengambil kompensasi (‘iwad)
dari barang tersebut. Misalnya ketika bekerja menghidupkan tanah mati,
menggali kandungan bumi, berburu, samsarah (makelar), Mudharabah,
Musaqat, Ijaratul-ajir. Sedangkan kepemilikan umum adalah ijin Asy-syari’ SHARE | Volume 5 | Number 1 | January - June 2016 0
Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 90 kepada suatu komunitas untuk bersama-sama memanfaatkan suatu benda. Benda-benda yang masuk dalam kategori kepemilikan umum adalah benda-
benda yang dinyatakan Asy-syari’ diperuntukan bagi suatu komunitas dan
mereka saling membutuhkan. Asy-Syari’ melarang benda tersebut hanya
dikuasai seorang saja (An-Nabbani, 1990). Untuk lebih jelasnya bisa kita
cermati dalam kerangka Sistem Ekonomi Islam berikut ini: Gambar 2. Kerangka Sistem Ekonomi Islam Madzhab Hamfara Gambar 2. Kerangka Sistem Ekonomi Islam Madzhab Hamfara Gambar 2. Kerangka Sistem Ekonomi Islam Madzhab Hamfara
SISTEM EKONOMI ISLAM
KEPEMILIKAN
INDIVIDU
KEPEMILIKAN UMUM
1. BARANG YANG MENJADI
KEBUTUHAN UMUM
2. TAMBANG DALAM JUMLAH BESAR
3. BARANG YANG TIDAK DAPAT
DIMILIKI INDIVIDU
KEPEMILIKAN
NEGARA
1. JIZYAH
2. KHARAJ
3. GHANIMAH
4. FA’I
5. ‘USYUR
6. 20% RIKAZ
7. HARTA TANPA AHLI
WARIS
8. HARTA ORANG MURTAD
9. BERBAGAI LAHAN,
BANGUNAN MILIK
NEGARA
SELURUH HARTA KEKAYAAN
MEKANISME
PASAR
SYARI’AH
DIKELOLA
OLEH NEGARA
DIKELOLA
OLEH NEGARA
? PENDEKATAN TEORI EKONOMI ISLAM MADZHAB HAMFARA Sumber: Condro, 2011
Harta kekayaan yang dimiliki umum meliputi barang-barang yang menjadi
kebutuhan umum, tambang dalam jumlah besar, dan barang-barang yang tidak
dapat dimiliki individu. Sedangkan harta kekayaan yang dimiliki Negara
adalah: Jizyah, Kharaj, Ghanimah, Fa’i, ‘Usyur, 20% Rikaz, harta tanpa ahli
waris, harta orang murtad dan berbagai lahan bangunan milik Negara. Dari
kedua jenis harta kekayaan umum dan kekayaan Negara itulah yang
seharusnya dikelola oleh Negara atas nama rakyat dan sebesar-besarnya untuk
kesejahteraan rakyat. Dengan sistem ekonomi Islam ini Negara memiliki
kewenangan penuh untuk mengelola, mengembangkan, menjaga agar bisa
digunakan untuk kepentingan Negara dalam rangka menyejahterakan
rakyatnya. Gambar 2. Kerangka Sistem Ekonomi Islam Madzhab Hamfara
SISTEM EKONOMI ISLAM
KEPEMILIKAN
INDIVIDU
KEPEMILIKAN UMUM
1. BARANG YANG MENJADI
KEBUTUHAN UMUM
2. TAMBANG DALAM JUMLAH BESAR
3. BARANG YANG TIDAK DAPAT
DIMILIKI INDIVIDU
KEPEMILIKAN
NEGARA
1. JIZYAH
2. KHARAJ
3. GHANIMAH
4. FA’I
5. ‘USYUR
6. 20% RIKAZ
7. HARTA TANPA AHLI
WARIS
8. HARTA ORANG MURTAD
9. BERBAGAI LAHAN,
BANGUNAN MILIK
NEGARA
SELURUH HARTA KEKAYAAN
MEKANISME
PASAR
SYARI’AH
DIKELOLA
OLEH NEGARA
DIKELOLA
OLEH NEGARA
? Sumber: Condro, 2011 SISTEM EKONOMI ISLAM SELURUH HARTA KEKAYAAN KEPEMILIKAN
INDIVIDU MEKANISME
PASAR
SYARI’AH 8. 9. DIKELOLA
OLEH NEGARA Sumber: Condro, 2011 Harta kekayaan yang dimiliki umum meliputi barang-barang yang menjadi
kebutuhan umum, tambang dalam jumlah besar, dan barang-barang yang tidak
dapat dimiliki individu. Sedangkan harta kekayaan yang dimiliki Negara
adalah: Jizyah, Kharaj, Ghanimah, Fa’i, ‘Usyur, 20% Rikaz, harta tanpa ahli
waris, harta orang murtad dan berbagai lahan bangunan milik Negara. Dari
kedua jenis harta kekayaan umum dan kekayaan Negara itulah yang
seharusnya dikelola oleh Negara atas nama rakyat dan sebesar-besarnya untuk
kesejahteraan rakyat. Dengan sistem ekonomi Islam ini Negara memiliki
kewenangan penuh untuk mengelola, mengembangkan, menjaga agar bisa
digunakan untuk kepentingan Negara dalam rangka menyejahterakan
rakyatnya. Harta kekayaan yang dimiliki umum meliputi barang-barang yang menjadi
kebutuhan umum, tambang dalam jumlah besar, dan barang-barang yang tidak
dapat dimiliki individu. Sedangkan harta kekayaan yang dimiliki Negara
adalah: Jizyah, Kharaj, Ghanimah, Fa’i, ‘Usyur, 20% Rikaz, harta tanpa ahli
waris, harta orang murtad dan berbagai lahan bangunan milik Negara. Dari
kedua jenis harta kekayaan umum dan kekayaan Negara itulah yang
seharusnya dikelola oleh Negara atas nama rakyat dan sebesar-besarnya untuk
kesejahteraan rakyat. Dengan sistem ekonomi Islam ini Negara memiliki
kewenangan penuh untuk mengelola, mengembangkan, menjaga agar bisa
digunakan untuk kepentingan Negara dalam rangka menyejahterakan
rakyatnya. PENDEKATAN TEORI EKONOMI ISLAM MADZHAB HAMFARA Oleh karena itu, pemerintah suatu negara perlu mengembangkan politik
ekonomi Islam sebagai landasan dalam melaksanakan amanah menerapkan
sistem ekonomi Islam Madzhab Hamfara untuk mengelola kekayaan milik SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 91 umum dan milik Negara. Dengan politik ekonomi Islam pemerintah bisa
membentuk sistem pengelolaan kekayaan lebih strategis dan terstruktur dengan
membentuk Baitul Mal sebagai bagian dari politik ekonomi Islam. Baitul Mal
tersebut memiliki 3 sektor yang masing-masing memiliki peran khusus yaitu: 1. Sektor kepemilikan individu 2. Sektor kepemilikan umum, dan 3. Sektor kepemilikan negara. Berikut ini skema politik ekonomi Islam Madzhab Hamfara pada Gambar 3. Gambar 3. Politik Ekonomi Islam Madzhab Hamfara
POLITIK EKONOMI ISLAM
KEPEMILIKAN
INDIVIDU
KEPEMILIKAN
UMUM
KEPEMILIKAN
NEGARA
SELURUH HARTA KEKAYAAN
MEKANISME
PASAR
SYARI’AH
DIKELOLA
OLEH NEGARA
DIKELOLA
OLEH NEGARA
BAITUL MAL (APBN):
1. SEKTOR KEPEMILIKAN INDIVIDU
2. SEKTOR KEPEMILIKAN UMUM
3. SEKTOR KEPEMILIKAN NEGARA
ZAKAT,
INFAQ,
SHODAQOH
KEBIJAKAN
FISKAL
KEBIJAKAN
MONETER
FUNGSI BAITUL MAL
Sumber: Condro, 2011
Politik ekonomi Islam Madzhab Hamfara ini penekanan kebijakan fiskal dan
kebijakan moneter terfokus kepada pengelolaan kepemilikan Individu,
kepemilikan umum dan kepemilikan negara. Sehingga setiap permasalahan
ekonomi bisa diselesaikan dengan baik melalui sektor-sektor tersebut. Peran
Baitul Mal sangat penting dalam rangka mengembangkan kepemilikan
individu, kepemilikan umum dan kepemilikan negara, karena merupakan
sentral dalam pengelolaan kekayaan, dan bertanggung jawab dalam melakukan
distribusi kekayaan kepada masyarakat. Gambar 3. Politik Ekonomi Islam Madzhab Hamfara Gambar 3. Politik Ekonomi Islam Madzhab Hamfara Gambar 3. Politik Ekonomi Islam Madzhab Hamfara Gambar 3. Politik Ekonomi Islam Madzhab Hamfara
POLITIK EKONOMI ISLAM
KEPEMILIKAN
INDIVIDU
KEPEMILIKAN
UMUM
KEPEMILIKAN
NEGARA
SELURUH HARTA KEKAYAAN
MEKANISME
PASAR
SYARI’AH
DIKELOLA
OLEH NEGARA
DIKELOLA
OLEH NEGARA
BAITUL MAL (APBN):
1. SEKTOR KEPEMILIKAN INDIVIDU
2. SEKTOR KEPEMILIKAN UMUM
3. SEKTOR KEPEMILIKAN NEGARA
ZAKAT,
INFAQ,
SHODAQOH
KEBIJAKAN
FISKAL
KEBIJAKAN
MONETER
FUNGSI BAITUL MAL
Sumber: Condro, 2011 Gambar 3. Politik Ekonomi Islam Madzhab Hamfara
POLITIK EKONOMI ISLAM
KEPEMILIKAN
INDIVIDU
KEPEMILIKAN
UMUM
KEPEMILIKAN
NEGARA
SELURUH HARTA KEKAYAAN
MEKANISME
PASAR
SYARI’AH
DIKELOLA
OLEH NEGARA
DIKELOLA
OLEH NEGARA
BAITUL MAL (APBN):
1. SEKTOR KEPEMILIKAN INDIVIDU
2. SEKTOR KEPEMILIKAN UMUM
3. PENDEKATAN TEORI EKONOMI ISLAM MADZHAB HAMFARA SEKTOR KEPEMILIKAN NEGARA
ZAKAT,
INFAQ,
SHODAQOH
KEBIJAKAN
FISKAL
KEBIJAKAN
MONETER
FUNGSI BAITUL MAL
Sumber: Condro, 2011
Politik ekonomi Islam Madzhab Hamfara ini penekanan kebijakan fiskal dan POLITIK EKONOMI ISLAM Sumber: Condro, 2011 Sumber: Condro, 2011 Politik ekonomi Islam Madzhab Hamfara ini penekanan kebijakan fiskal dan
kebijakan moneter terfokus kepada pengelolaan kepemilikan Individu,
kepemilikan umum dan kepemilikan negara. Sehingga setiap permasalahan
ekonomi bisa diselesaikan dengan baik melalui sektor-sektor tersebut. Peran
Baitul Mal sangat penting dalam rangka mengembangkan kepemilikan
individu, kepemilikan umum dan kepemilikan negara, karena merupakan
sentral dalam pengelolaan kekayaan, dan bertanggung jawab dalam melakukan
distribusi kekayaan kepada masyarakat. SHARE | Volume 5 | Number 1 | January - June 2016 SHARE | Volume 5 | Number 1 | January - June 2016 2
Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 92 Dalam politik ekonomi Islam ini fungsi Baitul Mal inilah yang bisa
memberikan jaminan kesejahteraan rakyat dengan mengembangkan potensi
kekayaan negara. Setiap pendapatan yang diperoleh Baitul Maal dalam periode
tertentu yang umumnya satu tahun dialokasikan untuk memenuhi kebutuhan
operasional negara yang meliputi belanja rumah tangga negara, kebutuhan
modal perusahaan milik negara, dan kebutuhan warga negara lainnya dengan
distribusi yang benar dan adil sesuai dengan mekanisme pasar syariah yang
berlaku. Kepemilikan Umum (Milkiyah ‘Ammah) Kepemilikan umum adalah izin Asy-Syari’ kepada suatu komunitas untuk
bersama-sama memanfaatkan suatu benda. Benda-benda yang termasuk dalam
kategori kepemilikan umum adalah benda-benda yang dinyatakan Asy-Syari’
diperuntukkan bagi suatu komunitas dan mereka saling membutuhkan. Asy-
Syari’ melarang benda tersebut hanya dikuasai seorang saja. Untuk lebih
jelasnya dapat kita pahami melalui Gambar 4 berikut. Gambar 4. Skema Kepemilikan Umum (Milkiyah ‘Ammah) Gambar 4. Skema Kepemilikan Umum (Milkiyah ‘Ammah)
KEPEMILIKAN UMUM
HARTA BENDA
KEBUTUHAN UMUM
TAMBANG YANG
BESAR
BARANG YANG TIDAK
MUNGKIN DIMILIKI
INDIVIDU
“Manusia bersekutu
dalam tiga hal: air,
padang rumput dan
api”. ‘Wahai Rasulullah, tahukah
engkau apa yang engkau
berikan kepadanya? Sesungguhnya engkau telah
memberikan sesuatu yang
bagaikan air mengalir’. Rasulullah kemudian
bersabda: ‘Tariklah
tambang tersebut darinya’.”
“Mina adalah
tempat tinggal
orang yang terlebih
dahulu datang”
Sumber: Condro, 2011
Kepemilikan umum menurut pandangan Sistem Ekonomi Islam Madzhab
Hamfara dapat dibagi lagi menjadi tiga yaitu:
1. Barang kebutuhan umum KEPEMILIKAN UMUM BARANG YANG TIDAK
MUNGKIN DIMILIKI
INDIVIDU HARTA BENDA
KEBUTUHAN UMUM TAMBANG YANG
BESAR “Manusia bersekutu
dalam tiga hal: air,
padang rumput dan
api”. “Mina adalah
tempat tinggal
orang yang terlebih
dahulu datang” ‘Wahai Rasulullah, tahukah
engkau apa yang engkau
berikan kepadanya? Sesungguhnya engkau telah
memberikan sesuatu yang
bagaikan air mengalir’. Rasulullah kemudian
bersabda: ‘Tariklah
tambang tersebut darinya’.” Sumber: Condro, 2011 Kepemilikan umum menurut pandangan Sistem Ekonomi Islam Madzhab
Hamfara dapat dibagi lagi menjadi tiga yaitu: 1. Barang kebutuhan umum SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 93 Barang kebutuhan umum adalah segala jenis barang atau harta yang masuk
kategori fasilitas umum, yang jika tidak ada dalam suatu negeri atau dalam
suatu komunitas tertentu, maka akan menimbulkan sengketa dalam
mencarinya. Dengan kata lain barang kebutuhan umum adalah apa saja
yang dianggap sebagai kepentingan manusia secara umum, seperti sumber-
sumber air, padang gembalaan, kayu-kayu bakar, energi listrik dan
sebagainya. Rasulullah SAW bersabda:”Kaum muslimin berserikat dalam
tiga hal yaitu air, padang gembalaan dan api (HR. Abu Dawud). dan
harganya adalah haram (HR. Imam Ibnu Majah). Makna dari tambahan
kalimat Hadits di atas adalah mengambil tsaman, yaitu keuntungan dari
harga yang diambil dengan menjual ketiga komoditas tersebut kepada
rakyat hukumnya adalah haram (Condro, 2011). Kepemilikan Umum (Milkiyah ‘Ammah) Hadits lain yang berhubungan dengan barang yang menjadi kebutuhan
hidup adalah hadits yang diriwayatkan oleh Ibnu Majah, dari Abu Hurairah
r.a, beliau berkata: “Bahwasannya Rasulullah SAW telah bersabda: Ada
tiga hal yang tidak akan pernah dilarang (untuk dimiliki siapapun): air,
padang gembalaan dan api” (HR. Ibnu Majah). Larangan Rasulullah SAW
sesungguhnya bukan terletak pada larangan memiliki ketiga jenis barang
tersebut, melainkan dari segi sifatnya, yaitu dari segi apakah barang
tersebut dibutuhkan oleh orang banyak dalam suatu komunitas tertentu
ataukah tidak. Sebagai ilustrasi misalnya dalam suatu daerah terdapat
sebuah danau dengan air yang berlimpah dan di sekelilingnya terhampar
sawah-sawah dan ladang yang subur dengan air dari danau tersebut, maka
negara wajib membantu dengan membuatkan sistem distribusi pengairan/
irigasi yang baik dan memastikan bahwa air bisa didistribusikan dan
digunakan
secara
luas
oleh
masyarakat
untuk
meningkatkan
kesejahteraannya. Oleh karena itu negara harus mencegah kemungkinan munculnya orang
atau sekelompok orang yang menguasai danau tersebut untuk kepentingan
sendiri dan kelompoknya dalam rangka memperkaya diri dan kelompoknya
saja. Sehingga menimbulkan pertentangan dalam masyarakat karena
mereka mengklaim bahwa danau itu adalah milik salah satu atau kelompok
orang tersebut. Dengan pengaturan oleh negara maka kepemilikan umum
tersebut bisa dinikmati oleh masyarakat secara luas untuk meningkatkan
kesejahteraannya. SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 94 2. Barang tambang yang besar Barang tambang dapat dikelompokkan menjadi dua: 1) barang tambang
yang jumlahnya terbatas, barang tambang yang terbatas jumlahnya
termasuk kepemilikan pribadi atau boleh dimiliki secara pribadi. Terhadap
tambang yang berjumlah kecil akan diberlakukan hukum rikaz, yaitu di
dalamnya ada seperlima (1/5) bagian harta yang harus dikeluarkan
zakatnya. 2) Barang tambang yang besar. Barang tambang yang besar atau
tambang tidak terbatas jumlahnya, yang tidak mungkin dihabiskan dari
Imam At-Thirmidzi, yang meriwayatkan Hadits dari Abyadh bin Hamal: ”Sesungguhnya ia pernah meminta Rasulullah SAW untuk mengelola
tambang garamnya. Lalu beliau memberikannya. Setelah ia pergi, ada
seorang majelis tersebut bertanya,”Wahai Rasulullah. Tahukah engku, apa
yang engkau berikan kepadanya? Sesungguhnya engkau telah memberikan
sesuatu yang bagaikan air mengalir (ma’ul-‘iddu): kemudian Rasul
bersabda: “Tariklah tambang tersebut darinya” (HR.At-Thirmidzi). Oleh karena itu barang tambang yang besar harus dikelola oleh Negara
dengan tujuan untuk kesejahteraan masyarakatnya. 3. Sumber daya alam, yang sifat pembentukannya menghalangi untuk dimiliki
individu. 3. Sumber daya alam, yang sifat pembentukannya menghalangi untuk dimiliki
individu. Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ Gambar 5. Skema Contoh Kepemilikan Umum Sumber: Condro, 2011 Sumber: Condro, 2011 3. Sumber daya alam, yang sifat pembentukannya menghalangi untuk dimiliki
individu. Sumber daya alam yang dimaksud di sini adalah sumber daya alam yang
sifat pembentukannya mencegah untuk dimiliki secara pribadi, Jenis barang
ini berbeda dengan kelompok jenis barang pertama, yang dari segi zatnya
memang boleh dimiliki oleh individu, seperti individu boleh memiliki
sumber air pribadi. Namun demikian kepemilikan sumber daya air itu
memiliki ‘illat, yaitu akan menjadi terlarang untuk dimiliki oleh individu
apabila sumber daya air itu dibutuhkan oleh suatu komunitas masyarakat
tertentu. Sebagai ilustrasi misalnya sumber air dalam suatu wilayah tertentu
yang digunakan sebagai sumber kehidupan masyarakat, maka sumber air
ini tidak boleh dimanfaatkan sendiri oleh individu maupun perusahaan
untuk pabrik minuman air kemasan dan dijual kepada masyarakat. Hal ini
sama saja memperkaya diri sendiri dan perusahaannya. Berikut ini Gambar
5 skema contoh kepemilikan umum yang meliputi harta benda kebutuhan
umum, tambang yang besar dan barang yang tidak mungkin dimiliki oleh
individu. Dari perincian gambaran benda-benda ini untuk mempertegas
bahwa benda-benda berikut ini membedakan mana-mana yang boleh
dimiliki secara umum dan benda yang tidak boleh dimiliki secara individu,
sehingga tidak menimbulkan ketimpangan dalam distribusi kekayaan ini. SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 95 SHARE | Volume 5 | Number 1 | January - June 2016 SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 96 Gambar 5. Skema Contoh Kepemilikan Umum
KEPEMILIKAN UMUM
HARTA BENDA
KEBUTUHAN UMUM
TAMBANG YANG
BESAR
BARANG YANG TIDAK
MUNGKIN DIMILIKI
INDIVIDU
CONTOH:
1. Sumber daya air
2. Sumber daya
hutan, padang
rumput. 3. Sumber daya
energi: minyak
bumi, gas, batu
bara, uranium. CONTOH:
1. Tambang emas. 2. Tambang perak. 3. Tambang tembaga
4. Tambang nikel
5. Tambang bauksit
6. Tambang bijih besi
7. Tambang timah
8. Tambang kuarsa
CONTOH:
1. Jalan
2. Jembatan
3. Sungai
4. Danau
5. Gunung
6. Bukit
7. Laut
8. Pantai
Sumber: Condro, 2011 Gambar 5. Skema Contoh Kepemilikan Umum Distribusi Kepemilikan Umum Infak
disalurkan untuk belanja pengembangan kepemilikan umum, misalnya
membiayai dewan kepemilikan umum, menggaji pakar, pekerja kepemilikan
umum, membeli sarana produksi kepemilikan umum. 2). Tauzi’ yaitu
pendistribusian langsung kepada rakyat, misalnya; dibagi secara gratis: air,
listrik dan minyak, menjual ke industri dalam negeri atau luar negeri,
membagikan hasil keuntungan kepada rakyat. 3). Hima, pengkhususan
kepemilikan umum untuk keperluan Negara dan rakyat, antara lain: untuk
keperluan jihad, pembiayaan instansi pemerintah, ta’wids (santunan penguasa),
gaji PNS (Pegawai Negeri Sipil) dan tentara, pembiayaan fasilitas umum, infaq
fakir, miskin, dan lain-lainnya. Untuk lebih jelaskan bisa dilihat pada
Gambar.6 tentang Distribusi kekayaan Umum: Distribusi Kepemilikan Umum Dalam Sistem Ekonomi Islam Madzhab Hamfara distribusi kepemilikan umum
dikelompokkan menjadi tiga bagian yaitu Infaq, Tauzi’ dan Hima. Dalam Sistem Ekonomi Islam Madzhab Hamfara distribusi kepemilikan umum
dikelompokkan menjadi tiga bagian yaitu Infaq, Tauzi’ dan Hima. Gambar 6. Skema Distribusi Kepemilikan Umum. SHARE | Volume 5 | Number 1 | January - June 2016
DISTRIBUSI KEPEMILIKAN UMUM
INFAQ
TAUZI’
BELANJA UNTUK
PENGEMBANGAN
KEPEMILIKAN UMUM
CONTOH:
1. Membiayai Diwan
kep. Umum
2. Menggaji pakar,
pekerja kep. Umum
3. Membeli sarana
produksi Kep. Umum
PENDISTRIBUSIAN
LANGSUNG KEPADA
RAKYAT
CONTOH:
1. Dibagi secara gratis: air,
listrik, minyak
2. Menjual ke industri
dalam negeri atau luar
negeri
3. Membagikan hasil
keuntungan kepada
rakyat
HIMA
PENGKHUSUSAN KEP. UMUM UNTUK KEPERL. NEGARA DAN RAKYAT
CONTOH:
1. Untuk kep. jihad
2. Pembiayaan instansi
pemerintah
3. Ta’widh (santunan)
penguasa
4. Gaji PNS dan tentara
5. Pemb. fasilitas umum
6. Infaq fakir, miskin dll. Sumber: Condro, 2011 DISTRIBUSI KEPEMILIKAN UMUM
INFAQ
TAUZI’
BELANJA UNTUK
PENGEMBANGAN
KEPEMILIKAN UMUM
CONTOH:
1. Membiayai Diwan
kep. Umum
2. Menggaji pakar,
pekerja kep. Umum
3. Membeli sarana
produksi Kep. Umum
PENDISTRIBUSIAN
LANGSUNG KEPADA
RAKYAT
CONTOH:
1. Dibagi secara gratis: air,
listrik, minyak
2. Menjual ke industri
dalam negeri atau luar
negeri
3. Membagikan hasil
keuntungan kepada
rakyat
HIMA
PENGKHUSUSAN KEP. UMUM UNTUK KEPERL. NEGARA DAN RAKYAT
CONTOH:
1. Untuk kep. jihad
2. Pembiayaan instansi
pemerintah
3. Ta’widh (santunan)
penguasa
4. Gaji PNS dan tentara
5. Pemb. fasilitas umum
6. Infaq fakir, miskin dll. Sumber: Condro, 2011 Sumber: Condro, 2011 SHARE | Volume 5 | Number 1 | January - June 2016 SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 97 Distribusi kepemilikan umum dikelompokkan menjadi 3 (tiga) yaitu: 1). Infak
disalurkan untuk belanja pengembangan kepemilikan umum, misalnya
membiayai dewan kepemilikan umum, menggaji pakar, pekerja kepemilikan
umum, membeli sarana produksi kepemilikan umum. 2). Tauzi’ yaitu
pendistribusian langsung kepada rakyat, misalnya; dibagi secara gratis: air,
listrik dan minyak, menjual ke industri dalam negeri atau luar negeri,
membagikan hasil keuntungan kepada rakyat. 3). Hima, pengkhususan
kepemilikan umum untuk keperluan Negara dan rakyat, antara lain: untuk
keperluan jihad, pembiayaan instansi pemerintah, ta’wids (santunan penguasa),
gaji PNS (Pegawai Negeri Sipil) dan tentara, pembiayaan fasilitas umum, infaq
fakir, miskin, dan lain-lainnya. Untuk lebih jelaskan bisa dilihat pada
Gambar.6 tentang Distribusi kekayaan Umum: Distribusi kepemilikan umum dikelompokkan menjadi 3 (tiga) yaitu: 1). Kekayaan Negara (Milkiyah Daulah) Harta milik Negara adalah harta yang tidak termasuk kategori milik umum,
melainkan milik pribadi, namun barang-barang tersebut terkait dengan hak
kaum muslimin secara umum. Pengelolaan sepenuhnya menjadi wewenang
kepala Negara (khalifah), yaitu menurut pandangan dan ijtihad khalifah. Untuk
lebih jelasnya lihat gambar.7 berikut ini (An-Nabhani, 1990). Gambar 7. Skema Kepemilikan Negara KEPEMILIKAN NEGARA
1. JIZYAH
2. KHARAJ
3. GHANIMAH
4. FA’I
5. ‘USYUR
6. 20% RIKAZ
7. HARTA TANPA AHLI WARIS
8. HARTA ORANG MURTAD
9. BERBAGAI LAHAN, BANGUNAN MILIK NEGARA
UNTUK KEPERLUAN
PENYELENGGARAAN
NEGARA
BOLEH JUGA DIBERIKAN
KEPADA INDIVIDU YANG
MEMERLUKANNYA
Sumber: Condro, 2011 BOLEH JUGA DIBERIKAN
KEPADA INDIVIDU YANG
MEMERLUKANNYA Sumber: Condro, 2011 Sumber: Condro, 2011 Dari skema tersebut terlihat bahwa di antara harta yang dapat dimasukkan
dalam kategori kepemilikan Negara ada 9 jenis. Harta yang masuk kategori
milik Negara dapat diberikan kepada individu tertentu sehingga menjadi hak SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 98 miliknya. Ketentuan ini tentu berbeda dengan ketentuan yang berlaku pada
kepemilikan umum. Harta milik umum pada dasarnya tidak dapat diberikan
oleh Negara pada individu tertentu, walaupun Negara dapat membolehkan
pada orang-orang untuk mengambilnya melalui pengelolaan oleh Negara, yang
memungkinkan bagi setiap individu untuk memanfaatkannya. Dari gambar di atas menjelaskan bahwa kepemilikan negara yang diperoleh
dari Jizyah, Kharaj, Ghanimah, Fa’i, ‘Usyur, Rikaz dan harta lainnya,
digunakan untuk keperluan penyelenggaraan negara dan bisa juga diberikan
kepada individu yang memerlukan dana untuk keperluan hidup masyarakat. ANALISIS DAN PEMBAHASAN Berdasarkan uraian dan penjelasan tentang sistem ekonomi Islam Madzhab
Hamfara ada beberapa analisis yang mendasar berkaitan dengan pengelolaan
kepemilikan umum dan kepemilikan Negara. Dari konsep kepemilikan yang
telah dijabarkan, menunjukkan bahwa Negara Indonesia telah banyak
melakukan kesalahan-kesalahan yang fatal terkait pengelolaan kepemilikan
umum dan kepemilikan Negara, antara lain:. 1. Kesalahan dalam memulai pengelolaan kekayaan negara bahwa Negara
Indonesia melakukan kerjasama dengan IMF untuk membangun dan
meningkatkan pertumbuhan ekonomi dengan berbasis Utang, sehingga
Utang Indonesia per Januari 2014 Bank Indonesia mencatat Utang
Indonesia mencapai USD$.269,27 miliar atau Rp.3.042,751 triliun dengan
tingkat kurs sebesar Rp11.300 per USD. Dengan Utang berbasis riba ini
adalah suatu kesalahan yang besar, karena hukum riba adalah Haram, bagi
pihak-pihak yang melakukan transaksi utang piutang tersebut, dan
hukumannya adalah kekal di neraka (dalam al-Baqarah 275). Jadi tidak ada
toleransi dalam hal ini, perilaku utang piutang dengan berbasis pada riba
harus dihapuskan. Selain melanggar hukum syarak dengan utang berbasis
riba, Negara Indonesia menjadi sangat tergantung dengan IMF dalam
mengelola sistem perekonomiannya dan tidak bisa independen dalam
menentukan kebijakan-kebijakan berkaitan dengan pengelolaan kekayaan
umum dan kekayaan Negara. Pada tahap inilah Negara memulai masuk
dalam perangkap Utang kepada IMF yang tujuan utamanya bagaimana
menguasai negara-negara makmur melalui Utang dan menjeratnya dengan
menaati aturan-aturan dan perjanjian-perjanjian yang menguntungkan IMF
dan negara-negara pendukungnya. 1. Kesalahan dalam memulai pengelolaan kekayaan negara bahwa Negara
Indonesia melakukan kerjasama dengan IMF untuk membangun dan
meningkatkan pertumbuhan ekonomi dengan berbasis Utang, sehingga
Utang Indonesia per Januari 2014 Bank Indonesia mencatat Utang
Indonesia mencapai USD$.269,27 miliar atau Rp.3.042,751 triliun dengan
tingkat kurs sebesar Rp11.300 per USD. Dengan Utang berbasis riba ini
adalah suatu kesalahan yang besar, karena hukum riba adalah Haram, bagi
pihak-pihak yang melakukan transaksi utang piutang tersebut, dan
hukumannya adalah kekal di neraka (dalam al-Baqarah 275). Jadi tidak ada
toleransi dalam hal ini, perilaku utang piutang dengan berbasis pada riba
harus dihapuskan. Selain melanggar hukum syarak dengan utang berbasis
riba, Negara Indonesia menjadi sangat tergantung dengan IMF dalam
mengelola sistem perekonomiannya dan tidak bisa independen dalam
menentukan kebijakan-kebijakan berkaitan dengan pengelolaan kekayaan
umum dan kekayaan Negara. Pada tahap inilah Negara memulai masuk
dalam perangkap Utang kepada IMF yang tujuan utamanya bagaimana
menguasai negara-negara makmur melalui Utang dan menjeratnya dengan
menaati aturan-aturan dan perjanjian-perjanjian yang menguntungkan IMF
dan negara-negara pendukungnya. 2. Dengan Utang yang besar ini menyebabkan pemerintah Indonesia memiliki
dalih atau alasan, untuk membayar utangnya dengan melakukan penjualan 2. ANALISIS DAN PEMBAHASAN Dengan Utang yang besar ini menyebabkan pemerintah Indonesia memiliki
dalih atau alasan, untuk membayar utangnya dengan melakukan penjualan SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 99 saham-saham milik perusahaan-perusahaan negara kepada pihak asing,
sehingga saham-saham bisa dikuasai oleh perusahaan asing dan pemilik
modal lainnya dengan jumlah lebih dari 50%, maka kepemilikan mayoritas
perusahaan-perusahaan adalah pihak asing dan para pemilik modal. Sebagai gambaran yang riil antara lain: Pertambangan Emas di Papua
diserahkan pengelolaan oleh PT. Freeport, dengan perolehan royalti emas
sebesar 1%, tembaga 1,5%, perak 1,25%, dan Indonesia tidak berdaya
untuk kembali menguasai perusahaan ini karena terikat dengan kontrak-
kontrak perjanjian yang telah dituangkan dalam undang-undang yang sudah
ditetapkan di Parlemen. Perusahaan-perusahaan asing yang menguasai
Indonesia Exxon Mobil, Chevron, PetroChine, dan lain-lainnya. Dalam hal
ini kepemilikan umum terhadap tambang emas tidak dikelola oleh
Indonesia, tetapi dikelola oleh PT. Freeport, sehingga keuntungan
perusahaan digunakan oleh perusahaan asing tersebut dan tidak digunakan
untuk kesejahteraan masyarakat Indonesia. Demikian juga perusahaan-
perusahaan tambang lainnya tidak dikelola oleh Pemerintah Indonesia,
sehingga masyarakat Indonesia tidak bisa menikmati kekayaan alam
Indonesia tersebut. 3. Menghapuskan kepemilikan umum atau kepemilikan negara artinya negara
melepaskan diri dari kewajiban-kewajibannya terhadap rakyat dalam
memberikan
kesejahteraan
rakyatnya. Kewajiban
negara
adalah
menyejahterakan rakyatnya, kalau kepemilikan umum dan kepemilikan
negara sudah diambil alih pengelolaan kepemilikannya, maka Indonesia
tidak memiliki sumber-sumber pendapatan yang akan digunakan untuk
membiayai kesejahteraan rakyat dan memenuhi kebutuhan dasar rakyat
Indonesia. Hal ini terbukti dari catatan Penduduk miskin, menurut Badan
Pusat Statistik (September 2013): 11,47% atau 28,55 juta orang penduduk
miskin. Anggota Komisi IX DPR, Poempida Hidayatulah mengatakan data
terbaru Tim Nasional Percepatan Penanggulangan Kemiskinan telah
menghitung, angka jumlah orang sampai tahun 2013 mencapai 96 juta jiwa. Angka pengangguran (BPS, 2013) mencapai 7,39 juta orang dari 118,19
juta. Dengan demikian tugas Negara dalam menyejahterakan masyarakat
Indonesia tidak bisa dilaksanakan karena Negara Indonesia dengan sengaja
melepaskan tanggung jawab dalam mengelola kepemilikan umum dan
kepemilikan Negara, yaitu dengan menyerahkan pengelolaan kekayaan
umum dan kekayaan Negara dikelola oleh pihak asing, pihak swasta dan
para pemilik modal lainnya. 3. 4. Tersentralisasinya kekayaan/ Aset hanya pada segelintir individu atau
perusahaan yang memiliki modal besar, dan pemilik modal besar yang bisa
menguasai aset-aset milik Negara dengan melakukan pengambilalihan aset. 4. ANALISIS DAN PEMBAHASAN Tersentralisasinya kekayaan/ Aset hanya pada segelintir individu atau
perusahaan yang memiliki modal besar, dan pemilik modal besar yang bisa
menguasai aset-aset milik Negara dengan melakukan pengambilalihan aset. SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 100 Maka ini berdampak luar biasa pada kesenjangan antara para pemilik
modal dengan yang tidak memiliki modal, sehingga perusahaan bermodal
besar bisa berkembang dengan pesat, sedangkan perusahaan-perusahaan
kecil semakin rawan mengalami penurunan aktivitas produksi, dan hal ini
berdampak pada pemutusan hubungan kerja (PHK), sehingga jumlah
pengangguran semakin meningkat. Maka dengan demikian distribusi
kekayaan menjadi tidak adil karena hanya dimiliki oleh pihak yang
memiliki modal saja yaitu kaum kapitalis. Sementara Negara tidak
bertanggung jawab terhadap pengelolaan kekayaan Umum dan Kekayaan
Negara. 5. Pada akhirnya Negara Indonesia akan disibukkan untuk mencari sumber-
sumber pendapatan baru untuk menggantikan sumber-sumber pendapatan
yang telah dijualnya, hal ini disebabkan tidak ada lagi sumber-sumber
pendapatan dari kekayaan umum maupun kekayaan Negara, sehingga
untuk membiayai APBN dan operasional Negara diperoleh dari
optimalisasi Pajak dan Utang. Dari jumlah APBN tahun 2014 sebesar
Rp1.842, 2 triliun, dibiayai dari Pajak yang merupakan sumber pendapatan
terbesar yaitu Rp 1.310,2 triliun, sekitar 80% dibiayai oleh Pajak. Dalam
sistem ekonomi Islam tidak dibenarkan Negara menarik pajak kepada
rakyatnya untuk membiayai belanja Negara, oleh karena itu ini merupakan
pemalakan terstruktur pemerintah kepada rakyatnya untuk membiayai
operasional Negara. 5. Pada akhirnya Negara Indonesia akan disibukkan untuk mencari sumber-
sumber pendapatan baru untuk menggantikan sumber-sumber pendapatan
yang telah dijualnya, hal ini disebabkan tidak ada lagi sumber-sumber
pendapatan dari kekayaan umum maupun kekayaan Negara, sehingga
untuk membiayai APBN dan operasional Negara diperoleh dari
optimalisasi Pajak dan Utang. Dari jumlah APBN tahun 2014 sebesar
Rp1.842, 2 triliun, dibiayai dari Pajak yang merupakan sumber pendapatan
terbesar yaitu Rp 1.310,2 triliun, sekitar 80% dibiayai oleh Pajak. Dalam
sistem ekonomi Islam tidak dibenarkan Negara menarik pajak kepada
rakyatnya untuk membiayai belanja Negara, oleh karena itu ini merupakan
pemalakan terstruktur pemerintah kepada rakyatnya untuk membiayai
operasional Negara. 6. Privatisasi telah menjerumuskan bangsa ini dalam cengkeraman
imperialisme ekonomi, karena ini merupakan bentuk penjajahan seperti
halnya yang dilakukan oleh Belanda, Inggris, Jepang pada zaman
penjajahan dulu, hanya saja ini lebih sistemik dan terstruktur dalam
peraturan dan perundang-undangan. Hal ini pencengkeraman pihak asing
dalam melakukan penjajahan menjadi lebih kuat, dan tidak mudah
dihindari serta melakukan pemindahan kekayaan Negara kepada pihak
asing dengan terbuka dan terstruktur. KESIMPULAN Berdasarkan analisis yang telah dilakukan diperoleh beberapa kesimpulan
dalam penelitian ini yaitu: 1. Negara Indonesia telah melakukan kesalahan dalam melaksanakan
pengelolaan kepemilikan umum (milkiyah ‘amah) dan kepemilika Negara
(milkiyah daulah), hal ini terlihat dari proses privatisasi kekayaan umum
dan kekayaan Negara, yang seharusnya dikelola oleh Negara Indonesia,
tetapi diserahkan kepada pihak asing dan swasta melalui penjualan saham-
saham milik perusahaan-perusahaan milik Negara. Sehingga tujuan untuk
mencapai kesejahteraan masyarakat tidak bisa terpenuhi. Hal ini tidak
sesuai dengan konsep dan sistem ekonomi Islam Madzhab Hamfara. 2. Dampak dari kesalahan dalam pengelolaan kekayaan umum dan kekayaan
Negara ini menyebabkan Negara tidak bisa memperoleh pendapatan secara
optimal dari perusahaan-perusahaan Negara, sehingga berdampak pada
upaya memperoleh pendapatan melalui pajak yang ditarik dari rakyat untuk
membiayai operasional Negara. Hal ini bertentangan dengan sistem
ekonomi Islam Madzhab Hamfara. 3. Dalam rangka meningkatkan pertumbuhan ekonomi Indonesia, untuk
pembangunan infrastruktur dan sarana prasarana dibiayai dengan
menggunakan Utang yang berbasis pada riba, sehingga Utang Negara
Indonesia selalu meningkat setiap tahunnya, dan tentu tidak diridhai Allah
SWT karena berbasis pada riba, yang hukumnya haram, karena
bertentangan dengan hukum Allah SWT bahwa riba adalah haram. ANALISIS DAN PEMBAHASAN Apabila dalam penjajahan terstruktur
ini tidak segera dihentikan dan dilakukan upaya pengambilalihan kembali
kekayaan umum dan kekayaan negara kepada Pemerintah Negara
Indonesia, maka ketimpangan-ketimpangan perekonomian di Indonesia
akan berlanjut terus dan ketidakadilan perekonomian akan berdampak pada
kemiskinan, kesenjangan sosial masyarakat, pengangguran, kerawanan
sosial, dan penyakit lainnya, karena tidak diterapkannya Sistem Ekonomi
Islam. 6. Privatisasi telah menjerumuskan bangsa ini dalam cengkeraman
imperialisme ekonomi, karena ini merupakan bentuk penjajahan seperti
halnya yang dilakukan oleh Belanda, Inggris, Jepang pada zaman
penjajahan dulu, hanya saja ini lebih sistemik dan terstruktur dalam
peraturan dan perundang-undangan. Hal ini pencengkeraman pihak asing
dalam melakukan penjajahan menjadi lebih kuat, dan tidak mudah
dihindari serta melakukan pemindahan kekayaan Negara kepada pihak
asing dengan terbuka dan terstruktur. Apabila dalam penjajahan terstruktur
ini tidak segera dihentikan dan dilakukan upaya pengambilalihan kembali
kekayaan umum dan kekayaan negara kepada Pemerintah Negara
Indonesia, maka ketimpangan-ketimpangan perekonomian di Indonesia
akan berlanjut terus dan ketidakadilan perekonomian akan berdampak pada
kemiskinan, kesenjangan sosial masyarakat, pengangguran, kerawanan
sosial, dan penyakit lainnya, karena tidak diterapkannya Sistem Ekonomi
Islam. 6. 7. Menghalangi masyarakat umumnya untuk memperoleh hak mereka,
karena akses untuk memperoleh kesejahteraan sudah dikuasai oleh SHARE | Volume 5 | Number 1 | January - June 2016 Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 101 kekuatan modal asing untuk menutup pendapatan-pendapatan Negara yang
seharusnya digunakan sebesar-besarnya untuk kesejahteraan rakyat. kekuatan modal asing untuk menutup pendapatan-pendapatan Negara yang
seharusnya digunakan sebesar-besarnya untuk kesejahteraan rakyat. SARAN Berdasarkan pada kesimpulan yang sudah diuraikan, maka saran yang bisa
diberikan kepada penanggungjawab Negara Indonesia adalah: 1. Negara Indonesia segera melakukan evaluasi dan restrukturisasi terhadap
kepemilikan umum dan kepemilikan Negara untuk bisa dikelola oleh
Negara Indonesia, sehingga bisa memberikan pemasukan pendapatan yang
signifikan untuk membiayai operasional Negara Indonesia. 2. Negara Indonesia melakukan pengkajian untuk menerapkan sistem
Ekonomi Islam dalam operasionalnya, sehingga pengelolaan kekayaan
umum dan kekayaan Negara bisa dilaksanakan sesuai dengan petunjuk dan 2. Negara Indonesia melakukan pengkajian untuk menerapkan sistem
Ekonomi Islam dalam operasionalnya, sehingga pengelolaan kekayaan
umum dan kekayaan Negara bisa dilaksanakan sesuai dengan petunjuk dan SHARE | Volume 5 | Number 1 | January - June 2016 2
Murtiyani, Sasono, Triono, & Zahra | Analisis dan Evaluasi Implementasi Pengelolaan Kepemilikan_ 102 perintah dari Allah SWT dalam menjalankan amanah sebagai pemimpin
Negara Indonesia, karena pemimpin akan diminta pertanggungjawaban di
Yaumul akhir kelak. perintah dari Allah SWT dalam menjalankan amanah sebagai pemimpin
Negara Indonesia, karena pemimpin akan diminta pertanggungjawaban di
Yaumul akhir kelak. 3. Negara Indonesia harus menghilangkan sistem pajak kepada rakyatnya, dan
lebih mengoptimalkan pendapatannya melalui pengelolaan kekayaan
umum dan kekayaan Negara untuk menyejahteraan rakyatnya. 3. Negara Indonesia harus menghilangkan sistem pajak kepada rakyatnya, dan
lebih mengoptimalkan pendapatannya melalui pengelolaan kekayaan
umum dan kekayaan Negara untuk menyejahteraan rakyatnya. 3. Negara Indonesia harus menghilangkan sistem pajak kepada rakyatnya, dan
lebih mengoptimalkan pendapatannya melalui pengelolaan kekayaan
umum dan kekayaan Negara untuk menyejahteraan rakyatnya. 4. Negara harus menghapuskan sistem riba dalam utang piutang, karena ini
bertentangan dengan syariat Allah SWT, dan kembali kepada sistem jual
beli
dan
shirkah
dalam
meningkatkan
kekayaan
Negara
untuk
kesejahteraan masyarakatnya. - (tt). Sistem Ekonomi Islam. Al-Azhar Press. Anonim. (2009). Menyongsong Sistem Ekonomi Anti Krisis, Cetakan 1,
Jakarta: Pustaka Thariqul Izzah. Aria Susman. (2009). Jejak Neoliberalisme di Indonesia part-2 REFERENSI Al-Qur’an Tajwid dan terjemah. (2010). Cetakan ke-10, CV. Penerbit
Diponegoro. Abdurrahman. AM. (2009). Politik Ekonomi Islam, Al azhar Press. An-Nabhani, Taqiyuddin. (2009). Membangun Sistem Ekonomi Alternatif
Perpektif Islam. Jakarta: Al-Azhar Press. - (tt). Sistem Ekonomi Islam. Al-Azhar Press. Aria Susman. (2009). Jejak Neoliberalisme di Indonesia part-2 Condro T, Dwi. (2011). Ekonomi Islam Madzhab Hamfara, Falsafah Ekonomi
Islam, Jilid 1, Irtikaz, David M. Smick, Kiamat Ekonomi Global (The
World is Curved), cetakan 1, Daras Books. Deliarnov (1995). Perkembangan Pemikiran Ekonomi, Jakarta: PT. Raja
Grafindo Persada. Indra Bastian. (2006). Privatisasi di Indonesia: Teori dan Implementasi. Jakarta: Salemba Empat. Riant Nugroho & Randy R. Wrihatnolo. (2008). Manajemen Privatisasi
BUMN. Jakarta: Elex Media Komputindo. SHARE | Volume 5 | Number 1 | January - June 2016
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https://openalex.org/W2332769882
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https://zenodo.org/records/2186268/files/article.pdf
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English
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XI. -Diffraction Rings due to a Circular Aperture
|
Journal of the Royal Microscopical Society
| 1,907
|
public-domain
| 770
|
405 405 XI.-Difiaction
Rings due to a Circular Aperture. (Read May 15th, 1907.) I N a paper by Mr. 3:. M. Nelson readin March 1906 and printed
in the October No. of the Society’s Journal, 2906, occurs the fol-
lowing sentence in reference to the diffraction rings formed
when light from practically a point source is received after passing
through a circular aperture : “ If the radius of the first dark ring
be experimentally determined, it will be found to be 32 p.c. smaller than its theoretical value. What would be thought of the
Newtonian theory of gravitation if the calculated distance of Mars
from tlie Sun was 32 p.c. too great ? ” This somewhat sweeping
criticism of the diffraction theory seemed to call for further experi-
mental tests before it could be accepted, especially as in the early
days of the subject experimental verification of the theory was
obtained. It is true that Fraunhofer’s measurements were made
with white light only, and in that respect are imperfect ; however,
when his values for rectangular and circular apertures are compared,
the factor of proportionality between the diameters of the first dark
ring in the two cases is found to be exactly 1 - 22, as required by the
theory. These experimental values are conveniently found quoted
in Knockenhauer’s Undulations-theorie des Lichtes. According to
Mr. Nelson, this factor turns out experimentally to be 0.8266. The question is therefore not merely one of disagreement between
theory and experiment, but also one of disagreement between
Fraunhofer’s and Mr. Nelson’s experimental values. p
The following measurements were made with the object of
obtaining a test possessing as great a precision as possible with the
moderu means at our disposal. p
The method adopted was that of direct measurement of the
diameters of the rings, using a micrometer eyepiece. An artificial
star was set up 23 metres away from a telescopic object-glass of
good quality of between 2 and 3 inches aperture giving an
image at 670 mm. behind the lens; and diffraction rings of a
suitable size were produced by placing a small aperture in front of
the telescope. The greatest difficulty was the faintness of the
rings when made large. An aperture of 2.23 mm. diameter was
found to be the most suitable, and the diameter of the first dark
ring was then about 0.2 mm. The artificial star used was a 40 6 Transaction.$ of the Society. pinhole 0 * 3 mm. (Read May 15th, 1907.) diameter in a piece of thin brass placed in front
of the condenser of an optical lantern, arranged so as to project a
parallel beam of light. The angular width of the bright object was
therefore only about 4 per cent. of the angular diameter of the
first dark ring, and the uncertainty of setting would therefore be
considerably less than this. The light was required as uearly as
possible monochromatic and very intense for obvious reasons. These results were obtained by using an electric arc, the positive
carbon having its core bored out and replaced by finely ground dry
salt; this carbon was placed below the negative carbon. This
arrangement gives an intense sodium light, consisting mainly of
the I)-lines, but containing also the other pairs of lines emitted by
sodium in the arc. The light from the latter was too feeble to
sensibly modify the diffraction figure formed by the much prepon-
derating I) light present. The accuracy of the micrometer eye-
pieces was well within the errors entailed in setting the cross-wire
on the rings. g
The measured diameters were reduced to give the constant for
each ring for comparison with the theoretic values. Of the first
dark ring 7 series of 4 measurements were taken; of the first
bright ring 2 series of 4 measurements each ; and of the second
dark ring, 3 series of 4 measurements each. The mean values of
these determinations are given below with Airy’s values for
comparison :- AIRY. PRESENT
EXPERIMEN
1st dark ring . . . . . . . . . . 1.22
1.21
1st bright ring
. . . . . . . . 1.64
1.68
2nd dark ring . . . . . . . . . . 2.23
2.20 The outcome of these measurements is to show practical ab
aree-
ment between theory and experiment ; this is especially so in the
case of the first dark ring, for which the possible precision of
measurement is certainly the greatest. The error is greatest in the
case of the first bright ring, but here amounts only to 24 per cent.
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https://link.springer.com/content/pdf/10.1007/s12975-021-00939-9.pdf
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English
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Beneficial Effect of Sodium Nitrite on EEG Ischaemic Markers in Patients with Subarachnoid Haemorrhage
|
Translational stroke research
| 2,021
|
cc-by
| 8,567
|
* Alexander Luettich
alexander.luettich@ndcn.ox.ac.uk Abstract Subarachnoid haemorrhage (SAH) is associated with long-term disability, serious reduction in quality of life and significant
mortality. Early brain injury (EBI) refers to the pathological changes in cerebral metabolism and blood flow that happen
in the first few days after ictus and may lead on to delayed cerebral ischaemia (DCI). A disruption of the nitric oxide (NO)
pathway is hypothesised as a key mechanism underlying EBI. A decrease in the alpha-delta power ratio (ADR) of the elec-
troencephalogram has been related to cerebral ischaemia. In an experimental medicine study, we tested the hypothesis that
intravenous sodium nitrite, an NO donor, would lead to increases in ADR. We studied 33 patients with acute aneurysmal
SAH in the EBI phase. Participants were randomised to either sodium nitrite or saline infusion for 1 h. EEG measurements
were taken before the start of and during the infusion. Twenty-eight patients did not develop DCI and five patients developed
DCI. In the patients who did not develop DCI, we found an increase in ADR during sodium nitrite versus saline infusion. In
the five patients who developed DCI, we did not observe a consistent pattern of ADR changes. We suggest that ADR power
changes in response to nitrite infusion reflect a NO-mediated reduction in cerebral ischaemia and increase in perfusion,
adding further evidence to the role of the NO pathway in EBI after SAH. Our findings provide the basis for future clinical
trials employing NO donors after SAH. Keywords Subarachnoid haemorrhage · Nitrite · Nitric oxide · EEG · Alpha-delta power ratio Alexander Luettich and Edit Franko contributed equally to the
study Alexander Luettich1 · Edit Franko1 · Desiree B. Spronk1 · Catherine Lamb2 · Rufus Corkill3 · Jash Patel4 ·
Martyn Ezra1 · Kyle T. S. Pattinson1 Received: 7 June 2021 / Revised: 26 July 2021 / Accepted: 6 August 2021
© The Author(s) 2021
/ Published online: 7 September 2021 https://doi.org/10.1007/s12975-021-00939-9
Translational Stroke Research (2022) 13:265–275 https://doi.org/10.1007/s12975-021-00939-9
Translational Stroke Research (2022) 13:265–275 ORIGINAL ARTICLE ORIGINAL ARTICLE Introduction DCI mostly occurs within 4–10 days after
the initial bleed [8] in approximately 30% of SAH patients
and is—in combination with infarction from DCI—among
the most unfavourable factors for outcome after SAH [6]. As angiographic vasospasm has been questioned as the most
important factor leading to the development of DCI, more
recent research increasingly considers a complex and mul-
tifactorial aetiology of DCI plausibly related to EBI disease
mechanisms originating before DCI will usually occur [4–7]. A disruption to the nitric oxide (NO) pathway may play a
pivotal role in the pathogenesis of EBI and DCI [9]. NO is an
endogenous vasodilator and signalling molecule, is part of
the neurovascular unit and is involved in regulating cerebral
blood flow [10, 11]. Following SAH, there are changes in
cerebral NO levels, yet the significance of these changes for
outcome remains inconclusive [12–15]. Endogenous nitrite
represents an important storage pool for NO, where nitrite
can be reduced to NO under hypoxic and acidic conditions
[16, 17]. Animal research points to the potential of NO to
act toward a decrease in ischaemic brain injury after cerebral
haemorrhage [11]. NO donor administration in rats suggests
that NO can increase cerebral blood flow and prevent early
ischaemic damage after experimental SAH [18, 19] and
can have a neuroprotective effect in ischaemia/reperfusion
models [20, 21]. Sodium nitrite infusion in monkeys was
shown to prevent cerebral arteriographic vasospasm after
SAH [22]. Importantly, sodium nitrite infusion can safely
modulate systemic nitrite levels in humans [23]. This raises
the question if sodium nitrite infusion can modulate or even
protect brain function after human cerebral insult. An uncon-
trolled electroencephalography (EEG) pilot study by Garry
et al. [24] suggested that the NO pathway after human SAH
is potentially manipulable by sodium nitrite infusion. Sample Size Sample size calculation was pre-registered (https://osf.io/
ywa9r/) and based on an uncontrolled pilot study by Garry
et al. [24] who investigated EEG responses to nitrite follow-
ing SAH. We focused the study on patients who did not sub-
sequently develop DCI, as a robust response to nitrite was
observed in this group, and this patient group was expected
to represent approximately 70% of cases. Garry et al. showed
large increases in ADR power after sodium nitrite infusion
in seven patients who did not develop DCI (baseline ADR:
Mean 0.037, SD 0.023; infusion ADR: Mean 0.063, SD
0.048). Based on these data, a calculation of the required
sample size to replicate this effect was conducted (mean of
paired differences: 0.027, SD of paired differences: 0.027). Assuming 80% power and a level of significance of 5% (two-
sided), there should be an effect with 11 patients who did not
develop DCI. As DCI is expected to occur in 30% of cases
[7] and there was a 1:1 allocation ratio of sodium nitrite and
saline infusion, recruitment of 32 patients should suffice to
replicate the effect. EEG provides immediate information on neuronal func-
tioning and connects with cerebral perfusion and brain
metabolism [25–28]. It is also a sensitive tool for detect-
ing ischaemic changes after SAH. Specifically, a decrease
in the alpha-delta power ratio (ADR) has been related to
cerebral ischaemia [29–32]. We conducted an experimental
medicine study probing the role of the NO pathway during
the EBI period after human SAH. We tested the hypothesis
that sodium nitrite infusion would lead to an increase in
ADR power. The sample size calculation was based on within patient
changes in ADR. The present investigation moved beyond
investigating within patient ADR changes, comparing ADR
changes over time between groups of sodium nitrite and
saline infusion. Introduction aneurysm [1]. Patients often remain highly disabled long
term, showing cognitive and emotional impairments, fail-
ure to return to work and a reduction in quality of life [2]. Accordingly, aneurysmal SAH accounts for a dispropor-
tionally high share of costs of all cerebrovascular diseases
in the UK and is associated with a serious decrease in life
expectancy [3]. Aneurysmal subarachnoid haemorrhage (SAH) is a sub-
type of stroke related to the accumulation of blood in the
subarachnoid space, caused by a rupture of an intracranial Alexander Luettich and Edit Franko contributed equally to the
study Pathophysiological changes after the ictus occur in two
stages. A phase of early brain injury (EBI) [4, 5] can be
followed by a stage of delayed neurological deterioration
often including delayed cerebral ischaemia (DCI) [6, 7]. EBI
describes the acute effects of intracranial haemorrhage and
transient global cerebral ischaemia within 72 h of the bleed
and includes (1) changes in physiology related to increased
intracranial pressure, decreased cerebral blood flow and
impaired cerebral autoregulation; (2) hydrocephalus,
mechanical stress on the subarachnoid space contributing
to vessel constriction; and (3) ionic, molecular and cellular
changes promoting oxidative stress, inflammation, platelet
aggregation, endothelial dysfunction, vessel constriction and 1
Nuffield Department of Clinical Neurosciences, Oxford
University, Oxford OX3 9DU, UK 2
Neuro Intensive Care Unit, John Radcliffe Hospital, Oxford
University Hospitals NHS Foundation Trust, Oxford, UK 3
Department of Neuroradiology, John Radcliffe Hospital,
Oxford University Hospitals NHS Foundation Trust, Oxford,
UK 4
Department of Neurosurgery, John Radcliffe Hospital,
Oxford University Hospitals NHS Foundation Trust, Oxford,
UK (0121 3456789)
3 Translational Stroke Research (2022) 13:265–275 266 the acute SAH admissions to the John Radcliffe Hospital in
Oxford. In order to obtain a maximally representative sam-
ple of patients, inclusion and exclusion criteria were inten-
tionally kept to a minimum (inclusion criteria: Age range
18–80 years, aneurysm secured by intravascular coiling, all
World Federation of Neurological Surgeons Subarachnoid
Haemorrhage Grading (WFNS) scores, study conducted
within a maximum of 5 days after the ictus; exclusion crite-
ria: Contraindications to sodium nitrite, pre-existing aneu-
rysmal clip). SAH was diagnosed via lumbar puncture or
CT. Informed consent was given either by the patient or
a personal consultee who acted in the best interest of the
patient. cytotoxicity [5]. Randomisation and Infusion The study was a double-blind (patients and experimenters)
and placebo-controlled experimental medicine study. Patients
were allocated 1:1 to either the experimental or control group
by random sealed envelope. Randomisation codes were used
by an independent doctor or nurse who accordingly prepared
either a sodium nitrite (10mcg/kg/min, experimental condi-
tion) or saline (0.9% sodium chloride, control condition) infu-
sion. Dosing schemes and timing of infusion were informed by
a pilot study in subarachnoid haemorrhage patients and a study
in healthy participants [24, 33]. Infusions were administered 1 3 Statistical Analysis We concentrated our analysis on patients who did not
develop DCI (see the “Sample Size” section). As patients
who develop DCI may react differently to nitrite infusion
[24] and their number was very low in our study, these
patients could not reasonably be included as a separate group
in a larger model together with patients who did not develop
DCI. Accordingly, we initially evaluated the effect of sodium
nitrite infusion across both patient groups and further inves-
tigated effects statistically for patients who did not develop
DCI and qualitatively for individuals who developed DCI. Patients The study protocol was approved by the South Cen-
tral–Oxford C NHS Health Research Authority Ethics Com-
mittee (reference number: 12/SC/0366). Patients were iden-
tified by members of their clinical team and recruited from 1 3 1 3 Translational Stroke Research (2022) 13:265–275 267 during EEG at an equivalent rate (0.12 ml/kg/h) by a suitably
trained clinician via a peripherally sited intravenous cannula
(Becton Dickinson Venflon, 20 gauge) in the patient’s arm
using a calibrated infusion pump (CareFusion Alaris PK). EEG data was analysed by a researcher who was unblinded
to patient randomisation codes in two stages. In a first step,
allocation of patients to two groups was unblinded after a sta-
tistical analysis plan was published (https://osf.io/ywa9r/). At
this stage, group identity was defined by a place holder. In
a second step, allocation of the two groups to either sodium
nitrite or saline infusion was unblinded after final analyses. Procedures The Stata 14 software package (StataCorp 2015) was
used for statistical analyses. In a first step, we conducted
simple linear regression investigating the effect of nitrite
infusion on the EEG controlling for potentially confound-
ing variables: The depedent variable was percentage EEG
change, independent variables included infusion type
(nitrite/saline), baseline EEG and the presence of sedation
(yes/no). In order to study the effect of sodium nitrite on the
EEG over time, we employed a multilevel linear regression
model. The dependent variable was percentage EEG change. Independent variables comparised infusion type, time win-
dow (0–20 min, 20–40 min, 40–60 min), the interaction of
these two variables, baseline EEG and sedation. Patients
were included as a random effect. For simple and multilevel
regression, sets of matched models were used to investigate
the effect of sodium nitrite on ADR, alpha and delta fre-
quency power. Patients received standard medical care independently of their
inclusion in the study. WFNS score on admission, Fisher scale
and Glasgow Coma Scale (GCS) on study day were collected
(for a more comprehensive description of assessments, see
supplemental methods, assessments). We conducted the study
as soon as possible after successful aneurysm treatment with
endovascular coiling within 5 days after the ictus at John Rad-
cliffe Hospital in Oxford. Medical records were assessed by
an independent consultant neurointensivist, according to the
definitions of Vergouwen et al. [8] to determine if patients had
developed DCI. Patients did not show evidence of DCI at or
before the time of the study. On the study day, the EEG was recorded for 20 min before
and 60 min during infusion of sodium nitrite or saline. Drugs
used for sedation were recorded as sedation is known to influ-
ence the EEG [34–38]. The EEG (for a detailed description,
see supplemental methods, EEG) was acquired from electrodes
Fp(1,2), F(3,4,z), T(3,4), Cz, P(3,4,z) and Oz/O1. The EEG
was referenced to the average left and right mastoid recording,
pre-processed, cleaned, decomposed and averaged within the
alpha (8–12 Hz) and delta (1–3.5 Hz) frequency power range,
and the alpha-delta power ratio (ADR) was calculated. The
alpha, delta and ADR signals were then averaged within a
20-min baseline time window before and within a 60-min or
three successive 20-min infusion time windows after the onset
of the infusion. percentage signal change = 100 x (power during infusion) −(power during baseline)
power during baseline Patients Individual characteristics for patients who developed DCI
were generally variable and are depicted in Table 2. Patient
2 was sedated (using fentanyl) and showed comparably high
delta power at baseline. Patients Table 1 Characteristics of patients without DCI grouped by infusion
type
Nitrite (n = 14)
Saline (n = 14)
Age, years, mean (range)
53 (31–69)
47 (29–61)
Female, n (%)
9 (64)
9 (64)
Time from bleed, hours, mean (SD)
64 (23)
68 (23)
Fisher scale, n (%)
1
1 (7)
1 (7)
2
3 (21)
3 (21)
3
6 (43)
3 (21)
4
4 (29)
7 (50)
WFNS score on admission, n (%)
1
10 (71)
10 (71)
2
2 (14)
4 (29)
4
1 (7)
0 (0)
5
1 (7)
0 (0)
GCS on study day, n (%)
E1VTM1
3 (21)
0 (0)
15
11 (79)
14 (100)
Sedation, n (%)
3 (21)
0 (0)
ADR at baseline, mean (SD)
0.28 (0.24)
0.35 (0.25)
Alpha at baseline, mean (SD)
4.07 (2.55)
5.84 (3.69)
Delta at baseline, mean (SD)
25.34 (24.94)
20.29 (12.82)
Table 2 Individual
characteristics of patients who
developed DCI
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Nitrite or saline, N/S
N
S
N
S
N
Age, years
51
66
43
53
63
Female or male, F/M
M
M
F
F
F
Time from bleed, hours
60
45
90
96
82
Fisher scale
2
4
4
2
2
WFNS score on admission
4
2
1
2
1
GCS on study day
14
15
15
15
15
Sedation, 0/1
0
1
0
0
0
ADR at baseline
0.27
0.05
0.44
0.23
0.2
Alpha at baseline
3.67
3.76
6.14
8.33
4.91
Delta at baseline
13.71
72.97
13.97
36.39
25.14 Table 1 Characteristics of patients without DCI grouped by infusion
type
Nitrite (n = 14)
Saline (n = 14)
Age, years, mean (range)
53 (31–69)
47 (29–61)
Female, n (%)
9 (64)
9 (64)
Time from bleed, hours, mean (SD)
64 (23)
68 (23)
Fisher scale, n (%)
1
1 (7)
1 (7)
2
3 (21)
3 (21)
3
6 (43)
3 (21)
4
4 (29)
7 (50)
WFNS score on admission, n (%)
1
10 (71)
10 (71)
2
2 (14)
4 (29)
4
1 (7)
0 (0)
5
1 (7)
0 (0)
GCS on study day, n (%)
E1VTM1
3 (21)
0 (0)
15
11 (79)
14 (100)
Sedation, n (%)
3 (21)
0 (0)
ADR at baseline, mean (SD)
0.28 (0.24)
0.35 (0.25)
Alpha at baseline, mean (SD)
4.07 (2.55)
5.84 (3.69)
Delta at baseline, mean (SD)
25.34 (24.94)
20.29 (12.82) depicted in Fig. Patients 1. No drug-related adverse events were
identified. Table 1 Characteristics of patients without DCI grouped by infusion
type As access to the back of the head was challenging for
practical and medical reasons, availability of electrodes was
biased toward frontal and medial electrodes but was com-
parable for the two infusion groups (supplemental table I). Summary patient characteristics for patients who did not
develop DCI are depicted in Table 1. Patients who received
the nitrite infusion were largely matched in baseline char-
acteristics of age, sex, time from bleed and Fisher scale
to patients who received the saline infusion. None of the
patients of the saline arm was sedated. Three patients of
the nitrite arm were sedated (using fentanyl and propofol or
midazolam), and this was explicitly respected in our statis-
tical models. These patients also featured a lower Glasgow
Coma Scale (GCS) on study day (E1VTM1) and accounted
for the two datasets with higher WFNS scores (4 and 5) on
admission. EEG power at baseline was largely comparable
between patient groups but was explicitly accounted for in
the statistical models. Individual characteristics for patients who developed DCI
were generally variable and are depicted in Table 2. Patient
2 was sedated (using fentanyl) and showed comparably high
delta power at baseline. Patients Forty-two patients were recruited. No patients were excluded
for any characteristic. However, for several patients, no
data or only an insufficient amount of clean data could be
obtained: Three patients did not tolerate EEG recording, and
there were technical problems or there were no data after
data cleaning with six patients, leaving 33 patients (mean
years of age 50.64 [29–69], 21 women, median WFNS score
1 [1–2]) for the analysis. Five of these patients developed
DCI (mean years of age 55.2 [43–66], 3 women, median
WFNS score 2 [1–2]), 28 did not develop DCI (mean years As the magnitude of the EEG signal depends heavily on
several factors (conductivity between electrodes and the
scalp, neural and non-neural tissue) that can vary substantially
between patients and experimenters, the signal was scaled
according to the following equation: of age 49.82 [29–69], 18 women, median WFNS score 1
[1–2]). A flow chart of recruited patients including patient
allocation to either sodium nitrite or saline infusion is of age 49.82 [29–69], 18 women, median WFNS score 1
[1–2]). A flow chart of recruited patients including patient
allocation to either sodium nitrite or saline infusion is 1 3 Translational Stroke Research (2022) 13:265–275 268 Fig. 1 Flow chart of recruited
patients Fig. 1 Flow chart of recruited
patients depicted in Fig. 1. No drug-related adverse events were
identified. As access to the back of the head was challenging for
practical and medical reasons, availability of electrodes was
biased toward frontal and medial electrodes but was com-
parable for the two infusion groups (supplemental table I). Summary patient characteristics for patients who did not
develop DCI are depicted in Table 1. Patients who received
the nitrite infusion were largely matched in baseline char-
acteristics of age, sex, time from bleed and Fisher scale
to patients who received the saline infusion. None of the
patients of the saline arm was sedated. Three patients of
the nitrite arm were sedated (using fentanyl and propofol or
midazolam), and this was explicitly respected in our statis-
tical models. These patients also featured a lower Glasgow
Coma Scale (GCS) on study day (E1VTM1) and accounted
for the two datasets with higher WFNS scores (4 and 5) on
admission. EEG power at baseline was largely comparable
between patient groups but was explicitly accounted for in
the statistical models. Patients Table 2 Individual
characteristics of patients who
developed DCI
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Nitrite or saline, N/S
N
S
N
S
N
Age, years
51
66
43
53
63
Female or male, F/M
M
M
F
F
F
Time from bleed, hours
60
45
90
96
82
Fisher scale
2
4
4
2
2
WFNS score on admission
4
2
1
2
1
GCS on study day
14
15
15
15
15
Sedation, 0/1
0
1
0
0
0
ADR at baseline
0.27
0.05
0.44
0.23
0.2
Alpha at baseline
3.67
3.76
6.14
8.33
4.91
Delta at baseline
13.71
72.97
13.97
36.39
25.14 Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Nitrite or saline, N/S
N
S
N
S
N
Age, years
51
66
43
53
63
Female or male, F/M
M
M
F
F
F
Time from bleed, hours
60
45
90
96
82
Fisher scale
2
4
4
2
2
WFNS score on admission
4
2
1
2
1
GCS on study day
14
15
15
15
15
Sedation, 0/1
0
1
0
0
0
ADR at baseline
0.27
0.05
0.44
0.23
0.2
Alpha at baseline
3.67
3.76
6.14
8.33
4.91
Delta at baseline
13.71
72.97
13.97
36.39
25.14 1 3 3 Translational Stroke Research (2022) 13:265–275 269 Statistical EEG Analyses Across All Patients delta. No effects of baseline were observed for any EEG
measure.f Results for simple linear regression (see supplemen-
tal figures I-III for model residuals) evaluating the effect
of nitrite across all patients showed relative percentage
change increases in ADR and alpha power with sodium
nitrite versus saline infusion (difference %-change ADR:
37.75, SE = 15.10, p = 0.018, 95% CI = [6.86, 68.64]; dif-
ference %-change alpha: 28.60, SE = 11.14, p = 0.016, 95%
CI = [5.82, 51.38]). No effect of sodium nitrite infusion on
percentage delta power change could be observed, and no
effect of baseline was found for any EEG measure. The effect of nitrite on ADR, alpha and delta power was
also investigated over time using multilevel regression (see
supplemental table II for number of data sets per time win-
dow, see supplemental figures VII-XII for model residuals
including model residuals for control analyses and supple-
mental tables III-V for control analyses).f There was a difference in ADR power change between
infusion groups for all three time windows (Fig. 2a), where
there was more ADR power change in the sodium nitrite
group (marginal effect %-change, time window 0–20 min:
44.27, SE = 17.22, p = 0.013, 95% CI = [9.34, 79.20]; mar-
ginal effect %-change, time window 20–40 min: 54.37,
SE = 18.05, p = 0.003, 95% CI = [19.00, 89.75]; marginal
effect %-change, time window 40–60 min: 54.39, SE = 18.28,
p = 0.003, 95% CI = [18.56, 90.21]). A control analysis on f There was a difference in ADR power change between
infusion groups for all three time windows (Fig. 2a), where
there was more ADR power change in the sodium nitrite
group (marginal effect %-change, time window 0–20 min:
44.27, SE = 17.22, p = 0.013, 95% CI = [9.34, 79.20]; mar-
ginal effect %-change, time window 20–40 min: 54.37,
SE = 18.05, p = 0.003, 95% CI = [19.00, 89.75]; marginal
effect %-change, time window 40–60 min: 54.39, SE = 18.28,
p = 0.003, 95% CI = [18.56, 90.21]). A control analysis on
ADR power differences between infusion groups in a sub-
set of patients that could be tested within 3 days after the
bleed showed comparable results (see supplemental table III
for marginal effects and supplemental figure VIII for model
residuals). Statistical EEG Analyses in Patients Who Did Not
Develop DCI Further statistical analyses concentrated on patients who did
not develop DCI. For simple linear regression (see supple-
mental figures IV-VI for model residuals), we observed rela-
tive percentage change increases in ADR and alpha power
with nitrite versus saline infusion (difference %-change
ADR: 51.57, SE = 17.68, p = 0.008, 95% CI = [15.08,
88.06]; difference %-change alpha: 37.46, SE = 12.37,
p = 0.006, 95% CI = [11.92, 63.00]). There was no effect
of nitrite infusion on percentage delta power change. Seda-
tion resulted in a decrease in alpha power change (differ-
ence %-change alpha: − 46.94, SE = 20.64, p = 0.032, 95%
CI = [− 89.55, − 4.33]), but had no effect on the ADR or Further analyses also investigated relative changes from
baseline. There was a relative increase in ADR from baseline
in the sodium nitrite arm for the first and third time win-
dow (predictive margin %-change, time window 0–20 min:
31.77, SE = 12.22, p = 0.009, 95% CI = [7.83, 55.72]; 1 3
Fig. 2 Statistical EEG analyses
in patients who did not develop
DCI. Predictive margins for
percentage change ADR (a),
alpha (b) and delta (c) power
during nitrite (red) and saline
(grey) infusion for individual
time windows. Marginal effects
between groups are marked
with stars: ***: p < 0.001, **:
p < 0.01, *: p < 0.05. Error bars
denote standard error 1 3 1 3 1 3 3 270 Translational Stroke Research (2022) 13:265–275 window 20–40 min: − 22.58, SE = 8.28, p = 0.006, 95%
CI = [− 38.81, − 6.35]). predictive margin %-change, time window 40–60 min:
35.80, SE = 13.00, p = 0.006, 95% CI = [10.33, 61.28]). predictive margin %-change, time window 40–60 min:
35.80, SE = 13.00, p = 0.006, 95% CI = [10.33, 61.28]). The saline arm showed a relative decrease in ADR from
baseline in the second time window (predictive margin
%-change, time window 20–40 min = − 33.19, SE = 12.18,
p = 0.006, 95% CI = [− 57.05, − 9.32]). There was a difference in changes in delta power between
infusion groups during the second time window (Fig. 2c),
where delta power change was larger within the saline group
(marginal effect %-change, time window 20–40 min: 28.50,
SE = 13.70, p = 0.037, 95% CI = [1.65, 55.35]). Statistical EEG Analyses in Patients Who Did Not
Develop DCI In line with the results for the ADR, there was a dif-
ference in alpha power change between the sodium nitrite
and the saline group for all time windows (Fig. 2b). There
was more alpha power change with sodium nitrite infu-
sion (marginal effect %-change, time window 0–20 min:
37.03, SE = 12.21, p = 0.002, 95% CI = [13.10, 60.95];
marginal effect %-change, time window 20–40 min: 46.51,
SE = 12.39, p < 0.001, 95% CI = [22.23, 70.78]; mar-
ginal effect %-change, time window 40–60 min: 28.76,
SE = 12.57, p = 0.022, 95% CI = [4.12, 53.41]). In addition, the saline arm showed a relative increase
in delta power from baseline for the second and the third
time window (predictive margin %-change, time window
20–40 min: 35.82, SE = 10.00, p < 0.001, 95% CI = [17.98,
53.65]; predictive margin %-change, time window
40–60 min: 19.81, SE = 10.00, p = 0.029, 95% CI = [1.97,
37.64]). Sedation was associated with marginally lower ADR
(difference %-change: − 52.75, SE = 29.37, p = 0.073, 95%
CI = [− 110.31, 4.82]) and significantly lower alpha (dif-
ference %-change: − 49.51, SE = 18.76, p = 0.008, 95%
CI = [− 86.28, − 12.74]) power changes. No effect of baseline
was observed on any EEG measure. The sodium nitrite arm showed a relative increase in
alpha power from baseline for all time windows (predic-
tive margin %-change, time window 0–20 min: 26.64,
SE = 8.32, p = 0.001, 95% CI = [10.33, 42.95]; predic-
tive margin %-change, time window 20–40 min: 23.93,
SE = 8.66, p = 0.006, 95% CI = [6.96, 40.90]; predic-
tive margin %-change, time window 40–60 min: 20.69,
SE = 8.95, p = 0.021, 95% CI = [3.15, 38.24]). Discussion Our key finding in our main study group (patients that did
not develop DCI) was that intravenous sodium nitrite led
to an increase in ADR and alpha power above baseline and
saline placebo. We suggest that these changes reflect the
action of NO and relate to a generally less ischaemic brain
pattern. Our findings confirm and extend animal studies
showing the importance of the NO pathway after SAH and
during ischaemic conditions. They provide a basis for future
studies investigating NO signalling after human SAH. p
We observed an increase in ADR for nitrite versus
saline infusion across the entire sample of patients. As
we expected a more robust effect of nitrite infusion in
patients who do not develop DCI (and the clear majority of
patients do not develop DCI [6]), our study was designed
and powered for this patient group. Individual datasets of
patients who developed DCI showed variable responses
with no obvious relation to infusion type. For patients who
did not develop DCI, we replicated a within-subject ADR
increase with sodium nitrite infusion relative to baseline. Most importantly, we also observed a between-subject dif-
ference in ADR changes with sodium nitrite versus saline
infusion, where sodium nitrite infusion was related to
relative ADR increases. Therefore, we clarified that EEG
changes were not merely an effect of time. We conducted
a randomised, double-blind mechanism study. Therefore,
we are confident that we did not introduce an experimenter
or patient-related bias of treatment or expectation. Patients
featured generally rather good-grade WFNS scores. They
were nevertheless representative of the subarachnoid
haemorrhage stroke population in Oxfordshire, as we did
not restrict patients to a specific grade of stroke sever-
ity. Importantly, it has been shown that even good-grade
patients can experience secondary deterioration [45] and
functional deficits which relate to less favourable outcome
[46]. More than 30% of good-grade patients do not return
to work after years [47].i Previous research has connected decreases in alpha and
increases in delta power to ischaemic conditions [30]. In
the context of DCI after SAH, similar relationships have
been shown [29], where increases or decreases in alpha
and ADR were even predictive of a less or more ischaemic
brain pattern [31, 32]. Higher alpha power and lower delta
power during rest have also both explicitly been linked to
increased cerebral perfusion and oxygen uptake [25–28, 30,
39]. Discussion Therefore, we suggest that increases in alpha and ADR
power with the present investigation could relate to nitrite-
mediated reductions of ischaemia and potentially increases
in cerebral perfusion. Case studies suggest that alpha and delta frequencies can
follow drug action aimed at improving cerebral ischaemia
after SAH [29, 40] and thrombolysis after ischaemic stroke
[41]. Infused sodium nitrite is well suited to alter brain states
after cerebral damage. It can safely be administered [23] and
is converted to NO in hypoxic and acidic conditions [16,
17], which can be observed after SAH [6, 42]. It can sup-
port cerebral autoregulation after SAH [43] and, crucially,
helps preserve and maintain cerebral microcirculation and
protect nerve cells after cerebral insult [11, 44]. Whereas
clinical studies on the effect of NO availability as measured
by NO metabolites after SAH are still inconclusive [12–15],
animal studies using experimental models suggest benefi-
cial effects of nitrite administration on cerebral angiographic
vasospasm after SAH [22] and neuroprotective effects with
ischaemic conditions [20, 21]. However, NO metabolites
mostly sampled from the cerebrospinal fluid represent an
indirect and regionally unspecific measure. They may not
be a good proxy for actual NO availability in injured brain
areas. Instead of concentrating on indirect measures of
global NO distribution, we probed the neural effects of NO
donor administration in SAH patients by using EEG markers
relating to immediate neural processing, cerebral ischaemia
and regional cerebral blood flow.i We further clarified nitrite action by investigating fre-
quency specific contributions to ADR power changes. Cru-
cially, we observed a within-subject increase in alpha power
from baseline during sodium nitrite infusion and a between-
subject difference in alpha changes, where sodium nitrite
infusion was related to a relative increase in alpha. Hence,
ADR power changes reflect alpha power changes. Previous research has found decreases in alpha power
with fentanyl, propofol and midazolam administration [36,
38]. In our study, alpha power changes from baseline were
decreased when patients were sedated. Therefore, the pre-
sent study shows the importance of controlling for sedation
in patient studies using EEG, even if relative EEG power is
investigated. Delta power showed an increase from baseline with saline
infusion and was temporarily higher with saline compared to
nitrite infusion. This was mirrored by a pattern of mean and
temporarily significant decreases in alpha and ADR power
with saline infusion. Individual EEG Data in Patients Who Developed DCI Data from the five patients who developed DCI are presented
on an individual basis. There was no obvious difference
between infusion groups for either time window with ADR,
alpha or delta power changes (Fig. 3a–c). p
There was a relative decrease from baseline in
alpha power with saline infusion in the second
time window (predictive margin %-change, time 1 3
Fig. 3 Individual EEG data in
patients who developed DCI. Percentage change ADR (a),
alpha (b) and delta (c) power
from baseline during nitrite
(red) and saline (grey) infusion
for individual time windows 1 3 3 271 Translational Stroke Research (2022) 13:265–275 patients who did not develop DCI, but a decrease in ADR
in those who subsequently developed DCI. However, Garry
et al.’s study was uncontrolled, and all patients were sedated,
intubated and mechanically ventilated, and thus sicker than
the present cohort. Discussion As absolute baseline power between the
two infusion groups was not obviously different, respectively
accounted for in our model, and patients were otherwise We extend and clarify the findings of Garry et al. [24]
who investigated a small cohort of critically ill patients
with SAH in an intensive care environment using an identi-
cal sodium nitrite infusion. The main findings were robust
within-subject increases in the ADR from baseline in those 1 Translational Stroke Research (2022) 13:265–275 272 resting, we speculate that the changes in alpha and delta
activity from baseline during saline infusion could relate to
declining vigilance. The first infusion time window already
included time points from at least 20–40 min of recording
after EEG setup. A decrease in alpha activity and more
prominent delta activity have been related to lower levels
of vigilance [48, 49]. may use denser electrode setups, potentially in combination
with other techniques [51], to further characterise the site
of nitrite action. While none of our patients was diagnosed to suffer from
DCI at or before the time of our study, we conducted the
study within a maximum of 5 days after the bleed. The
time frame reflects our effort to recruit as many patients
as possible in order to obtain a representative sample of
subarachnoid haemorrhage patients. We believe that the
results from our group analyses nevertheless reflect EBI
disease mechanisms, as patients were on average tested
within 3 days after the ictus. A focussed control analysis
in a subset of patients who could be tested within 3 days
after the bleed showed comparable results. Unlike alpha power, delta power was comparably less
sensitive to nitrite infusion with the present investigation,
and ADR power changes did not show superior responsiv-
ity to alpha power changes. Stroke severity in our patient
population was lower compared to patients in other investi-
gations where a prominent role of delta or ADR in ischaemia
was suggested. There, patients were stuporous or comatose
[29] or featured higher WFNS scores [24, 32]. Machado
et al. [50] put forward for consideration that delta power in
ischaemic stroke patients would rather relate to an ischaemic
core region, while alpha power would reflect oedema, tissue
at risk and the penumbra. Accordingly, delta power changes
have been related to more prominent reductions in cerebral
blood flow compared to alpha power changes [30]. Discussion It could
thus be argued that patients in our study featured comparably
more residually intact tissue, which could be reflected in a
lack of delta reactivity. Alternatively, nitrite might generally
only act in less damaged and residually functioning tissue. Future studies comparing alpha and delta responses to nitrite
infusion in higher versus lower grade SAH patients will need
to clarify the possibility of an interaction between nitrite
action and SAH lesion severity. To complement this research, it will be necessary to
study the effect of nitrite infusion in a larger group of
SAH patients who develop DCI. In particular, it will be
mechanistically informative to determine if EEG mark-
ers of ischaemia and perfusion can also be modulated in
this population of SAH survivors and if a different dosing
scheme of sodium nitrite infusion will be necessary. Other studies aiming toward the use of sodium nitrite
as a therapeutic have infused sodium nitrite in healthy
participants for 2 days [52] or SAH patients for 2 weeks
[23]. Although sodium nitrite was infused at comparably
lower doses in these investigations, the long-term infusion
dosing scheme in SAH patients [23] suggested that blood
nitrite levels were at ceiling with the highest dose admin-
istered. We cannot exclude an effect of short-term sodium
nitrite infusion on outcome with the present research as
our study was not powered for investigating patients who
develop DCI. However, we deliberately administered
sodium nitrite only comparably very briefly and for 1 h,
as we did not aim to treat patients but merely probe the
cerebral response to an NO donor after SAH. Crucially,
we could indeed show a beneficial change in electrophys-
iological markers of ischaemia in patients who did not
develop DCI. Future clinical trials will need to investigate
the diagnostic [24] and therapeutic potential of short- and
long-term sodium nitrite infusion.i Importantly, Franko et al. [33] did not find a change
in alpha, delta or ADR power with sodium nitrite versus
saline infusion in healthy participants suggesting that the
EEG changes observed with the present investigation are
disease related. As already discussed, nitrite is reduced to
NO specifically well in hypoxic and acidic environments
[16, 17]. Therefore, infused nitrite could preferentially be
converted to NO in the injured brain, e.g. after SAH. Discussion In
injured but residually functional tissue, NO could best exert
its neuroprotective effects, including an increase in regional
cerebral perfusion, which is reflected in EEG. In contrast, an
increased level of nitrite in normoxic tissue of healthy brains
would not have a large effect on brain perfusion and the EEG
would not change accordingly. Our findings may help render such studies safer. In
addition to beneficial and neuroprotective effects of NO
after cerebral insult, detrimental and cytotoxic effects
relating to secondary brain injury are also known, and
these depend on timing and regional availability of NO [9,
53]. Whereas increased NO at the vascular bed of residu-
ally functioning tissue after SAH could be neuroprotective,
excessive NO in critically injured brain tissue may further
cell damage. Therefore, clinical investigations using NO
donors after SAH should tightly monitor NO action on
neuronal functioning. EEG is safe, non-invasive, cheap
and not dependent on active patient involvement. Most
importantly, EEG directly reflects neuronal processing and Despite the fact that NO has been proposed to promote
various beneficial effects on damaged tissue, including an
improvement of cerebral blood flow at the microcirculatory
[11, 44] level and with larger vessels [43], and a reduction in
platelet aggregation, inflammation and Ca2+-mediated cyto-
toxicity [16], our experimental methods do not allow for
an explanation of the effects of nitrite infusion beyond the
level of a reduction in ischaemic processes and an increase
in cerebral perfusion [25–30]. Subsequent investigations 3 3 273 Translational Stroke Research (2022) 13:265–275 is related to ischaemia and cerebral perfusion. Therefore,
EEG might help to determine optimal levels of nitrite infu-
sion to maximise patient safety [23]. Our research shows
that the EEG can be used to study effects of short-term NO
donor infusion after SAH and provides a mechanistic basis
for future EEG-aided clinical trials employing NO donors. Competing Interests Martyn Ezra and Kyle Pattinson are named as
coinventors on a provisional UK patent application titled “Use of cer-
ebral nitric oxide donors in the assessment of the extent of brain dys-
function following injury”. Competing Interests Martyn Ezra and Kyle Pattinson are named as
coinventors on a provisional UK patent application titled “Use of cer-
ebral nitric oxide donors in the assessment of the extent of brain dys-
function following injury”. Discussion 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/. Conclusions To conclude, we studied the effects of short-term sodium
nitrite infusion during EBI after SAH using EEG. Sodium
nitrite infusion compared to saline control infusion resulted in
clear relative increases in the ADR and alpha power in patients
who did not develop DCI. We suggest that these EEG changes
reflect NO action and relate to a change toward a less ischae-
mic brain pattern. In future studies, using EEG to determine
the effect of NO donors after SAH should improve mechanistic
understanding of the NO pathway and could further the devel-
opment of novel diagnostics and therapeutics. Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s12975-021-00939-9. Acknowledgements We are extremely grateful to Dr. Jill Mollison for
providing statistical support and Dr. Lara Prisco for determining out-
comes. We are equally grateful to our patients whose participation is
the very basis of this research. 3. Rivero-Arias O, Gray A, Wolstenholme J. Burden of disease and
costs of aneurysmal subarachnoid haemorrhage (aSAH) in the
United Kingdom. Cost Eff Resour Alloc. 2010;8:1–12. 4. Fujii M, Yan J, Rolland WB, Soejima Y, Caner B, Zhang JH. Early brain injury, an evolving frontier in subarachnoid hemor-
rhage research. Transl Stroke Res. 2013;4:432–46. Funding This research was funded by the NIHR Oxford Biomedical
Research Centre (BRC) based at Oxford University and Oxford Uni-
versity Hospitals NHS Foundation Trust. This research was funded in
part by the Wellcome Trust (grant ref 203139/Z/16/Z). For the purpose
of Open Access, the author has applied a CC BY public copyright
licence to any Author Accepted Manuscript version arising from this
submission. 5. Sehba FA, Hou J, Pluta RM, Zhang JH. The importance of early
brain injury after subarachnoid hemorrhage. Prog Neurobiol. 2012;97:14–37. 6. Macdonald RL. Delayed neurological deterioration after suba-
rachnoid haemorrhage. Nat Rev Neurol. 2014;10:44–58. https://
doi.org/10.1038/nrneurol.2013.246 (Nature Publishing Group). 7. Rowland MJ, Hadjipavlou G, Kelly M, Westbrook J, Pattin-
son KTS. Delayed cerebral ischaemia after subarachnoid
haemorrhage: looking beyond vasospasm. Br J Anaesth. 2012;109:315–29. Availability of Data and Material The data that support the findings of
this study are available from the corresponding author upon reason-
able request. 8. Vergouwen MDI, Vermeulen M, van Gijn J, Rinkel GJE,
Wijdicks EF, Muizelaar JP, et al. Definition of delayed cerebral
ischemia after aneurysmal subarachnoid hemorrhage as an out-
come event in clinical trials and observational studies.Stroke. 2010;41:2391–95. Available from: https://www.ahajournals.
org/doi/10.1161/STROKEAHA.110.589275. Accessed 19 Sept
2019. Code Availability Data processing scripts for this study are available
from the corresponding author upon reasonable request. References 1. Macdonald RL, Schweizer TA. Spontaneous subarachnoid
haemorrhage. Lancet. 2017;389:655–66. https://doi.org/10.
1016/S0140-6736(16)30668-7 (Elsevier Ltd). 2. Al-Khindi T, Macdonald RL, Schweizer TA. Cognitive and
functional outcome after aneurysmal subarachnoid hemorrhage. Stroke. 2010;41:e519–e536. Available from: https://www.ahajo
urnals.org/, https://doi.org/10.1161/STROKEAHA.110.581975. Accessed 27 Oct 2020. Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s12975-021-00939-9. Declarations Woszczyk A, Deinsberger W, Böker DK, Raabe A, Seifert V,
Dóczi T. Nitric oxide metabolites in cisternal CSF correlate
with cerebral vasospasm in patients with a subarachnoid haem-
orrhage. Acta Neurochir (Wien). 2003;145:257–64. 32. Rots ML, van Putten MJAM, Hoedemaekers CWE, Horn J. Con-
tinuous EEG monitoring for early detection of delayed cerebral
ischemia in subarachnoid hemorrhage: a pilot study. Neurocrit
Care. 2016;24:207–16.f 16. Kapil V, Khambata RS, Jones DA, Rathod K, Primus C, Mas-
simo G, et al. The noncanonical pathway for in vivo nitric oxide
generation: the nitrate-nitrite-nitric oxide pathway. Pharmacol
Rev. 2020;72:692–766. 33. Franko E, Ezra M, Crockett DC, Joly O, Pattinson K. Effect of
nitrite on the electroencephalographic activity in the healthy
brain. Nitric Oxide Biol Chem. 2019;90:47–54. https://doi.org/
10.1016/j.niox.2019.06.002 (Elsevier). 17. Shiva S. Nitrite: A physiological store of nitric oxide and mod-
ulator of mitochondrial function. Redox Biol. 2013;1:40–4. https://doi.org/10.1016/j.redox.2012.11.005 (Elsevier).f 34. Feshchenko VA, Veselis RA, Reinsel RA. Comparison of the EEG
effects of midazolam, thiopental, and propofol: the role of under-
lying oscillatory systems. Neuropsychobiology. 1997;35:211–20. p
g
j
18. Sehba FA, Ding WH, Chereshnev I, Bederson JB. Effects of
S-nitrosoglutathione on acute vasoconstriction and glutamate
release after subarachnoid hemorrhage. Stroke. 1999;30:1955–61. 35. Glass PS, Bloom M, Kearse L, Rosow C, Sebel P, Manberg P. Bispectral analysis measures sedation and memory effects of
propofol, midazolam, isoflurane, and alfentanil in healthy volun-
teers. Anesthesiol J Am Soc Anesthesiol. 1997;86:836–47 (The
American Society of Anesthesiologists). 19. Sehba FA, Friedrich V, Makonnen G, Bederson JB. Acute cer-
ebral vascular injury after subarachnoid hemorrhage and its pre-
vention by administration of a nitric oxide donor. J Neurosurg. 2007;106:321–9. 36. Numan T, van Dellen E, Vleggaar FP, van Vlieberghe P, Stam CJ,
Slooter AJC. Resting state EEG characteristics during sedation
with midazolam or propofol in older subjects. Clin EEG Neurosci. 2019;50:436–43. Available from: http://journals.sagepub.com/doi/
10.1177/1550059419838938. Accessed 19 Oct 2010. 20. Dezfulian C, Alekseyenko A, Dave KR, Raval AP, Do R, Kim F,
et al. Nitrite therapy is neuroprotective and safe in cardiac arrest
survivors. Nitric Oxide Biol Chem. 2012;26:241–50. https://doi.
org/10.1016/j.niox.2012.03.007 (Elsevier Inc). 37. Veselis RA, Reinsel RA, Marino P, Sommer S, Carlon GC. The
effects of midazolam on the EEG during sedation of critically ill
patients. Anaesthesia. 1993;48:463–70.f 21. Jung KH, Chu K, Ko SY, Lee ST, Sinn DI, Park DK, et al. Early
intravenous infusion of sodium nitrite protects brain against
in vivo ischemia-reperfusion injury. Stroke. Declarations Ethics Approval The study protocol was approved by the South Cen-
tral–Oxford C NHS Health Research Authority Ethics Committee (ref-
erence number: 12/SC/0366). The study was conducted in accordance
with the ethical standards as laid down in the Declaration of Helsinki. 9. Garry PS, Ezra M, Rowland MJ, Westbrook J, Pattinson KTS. The role of the nitric oxide pathway in brain injury and its treat-
ment - from bench to bedside. Exp Neurol. 2015;263:235–43. https://doi.org/10.1016/j.expneurol.2014.10.017 (Elsevier B. V.). Consent to Participate Informed consent for participation in the study
was given either by the patient directly or a personal consultee who
acted in the best interest of the patient. 10. Attwell D, Buchan AM, Charpak S, Lauritzen M, MacVicar BA,
Newman EA. Glial and neuronal control of brain blood flow. Nature. 2010;468:232–43.l Consent for Publication All authors have read the manuscript and
approved it for submission. 11. Toda N, Ayajiki K, Okamura T. Cerebral blood flow regula-
tion by nitric oxide: recent advances. Cereb Blood Flow Regul
ASPET. 2009;61:1–221. 1 3 274 Translational Stroke Research (2022) 13:265–275 28. O’Gorman RL, Poil SS, Brandeis D, Klaver P, Bollmann S,
Ghisleni C, et al. Coupling between resting cerebral perfusion
and EEG. Brain Topogr. 2013;26:442–57. 12. Jung CS, Oldfield EH, Harvey-White J, Espey MG, Zimmermann
M, Seifert V, et al. Association of an endogenous inhibitor of nitric
oxide synthase with cerebral vasospasm in patients with aneurys-
mal subarachnoid hemorrhage. J Neurosurg. 2007;107:945–50. 29. Claassen J, Hirsch LJ, Kreiter KT, Du EY, Sander Connolly E,
Emerson RG, et al. Quantitative continuous EEG for detecting
delayed cerebral ischemia in patients with poor-grade subarach-
noid hemorrhage. Clin Neurophysiol. 2004;115:2699–710. 13. Rejdak K, Petzold A, Sharpe MA, Kay AD, Kerr M, Keir G,
et al. Cerebrospinal fluid nitrite/nitrate correlated with oxy-
hemoglobin and outcome in patients with subarachnoid hem-
orrhage. J Neurol Sci. 2004;219:71–6. 30. Foreman B, Claassen J. Quantitative EEG for the detection of
brain ischemia. Critical Care. 2012;16:216. 14. Suzuki M, Asahara H, Endo S, Inada K, Doi M, Kuroda K,
et al. Increased levels of nitrite/nitrate in the cerebrospinal fluid
of patients with subarachnoid hemorrhage. Neurosurg Rev. 1999;22:96–8. 31. Gollwitzer S, Groemer T, Rampp S, Hagge M, Olmes D, Huttner
HB, et al. Early prediction of delayed cerebral ischemia in suba-
rachnoid hemorrhage based on quantitative EEG: a prospective
study in adults. Clin Neurophysiol. 2015;126:1514–23. 15. Declarations 2006;37:2744–50.i 22. Pluta RM, Dejam A, Grimes G, Gladwin MT, Oldfield EH. Nitrite
infusions to prevent delayed cerebral vasospasm in a primate
model of subarachnoid hemorrhage. Jama. 2005;293:1477–84. American Medical Association Available from: http://jama.
jamanetwork.com/article.aspx?doi=10.1001/jama.293.12.1477. Accessed 13 Oct 2020.i 38. Wauquier A, Bovill JG, Sebel PS. Electroencephalographic effects
of fentanyl-, sufentanil- and alfentanil anaesthesia in man. Neu-
ropsychobiology. 1984;11:203–6 Available from: https://www.
karger.com/Article/FullText/118078. Accessed 19 Oct 2020. 39. Tolonen U, Sulg IA. Comparison of quantitative EEG parameters
from four different analysis techniques in evaluation of relation-
ships between EEG and CBF in brain infarction. Electroencepha-
logr Clin Neurophysiol. 1981;51:177–85. 23. Oldfield EH, Loomba JJ, Monteith SJ, Crowley RW, Medel R,
Gress DR, et al. Safety and pharmacokinetics of sodium nitrite
in patients with subarachnoid hemorrhage: a phase IIA study. J
Neurosurg. 2013;119:634–41. 40. Rathakrishnan R, Gotman J, Dubeau F, Angle M. Using con-
tinuous electroencephalography in the management of delayed
cerebral ischemia following subarachnoid hemorrhage. Neurocrit
Care. 2011;14:152–61. 24. Garry PS, Rowland MJ, Ezra M, Herigstad M, Hayen A, Sleigh
JW, et al. Electroencephalographic response to sodium nitrite
may predict delayed cerebral ischemia after severe subarachnoid
hemorrhage. Crit Care Med. 2016;44:e1067–73 . Available from:
http://insights.ovid.com/crossref?an=00003246-201611000-
00032. Accessed 4 Feb 2019. 41. Finnigan SP, Rose SE, Chalk JB. Rapid EEG changes indicate
reperfusion after tissue plasminogen activator injection in acute
ischaemic stroke. Clin Neurophysiol. 2006;117:2338–9.f 42. Hoffman WE, Wheeler P, Edelman G, Charbel FT, Torres NJ,
Ausman JI. Hypoxic brain tissue following subarachnoid hemor-
rhage. Anesthesiology. 2000;92:442–6. 25. Diedler J, Sykora M, Bast T, Poli S, Veltkamp R, Mellado P, et al. Quantitative EEG correlates of low cerebral perfusion in severe
stroke. Neurocrit Care. 2009;11:210–6.i 43. Ezra M, Garry P, Rowland MJ, Mitsis GD, Pattinson KT. Phase
dynamics of cerebral blood flow in subarachnoid haemorrhage
in response to sodium nitrite infusion. Nitric Oxide Biol Chem. 2021;106:55–65. https://doi.org/10.1016/j.niox.2020.10.004
(Elsevier Inc).l 26. Stragapede L, Furlanis G, Ajčević M, Ridolfi M, Caruso P, Nac-
carato M, et al. Brain oscillatory activity and CT perfusion in
hyper-acute ischemic stroke. J Clin Neurosci. 2019;69:184–9. 27. Nagata K, Tagawa K, Hiroi S, Shishido F, Uemura K. Electro-
encephalographic correlates of blood flow and oxygen metabo-
lism provided by positron emission tomography in patients
with cerebral infarction. Electroencephalogr Clin Neurophysiol. 1989;72:16–30. 44. Dalkara T, Morikawa E, Panahian N, Moskowitz MA. Blood flow-
dependent functional recovery in a rat model of focal cerebral
ischemia. Am J Physiol Circ Physiol. Declarations 1994;267:678–83. Avail-
able from: https://www.physiology.org/doi/10.1152/ajpheart.1994.
267.2.H678. Accessed 18 Oct 2020. 1 3 Translational Stroke Research (2022) 13:265–275 275 frequencies in the human resting awake electroencephalogram. Neurosci Lett. 2003;340:17–20. 45. Veldeman M, Albanna W, Weiss M, Conzen C, Schmidt TP,
Clusmann H, et al. Treatment of delayed cerebral ischemia in
good-grade subarachnoid hemorrhage: any role for invasive neu-
romonitoring? Neurocrit Care. 2020;35:172–83. https://doi.org/
10.1007/s12028-020-01169-x (Springer US). 50. Machado C, Cuspineda E, Valdés P, Virues T, Liopis F, Bosch J,
et al. Assessing acute middle cerebral artery ischemic stroke by
quantitative electric tomography. Clin EEG Neurosci [Internet]. 2004;35:116–24. ( p
g
)
46. Leira EC, Davis PH, Martin CO, Torner JC, Yoo B, Weeks JB,
et al. Improving prediction of outcome in “good grade” subarach-
noid hemorrhage. Neurosurgery. 2007;61:470–4 (Available from:
https://academic.oup.com/neurosurgery/article/61/3/470/2593946. Accessed 8 July 2021. 51. Fantini S, Sassaroli A, Tgavalekos KT, Kornbluth J. Cerebral
blood flow and autoregulation: current measurement techniques
and prospects for noninvasive optical methods. Neurophotonics. 2016;3:031411. 52. Pluta RM, Oldfield EH, Bakhtian KD, Fathi AR, Smith RK, DeV-
room HL, et al. Safety and feasibility of long-term intravenous
sodium nitrite infusion in healthy volunteers. PLoS One. 2011;6 y
47. Seule M, Oswald D, Muroi C, Brandi G, Keller E. Outcome,
Return to Work and health-related costs after aneurysmal suba-
rachnoid hemorrhage. Neurocrit Care. 2020;33:49–57. https://doi.
org/10.1007/s12028-019-00905-2 (Springer US). 53. Hänggi D, Steiger HJ. Nitric oxide in subarachnoid haemor-
rhage and its therapeutics implications. Acta Neurochir (Wien). 2006;148:605–13. 48. Roth B. The clinical and theoretical importance of EEG rhythms
corresponding to states of lowered vigilance. Electroencephalogr
Clin Neurophysiol. 1961;13:395–9. Available from: https://linki
nghub.elsevier.com/retrieve/pii/0013469461900086. Accessed 10
Oct 2020. Publisher's Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. 49. Strijkstra AM, Beersma DGM, Drayer B, Halbesma N, Daan
S. Subjective sleepiness correlates negatively with global alpha
(8–12 Hz) and positively with central frontal theta (4–8 Hz) 1 3 1 3 3
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1. INTRODUCTION within the CityGML specification), utility networks (Becker et
al, 2010; 2013), real estate management, robotics, building
information models (BIM), and hydrography (see CityGML,
2013). Over the past ten years technologies for generating, maintaining
and using 3D geo-information have matured. Many local
governments have 3D models of their area, a large number of
companies are providing services for creating or disseminating
3D models, and universities and research organisations are
investigating 3D technologies, from capture to data services. In
addition, the Open Geospatial Consortium defined the 3D
standard CityGML 1.0 in 2008 and released a 2.0 version in
2012 (OGC, 2012). This has grown to a well-established
standard for 3D geo-information. It is clear that 3D applications are becoming increasingly
important and therefore production of 3D information by
governmental organisation gets more and more mandatory. For
national mapping agencies, who have a long history in
maintaining and incremental updating countrywide 2D-datasets,
the major challenge is how to best convert this 2D data into 3D
data, while supporting the established process of continuous
incremental updating and making best use of research results
and available technologies. Some mapping organisations are
already making serious progress in this step (i.e. Swisstopo,
ICC, IGN France, The Netherlands, Ordnance Survey GB). CityGML originated in academia in Germany (Bonn, TU
Berlin) and was originally defined as an exchange format. But it
is also - and perhaps more importantly- an information model
for representing 3D spatial objects. Although it started as a
standard to model objects that occur in cities, the current
version (2.0) has been expanded and now supports a variety of
objects in landscape and city models. The question addressed in this paper is how research results
achieved in the past ten years in the 3D domain are applied in
3D mapping activities at national level in practice and what
research problems remain. In addition, the paper explores the
potential of the OGC 3D standard CityGML for 3D national
mapping and what further developments are required to make
the standard more appropriate for this purpose. j
p
y
Many projects use CityGML for 3D modelling. For example
North-Rhine Westphalia in Germany has been the first state that
provides a statewide 3D model consisting of 3D roads, railways,
DTM, and 3D buildings employed for noise dispersion mapping
(Czerwinski et al 2006; 2007). ABSTRACT:
h Over the past ten years technologies for generating, maintaining and using 3D geo-information have matured. For national mapping
agencies one of the challenges is how to best extend 2D data into 3D data, making best use of research results and available
technologies. Some mapping organisations are making serious progress. The question addressed in this paper is how research results
achieved in the past ten years are applied in practice and what research problems remain. In addition, the paper explores the
potentials of the OGC 3D standard (i.e. CityGML) for 3D national mapping and what developments are further required to make the
standard better fit for this purpose. The main conclusions of the paper are that 3D data is more and more available but still suffers
from a low level of usage (mainly visualisation) and standards and formats based on CityGML have been stabilised although
software support is still in the early stage. Several recommendations are made to meet these problems, including the definition of
European CityGML profiles (as the INSPIRE Building profile) to harmonise 3D needs and standardise 3D implementations at
international level. KEY WORDS: 3D national mapping; 3D standard, CityGML KEY WORDS: 3D national mapping; 3D standard, CityGML a TU Delft & Geonovum & Kadaster, j.e.stoter@tudelft.nl
b Swisstopo Andre.Streilein@swisstopo.ch
c ICC, Catalonia, m.pla@icc.cat, b.baella@icc.cat
d Ordnance Survey, GB, dave.capstick@ordnancesurvey.co.uk, rolo.home@ordnancesurvey.co.uk,
Carsten.Roensdorf@ordnancesurvey.co.uk
eIGN, France jean-philippe.lagrange@ign.fr a TU Delft & Geonovum & Kadaster, j.e.stoter@tudelft.nl
b Swisstopo Andre.Streilein@swisstopo.ch
c ICC, Catalonia, m.pla@icc.cat, b.baella@icc.cat
d Ordnance Survey, GB, dave.capstick@ordnancesurvey.co.uk, rolo.home@ordnancesurvey.co.uk,
Carsten.Roensdorf@ordnancesurvey.co.uk
eIGN, France jean-philippe.lagrange@ign.fr a TU Delft & Geonovum & Kadaster, j.e.stoter@tudelft.nl
b Swisstopo Andre.Streilein@swisstopo.ch
c ICC, Catalonia, m.pla@icc.cat, b.baella@icc.cat
d Ordnance Survey, GB, dave.capstick@ordnancesurvey.co.uk, rolo.home@ordnancesurvey.co.uk,
Carsten.Roensdorf@ordnancesurvey.co.uk
eIGN, France jean-philippe.lagrange@ign.fr ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey APPROACHES OF NATIONAL 3D MAPPING: RESEARCH RESULTS AND
STANDARDISATION IN PRACTICE J.E. Stotera,*, A. Streileinb, M. Plac, B. Baellac, D. Capstickd, R. Homed, C. Roensdorfd, J.P. Lagrangee ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey services by NMAs, explore the relationship between 2D and 3D
data, identify the potentials of the CityGML standard and define
requirements for future developments of the standard. Th
k h
t i l
b f
d t G
(2013) services by NMAs, explore the relationship between 2D and 3D
data, identify the potentials of the CityGML standard and define
requirements for future developments of the standard. The workshop materials can be found at Geonovum (2013). orthophotos and for deriving automatically LOD1 city
models (see Figure 2). All the existing data collected
according to the previous data model was reused. Fig 2. Example of topographic data at 1:1.000 scale and the derived
city model 2.1 3D Topographic Data in urban areas The topographic data set at 1:1000 scale (CT-1M) is the most
detailed data covering the urban areas and the areas planned for
urbanization, a total surface of 330.000 hectares. The accuracy
of this product is 20 centimeters for X and Y and 25
centimeters for height. The data is compiled using
photogrammetric systems, completed by data surveyed on the
field, stored in an ORACLE database and distributed as
geoservices and several GIS and CAD formats, including KMZ
for buildings (see Figure 1). The updating cycle is between 4
and 6 years, depending on the number and importance of the
changes on the territory. y
Fig 3: Elevation data from BT-5M, DTM at the left and DSM at the
right Fig 3: Elevation data from BT-5M, DTM at the left and DSM at the
right Fig 1. KMZ data from the BT-5M buildings Data are distributed as geoservices and in several CAD, GIS
and raster formats, including KMZ for buildings. 2. 3D MAPPING AT ICC: 3D DATA FOR
TOPOGRAPHIC PRODUCTS The Institut Cartogràfic de Catalunya (ICC) is the regional
mapping agency of the Government of Catalonia. From its
creation in late 1982, the ICC has focused improving the
cartographic production workflows. Fig 2. Example of topographic data at 1:1.000 scale and the derived
city model Topographic data, ranging from scales 1:1000 to 1:25.000, are
mainly collected using photogrammetric systems and stored
using 2.5D data models. Elevation models are derived from
these models. Smaller scales such as 1:50.000 and 1:250.000 are
collected in 2D, digitized on top of orthophotoimages. Since
2002, LIDAR systems are an additional source for elevation
data. A full coverage of Catalonia is available with 1point/2m2
density. 2.2 The Topographic Database at 1:5000 scale The Topographic Database at 1:5000 scale (BT-5M) is the
most detailed database that covers complete Catalonia. The
data is compiled using photogrammetric systems according to a
2.5D data model and stored in an ORACLE database. The
accuracy is 1 meter for X and Y and 1.5 meters for the height. During the stereoplotting process all the features required to
generate a digital terrain model (DTM) and a digital surface
model (DSM) are compiled together with the topographic
objects (Figure 3). The updating cycle is 5 years over all the
country and more frequently over the most dynamic areas,
located mainly in the coast. ICC products, including data, metadata and specifications, are
distributed free-of-charge. 1. INTRODUCTION Another important achievement
in CityGML implementation is the extension of the German
national cadastre model ALKIS by 3D building models. In this
project a CityGML profile has been defined and most states in
Germany provide 3D building models according to this profile. In addition INSPIRE data specifications for buildings contain a
3D building profile in line with the CityGML specifications for
buildings. Finally, different Application Domain Extensions
have been developed. Some examples are noise (as used in the
German state North-Rhine Westphalia and as documented The paper first summarises the approaches and open issues of
3D nationwide mapping activities of the five above mentioned
mapping authorities (sections 2 till 6). Then section 7
summarises the main findings, draws conclusions and identifies
topics that require further (research) attention. The material in this paper is based on the EuroSDR/OGC
Workshop on “CityGML in National Mapping” that took place
in January 2013, at IGN France. The objectives of the workshop were to discuss the benefits and
barriers to the provision of 3D geospatial data and related ribution has been peer-reviewed. The double-blind peer-review was conducted on the basis of the full paper. 269 ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey 2.4 Work in progress Existing and new products anticipated to have a 3D
element Other line of work is the analysis of the compatibility between
ICC data models and other models and standards, as
CityGML and INSPIRE. Finally, aspects related to dissemination of the information,
are also under study. Although there is a huge potential of city
model applications (urban planning, telecommunications,
energy,
environment,
smart
cities,
simulations,
etc.),
visualization is the most common while the other ones are not
yet enough developed. A 3D viewer for ICC data, considering
continuous zoom using the CT-1M, BT-5M and BT-25M
information, is currently in development. Also, to facilitate and
spread the use of city models, an ongoing project focuses on
analyzing in depth the requirements related to the use of ICC
data in some specific 3 D city model applications, in order
to define some user profiles and provide data better adapted to
them. OSGB’s approach to 3D capture and design is guided by a set of core
principles, as outlined in Table 2. Principle
Narrative
Survey
standards
Data
will
be
captured
to
defined
standards
of
geometric
and
attribute
accuracy
Maintained
Features
will
be
revised
and
updated
throughout
their
lifecycle
through
a
combination
of
continuous
and
cyclic
processes
Nationally
consistent
Equivalent
geographies
will
(over
time)
be
published
to
an
equivalent
specification
Intelligent
The
data
will
enable
spatial
query
and
analysis
Interoperable
The
data
will
be
usable
in
association
with
both
Ordnance
Survey
and
external
datasets. Accessible
Products
will
be
made
available
in
formats
and
delivery
methods
which
encourage
adoption
for
sustained
application
Table 2. Core principles for Ordnance Survey 3D products 2.3 The Topographic Database at 1:25.000 scale 270 ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey Fig 4. Example of 3D building generalization, from the BT-5M to
the BT-25M urban change, asset management, civil contingency planning
and risk assessment. This section outlines how OSGB anticipates meeting their
customers’ 3D needs. It will discuss issues that are being faced
when moving towards a 3D-centric view. Finally, it will
consider the usages and challenges being faced regarding the
implementation of CityGML. Fig 4. Example of 3D building generalization, from the BT-5M to
the BT-25M 2.4 Work in progress The long-term plan is to ‘3D-enable’ the core databases,
products and services. This will involve three broad types of
change (see Figure 5): The first line of the 3D work in progress is focused in the
enrichment of the CT-1M data, adding the inclined roofs for
obtaining a LOD2 city models. After some tests on
commercial
software
for
obtaining
inclined
roofs
automatically, based on image correlation and building
boundaries, the results are promising although manual tasks
will be required to refine them. •
Creating a new generation of elevation models
(completed) •
Adding ground height values to 2D vertices and features
in the existing 2D datasets, including topographic features
and routing networks (underway) •
Generating elevations for features that extrude from the
ground surface, including buildings, structures and
vegetation The second line of 3D work is the use of LIDAR data to
improve the elevation data derived from the BT-5M, mainly
in dense forests areas, where ground is not visible in the aerial
images. Although the accuracy of LIDAR data is 0.5 m in X
and Y and 6-10 cm in Z, it must be taken into account that in
cliffs and areas of high slope LIDAR data requires fine
filtering. Moreover, the potential problems of incoherence
between planimetric features and LIDAR data will be analyzed,
for example in talwegs. Fig 5. Categories of ‘3D-enabled’ data The third line of work is related to maintenance and updating
of the information, crucial for optimizing the production
costs. The related tasks in development are the coordination
with municipalities and other government organizations to
share information to speed the change detection. Table 1 shows the 3D-enabled datasets that are under active
consideration or development. Datasets
New
elevation
models
DTMs
(complete)
DSMs
Ground
heighting
of
existing
2D
datasets
Topographic
features
Road
network
Hydrology
network
(complete)
Rail
network
(complete)
Tracks
and
Paths
network
Address
points
Additional
capture
for
elevated
features
Building
heights
(LOD
1)
3D
buildings
and
structures
(LOD2)
Vegetation
Table 1. Existing and new products anticipated to have a 3D
element Datasets
New
elevation
models
DTMs
(complete)
DSMs
Ground
heighting
of
existing
2D
datasets
Topographic
features
Road
network
Hydrology
network
(complete)
Rail
network
(complete)
Tracks
and
Paths
network
Address
points
Additional
capture
for
elevated
features
Building
heights
(LOD
1)
3D
buildings
and
structures
(LOD2)
Vegetation
Table 1. 2.3 The Topographic Database at 1:25.000 scale Although from the beginning of 2000’s a complete coverage of
BT-5M was available, the customers were asking for a
database at smaller scale for new GIS applications and
mapping. This database should be manageable, like the
1:50.000 database, but with the topographic information as
detailed as possible, like the 1:5.000. Moreover, elevation data
was considered essential for visualization and analysis
applications. A new topographic database at 1:25.000 scale and
2.5D (BT-25M) was designed to fulfill these user requirements. Fig 1. KMZ data from the BT-5M buildings The first version of the data model at 1:1000 scale modelled
the topographic data in 2D and used contour lines and spot
heights to represent elevation. In 1999 a new data model
was designed to collect data in 2.5D, where each vertex is
defined by X, Y and Z coordinates. This new data model
allowed new exploitations related to the three dimensionality of
the information. In 2009 the third version of the data model
introduced changes related to the use of the new reference
system ETRS89, the adaption of the complete production
process to digital technology, the adoption of standards to
warrant the data and metadata interoperability and the
collection of new features. The new features were added to
obtain a digital terrain model (DTM) and a digital surface
model (DSM), mainly used in the rectification of true The existence of a production program for the BT-5M and the
experiences at the ICC in implementing generalization
workflows, allowed to start producing the first version of the
BT-25M using generalization processes in the year 2003. Comparing with previous ICC generalization experiences, the
workflow entailed two challenges: to obtain a topographic
database, not only a map, and to generalize 2.5D data instead
of 2D data (Figure 4). The complete coverage was finished in
2011 and, nowadays, the database is being updated
independently of the original database, because the updating
cycle planned for the BT-25M will be approximately 2 years,
much shorter than the 5 years updating cycle of the BT-5M. ribution has been peer-reviewed. The double-blind peer-review was conducted on the basis of the full paper. ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey city. Its semantic interoperability aligned to the formal
description of 3D features is one of its key advantages. city. Its semantic interoperability aligned to the formal
description of 3D features is one of its key advantages. logical upgrade from OS MasterMap Topography Layer. This
product will build on the 2D geometry and feature identities that
are already in place. Therefore the 3D geometry will heavily
rely upon the 2D data. Initially, the focus for product generation
will be on urban geographies. Ordnance Survey already supplies its detailed 2D data products
in GML format, and so CityGML would seem to be a logical
extension of this. Ordnance Survey already supplies its detailed 2D data products
in GML format, and so CityGML would seem to be a logical
extension of this. There are several ways in which OSGB could use CityGML. These include: There are several ways in which OSGB could use CityGML. These include: 3.2 Barriers to moving from a 2D- to a 3D-centric view
OSGB considers the challenges for the step from 2D to 3D to be
two-fold: technical and organisational barriers. •
As a basis for a 3D database model •
As a transfer format between internal systems e.g. from
data capture to database environment. 3.2.1
Technical barriers
Firstly, automated data capture techniques are preferred for
minimising time and cost. This is especially relevant when
scaling up to a city or national level. 3D data must also be
captured during the same revision cycles as the existing 2D
datasets in order to ensure synchronicity and interoperability. Since extensive use of photogrammetric techniques is being
made for 2D capture, there is an obvious incentive to use similar
techniques for 3D data capture to ensure this. •
As a transfer format for data supply to our customers While there are many advantages to using CityGML, OSGB
also perceive some challenges, in particular: While there are many advantages to using CityGML, OSGB
also perceive some challenges, in particular: •
The need to define an accepted national profile for
CityGML that is •
Fully supported by software vendors in order to exploit
the richness of the information •
Fully supported by software vendors in order to exploit the richness of the information Secondly, data capture trials have shown that there are issues
related to matching up of existing 2D footprints and 3D roof
shapes. 3.4 Future plans OSGB sees increased interest and expectation of 3D in a variety
of forms from our customers. Since existing customers rely
heavily on the 2D products, the main challenge is how to
transition to a truly 3D-enabled world while maintaining
continuity for customers who need support in evolving towards
3D. This also requires a balance between the growing
requirement for rich 3D information which is more than just
heighted geometry and the reluctance by customers to have to
manage complexity. Above all, 3D needs to be sustainable, both
in terms of production and maintenance feasibility, but also in
terms of acceptability and usability by customers for solving
real problems. Fig 6. The 3D roof level structures clearly do not match with the 2D
footprint 4.1 Large scale 3D mapping in The Netherlands The national 3D standard for large-scale topography mapping in
the Netherlands was established in 2011 during the first phase
of a pilot which aimed to push 3D developments in The
Netherlands by collaborating with all stakeholders The
Kadaster, the Ministry of Infrastructure and Environment, the
Netherlands Commission for Geodesy and Geo-information and
Geonovum initiated the 3D pilot (Stoter et al, 2011; Stoter et al,
2013). This ‘learned behaviour’ idea can also be extended to our
customers. For most of OS customers 3D is a very new concept
operationally. The experience of OS is that customers have a
generally low level of experience and understanding of the
implications and cost of using 3D data. 3. 3D MAPPINT AT ORDNANCE SURVEY: FROM 2D
TO 3D – EXPERIENCES AND CHALLENGES As the National Mapping Agency for Great Britain, Ordnance
Survey’s products include address data, digital terrain models
(DTMs), road networks, imagery and topographic mapping
(vector and raster), including the flagship topographic vector
mapping product, OS MasterMap® Topography Layer. With the exception of the DTM products, Ordnance Survey’s
portfolio is largely 2D-centric. However, many customers are
using 2D data to help make decisions that are inherently three-
dimensional in nature; for instance, planning and consulting on In 2012 OSGB commenced the automated capture of height
values for all urban buildings and structures. Customers see this
as an important evolutionary step. However, a major goal and
challenge is the development of a 3D topography product as a In 2012 OSGB commenced the automated capture of height
values for all urban buildings and structures. Customers see this
as an important evolutionary step. However, a major goal and
challenge is the development of a 3D topography product as a tion has been peer-reviewed. The double-blind peer-review was conducted on the basis of the full paper. 271 ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey The existing 2D capture specification is oriented
towards building function rather than roof form. Thought needs
to be employed on how the two representations can be brought
closer together. See Figure 6 for more details. Both of these carry the risk of resistance to the take-up of
CityGML across all involved parties. Therefore the wide
support of CityGML by data providers, software vendors as
wells as users needs to be addressed. Finally, continuing investment and technical development is
necessary in order to build end-to-end 3D production and
supply system. 4. 3D MAPPING IN THE NETHERLANDS In The Netherlands two initiatives are on-going for nation-wide
3D mapping. The first initiative focuses on large scale data
collected by local governments (mainly municipalities). The
second initiative is led by the Kadaster (who also holds the
mapping agencies) and aims at automatically generating a 3D
version of the 1:10k object oriented data set based on a smart
combination of the 2D topographic data and high-resolution
laser data. In The Netherlands two initiatives are on-going for nation-wide
3D mapping. The first initiative focuses on large scale data
collected by local governments (mainly municipalities). The
second initiative is led by the Kadaster (who also holds the
mapping agencies) and aims at automatically generating a 3D
version of the 1:10k object oriented data set based on a smart
combination of the 2D topographic data and high-resolution
laser data. Fig 6. The 3D roof level structures clearly do not match with the 2D
footprint 3.2.2
Organisational barriers Ordnance Survey has for over 200 years produced 2D data. This
has resulted in a certain level of learned behaviour and in a
legacy of products defined in terms of 2D data specifications
and representations. Consequently a process of review is
necessary internally in order to gain a better understanding of
3D in its end-to-end sense, from capture to customer. ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey Although IMGeo has been fully prepared for extension into 3D
by integration of the information model with the OGC
CityGML standard, 3D TOP10NL will serve other objectives. TOP10NL data is available nationwide while 2D IMGeo data
will only be generated in the coming years. In addition,
TOP10NL is less detailed than IMGeo and therefore better
suitable for fully automatic 3D object reconstruction since fewer
details are present (therefore less special cases). We assume that
the resulting dataset is sufficient for a nationwide dataset (i.e. acceptable performance for nationwide applications), and that it
can be further refined (for example with 3D IMGeo data) when
applied in future projects. facilitates extensions to 2.5D representations (i.e. as height
surfaces; equivalent to CityGML LOD0) and 3D (i.e. volumetric;
i.e. CityGML
LOD1,
LOD2
and
LOD3)
representations of the objects according to geometric and
semantic principles of CityGML. facilitates extensions to 2.5D representations (i.e. as height
surfaces; equivalent to CityGML LOD0) and 3D (i.e. volumetric;
i.e. CityGML
LOD1,
LOD2
and
LOD3)
representations of the objects according to geometric and
semantic principles of CityGML. Although the 3D standard is an important prerequisite for 3D
applications, the implementation of the 3D standard required
further agreements. Therefore, in the second phase of the 3D
Pilot (run from September 2011 till December 2012) the
established 3D community in The Netherlands developed ready-
to-use tools and best practice documents to support wide and
easy implementation of the 3D standard. The deliverables of 3D
Pilot NL, Phase II were published by the end of 2012 as an open
source toolkit (Geonovum, 2012; partly in English). This toolkit
consists of: Some tests on generating 3D TOP10NL fully automatically,
have been done in the past years, see Figure 8 (Oude Elberink et
al, 2012). Currently, the research results will be made further
ready for practice. The developments of 3D TOP10NL builds
on the insights obtained by the pilots on 3D IMGeo, since the
techniques to obtain LOD0 and LOD1 representations from a
combination of 2D data and high-resolution height points are
similar. 1. Implementation specifications for the national CityGML
extension that explains all technical details of the standard,
for all classes. 2. FME workbench that automatically generates 3D data
accordingly (LOD0 for all classes as well as LOD1 and
LOD2 for buildings and vegetation), based on a
combination of 2D IMGeo data and high-resolution
laserdata. ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey The FME Workbench incorporates the Open
Source tools developed in Oude Elberink (2010) Fig. 8 3D TOP10NL data 3. Example standard-compliant 3D data (see Figure 7) to help
newcomers to understand the national 3D standard and to
provide a source to experiment with 3D data. Fig. 8 3D TOP10NL data 4. 3D validator, an internationally unique, open source tool
that checks 3D geometries according to ISO19107 and
GML. Several challenges need further attention before 3D TOP10NL
can be put in production. These are: Several challenges need further attention before 3D TOP10NL
can be put in production. These are: 5. A document that describes a variety of update processes of
3D data sets based on tests with commercial software. 1. How to reconstruct the data for the whole of the Netherlands
given the huge amount of laserdata? How to upscale to larger
areas? Generate per partition and glue afterwards? 6. A Website that collects and portrays 3D showcases to
demonstrate the added value of 3D to policy makers and
newcomers in the field 2. How to maintain and update the 3D data: in a database
environment or regenerate complete areas in case of updates? How to assure that 3D TOP10NL is consistent with 2D
TOP10NL? 7. A strategy for aligning CityGML to data generated in
Building Information Models. 3. How to deliver the data? As files (GML? Shape?) or in a
Webviewer (WebGL? KML?)? Is it possible to make a dataset
of the whole country available at once or should we deliver data
sets of specific areas on demand? Fig. 7: 3D CityGML compliant data 5. 3D MAPPING IN SWITZERLAND The Swiss Federal Office of Topography (swisstopo) offers a
wide range of products ranging from traditional paper maps to
digital multimedia products. Since 2008 swisstopo has been
developing and updating the Topographic Landscape Model
(TLM). TLM is the basis landscape model for Switzerland. It is a
seamless GIS containing 3D primary geometry without
generalisation with an accuracy of better than 1meter in x, y &
z. TLM consists of 10 themes, which in turn contain several
independent datasets previously maintained separately such as
boundaries, names and terrain (see Figure 9). It also has a
revised extended data model to meet the needs of reference
partners such as road and water authorities. Fig. 7: 3D CityGML compliant data 3.3 CityGML for Ordnance Survey Feedback has shown that customers see Ordnance Survey as a
prime candidate for promoting the use of 3D data through
internationally recognised standards. CityGML fits this bill very
neatly. The OGC standard CityGML is one of the few (if not
only) internationally recognised standards for 3D geo-
information. It has a rich semantic description of terms and
allows for the modelling of most of the objects found within a The new 3D standard is embedded in an information model,
called “Information Model Geography”, i.e. IMGeo, which is
modelled as an Application Domain Extension of the OGC 3D
standard CityGML (Van den Brink 2011; 2013). IMGeo
contains object definitions for large-scale representations of
roads, water, land use/land cover, bridges, tunnels etc. and
prescribes 2D point, curve or surface geometry for all objects. As the new version of IMGeo is modelled as CityGML ADE, it ribution has been peer-reviewed. The double-blind peer-review was conducted on the basis of the full paper. 272 ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey 4.2 3D TOP10NL The second initiative for national 3D mapping is the project 3D
TOP10NL of the Dutch Kadaster (Oude Elberink et al, 2013). The project aims at automatically reconstructing a 3D version of
the object oriented 1:10k data set in a fully automatic manner
and delivering this 3D data as open data to the community. This
is done in collaboration with the universities of Twente and
Delft. The automated reconstruction also uses the Open Source
tools developed in Oude Elberink (2010). The TLM describes around 10 million natural and artificial
landscape features, complete with their position and shape, the landscape features, complete with their position and shape, the
corresponding landscape feature type and many other attributes: landscape features, complete with their position and shape, the
corresponding landscape feature type and many other attributes: landscape features, complete with their position and shape, the
corresponding landscape feature type and many other attributes: corresponding landscape feature type and many other attributes: •
Roads and Tracks (Road and path network, cycling paths
and footpaths) •
Public Transportation (Railway tracks and other rail
systems, shipping routes and public transport stops) tion has been peer-reviewed. The double-blind peer-review was conducted on the basis of the full paper. 273 ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey •
Buildings (Buildings, walls, dams, constructions relating
to transport, communication, energy, sport and leisure) The existing data was migrated into the TLM as a basis, mainly
due to the rich information in attributes and topology
maintained over the years. The data is then updated based on an
update cycle of 6 years and improvements and additions are
made to the geometries and attributes. •
Areas (Plots with special land usage (transport, habitat,
industry etc.)) •
Land cover (Ground coverage independent from land
usage) Parallel to the six-year update process, an annual update is
carried out through the integration of data from so-called
reference partners for selected topics. Initially, this annual
update is implemented mainly for the administrative boundaries,
buildings and road links. The official cadastral survey plays an
important role as a reference partner in this process. 5.1 Production and updating The systematic improvement of the data will mean that a
Version 2.0 of the various products can be made available
alongside Version 1.0. Version 2.0 will be made available sector
by sector in accordance with the update blocks. It will contain
significantly more landscape features, offer a higher geometric
accuracy for all data and will therefore fulfil the requirements
with regard to TLM quality. The ever-increasing demand for better, more up-to-date diverse
digital data has meant that the rapid updating of swisstopo
products has taken on a high importance. With the new
established
production
process
for
TLM
an
updating
infrastructure for a central data set from which all products
could be then produced was established. Reference partners (such as the cadastre, road and rail
authorities among others) deliver their up-to-date data for rapid
insertion into the TLM in order to maintain top up-to-dateness. The updating process itself is performed in a modern database
based system comprising of hardware and software for the
capture, editing, management, storage and distribution of the
TLM. The associated Digital Terrain Model will also be edited
using this infrastructure. The TLM is also used as the basis at swisstopo to create the
national (2D) maps to a scale of 1:25k, 1:50k and 1:100k. 4.2 3D TOP10NL The
nomenclature for updating names are also integrated from the
official cadastral survey. •
Hydrography (Watercourses and lake contours) •
Single Point Objects (Selection of prominent landscape
features which aid orientation (crossings, sources, caves)) •
Names (Various names (mountains, areas, towns)) •
Names (Various names (mountains, areas, towns))
•
Terrain Names (Various names (mountains, areas, towns))
•
Terrain Fig. 9: Representation of swissTLM3D 6.1 Early steps
G
did 6.1 Early steps
IGN France did quite a bit of 3D city modelling from the late
70s until the early 90s with the TRAPU product line (Egeles,
1989), based on an in house developed analytic stereoplotting
and 3D (actually 2.5D) data management system. Several city
LOD2 models were produced in this early period (Figure 10). This was however a quite expensive product in such times,
limited to main city centres, not maintained over time, difficult
to use with available GISs of the 80s, and finally discontinued
when a new nationwide product was launched. IGN France did quite a bit of 3D city modelling from the late
70s until the early 90s with the TRAPU product line (Egeles,
1989), based on an in house developed analytic stereoplotting
and 3D (actually 2.5D) data management system. Several city
LOD2 models were produced in this early period (Figure 10). There is one single Corporate Editor enabling staff trained on
one part of the production line to easily move to another part of
the production line without any technical retraining being
required. This was however a quite expensive product in such times,
limited to main city centres, not maintained over time, difficult
to use with available GISs of the 80s, and finally discontinued
when a new nationwide product was launched. 5.2 3D Products 5.2 3D Products
Various 3D data sets extracted from the TLM are available
since autumn 2010. They comprise the entire territory in
reduced form - measured according to the final requirements of
the TLM. To be more precise, the number of landscape features
on offer is reduced compared with the TLM landscape feature
catalogue and the geometric accuracy in the areas where the
systematic improvement has not yet been made corresponds to
that of data set VECTOR25. This is an initial version (1.0) of
the products extracted from the TLM. Terrain
i i Existing data is used as a basis: For areas less than 2000 m
above sea level data is used from the laser point survey flights
2000 – 2008. For areas more than 2000 m above sea level
DHM25 was used. The laser point data is updated in accordance with the TLM
update cycle of 6 years (photogrammetric restitution). During
this process, the dots are deleted and new points, breaklines and
areas are set. For example, incorrect points in areas around
bridges are removed and an enclosing breakline (enclosure) is
implemented for lakes. In order to meet the requirements of the TLM with regard to
accuracy in areas lying more than 2000 m above sea level, the
DHM25 was replaced in 2010-2012 with more accurate data
gained via autocorrelation of aerial photographs. This newly
generated data will also be updated in the TLM update cycle. Fig. 9: Representation of swissTLM3D The content of the TLM and the landscape feature definitions
were developed in cooperation with various professional
institutions from both the federal government and the cantons. Geometries and a wide range of attributes are provided for all
landscape features. All objects have x, y and z coordinates
which also means that objects in TLM maintain consistency
with DTM-TLM, for example 3D roads should lie on the DTM. TLM is not a product, but rather will be the basis for a wide
variety of products including the derived 1:25k cartographic
base maps. It serves also the bases for Switzerland’s National
Spatial Data Infrastructure (NSDI). 6.3 Closer to full 3D In parallel with the completion of the RGE and BD Topo
national coverage, IGN launched in late 2007 an innovation
project which consisted in industrializing research results
pertaining to 3D data automated extraction, and chose from the
beginning to primarily rely upon CityGML and a pivot format
in this venture. Fig 11 - A view of BD Topo This innovation project was completed by the end of 2009, and
resulted in au automated workflow currently used for two
production activities: •
Creation of high resolution DTMs from dedicated aerial
coverages in rural areas (25cm ground pixel size, typically
4,000 to 10,000 km²) •
Creation of 3D city models (LOD 2 level) with texturation
extracted from images, from higher resolution and denser
aerial coverages: that is to say the Bati3D product (Figure
13) Fig 11 - A view of BD Topo Fig 11 - A view of BD Topo Each feature comes with a Zmin and Zmax value, the Zmin
value for a building being taken at the gutter level. The BD
Topo provides a description for over 40 millions buildings
across the country. Fig 13 – Marseille old downtown Bati3D Several issues with usage of such data were assessed early on
(e.g., see Certu 1999; 2002): data volume and heavy load on
computers, limited functionalities of GIS software packages to
properly handle 3D data, staff training, but also content issues:
buildings as topographical features do not always match
requirements (which may include an interest in ownership
divisions, so that BD Topo buildings are progressively merged
with cadastral buildings since a few years), visualization of
coloured boxes is not always sufficient for communication etc. An interesting development which followed in the past years is
a co-edited product, in partnership with an SME (Archivideo,
recently
acquired
by
Dassault
Systèmes):
Archivideo
reprocessed BD Topo buildings to add synthetic textures to
roofs and facades, based on the geographic area and building
characteristics (Figure 12). Fig 13 – Marseille old downtown Bati3D Bati3D is much cheaper to produce than most pre-existing 3D
products, in that the bulk of the cost lies in the dedicated aerial
coverage, thanks to the research results which were input into
the production system (automated building and tree 3D
capture). 6.2 A first nationwide coverage The next step, which marked the discontinuation of TRAPU
was the BD Topo product, launched in 1994 (Figure 11). This
product provides a LOD1 model of buildings as part of a 2.5D
geographic information framework, the Référentiel à grande
échelle (RGE, for large scale reference framework). The first
French department to be covered was the Vaucluse in 1994,
while the nationwide coverage was completed in 2007 only. Fig 12– Territoire3D© Natural and artificial landscape features Compared with the formerly existing geodata (VECTOR25,
SwissNames, etc.) the TLM offers higher geometric accuracy,
but is also more extensive and up-to-date. ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
II
/W
ISPRS
h
DG
I f
C
f
WG II/
W
k h
N
b
I including client/server architectures, so as to allow a growth of
3D data usage. including client/server architectures, so as to allow a growth of
3D data usage. Fig 10 - A TRAPU city model Fig 12– Territoire3D© Fig 10 - A TRAPU city model ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey usage entails a need for much higher modelling of specific
areas (e.g., a new streetcar project impact study will
induce a need for a finer modelling of buildings in the
considered streets, both in terms of 3D modelling of
features that appear in these street and in terms of images
used to texture the facades) being said, many users still require data to be provided in a
different data model, because of limited support on the side of
software vendors. IGN-F will keep supporting this standard, and
contributing to its evolution, in the context of the anticipated
development of LOD2 (or higher) data production at the
national level. 7.1 Conclusions
h From the approaches described in this paper, several
conclusions can be drawn. Firstly, 3D data is more and more available but still suffers from
a low level of usage (mainly visualisation). In addition
standards and formats based on CityGML have been stabilised
although software support is still in the early stage. We also
observe low level of European harmonisation. A cross-
European profile for CityGML would be beneficial for several
reasons. It would raise the level of adoption of CityGML 3D
data, and hence raise usage level; it would push software
developers to provide better support for the standard; it would
be an important step towards European-wide harmonized data
and finally, a better support of the standard would lower the
costs needed for data conversion. Fig 14 – Marseille old downtown Caro3D Another conclusions that can be drawn from the approaches
described in this paper, is that for 3D mapping at the national
level, two approaches can be distinguished. Fig 14 – Marseille old downtown Caro3D It turns out to be that this later product better meets the
requirements of users, but still mostly requirements mostly
focused on visualisation of urban areas (in the example above,
the whole city of Marseille is available as Bati3D data, while a
more limited area in the city centre is provided as Carto3D
data). The first approach is followed by ICC, IGN and swisstopo and
implements 3D mapping at the most fundamental level. That is,
the 3D data is considered as the prime source of data and 2D
products and services are implemented as derivation of the 3D
data. This approach is most sustainable for the future. The second approach (still) considers the 2D data as prime
source of data and extends these data into the third dimension to
obtain 3D products and services. This approach is followed by
The Netherlands and OSGB. The advantages of this approach
are that the transition from 2D to 3D is less drastic, the
consistency between 2D and 3D is assured and that the
semantics acquired in 2D can serve as input for automatic
reconstruction procedures. The disadvantage is that 3D
information is derived from 2D and therefore the 3D mapping is
highly driven by 2D traditions and thinking. In addition, the
derivation process of 3D from 2D is much more complex than
deriving 2D from 3D as in the first approach. 7. CONCLUSIONS AND FURTHER WORK This activity led to a partnership with another SME, a both
technological and editorial partnership: This activity led to a partnership with another SME, a both
technological and editorial partnership: This paper presents the approaches of national 3D mapping in
five states and countries. These approaches show that 3D
mapping activities are no longer limited to project-based, ad hoc
and prototype applications. Instead 3D is considered as an
important
aspect
of
existing
topographic
information
workflows. •
Co-development of software components, starting with a
generic 3D I/O library which can cope with most 3D data
formats (CityGML, 3DS, KML, Collada), with IGN in
charge of CityGML I/O •
Development, here again, of a co-edited enhanced
product: the Carto3D city model line, characterised by
some level of additional editing and, in the first place, a
texturation of facades which is based on images captured
by a mobile mapping vehicle (Figure 14) 6.4 Pending issues as for true 3D data models As mentioned above, automated processes are good at creating
the bulk of a city model, but failures are costly to identify, and
corrective editing can be painstakingly long. Further work on
automated identification of failures, and on assisted editing
tools are therefore underway, to provide better quality models at
an affordable cost. Compression of textures is another issue, since texture atlases
can represent a too heavy load when loading a CityGML
dataset, this may be linked with both limitation of the chosen
encoding, and of available tools on the user side. Last but not least, comes the issue of 3D data model
maintenance: How to appropriately update such data sets at an
affordable cost? There is a whole range of R&D matters to
consider, from aerial coverage options (including UAVs or
ultralight aviation) to exploiting images captured from the
ground by light cameras (e.g., smartphone cameras). As long as
3D data is used only for short term communication projects,
data maintenance is not an issue, but will turn to be crucial
when more elaborated usages develop. Another important conclusion is that, despite maturing 3D
technologies, still these are not fully exploited in practice. This
has several reasons. Firstly, 3D is outside the expert and knowledge domain of many
organizations. The unfamiliarity with 3D causes 3D to be
considered as something complicated. This limits actual
implementations
beyond
prototypes
and
project-based
initiatives. Secondly, there is a big question of who will be the users of 3D
and, with that, what will be the user requirements and what is
the business case for the needed investments. While these are
valid questions for an organisation when considering 3D
investments, these questions cannot easily be answered. For the 6.3 Closer to full 3D However two lessons seem to emerge: With the addition of the BD Ortho IGN orthoimagery wrapped
onto the national DTM, and of 3D synthetic vegetation, such a
product provides a much more attractive and realistic
visualisation of a geographical area, at low additional cost. It is
therefore increasingly used for impact studies, in the first place
by local authorities. •
Once again issues with the software packages available to
users, particularly in terms of data load (as for a big city
such as Marseille with a large number of buildings, true
textures are a challenge for many visualisation packages,
this was exemplified in many instances, including the
OGC 3DPIE 2011 testbed, see OGC 2012b) This development is interesting on at least two grounds: Firstly
because it shows that intermediate, but not ‘fully real’ products
can meet quite a range of requirements, short of more
elaborated and complex 3D products secondly, this example
shows that tools available on the user side are maturing, •
Some usage requirements are not met with such a product,
either because automated process failures (wrongly
depicted roofs) are not acceptable, or because the intended has been peer-reviewed. The double-blind peer-review was conducted on the basis of the full paper. 275 ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey 7.2 Further work Now 3D techniques have matured and OGC has established a
standard for 3D data which is well stabilised, in many countries
it is no longer a question if countrywide 3D mapping should be
realised, but “when” and “how”. Czerwinski, A., T. Kolbe, L. Plümer, E. Stöcker-Meier (2006). Interoperability
and
accuracy
requirements
for
EU
environmental
noise
mapping,
proceedings,
InterCarto,
InterGIS, Berlin 2006. From the findings in this paper several issues can be identified
that require further (research) attention to progress in the
domain of 3D (no ranking): Czerwinski, A., Sandmann, S., Stšcker-Meier, E. and Pluemer,
L. (2007). "Sustainable SDI for EU noise mapping in NRW –
best practice for INSPIRE." International Journal of Spatial
Data Infrastructures Research 2: 90-111. •
More insights in real customer needs of 3D data sets are
required (i.e. what topics are relevant and to what extent). These needs should be formulated in terms of what
problems 3D can solve. This will stimulate other use of
3D data than visualisation. Geonovum, 2013, EuroSDR/OGC Workshop CityGML in
National Mapping,
http://www.geonovum.nl/content/programme-workshop-
national-mapping •
More research is needed on how to fuse data, combining
existing data into 3D databases can create problems, i.e. integrating existing non-3D datasets (addresses,
boundaries, etc.) with 3D datasets. Geonovum 2012, www.geonovum.nl/3d/toolkit Geonovum 2012, www.geonovum.nl/3d/toolkit •
Consistency between 2D and 3D needs further attention. For most mapping authorities 3D geo-information
originates from their legacy datasets in 2D. In using this
as a basis many associated issues emerge. Egels, Y. (1989) TRAPU, un outil de saisie et de visualisation
du
tissu
urbain. Metropolis,
pages
21-27
http://archives.cerma.archi.fr/sig/pdf/paparoditis.pdf •
Maintenance and incremental updating of sustainable 3D
datasets (sustainability vs. visualization) requires further
attention. iScope, 2013, http://www.iscopeproject.net/iscopeNew/ OGC (2012). OpenGIS® City Geography Markup Language
(CityGML)
Encoding
Standard,
version
2.0,
www.opengeospatial.org/standards/citygml •
The need of national and European policies on spatial
information to stimulate the use of available 3D
technologies. OGC (2012b) OGC 3D Portrayal Interoperability Experiment
Final Report, 27/08/2012 For CityGML, the following recommendations can be
formulated for future directions. Oude Elberink, Sander, Stoter, Jantien, Ledoux, Hugo,
Commandeur, Tom, 2013, Generation and Dissemination of a Oude Elberink, Sander, Stoter, Jantien, Ledoux, Hugo,
Commandeur, Tom, 2013, Generation and Dissemination of a
National Virtual 3D City and Landscape Model for the
Netherlands
Photogrammetric engineering and remote
sensing
79 2
147-158 2013
American
Society for Photogrammetry and Remote Sensing Firstly, CityGML needs further support in software systems. ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey digitising of maps resulting in 2D databases, the benefits were
clear: initially production lines were improved and later the
digitised point, lines and areas could be upgraded to 2D
information needed in spatial analyses. In contrast, 3D does not
contribute to the improvement of an existing process. Instead
3D makes new applications possible and therefore it is much
easier to identify the costs to acquire 3D than the benefits of
having 3D information available. Many benefits will only
become clear after having the 3D information available for large
areas. 8. REFERENCES Becker T., C. Nagel, T.H. Kolbe (2010). Integrated 3D
modeling of multi-utility networks and their interdependencies
for critical infrastructure analysis, 5th International 3D GeoInfo
Conference, November 2010, Berlin Becker, Thomas, Claus Nagel, and Thomas H. Kolbe (2013). "Semantic 3D Modeling of Multi-Utility Networks in Cities for
Analysis and 3D Visualization." Progress and New Trends in
3D Geoinformation Sciences. Springer Berlin Heidelberg, 2013. 41-62. Finally, a reluctance to make the step towards 3D mapping can
be explained by the lack of a widely adopted 3D standard. While CityGML is a good candidate, software support is
limited. In addition, more agreements need to be made at
international level on how to implement the standard in specific
applications or domains. The 3D profile of the INSPIRE Data
Specifications for Buildings, which is based on CityGML, is a
good example. We need more of such implementation profiles
to offer a solid investment foundation for both organisations and
the market. Certu 1999, Evaluation BD Topo® - Synthèse – CERTU, La 3e
dimension géographique – Utilisation des modèles numériques
de terrain, illustrée par la BD Alti® de l’IGN Certu,
2002,
Evaluation
BD
Topo®,
online:
http://lara.inist.fr/bitstream/handle/2332/1061/CERTU_bdtopo. pdf%3Fsequence%3D4 CityGML, 2013, Application Domain Extensions, [online]
www.citygmlwiki.org/index.php/CityGML-ADEs 6.5 CityGML and IGN-F 3D data products y
p
Insofar CityGML has proved to be up to the task, robust enough
to provide a good basis for 3D data exchanges. Only ongoing
research suggests that further developments are needed. This ribution has been peer-reviewed. The double-blind peer-review was conducted on the basis of the full paper. 276 7.2 Further work This includes implementation of standard products and
development of open source tools to support operations. In
addition, as a generic standard CityGML needs further
agreements defined in profiles. Therefore European CityGML
profiles (as the INSPIRE Building profile) need to be defined to
harmonise 3D needs and standardise 3D implementations at
international level. Relationships with INSPIRE portfolio
beyond buildings need to be investigated. ,
,
,
National Virtual 3D City and Landscape Model for the
Netherlands
Photogrammetric engineering and remote
sensing
79 2
147-158 2013
American
Society for Photogrammetry and Remote Sensing Oude Elberink, S.J. (2010) Acquisition of 3D topography :
automated 3D road and building reconstruction using airborne
laser scanner data and topographic maps. Enschede, University
of Twente Faculty of Geo-Information and Earth Observation
(ITC), 2010. ITC Dissertation 167, ISBN: 978-90-6164-288-6 Finally, interfaces with other standards are needed to exchange
3D data with other domains. For example the link with BIM-
IFC (at least in practical uses) needs to be further investigated. contribution has been peer-reviewed. The double-blind peer-review was conducted on the basis of the full paper. 277 his contribution has been peer-reviewed. The double-blind peer-review was conducted on the basis of the full paper. 27 ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey his contribution has been peer-reviewed. The double-blind peer-review was conducted on the basis of the full paper. 27 This contribution has been peer-reviewed. The double-blind peer-review was conducted on the basis of the full paper. 278 ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences,
Volume II-2/W1, ISPRS 8th 3DGeoInfo Conference & WG II/2 Workshop, 27 – 29 November 2013, Istanbul, Turkey Stoter, J.E., Vosselman, G, Goos, J, S. Zlatanova, E. Verbree,
R.Klooster, M. Reuvers, 2011, Towards a National 3D Spatial
Data Infrastructure: Case of The Netherlands. – PFG –
Photogrammetrie, Fernerkundung, Geoinformation 2011(6):
405–420. Stoter, J.E., L. van den brink, J. Beetz, H. Ledoux, M. Reuvers,
R. Klooster, P. Janssen, F. Penninga, G. Vosselman, 2013,
Establishing and implementing a national 3D standard, Journal
for Photogrammetry and Geoinformation (PFG), In press Van den Brink, L., Jantien Stoter2, Sisi Zlatanova, 2013, UML-
Based Approach to Developing a CityGML Application
Domain Extension, Transactions in GIS, Article first published
online: 22 May 2013 DOI: 10.1111/tgis.12026 Van Den Brink, Linda, Stoter, Jantien en Zlatanova, Sisi, 2012,
Establishing a national standard for 3D topographic data
compliant
to
CityGML,
in:
International
Journal
of
Geographical Information Science, in press. URL:
http://www.tandfonline.com/doi/abs/10.1080/13658816.2012.66
7105
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Social network inheritance and differentiation in wild baboons
|
Royal Society open science
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cc-by
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Alecia Carter1 1Anthropology Department, University College London, London WC1E 6BT, UK
2Zoological Society of London, Institute of Zoology, London NW1 4RY, UK
3Institut des Sciences de l’Evolution de Montpellier, CNRS, University of Montpellier, 34095
Montpellier Cedex 5, France 1Anthropology Department, University College London, London WC1E 6BT, UK VR, 0000-0002-9992-8524; AC, 0000-0001-5550-9312 VR, 0000-0002-9992-8524; AC, 0000-0001-5550-9312 Immatures’
social
development
may
be
fundamental
to
understand important biological processes, such as social
information transmission through groups, that can vary with
age and sex. Our aim was to determine how social networks
change with age and differ between sexes in wild immature
baboons, group-living primates that readily learn socially. Our results show that immature baboons inherited their
mothers’ networks and differentiated from them as they
aged, increasing their association with partners of similar age
and the same sex. Males were less bonded to their matriline
and became more peripheral with age compared to females. Our results may pave the way to further studies testing a
new hypothetical framework: in female-philopatric societies,
social information transmission may be constrained at the
matrilineal level by age- and sex-driven social clustering. Vittoria Roatti1,2, Guy Cowlishaw2, Elise Huchard3 and
Alecia Carter1 Vittoria Roatti1,2, Guy Cowlishaw2, Elise Huchard3 and
Alecia Carter1 Social network inheritance
and differentiation in wild
baboons royalsocietypublishing.org/journal/rsos Research
Cite this article: Roatti V, Cowlishaw G,
Huchard E, Carter A. 2023 Social network
inheritance and differentiation in wild baboons. R. Soc. Open Sci. 10: 230219. https://doi.org/10.1098/rsos.230219 Research
Cite this article: Roatti V, Cowlishaw G,
Huchard E, Carter A. 2023 Social network
inheritance and differentiation in wild baboons. R. Soc. Open Sci. 10: 230219. https://doi.org/10.1098/rsos.230219 1. Introduction For group-living species, social connections can affect fitness by
influencing health and survival [1]. For example, strong social
bonds enhance longevity, and both adult and offspring survival in
wild baboons (Papio spp.) [2–6], killer whales (Orcinus orca) [7] and
spotted hyenas (Crocuta crocuta) [8]. Yet despite the importance of
an individuals’ social connectedness in many social species (but
see eastern grey kangaroos Macropus giganteus [9]), the mechanism
driving this relationship is unclear. An individual’s position in the
social network could affect a range of biological processes
influencing fitness, such as the acquisition of information from
knowledgeable group members (i.e. social learning) [10]. Social
connections may be particularly important for immature age
classes, who could benefit from social information more than
adults due to their inexperience of the environment [11]. Author for correspondence:
Vittoria Roatti
e-mail: vittoria.roatti.19@ucl.ac.uk Electronic supplementary material is available
online at https://doi.org/10.6084/m9.figshare.c. 6631226. © 2023 The Authors. Published by the Royal Society under the terms of the Creative
Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits
unrestricted use, provided the original author and source are credited. Social learning can be adaptive because it allows access to vital information without expending the
time and energy needed for asocial learning [11,12] and can lead to variations in fitness [13,14]. For
instance, older African elephant (Loxodonta africana) matriarchs have relatively greater conspecific call
discrimination abilities, which can affect the behavioural responses of their family group and,
consequently, enhance their reproductive success [13]. Social learning is closely tied to social
connections: the number and characteristics of individuals’ connections set an upper limit on social
learning opportunities [15–18] and often predict information transmission. For example, socially central
chacma baboons (P. ursinus) have better access to social information (e.g. about the location of food)
because they have a larger number of social partners to provide that information compared to
peripheral ones [19]. Recent studies have quantified social connections using social network analysis,
which can account simultaneously for all the interactions occurring in a group, determining both an
individual’s direct and indirect connections in the network [20]. royalsocietypublishing.org/journal/r
2 2 Social connections can be characterized through proximity (i.e. individuals are associating if they share
similar spatial positions) or interactions (i.e. individuals are associating if they are observed interacting) [21]. Proximity may be important because individuals could acquire information more easily from others that are
in visual contact. 1. Introduction For example, spatial associations predict the discovery of foraging tasks by threespine
sticklebacks (Gasterosteous aculeatus) [22] and experimental food patches in sympatric tits (Family
Paridae) [23]. Alternatively, or concurrently, individuals may be more attentive to those with whom they
share strong bonds. For instance, ring-tailed lemurs (Lemur catta) [24] and ravens (Corvus corax) [25] who
are more central in an affiliation network are more likely to acquire information about the solution of a
foraging task. Although our knowledge of social networks has increased dramatically in recent years
[26], few studies have focused specifically on immature age classes, and little is known about how their
social networks, and social learning opportunities, develop with age [15]. os
R. Soc. Open Sci. 10: 230219 Understanding how social networks change during immatures’ development may give important
insights into which factors constrain or enhance group-level social learning. Immature individuals may
both be more socially active than adults and rely more on social learning than asocial learning compared
to adults [11,15]. A greater tendency to seek social opportunities was reported in juvenile mantled
howler monkeys (Alouatta palliata) [27], rhesus macaques (Macaca mulatta) [28] and bottlenose dolphins
(Tursiops aduncus) [29]. Because immature individuals may have social networks differentiated from
those of adults (e.g. in Colombian spider monkeys Ateles fusciceps rufiventris [30]), they can play an
important role in the group’s network, to the point that their analytical exclusion changes the group’s
structure, as reported for olive baboons (P. anubis) [31]. In addition, juvenile individuals from various
taxa show greater evidence of social learning than adults (e.g. chacma baboons [32], meerkats (Suricata
suricatta) [33] and Australian skinks (Eulamprus quoyii) [34]). Despite the importance of immature
animals’ social connections for a group’s ability to disseminate information [35], few studies have
characterized changes in immature individuals’ social bonds through time, in the wild, and in both sexes. In this study, we draw on previous knowledge of primate social development to analyse immatures’
social networks in a wild population of a female-philopatric primate species, the chacma baboon. Primates offer wider opportunities to explore immatures’ social connections compared to other taxa
because they experience a relatively long period of juvenescence [36]. Because taxa that have a later
age at first reproduction have been linked with higher social learning abilities [37], studying primate
immatures’ social networks may set the bases for future studies on how these connections enhance or
constrain social information transmission. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 Social bonds could also be affected by age. Immatures tend to spend less time with their mothers and
socialize with same-sex age peers as they grow older, thus expanding and differentiating their social
network from the maternal one [43,46]. This social exploration phase may lead to an increase in the
number of partners immatures associate with [28,47], which may also derive from the greater
tolerance that primates show towards immature individuals compared to adults [48,49]. Association
with age peers (baboons [50]; macaques [51]; vervet monkeys [52]), same-sex individuals (baboons
[49]; macaques [53]) or individuals who are matched by both age and sex (baboons [40]; blue
monkeys Cercopithecus mitis [54]; diademed sifakas Propithecus diadema [55]) seems to be common to
social primates when immature, although some exceptions occur (e.g. a decrease in association with
peers [56]; or association with the opposite sex [55]). Finally, the development of social connections could depend on sex. In general, immature females
spend more time with the mother and maternal relatives during development than males [57,58] (but
see [59]), both in female-philopatric species (baboons [49]; geladas Thercopithecus gelada [60]; macaques
[61]; vervet monkeys [62]) and male-philopatric species (chimpanzees [63]; spider monkeys A. geoffroyi
[64]). Daughters’ social connections may also resemble maternal ones more than sons’ (macaques
[53]). A species’ social system could drive sex differences in integration in the social network, in terms
of both the number and strength of social bonds and the diversity of social partners [57,58]. In female-philopatric species, juvenile females typically dedicate more time to grooming and associate
with more social partners than males (baboons [65]; macaques [66]; blue monkeys [54]; vervet
monkeys [42]; but see [60,67]). Despite a general tendency to associate with same-sex peers with age,
this becomes more marked in males than females. In fact, females tend to associate with a wider
variety of partners, thus remaining more central in the group’s network, while males tend to become
more peripheral prior to dispersal by preferring male peers (baboons [68]; macaques [69]; blue
monkeys [54]). Opposite tendencies were reported in male-philopatric species, in which grooming is
equally common in males as in females and males have a wider social network than females
(chimpanzees [70]; spider monkeys [64]). R. Soc. Open Sci. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 10: 230219 Although most studies agree on how social networks change with age and sex, they largely focus on
cross-sectional comparisons [54,69], rather than following the same cohort of individuals longitudinally
(but see [42,66]). Compared to cross-sectional comparisons, longitudinal studies have the potential to
identify developmental trends. In addition, many studies concern food-provisioned populations (e.g. at
Cayo Santiago [66]), with a few exceptions targeting wild populations [42,54]. Wild populations may
better represent the constraints faced by immature individuals in establishing social bonds. This is
because primates trade grooming for tolerance during foraging, meaning that social bonds can be
affected by competition for food [71,72], which may be different in food-provisioned populations
compared to wild populations [73]. Finally, the transmission of maternal networks in female-philopatric
species has been explored mainly in daughters [41,42] because of the stronger bond shared with the
mother compared to sons [74]. However, males’ integration in the maternal network is important to
better understand possible constraints on information transmission across the group. For example, given
that males tend to learn asocially, while females rely more on social learning [75], social information flow
may be constrained if individuals who tend to produce information (males) have poor connections with
the ones who tend to acquire it (females) [76]. For male primates, who are generally larger than females,
it may be relatively more advantageous to explore, for example, a new, potentially higher pay-off, food
patch or item because they can supplant a smaller individual, acquiring its resource, if unsuccessful [75]. Instead, females, who bear relatively higher parental investment costs [77], may be less willing to forego
known foraging opportunities for an uncertain reward [19]. With this study, we aimed to contribute to the understanding of how animal social networks, and
thus opportunities for social learning, change with age throughout the immature period, and how
they differ between the sexes. We tested two main hypotheses about the development of social
connections in wild immature baboons, using social network analysis in the context of a longitudinal
study. Our first hypothesis (H1) was that immatures would ‘inherit’ their mothers’ social networks at
birth [45] and that these would differentiate during development [51]. We had three predictions for
this hypothesis. P1a: as reported in other female-philopatric primate species [58], the time spent with
the mother will decrease with age and this decrease will be greater for males than for females. 1. Introduction Baboons are ideal models for this study because they live in
large social groups [38], readily learn socially [19], and information transmission is predicted by both
the proximity and grooming social networks [19]. Chacma baboons live in multi-male–multi-female
societies [38]. Females are philopatric and form matrilines (i.e. social units including a mother and her
offspring) within a linear and stable hierarchy, with offspring inheriting their mother’s rank [39]. Males disperse at maturity (around 8 years old) [19]. Baboons learn socially about the location of new
food patches [19] and may acquire information on what to eat by observing others while foraging
and/or by inspecting the same food items others are feeding upon [16]. Juveniles’ networks may be shaped by the process of social inheritance and two individual traits: age
and sex. Social inheritance suggests that offspring inherit their mother’s social connections in a similar
manner to how they inherit her dominance rank (baboons [40]; macaques [41]; vervet monkeys
Chlorocebus pygerythrus [42]). For example, young infant rhesus macaques who do not move
independently from their mother share her network [43], which can last up to seven months of age. When older, infants’ social networks may remain linked to that of their mother even after they start to
move independently. Such social preferences may be passed passively through social learning (i.e. observation of maternal social preferences) and/or familiarity (i.e. exposure to maternal social partners) [41], or through active maternal intervention (i.e. encouraging relationships with certain individuals) [44]. A modelling approach confirmed that a purely genetic explanation is not necessary [45]. [41], or through active maternal intervention (i.e. encouraging relationships with certain individuals) [44]. A modelling approach confirmed that a purely genetic explanation is not necessary [45]. 3 2.1. Study site and species The study was carried out at the Tsaobis Baboon Project [80], a long-term (since 2000), individual-based
study located on the edge of the Namib Desert, in Namibia (22°220S, 15°440E). The study area includes the
large ephemeral Swakop River and its tributaries, with dense patches of riparian woodland surrounded
by rocky hills, where vegetation is scarce and limited to dwarf trees and small shrubs (see [81] for a full
description of the area). Adult female chacma baboons give birth to an infant approx. every 2 years and infants are weaned
around 12–18 months of age at Tsaobis [82]. For the purposes of this study, individuals less than or equal
to 5 years old were considered immatures. We chose this threshold because, in most classifications, males
are considered juveniles until 5 years old, entering sub-adulthood (before reaching sexual maturity) after
5 years old; females are considered juveniles until 4–5 years old and conceive their first infant around
6 years old [83–86]. Immature individuals less than or equal to 12 months old are referred to as
infants, while those greater than 12 months old are considered juveniles [46]. Downloaded from https://royalsocietypubli royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 Our second hypothesis (H2) was that an individual’s social network characteristics would change
during development in terms of both immatures’ integration in the troop’s network and the type of
social partners with whom immatures associate [58]. We had three predictions for this hypothesis. P2a: the association with different social partners will increase with age as immatures widen their
social network beyond the maternal network [47]. As observed in other female-philopatric primate
species [58], this increase will be relatively lower in males, who will also have fewer social partners
than females [42]. P2b: if immatures inherit their mothers’ networks (P1b and P1c), their positions in
the social network will remain stable throughout development for females, while males will become
more peripheral with age [69]. P2c: the characteristics of immatures’ social partners will shift from a
majority of adult partners (deriving from the maternal network) towards including more same-sex age
peers or other immature individuals. This tendency will be greater for males than for females [54]. blishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 os
R. Soc. Open Sci. 10: 230219 ed from https://royalsocietypublishing.org/ on 30 May 2023 royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 P1b:
while mother–offspring social networks will be positively correlated [8], the strength of the correlation
will decrease with age as immatures become more independent from the mother [43]. The decrease
will be greater for males than for females [61]. P1c: because the mother and maternal relatives have a special importance in matrilineal species [78,79], immatures’ networks will be more correlated to the
mothers’ networks than to those of other individuals [60]. This pattern will decrease with age and the
decrease will be greater for males than for females [66]. 4 2.2. Data collection Observations were conducted on three troops (J, L and M) during six annual field seasons between 2014
and 2019. Data collection occurred over two- to three-month periods in each field season (approx. two
months in 2014, 2016 and 2017; five months in 2015; three months in 2018 and 2019). Troops J and L
were followed during all years, while M, which fissioned from J troop between 2016 and 2017, was
followed only in 2017 and 2019. During the study periods, baboons were usually followed daily from
dawn to dusk, and data were recorded on all recognizable individuals (see below) with the
application CyberTracker [87]. The study population is habituated to the presence of observers on foot and the majority of
the individuals were individually recognizable, apart from the unmarked juveniles and infants (the
proportions of recognizable individuals were: 0.8 in 2014 and 2017; 0.7 in 2015; 1 in 2016 and 2019;
and 0.9 in 2018). We quantified individuals’ associations based on a proximity and an interaction (i.e. grooming) rule. Networks built with different rules may provide different information [88,89] and
thus allow a more comprehensive description of baboons’ social network development. In addition,
both spatial and grooming networks predicted social information use in this population [19]. Proximity associations were characterized through instantaneous (scan) sampling [90]. Because
baboon troops at Tsaobis can spread over large distances with frequent mixing of individuals, it is
difficult to perform surveys of the troop (the whole social group) at one point in time, as is done for
some other species [91,92]. To guarantee sampling independence, we instead recorded the neighbours
of ‘focal’ individuals, who were chosen from a randomized list including all the baboons present in
the troop. All individuals within 10 m of a focal individual were recorded as a proximity ‘subgroup’. Individuals who did not have a neighbour within 10 m were recorded as alone. When a focal
individual was sampled, s/he was not re-sampled within an hour if appearing again in a (new) list. Similarly, when an individual was recorded as a neighbour of a focal individual, s/he was not re-sampled as a focal individual within an hour. If group membership did not change substantially
within an hour (during long resting periods, for example), observations ceased until group movement
(and thus new subgroups) started again. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 In each month, each individual groomed 5–34
times (averaged per year) in J troop (129–706 total grooming events per month—averaged per year),
12–52 times in L troop (444–1372 total per month) and 21–31 times in M troop (272–378 total per month). Immature individuals’ ages (in months) were determined by known and estimated dates of birth. Known dates were from births that occurred during fieldwork. If the birth occurred outside of the field
season, the date of birth was estimated based on (i) possible conception dates from consortship records
(i.e. mate-guarding events at the peak of a female’s fertility) [93] after which the female did not
menstruate, and assuming a pregnancy duration of six months [94]; or (ii) if the mother’s conceptive
cycle was not observed, through observation of the infant’s coloration [95]. The identity of an infant’s
mother was determined through direct behavioural observations (nursing). The mothers’ numbers of
offspring in the troop during each season were calculated based on long-term troop composition data. Dominance ranks were determined for adult females through dominance interactions recorded ad
libitum. These included displacements, supplants, threats, chases and attacks for which a giver and
recipient were identified. Only one interaction was recorded even if this involved several dominance
behaviours in sequence. Female ordinal ranks were computed at the end of each season through the ‘I &
SI’ method [96] using Matman 1.1.4 (Noldus Information Technology 2003). The method is appropriate
for linear dominance hierarchies [96,97], and it has been applied to other studies on this population (e.g. [98]). The dominance hierarchies obtained for each season were strongly linear. We standardized
absolute ranks by controlling for group size using the formula 1 −[(1 −r)/ (1 −n)], where r is an
individual’s absolute rank and n is the group size. Relative ranks range from 0 (lowest) to 1 (highest) [98]. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 Grooming interactions were recorded ad libitum [90] while moving through the troops. In this way, the
ad libitum-collected data were not biased towards the individuals who were easier to find and, as such,
we did not account for individual baboon observability. We collected data on the identities of the
baboons grooming and the directionality of grooming events (giver to recipient). To avoid pseudo-
replication, a grooming dyad was not recorded more than once within the same half-hour [19]. The
number of grooming individuals varied across the study years, ranging from 36 to 62 for J troop, from
42 to 81 for L troop and from 21 to 24 for M troop. In each month, each individual groomed 5–34
times (averaged per year) in J troop (129–706 total grooming events per month—averaged per year),
12–52 times in L troop (444–1372 total per month) and 21–31 times in M troop (272–378 total per month). Immature individuals’ ages (in months) were determined by known and estimated dates of birth. Known dates were from births that occurred during fieldwork. If the birth occurred outside of the field
season, the date of birth was estimated based on (i) possible conception dates from consortship records
(i.e. mate-guarding events at the peak of a female’s fertility) [93] after which the female did not
menstruate, and assuming a pregnancy duration of six months [94]; or (ii) if the mother’s conceptive
cycle was not observed, through observation of the infant’s coloration [95]. The identity of an infant’s
mother was determined through direct behavioural observations (nursing). The mothers’ numbers of
offspring in the troop during each season were calculated based on long-term troop composition data. Grooming interactions were recorded ad libitum [90] while moving through the troops. In this way, the
ad libitum-collected data were not biased towards the individuals who were easier to find and, as such,
we did not account for individual baboon observability. We collected data on the identities of the
baboons grooming and the directionality of grooming events (giver to recipient). To avoid pseudo-
replication, a grooming dyad was not recorded more than once within the same half-hour [19]. The
number of grooming individuals varied across the study years, ranging from 36 to 62 for J troop, from
42 to 81 for L troop and from 21 to 24 for M troop. 2.2. Data collection Since very young infants are carried exclusively by their
mothers, they were included in the scans only from when they were observed without the mother,
indicating that they could move independently from her [19]. The number of individuals sampled in
proximity scans varied across the study years, ranging from 44 to 59 for J troop, from 41 to 74 for L
troop and remaining stable at 21 for M troop. In each month, each individual was sampled 4–13 times
(averaged per year) in J troop (300–701 total observations per month—averaged per year), 8–18 times
in L troop (423–1037 total per month) and 13–20 times in M troop (273–427 total per month). 5 2.3. Data analyses Data analyses were performed with R v.4.0.2 [99]. Following previous studies with sparse network data
[100–103], we used multiple static networks (i.e. describing a network sampled over a particular length of
time) aggregated in approx. 30-day time periods. The periods chosen for time-aggregated networks
should be appropriate for the kind of questions investigated and the available data resolution [20,104]. We chose these periods of time because immatures undergo rapid developmental changes during
their first year of life [46]: this 30-day resolution allowed us to capture social network transformations
during infancy as well as juvenile years. Proximity and grooming data were treated separately. The sampling periods were generated starting
from the first day in which data were collected for a given field season; these dates were different for
proximity and grooming data, thus generating different windows and numbers of periods overall. In J
troop, this resulted in 12 and 16 30-day periods for proximity and grooming data, respectively (1–4
periods per year depending on field seasons’ lengths); in L troop, there were 11 and 17 30-day periods
for proximity and grooming data, respectively (1–5 periods per year); in M troop, we produced two and
three 30-day periods for proximity and grooming data, respectively (1–2 per year). To have a sufficient
number of observations per individual to build robust and accurate networks, we removed any period
with less than 95 observations. We also removed any individual who was seen less than five times per
season (e.g. disappeared mid-season due to death or emigration). The removal of individuals for whom
few data are collected is recommended because their network may not be represented accurately [104]. Social networks were produced for each sampling period, within each troop. We built weighted
networks in which network values indicate both the presence and strength of a connection between two individuals [105], and, in the case of grooming, weighted and directed networks. We did not filter
associations based on edge weight because thresholding (i.e. removing the associations that have a
weight lower than the threshold) eliminates weak ties, which can nevertheless be important [106,107],
and may generate errors in understanding the determinants of network structure [104]. We used a
simple ratio index (SRI) to calculate weighted connections in the proximity network. SRI is the
proportion of time two individuals associated (ranging between ‘0’—never associated—and ‘1’—
always associated). 2.3. Data analyses This index is recommended only when individuals can be detected and identified
correctly [108] as is the case at Tsaobis where group membership is stable, and visibility is usually
excellent due to low vegetation cover. For the grooming network, we used grooming counts or
proportions depending on the model tested (see below). Network graphs were computed from
association networks and matrices of grooming counts using igraph’s graph_adjacency function. The
grooming network was divided into its out (given) and in (received) components, which were treated
both separately and united (total). Although proximity and grooming networks were analysed
separately, similar models were applied in both cases, with the dependent variable differing
depending on the network. All analyses were run on ego-networks: the variables tested were
calculated for each immature individual in each sampling period [100,103]. royalsocietypublishing.org/journal/rsos
R. Soc. 6 6 To test our predictions, we used both linear mixed effects models (LMMs) and generalized linear mixed
effects models (GLMMs) using the function (g)lmer from the package lme4. The package lmerTest was
required to obtain p-values from models’ summaries. The models are summarized in table 1. All models
included the following fixed and random factors. The independent variables (fixed factors) of interest
were age, sex and the interaction between age and sex. We then controlled as a fixed factor the mothers’
relative dominance rank because rank is known to affect social bonds in primates [39,109,110]. We also
controlled as a fixed factor the mothers’ number of offspring in the troop because the mothers’ grooming
time might have to be shared among her offspring [111]. In addition, the number of maternal siblings
may influence, for instance, immatures’ grooming allocations in case they prefer maternal kin to other
individuals [66,112]. In order to control for repeated measures from the same troops, we added troop as
a fixed factor (because only three troops were followed in this study, it could not be included as a
random factor [113]). Because our data have repeated measures from the same individuals through time,
in each model, we included as a fixed factor the individuals’ identity (immature and mother) [101–103]. We nested immatures’ identities within their mothers’ identities because maternal behaviour could affect
the offspring’s sociality [114]. Finally, we added year as a random factor to consider between-years
variation in environmental factors. 2.3. Data analyses We did not control for season in our analyses because most
observations were made in the austral winter and baboons have no distinct breeding and mating seasons
like, for example, macaques [86,106]. The models (described in further detail below) were presented as ‘full models’: the models did not
undergo any simplification procedure after the a priori variable selection based on the current
literature (see above and table 1) [115,116]. We obtained R2 values (coefficient of determination) from
the package MuMIn and the function r.squaredGLMM. We checked for multi-collinearity using
variance inflation factors (VIFs) with the package car and the function vif. For LMMs, we checked
residuals’ homoscedasticity. For GLMMs, we verified overdispersion with the package DHARMa and
the function testDispersion. In all cases, VIF values were less than or equal to 3, indicating low
collinearity [117,118]; LMM models’ residuals were normally distributed and GLMMs’ under/
overdispersion, when significant, was within the range of 0.7–2, indicating a normal degree of
variation for a binomial distribution [119]. For binomial models, we obtained the (i) odds ratios (OR)
by exponentiating the variables’ estimates and (ii) 97.5% confidence intervals through the function
confint.merMod (method = ‘Wald’). We then calculated the percentage variation in odds per each unit
variation in a dependent variable as (OR −1) × 100 [120]. We expressed differences in OR changes
from one age stage (t0) to the next (t1) as increase/decrease percentages, computed through the
formula: [(ORt1 −ORt0)/ORt0)] × 100. For LMMs, estimates (beta coefficients) were described as
increase/decrease percentages over a dependent variable’s range. In the presence of an interaction, the
main effects were interpreted as conditional effects [121,122]. To compute age changes in effect sizes,
we chose two specific periods based on the literature: (i) 1 year old, when immatures are generally
weaned but still spend most of the time with the mother [46]; (ii) 4 years old, when immatures should
have established their own social relationships, mostly independently from maternal presence [66]. To
obtain differences in effect sizes between sexes, we calculated the effect of sex at 30 months of age
(the middle of the immatures’ age range). Finally, to compare the effects of different independent
variables (within a model), we derived standardized (i.e. all the variables are on the same scale) riable, the models’ fixed and random factors, and the type of test used. Under model, the
t or only partially how (Continued.) Table 1. 2.3. Data analyses Summary of the models used to test the predictions of hypotheses 1 (H1) and 2 (H2), including the dependent variable, the models’ fixed and random factors, and the type of test used. Under model, the
asterisk () means that a permutation test was performed. Support indicates whether the prediction was supported and, if not or only partially, how. hypothesis
prediction
model
dependent variable
network
support
test
fixed/random factors
H1: immatures would inherit their
mothers’ social networks at birth,
and these would differentiate
throughout development
P1a: time spent with the mother will
decrease with age and this decrease will
be greater for males than for females
MP1a
proportion of time with
mother
proximity
yes
GLMM
binomial
fixed: age, sex, age:sex,
nb. offspring,
relative rank, troop
random: year,
immatures’ identities
nested within their
mothers’ identities
grooming
total: yes
P1b: while mother–offspring networks will
be positively correlated, the strength of
the correlation will decrease with age. This decrease will be greater for males
than for females
MP1b
mother–offspring network
correlation
proximity
yes
LMM
grooming
given: partial (↑with age
in females)
received: partial (age:sex
not significant)
total: yes
P1c: immatures’ networks will be more
correlated to the mothers’ networks
than to those of other individuals. This
pattern will decrease with age and the
decrease will be greater for males than
for females
MP1c.1
0–1 score: 1 = correlation
with mother’s
network > correlation
with random
individual’s network;
0 = vice versa
proximity
no (age and age:sex not
significant)
GLMM
binomial
grooming
given: no (↑with age,
age:sex not significant)
received: no (age and
age:sex not significant)
total: no (age and age:sex
not significant)
MP1c.2
0–1 score: 1 = correlation
with mother’s
network > mean
correlation with other
individuals; 0 = vice
versa
proximity
no (↑with age, age:sex
not significant)
grooming
given: no (↑with age,
age:sex not significant)
received: no (age and
age:sex not significant)
total: no (↑with age,
age:sex not significant) prediction
model
dependent variable
network
support
test
fixed/random factors
teristics
hout
s of both the
rk and the
with whom
P2a: the associations with different social
partners will increase with age as
immatures widen their social network. This increase will be relatively lower in
males, who will also have fewer social
partners than females
MP2a
strength
proximity
no (↓with age, sex and
age:sex not significant)
LMM
fixed: age, sex, age:sex,
nb. 2.3. Data analyses offspring,
relative rank, troop,
strength mother
random: year,
immatures’ identities
nested within their
mothers’ identities
grooming
given: yes
received: partial (sex and
age:sex not significant)
total: partial (sex not
significant)
P2b: immatures’ positions in the social
network will remain stable throughout
development for females, while males
will become more peripheral with age
MP2b.1
eigenvector centrality
proximity
no (↓with age for
females and age:sex
not significant)
LMM
fixed: age, sex, age:sex,
nb. offspring,
relative rank, troop,
centrality mother
random: year,
immatures’ identities
nested within their
mothers’ identities
grooming
total: no (↑with age for
both sexes and age:sex
not significant)
MP2b.2
betweenness centrality
proximity
no (age and age:sex not
significant)
LMM
fixed: age, sex, age:sex,
nb. offspring,
relative rank, troop,
betweenness mother
random: year,
immatures’ identities
nested within their
mothers’ identities
grooming
total: no (↑with age for
both sexes)
(Continued.)
royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219
8 8 prediction
model
dependent variable
network
support
test
fixed/random factors
P2c: the characteristics of immatures’ social
partners will shift from a majority of
adult partners to a majority of same-sex
age peers or other immature individuals. This tendency will be greater for males
than for females
MP2c.1
proportion of immature
(less than or equal to 5
years old) partners
proximity
partial (age not
significant)
GLMM
binomial
fixed: age, sex, age:sex,
nb. offspring,
relative rank, troop
random: year,
immatures’ identities
nested within their
mothers’ identities
grooming
given: partial (age:sex not
significant)
received: partial (age:sex
not significant)
total: partial (age:sex not
significant)
MP2c.2
proportion of peer (± 6
months) partners
proximity
no (age and age:sex not
significant)
grooming
given: partial (age:sex not
significant)
received: partial (age:sex
not significant)
total: partial (age:sex not
significant)
MP2c.3
proportion of same-sex
partners
proximity
yes
grooming
given: partial (age:sex not
significant)
received: yes
total: yes royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219
9 9 binomial Table 1. (Continued.) estimates (see electronic supplementary material, §6). In all cases, p-values ≤0.05 were considered
significant [115,123]. 10 g
Social network data are non-independent because the data are relational. Data permutations serve to
solve the issue of data non-independence when performing hypothesis testing with network data
[104,105]. The permuted data are used to re-run the model to obtain a ‘null’ effect size against which
the observed effect size can be compared. 2.4. H1. Inheritance of maternal social networks From the base model, we tested our hypotheses and predictions using the following specific models (table 1). For H1, we ran a total of four sets (applied to the different networks) of models across our three predictions. First, we quantified changes in the time immatures spend with the mother (P1a), using binomial GLMMs
(family = binomial, link = logit) [125] in which the dependent variable was the proportion of time spent with
the mother (MP1a). For proximity, we used the proportion of observations in which each immature was
observed with the mother (within 10 m) in each observation period; for grooming, we used the
proportion of observations in which each focal immature groomed with the mother, considering only
the total grooming. Permutations were not run for these models because the dependent variable (i.e. proportion of observations with the mother) is not a social network value or metric. We next tested whether there was a positive correlation between mothers’ and immatures’ social
networks and how this changed through time (P1b). To this end, we calculated two measures of
similarity between the mothers’ and immatures’ ego-networks for each period (i.e. the vectors that
correspond to the square matrix rows of each immature focal individual and her/his mother). The
two measures were (i) the test statistic from a Pearson’s correlation (bounded between −1 and 1) of
the mother’s and offspring’s vectors [8] and (ii) the cosine similarity between the mother’s and
immature’s vectors [42]. Because these two network similarity measures were positively correlated
and the results of the models using either one of these dependent variables were qualitatively similar,
we report here only the results for models using the Pearson’s correlation (MP1b) (see electronic
supplementary material, §1, for results using the cosine similarity). For this analysis, we used the
proportion matrix for grooming because we were interested in similarities between networks, and
proportions better represent the distribution of grooming to/from different partners, independent of
the total number of events. Immatures’ grooming networks may be constrained by partner availability
if they tend to be in proximity to the mother and could thus choose only among her social partners. Controlling for the time spent in the mother’s proximity would potentially solve this issue [126]. However, there was no significant effect of the proportion of time spent in proximity with the mother
(see electronic supplementary material, §2). 2.3. Data analyses For the proximity data, in most cases, we permuted the
datastream
using
the
network_swap
function
from
the
asnipe
package;
however,
node-based
permutations (see below) were used for three of the models (as described further on). Datastream
permutations randomize subgroup membership by swapping observations between individuals in the
N × K matrices (i.e. N individuals per K observations) [124] and are recommended for group
membership data [123]. The original datastream was used to make the first permutation, swapping 20
observations in each 30-day period. For each successive permutation, the previous permuted matrix
was re-permuted. In this way, matrices become more and more randomized compared to the original
datastream. For this reason, a set of initial permutations was discarded. We made 300 permutations
per period, discarded the first 200 permuted matrices, retaining 100 randomized networks/period to
be used in the ‘randomized’ models. These permuted matrices represent the null hypothesis of
random associations among individuals [123]. For the grooming data and three models from
proximity data (P2c models; see details below), we applied node-based permutations, which are
commonly used for grooming networks [28,118], with the function rmperm from the package sna. Node-based permutations re-distribute the nodes (individuals) while leaving the same number of
associations [104]. As for the proximity data, we made 300 permutations per sampling period and
selected the last 100 for each period. To determine the statistical significance of the estimated social
network effect, we re-ran each model (see below) using the 100 permuted matrices. For each model
and each network variable, the two-tailed p-value was calculated as the number of times that the
observed effect size was larger or smaller than the distribution of effect sizes from the permuted data
(resulting as either p-value = 0.05 or not significant) [123]. 2.5. H2. Changes in social network characteristics throughout development For H2, we ran a further six sets of models. Predictions P2a and P2b predicted changes in individuals’ node-
level social network metrics that reflect the level of integration in the troop’s network. We chose to calculate
individuals’: strength (MP2a), eigenvector centrality (MP2b.1) and betweenness centrality (MP2b.2). Strength is the sum of the edge weights connected to a node, representing the relative association rate
within a sampling period and thus an individual’s opportunities to collect social information. Eigenvector centrality (hereafter centrality) is the sum of the centralities of an individual’s social partners. An individual can have high centrality either by having a large number of partners or associating with
individuals who have numerous connections (or both) [104,127]. This metric also describes an
individual’s relative opportunities to acquire and propagate social information in a network [19]. Betweenness centrality (hereafter betweenness) is the number of shortest paths that cross a node. This
measures an individual’s importance in connecting otherwise unconnected individuals [104]. For both
proximity SRI and grooming count data, we calculated the metrics using igraph (functions strength,
eigen_centrality, betweenness). Grooming given, received and total were computed for strength, while
given/received components cannot be separated for centrality and betweenness. We analysed these five
metrics using LMMs. Proximity betweenness and grooming strength given were log-transformed (log1p
function) to make them normally distributed; grooming centrality and betweenness were square root
transformed (sqrt function). In these models, we additionally controlled as a fixed factor the
corresponding metric of the mother. This is because, if immatures inherit their mothers’ networks, the
mother’s social network position may affect that of her offspring [42]. To make metrics comparable
across networks of different sizes, metrics were standardized within each sampling period by mean-
centring them around zero with a standard deviation of one through the function scale [128]. To test P2c, that immatures will shift from associating with adults to peers as they age, we counted the
number of immature, peer and same-sex partners each immature associated with. An individual’s
number of social partners is their degree [104]. A peer was defined as an individual whose age was
±6 months of a focal individual’s age. We distinguished between immatures and peers to understand
whether immatures were generally attracted by other non-adult individuals (less than or equal to 5
years old) or if they specifically targeted partners of their same age. 2.5. H2. Changes in social network characteristics throughout development To test the tendency to associate
with different partner categories with age, we ran binomial GLMMs with the proportion of immature
(MP2c.1), peer (MP2c.2) and same-sex (MP2c.3) partners as dependent variables. Using the proportion
of partners belonging to a certain category over the total number of partners of that category in the
troop, we could control for partner availability [66]. For the P2c models, we ran node-based
permutations for the proximity network. These were chosen over datastream permutations because, as
mentioned, they keep the number of associations constant [104] and were thus appropriate to test
whether there was a shift in partner preferences based on their phenotype rather than number. 2.4. H1. Inheritance of maternal social networks Because including this variable reduced our dataset (since
proximity data were not available for every grooming period), it was not included in the final models. We next tested whether immatures’ ego-networks were more similar to their mothers’ ego-networks
than to those of other group-mates and how this changed with age (P1c). Because there is no agreed method to test this, we adopted three different approaches, all of which compared the Pearson’s
correlation coefficient (r) of the mother–immature dyad with combinations of other dyads from the
proximity networks or grooming proportions networks. We present two of these approaches here and
discuss the third one in electronic supplementary material, §5. In the first approach, for each immature
individual, we selected a random non-mother individual from the troop and created a new variable,
assigning a score of ‘1’ when the mother–immature’s r was higher than the random–immature’s r, and
‘0’ when the reverse was true. We ran a binomial GLMM with the 1/0 score as the dependent variable
(MP1c.1). In the second approach, we applied the same 1/0 scoring method and a binomial GLMM,
using the mean r for all the non-mother individuals instead of a random individual (MP1c.2). 11 royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 3. Results We collected a total of 12 592 proximity records (J: 5862; L: 6030; M: 700) and 19 597 grooming records (J:
6277; L: 12 398; M: 922). The number of immature individuals followed (i.e. sample size) were 109 to 116
for the proximity network and 93 to 142 for the grooming network, born from 32 to 43 mothers (see
electronic supplementary material, §3, for the number of observations and the sample size of each
model) [129]. In the following sections, we present the significant results (following each prediction) on our main
variables of interest: age, sex and their interaction (tables 2 and 3; figures 1–6). Full tables are reported in
the electronic supplementary material, §5 (together with the results of the third approach used to test P1c; Table 2. Models’ results on the age and sex (and their interaction) variables for the hypothesis 1 models (tables with all the
independent variables are available in electronic supplementary material, §5). Under variable, age:sex indicates the presence of
an interaction. The values reported for sex refer to males. The estimate, standard error (s.e.), p-value and conditional (delta) R2
are reported for each variable/model. The names of models that have significant variables and the rows of those significant
variables are marked in italics. 12 variables are marked in italics. model
variable
estimate
s.e. 3. Results p-value
R²
P1a proximity
age
−0.0112
0.0051
< 0.05
0.27
sex
0.2702
0.2529
NS
age:sex
−0.0135
0.0067
< 0.05
P1a grooming total
age
−0.0232
0.0035
< 0.001
0.84
sex
1.0043
0.2027
< 0.001
age:sex
−0.0173
0.0038
< 0.001
P1b proximity
age
−0.0029
0.0013
0.05
0.20
sex
−0.0007
0.0670
NS
age:sex
−0.0021
0.0018
0.05
P1b grooming given
age
0.0022
0.0010
0.05
0.25
sex
−0.0227
0.0607
NS
age:sex
−0.0027
0.0015
0.05
P1b grooming received
age
−0.0042
0.0013
0.05
0.44
sex
−0.0222
0.0575
NS
age:sex
0.0003
0.0016
NS
P1b grooming total
age
−0.0010
0.0010
0.05
0.28
sex
−0.0147
0.0449
NS
age:sex
−0.0014
0.0012
0.05
P1c.1 proximity
age
0.0053
0.0090
NS
0.03
sex
−0.3070
0.4550
NS
age:sex
0.0017
0.0120
NS
P1c.1 grooming given
age
0.0198
0.0091
0.05
0.08
sex
−0.1326
0.4849
NS
age:sex
−0.0172
0.0126
NS
P1c.1 grooming received
age
−0.0088
0.0092
NS
0.13
sex
−0.4860
0.3657
NS
age:sex
0.0108
0.0115
NS
P1c.1 grooming total
age
0.0096
0.0072
NS
0.17
sex
−0.3061
0.3116
NS
age:sex
−0.0022
0.0092
NS
P1c.2 proximity
age
0.0219
0.0097
0.05
0.08
sex
0.3355
0.4720
NS
age:sex
−0.0168
0.0128
NS
P1c.2 grooming given
age
0.0161
0.0100
0.05
0.19
sex
−0.9799
0.5681
0.05
age:sex
−0.0024
0.0143
NS
P1c.2 grooming received
age
−0.0112
0.0114
NS
0.25
sex
−0.1466
0.4497
NS
age:sex
0.0021
0.0141
NS
(Continued.)
etypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 Table 2. (Continued.)
model
variable
estimate
s.e. p-value
R²
P1c.2 grooming total
age
0.0198
0.0079
0.05
0.24
sex
−0.0312
0.3522
NS
age:sex
−0.0134
0.0101
NS
y
yp
g
1 13 Table 3. Models’ results on the age and sex (and their interaction) variables for the hypothesis 2 models (tables with all the
independent variables are available in electronic supplementary material, §5). Under variable, age:sex indicates the presence of
an interaction. The values reported for sex refer to males. The estimate, standard error (s.e.), p-value and conditional (delta) R2
are reported for each variable/model. The names of models that have significant variables and the rows of those significant
variables are marked in italics. model
variable
estimate
s.e. 3. Results p-value
R²
P2a proximity
age
−0.0211
0.0039
0.05
0.41
sex
−0.3603
0.1936
NS
age:sex
0.0069
0.0050
NS
P2a grooming given
age
0.0455
0.0019
0.05
0.72
sex
−0.2985
0.0893
0.05
age:sex
−0.0199
0.0025
0.05
P2a grooming received
age
0.0069
0.0020
0.05
0.40
sex
−0.0613
0.0922
NS
age:sex
−0.0022
0.0025
NS
P2a grooming total
age
0.0341
0.0019
0.05
0.64
sex
−0.0125
0.0863
NS
age:sex
−0.0235
0.0023
0.05
P2b.1 proximity
age
−0.0211
0.0036
0.05
0.49
sex
−0.2964
0.1831
NS
age:sex
0.0047
0.0047
NS
P2b.1 grooming total
age
0.0097
0.0022
0.05
0.49
sex
−0.0195
0.1005
NS
age:sex
−0.0068
0.0028
NS
P2b.2 proximity
age
0.0054
0.0042
NS
0.07
sex
0.1166
0.2167
NS
age:sex
−0.0055
0.0057
NS
P2b.2 grooming total
age
0.0355
0.0023
0.05
0.52
sex
−0.2042
0.1020
NS
age:sex
−0.0126
0.0029
0.05
P2c.1 proximity
age
0.0007
0.0013
NS
0.14
sex
−0.0922
0.0641
0.05
age:sex
0.0021
0.0017
0.05
P2c.1 grooming given
age
0.0346
0.0032
0.05
0.64
sex
−1.1815
0.1887
0.05
age:sex
0.0060
0.0044
NS
(Continued.) 14 model
variable
estimate
s.e. p-value
R²
P2c.1 grooming received
age
0.0119
0.0027
0.05
0.36
sex
−0.1137
0.1339
NS
age:sex
0.0023
0.0036
NS
P2c.1 grooming total
age
0.0227
0.0022
0.05
0.56
sex
−0.4327
0.1195
0.05
age:sex
−0.0010
0.0030
NS
P2c.2 proximity
age
−0.0001
0.0026
NS
0.01
sex
−0.0362
0.1412
NS
age:sex
0.0018
0.0036
NS
P2c.2 grooming given
age
0.0303
0.0051
0.05
0.31
sex
−1.1021
0.3042
0.05
age:sex
0.0137
0.0073
NS
P2c.2 grooming received
age
0.0261
0.0054
0.05
0.23
sex
−0.0887
0.2828
NS
age:sex
0.0067
0.0070
NS
P2c.2 grooming total
age
0.0275
0.0042
0.05
0.33
sex
−0.5450
0.2297
0.05
age:sex
0.0090
0.0057
NS
P2c.3 proximity
age
0.0010
0.0012
0.05
0.18
sex
−0.1528
0.0627
0.05
age:sex
0.0032
0.0016
0.05
P2c.3 grooming given
age
0.0364
0.0029
0.05
0.70
sex
−1.5735
0.1916
0.05
age:sex
0.0078
0.0044
NS
P2c.3 grooming received
age
0.0080
0.0023
0.05
0.57
sex
−1.9513
0.1537
0.05
age:sex
0.0210
0.0038
0.05
P2c.3 grooming total
age
0.0197
0.0020
0.05
0.72
sex
−1.8453
0.1327
0.05
age:sex
0.0158
0.0032
0.05
royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 see Methods). Comparisons among models’ variables through standardized estimates are shown in
electronic supplementary material, §6. see Methods). Comparisons among models’ variables through standardized estimates are shown in
electronic supplementary material, §6. 3.1. H1. Inheritance of maternal social networks Open Sci. 10: 230219
15 15 proportion time with mother Figure 1. Changes in the immatures’ (a,c) odds of being with the mother (model-predicted values, MP1a) and (b,d) proportion of
time spent with the mother (raw data) with age in the two sexes (F = female, M = male) for the proximity and grooming (gr.)
networks. Higher OR means a relatively higher likelihood of an event (y-axis variable) to occur. Ribbons represent 97.5% confidence
intervals. females (not predicted): at 30 months old, the odds of grooming (total) with the mother were 62.46%
higher in males compared to females. Our second prediction for H1, P1b, was partially confirmed, depending on the network (table 2 and
figure 2). The mean correlation values between the mothers’ and offspring’s networks were positive for
both the proximity and grooming networks (see electronic supplementary material, §4). As predicted for
the proximity network, the immatures’ networks became significantly less similar to their mothers’ with
age and this decrease was significantly greater in males compared to females. Between 1 and 4 years old,
the mother–offspring proximity network correlation decreased by 7.2% and 12.6% in females and males,
respectively. For the grooming network, the support to P1b was less clear compared to the proximity
network. According to our prediction for grooming received and total (as for the proximity network),
the immatures’ networks became significantly less similar to their mothers’ with age. This pattern was
significantly more marked in males than in females only for grooming total. Between 1 and 4 years
old, the mother–offspring grooming network correlation decreased by 12.24% and 2.88% (received
and total, respectively) in females, and by 11.52% and 6.84% (received and total, respectively) in
males. Instead, contrary to our prediction for females, age had significant opposite effects in the two
sexes for grooming given: between 1 and 4 years old, the mother–offspring network correlation
increased by 6.84% in females and decreased by 1.08% in males. Our third prediction for H1, P1c, had contrasting support depending on the network and the model
(table 2 and figure 3). The observations in which the immatures’ proximity and grooming networks were
more similar to those of their mothers (compared to the networks of other individuals) were higher than
the observations in which the opposite was true (less similar to their mothers’ networks) in nine (of 16)
models (see electronic supplementary material, §4). 3.1. H1. Inheritance of maternal social networks To address our first hypothesis, we tested how the time spent with the mother (P1a) and the correlation
between the offspring’s and mothers’ social networks (P1b and P1c) changed through time. Our first
prediction for H1, P1a, was confirmed both for the proximity and grooming total networks (table 2
and figure 1): immatures spent significantly less time with their mothers as they grew older, and this
decrease was significantly greater in males than in females. Between 1 and 4 years old, the odds of
being in proximity with the mother decreased by 33.27% and 58.89% in females and males,
respectively; the odds of grooming (total) with the mother decreased by 56.63% and 76.74% in
females and males, respectively. However, males groomed significantly more with their mothers than proximity
gr. total
age (months)
gr. total
proximity
odds being with mother
proportion time with mother
1.0
(a)
(b)
(c)
(d)
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
10
20
30
40
50
60
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
10
20
30
40
50
60
sex
F
M
sex
F
M
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Figure 1. Changes in the immatures’ (a,c) odds of being with the mother (model-predicted values, MP1a) and (b,d) proportion of
time spent with the mother (raw data) with age in the two sexes (F = female, M = male) for the proximity and grooming (gr.)
networks. Higher OR means a relatively higher likelihood of an event (y-axis variable) to occur. Ribbons represent 97.5% confidence
intervals. months)
gr. total
proximity
proportion time with mother
(b)
(d)
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
10
20
30
40
50
60
sex
F
M
0
10
20
30
40
50
60
royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219
15 proximity
gr. total
age
odds being with mother
1.0
(a)
(c)
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
10
20
30
40
50
60
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
sex
F
M
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60 royalsocietypublishing.org/journal/rsos
R. Soc. 3.1. H1. Inheritance of maternal social networks Among the six models tested for the grooming network, age and the age/sex
interaction were significant in three models (not significant in MP1c.1 and MP1c.2 received, and in
MP1c.1 total). In contrast with our prediction (as for the proximity network), the immatures’
grooming given (MP1c.1 and MP1c.2) and total (MP1c.2) networks became significantly more similar
to their mothers’ compared to other individuals. This pattern was less marked in males, although the
age/sex interaction was not significant. Between 1 and 4 years old, the odds of having a network
relatively more similar to the mother’s one increased between (hereafter—range of values) 78.44 and
104.32% (MP1c.2 given and MP1c.2 total, respectively) in females and between 9.89 and 63.67%
(MP1c.1 and MP1c.2 given, respectively) in males. Finally, in MP1c.2, males’ grooming given networks
matched those of their mothers significantly less than the ones of other individuals compared to
females (not predicted): at 30 months of age, the odds of having a network relatively more similar
to the mother’s one were 65.07% lower in males than in females. in MP1c.1. For the grooming network, the models’ support on age changes was less clear compared to
the proximity network. Among the six models tested for the grooming network, age and the age/sex
interaction were significant in three models (not significant in MP1c.1 and MP1c.2 received, and in
MP1c.1 total). In contrast with our prediction (as for the proximity network), the immatures’
grooming given (MP1c.1 and MP1c.2) and total (MP1c.2) networks became significantly more similar
to their mothers’ compared to other individuals. This pattern was less marked in males, although the
age/sex interaction was not significant. Between 1 and 4 years old, the odds of having a network
relatively more similar to the mother’s one increased between (hereafter—range of values) 78.44 and
104.32% (MP1c.2 given and MP1c.2 total, respectively) in females and between 9.89 and 63.67%
(MP1c.1 and MP1c.2 given, respectively) in males. Finally, in MP1c.2, males’ grooming given networks
matched those of their mothers significantly less than the ones of other individuals compared to
females (not predicted): at 30 months of age, the odds of having a network relatively more similar
to the mother’s one were 65.07% lower in males than in females. 3.1. H1. Inheritance of maternal social networks Contrary to our prediction for the proximity
network, we found evidence that immatures’ networks became significantly more similar to their
mothers’ compared to those of other individuals with age in MP1c.2. This pattern was less marked in
males than in females, but the age/sex interaction was not significant. Between 1 and 4 years old, the
odds of having a proximity network relatively more similar to the mother’s one increased by 119.79%
and 20.07% in females and males, respectively. Age and the age/sex interaction were not significant proximity
gr. received
gr. total
gr. given
mother–offspring network correlation
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Figure 2. Changes in the mother–offspring social network correlation with age in the two sexes (F = female, M = male) for the
proximity and grooming (gr.) networks (MP1b). Ribbons represent 97.5% confidence intervals. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219
16 proximity
network correlation
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R. Soc. Open Sci. 10: 230219
16 16 gr. total
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mother–offspring n
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age Downloaded from https://royalsocietypublish Figure 2. Changes in the mother–offspring social network correlation with age in the two sexes (F = female, M = male) for the
proximity and grooming (gr.) networks (MP1b). Ribbons represent 97.5% confidence intervals. in MP1c.1. For the grooming network, the models’ support on age changes was less clear compared to
the proximity network. 3.2. H2. Changes in social network characteristics throughout development To address our second hypothesis, we explored how immatures’ integration in the troop’s network
changed during development (relative association rates with different social partners—P2a—and
overall network position—P2b) and modelled shifts in social partner preferences (P2c). Our first and
second predictions for H2, P2a and P2b, were partially confirmed, depending on the network and the
metric involved (table 3; figures 4 and 5). Contrary to what we predicted for the proximity network,
both strength (MP2a) and centrality (MP2b.1) significantly decreased as immatures grew older and proximity—MP1c.2
gr. given—MP1c.2
gr. total—MP1c.2
gr. given—MP1c.1
odds network similar to mother
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sex
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age (months)
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igure 3. Changes in the odds that the correlation with the mother’s network was higher than the correlation with a random
dividual’s network (MP1c.1) and the mean correlation with other individuals’ networks (MP1c.2). These changes are related
o age and divided between the two sexes (F = female, M = male) for the proximity and grooming (gr.) networks. Higher OR
means a relatively higher likelihood of an event (y-axis variable) to occur. Ribbons represent 97.5% confidence intervals. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219
17 proximity—MP1c.2
similar to mother
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s 17 royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219
17 gr. total—MP1c.2
1.0
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months)
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odds network s
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age Figure 3. Changes in the odds that the correlation with the mother’s network was higher than the correlation with a random
individual’s network (MP1c.1) and the mean correlation with other individuals’ networks (MP1c.2). 3.2. H2. Changes in social network characteristics throughout development These changes are related
to age and divided between the two sexes (F = female, M = male) for the proximity and grooming (gr.) networks. Higher OR
means a relatively higher likelihood of an event (y-axis variable) to occur. Ribbons represent 97.5% confidence intervals. this pattern was more marked in females, although the age/sex interaction was not significant. Between 1
and 4 years old, proximity strength and centrality decreased, respectively, by 14.4% and 12.96% in
females, and by 9.72% and 10.08% in males. Finally, proximity strength was not significantly
predicted by sex, and betweenness (MP2b.2) was not significantly predicted by age and the age/sex
interaction. Instead, for the grooming network, immatures’ strength (all components, as predicted),
centrality and betweenness (total, contrary to our prediction) significantly increased with age. According to our prediction, the increases in grooming strength (all components), centrality and
betweenness (total) were less marked in males. However, the age/sex interaction was not significant
for strength received and centrality total. Between 1 and 4 years old, grooming strength increased by
37.44% and 5.76% (given and received, respectively) in females, and by 20.88% and 3.96% (given and
received, respectively) in males. At the same time, grooming centrality and betweenness (total)
increased, respectively, by 5.76% and 30.24% in females, and by 1.8% and 19.44% in males. Finally, as
predicted, males had a lower grooming given strength than females, while sex was not significant for
grooming received and total: at 30 months old, males’ strength given was 6.83% lower than females’. O
hi d
di i
f
H2 P2
i ll
fi
d d
di
h
k (
bl
3
d Our third prediction for H2, P2c, was partially confirmed, depending on the network (table 3 and
figure 6). As predicted for the proximity network, immatures shifted their social preferences towards
immature (MP2c.1) and same-sex (MP2c.3) partners while growing older. This age change was
significant only for MP2c.3, while the pattern was significantly more marked in males than in females
in both models. Between 1 and 4 years old, the odds of being in proximity with an immature and a
same-sex partner increased, respectively, by 2.43% and 3.51% in females, and by 10.57% and 16.28%
in males. Instead, the change in preference for peer partners (MP2c.2) with age was not significant for proximity
gr. received
gr. total
gr. 3.2. H2. Changes in social network characteristics throughout development Finally, sex
had a significant effect in all MP2c models but in MP2c.2 proximity, and in MP2c.1 and MP2c.2
grooming received (not predicted). Males were less in proximity and groomed less with partners of
similar age and of the same sex than females. At 30 months of age, the odds of being in proximity
with an immature and a same-sex partner were, respectively, 2.80% and 5.43% lower in males than in
females; the odds of grooming with an immature, a peer or a same-sex partner were lower in males
than in females between 24.04 and 74.64% (MP2c.2 and MP2c.3 total, respectively). the proximity network. As predicted for the grooming network, immatures developed a significant
preference for grooming immature and peer partners with age (all components). This pattern was
relatively more marked in males, but the age/sex interaction was not significant. Between 1 and 4
years old, the odds of grooming with an immature and a peer partner increased between (hereafter—
range of values) 53.57 and 247.3% (MP2c.1 received and given, respectively) in females and between
66.93 and 387.93% in males (MP2c.1 received and MP2c.2 given, respectively). Moreover, as predicted,
immatures groomed significantly more with partners of the same sex as they aged (all components). This pattern was more marked in males than in females, although the age/sex interaction was
significant only for grooming received and total. Between 1 and 4 years old, the odds of grooming
with a partner of the same sex increased between 33.52 and 270.69% (received and given, respectively)
in females and between 184.75 and 391.22% (received and given, respectively) in males. Finally, sex
had a significant effect in all MP2c models but in MP2c.2 proximity, and in MP2c.1 and MP2c.2
grooming received (not predicted). Males were less in proximity and groomed less with partners of
similar age and of the same sex than females. At 30 months of age, the odds of being in proximity
with an immature and a same-sex partner were, respectively, 2.80% and 5.43% lower in males than in
females; the odds of grooming with an immature, a peer or a same-sex partner were lower in males
than in females between 24.04 and 74.64% (MP2c.2 and MP2c.3 total, respectively). 3.2. H2. Changes in social network characteristics throughout development given
social network strength
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Figure 4. Changes in immatures’ social network strength with age in the two sexes (F = female, M = male) for the proximity and
grooming (gr.) networks (MP2a). Ribbons represent 97.5% confidence intervals. y
yp
g
g j
p
1 proximity
ork strength
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s 18 royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219
18 gr. total
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M
(months) gr. received
social networ
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age Figure 4. Changes in immatures’ social network strength with age in the two sexes (F = female, M = male) for the proximity and
grooming (gr.) networks (MP2a). Ribbons represent 97.5% confidence intervals. the proximity network. As predicted for the grooming network, immatures developed a significant
preference for grooming immature and peer partners with age (all components). This pattern was
relatively more marked in males, but the age/sex interaction was not significant. Between 1 and 4
years old, the odds of grooming with an immature and a peer partner increased between (hereafter—
range of values) 53.57 and 247.3% (MP2c.1 received and given, respectively) in females and between
66.93 and 387.93% in males (MP2c.1 received and MP2c.2 given, respectively). Moreover, as predicted,
immatures groomed significantly more with partners of the same sex as they aged (all components). This pattern was more marked in males than in females, although the age/sex interaction was
significant only for grooming received and total. Between 1 and 4 years old, the odds of grooming
with a partner of the same sex increased between 33.52 and 270.69% (received and given, respectively)
in females and between 184.75 and 391.22% (received and given, respectively) in males. 4. Discussion In this paper, we explored how wild chacma baboon social networks are shaped throughout the
immature period, focusing on age changes and sex differences. Here, we first discuss the effects of age proximity—MP2b.1
gr. total—MP2b.1
gr. total—MP2b.2
sex
F
M
centrality
centrality
betweenness
social network
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Figure 5. Changes in immatures’ social network centrality (MP2b.1) and betweenness (MP2b.2) with age in the two sexes
(F = female, M = male) for the proximity and grooming (gr.) networks. Ribbons represent 97.5% confidence intervals. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219
19 19 social network gr. total—MP2b.2
F
M
betweenness
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centrality
social n
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ag betweenness Downloaded from https://royalsocietypublis Figure 5. Changes in immatures’ social network centrality (MP2b.1) and betweenness (MP2b.2) with age in the two sexes
(F = female, M = male) for the proximity and grooming (gr.) networks. Ribbons represent 97.5% confidence intervals. and sex (and their interaction) on the inheritance of maternal networks (H1) and on the changes in the
immatures’ social network integration and social partners’ characteristics (H2). We then comment on the
potential implications of these developmental changes and sex differences in network patterns for social
information transmission. and sex (and their interaction) on the inheritance of maternal networks (H1) and on the changes in the
immatures’ social network integration and social partners’ characteristics (H2). We then comment on the
potential implications of these developmental changes and sex differences in network patterns for social
information transmission. Immature baboons inherited their mothers’ social connections (H1). As they aged, they reduced the
strength of their relationship with the mother and maternal social partners, but a mother’s bonds
continued to influence her offspring’s throughout their development. The gradual acquisition of
ecological competence (e.g. knowing what to eat) may decrease immatures’ reliance on maternal care
[16,130] and consequently the time spent with the mother, as is common among post-weaning
mammals [131,132]. 4. Discussion We found that this was the case, as immature baboons spent less time with their
mothers with age. Perhaps as a result, immatures’ networks tended to diverge from those of their
mothers with age. Nevertheless, mother–offspring proximity and grooming networks were positively
correlated and immatures’ networks generally became more similar to their mothers’ compared to
those of other individuals with age, thus supporting a social inheritance explanation [8,41]. Although
the mean mother–offspring network correlations were relatively low (less than 0.5; see electronic
supplementary material, §4), these values may be normal given that (i) immatures’ grooming
integration increased with age (see Results §H2), being thus initially lower than that of their mothers,
and (ii) in wild populations social networks are affected by demographic changes [133,134]. Our mean
cosine similarity values for immature female grooming networks were qualitatively similar to those
reported for vervet monkeys [42] (see electronic supplementary material, §1). The relatively low cosine
similarity values in vervet monkeys were hypothesized to derive from the changes in the mothers’
own grooming networks, which constituted a moving target for their daughters [42]. The same 1.0
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gr. 4. Discussion total—MP2c.3
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Figure 6. Changes in immatures’ odds of being with a particular social partner (another immature, MP2c.1; a peer, MP2c.2; a same-
sex individual, MP2c.3) with age in the two sexes (F = female, M = male) for the proximity and grooming (gr.) networks. Higher
OR means a relatively higher likelihood of an event (y-axis variable) to occur. Ribbons represent 97.5% confidence intervals. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219
20 1.0
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R. Soc. Open Sci. 10: 230219
20 gr. received—MP2c.1
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h social partner gr. total—MP2c.2
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gr. total—MP2c.2
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60 Figure 6. Changes in immatures’ odds of being with a particular social partner (another immature, MP2c.1; a peer, MP2c.2; a same-
sex individual, MP2c.3) with age in the two sexes (F = female, M = male) for the proximity and grooming (gr.) networks. Higher
OR means a relatively higher likelihood of an event (y-axis variable) to occur. Ribbons represent 97.5% confidence intervals. process may contribute to explain our results, given that the mothers’ grooming networks changed
between the study years (see electronic supplementary material, §7). Although immature baboons inherited their mothers’ social networks, our results showed that
their social network integration and social partner preferences changed throughout development (H2). Age was an important determinant of this process (see electronic supplementary material, §6). Given
that the mother’s presence constrains the number and kind of partners with whom immatures can
associate [135,136], age, which determines the extent of an immature’s dependence on the mother [46],
could be an important predictor of these network changes. In particular, as they aged, immatures had
lower associations in their proximity network but were more active in grooming. First, the decrease in
proximity network integration may be due to both external factors and immatures’ partner choices. External factors could include primates’ attraction towards infants [46,51] and the greater tolerance
that adults have towards younger versus older immatures. The infant network may shrink as older
infants lose their attractiveness [137,138]. In addition, younger immature primates are generally granted relatively higher proximity tolerance [15], which could aid their survival through the acquisition
of ecological knowledge [48,139] and protection [39,114]. On the other hand, older immatures may need
relatively less support and be willing to associate with fewer partners [49,140]. Alternatively (or
concurrently), immatures’ proximity integration may decrease because they become more ‘socially
selective’. Social selectivity has been reported in mammals during senescence and may result from
several factors, such as reduced energetic and cognitive capacities [141]. For example, older
chimpanzees [142] and killer whales [143] associate with fewer partners than younger adults. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 os
R. Soc. Open Sci. 10: 230219 The results on changes in partner preferences with age confirmed other studies’ findings [66,146]. Immatures groomed more with partners of similar age (both other immatures, ≤5 years old; and
peers, ± 6 months age difference) while growing older. In addition, they both spent more time in
proximity and groomed more with partners of the same sex as they aged, indicating strong social
selectivity with age. Immatures could favour similar-age and same-sex partners over other group
members because of their increasing independence from adult social partners and the benefit
of forming (potentially) long-lasting bonds. In the first case, while adults are important sources of
knowledge on what to eat [15], baboons are generally weaned and thus able to forage independently
within the first year of life [16,46], which could allow greater social selectivity. In addition, while
young immatures could benefit from the proximity of adult kin to receive agonistic support, this may
become less important with age as older immatures have established their social rank [114]. Finally,
juveniles may need less foraging time compared to adults (due to a relatively smaller body size),
having thus more time to socialize with other immatures [136,143]. In the second case, long-lasting
bonds are important because they can enhance fitness [5,6]. Bonding with individuals of similar age
(e.g. paternal kin [147]) and the same sex could translate into stable social bonds [148–150]. Philopatric females could share a large part of their lives with similarly aged females [148], while
males could disperse together at maturity [151] (see below). Age changes in immature baboon social networks differed by sex, which had generally a strong effect in
our models compared to the other variables (see electronic supplementary material, §6). The results of our
study are consistent with males’ gradual social peripheralization prior to dispersal in female-philopatric
species [57,58]. In our first analyses (H1), we found that males formed relatively weaker bonds with their
mothers and with maternal partners. First, the decrease in time spent with the mother was more
pronounced for males than for females. Male primates’ physical distancing from their mothers could
arise from the mothers directing more agonism towards sons than daughters [74] or males being more
physically precocious than females (e.g. in play [63]). These patterns may lead males to spend time
farther from the mother earlier than females. 4. Discussion It is
possible that a similar process, although for different reasons, happens during development, when
immatures narrow their association preferences from a variety of adult and immature partners to
individuals of similar age and the same sex (see below). Second, the increase in immatures’ grooming
integration with age (contrary to the proximity network) may have two possible explanations. As they
age, immatures could dedicate more time to grooming and less to play [47,144], allowing them to
groom more frequently or a wider network of troop members. Concurrently, they may become more
proficient groomers, thus becoming more desirable grooming partners [145]. 21 royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 Nevertheless, males groomed proportionally more with
their mothers than females. This pattern may derive from males’ lower grooming integration, leading
mothers to be important grooming partners for their sons. Rates for grooming given were qualitatively
higher in females than in males, but both sexes received similar amounts of grooming (see electronic
supplementary material, §8). If females receive grooming from more partners than males [146] (see
below), they may have less time to dedicate to the mother. Second, males’ proximity and grooming
networks resembled their mothers’ less closely compared to females. In female-philopatric species,
maternal kin form close bonds with each other [78,79]. These are more important for females compared
to males for two reasons: maternal kin bonds will help females inherit and maintain their social rank
(through agonistic support [152]), and they will last throughout their lives, potentially leading to fitness
benefits [2,5]. Instead, when males reach a body size greater than that of adult females (at approx. 6
years old), they attain their social rank through agonistic interactions, without the support of their
matriline [153]. Males also disperse at adulthood, leaving behind the social bonds formed in their natal
troop [38], and thus can ‘afford’ to invest less in them during juvenescence. Our second analyses (H2) showed that males became relatively less integrated in the grooming
network as they aged. Males’ relatively lower grooming network integration could derive from both
their lower tendency to groom and their higher sexual segregation compared to females. In female-
philopatric primate species, immature males groom less than females, but spend more time playing
[58]. Play has been hypothesized to be important to acquire fighting skills for immature males, who will compete for dominance [135]. Focusing on same-sex grooming partners, males’ integration may
diminish also because of lower availability of potential partners, while females share grooming more
evenly with both sexes. Juvenile males may prefer to groom with each other because male bonding
could lead them to emigrate together, increasing their probability of survival during dispersal and
integration in a new group [151,154]. Despite males’ gradual peripheralization, immatures’ grooming
integration increased with age in both sexes. This is partly explained by the fact that infants of both
sexes dedicate very little time to grooming, given that it is a behaviour learnt with time [144,145]. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 In
addition, the increase in males’ grooming activity may be explained by a similar increase in their
sexual activity during development [155]: older juveniles may copulate with adult females at the early
or late stages of their cycle (when fertility is low and females are not monopolized by a dominant
male [156]) and exchange grooming with them [157,158]. 22 The age and sex differences in network structure highlighted by our results may have important
implications for social learning, and thus the formation of culture (i.e. behaviours shared by most
members of a group and maintained through time [159]). In particular, we suggest that the emergence
of culture, according to its most common definition (i.e. at the group level; but see [160]), in female-
philopatric societies may be limited by age- and sex-driven social clustering [161]. Matrilines could be
pivotal centres of information transmission for young immatures (e.g. in vervet monkeys [162,163]; in
Japanese macaques M. fuscata [164,165]). However, the presence of social clusters around matrilines, as
our results show, could diminish social information transmission across a group and instead cause it
to cluster in matrilineal ‘subgroups’. This is because the individuals who tend to acquire information
are socially separated from those who are more likely to produce it: (i) immatures, who rely more on
social learning, and adults, who have a greater ability to produce information [15]; (ii) females, who
learn more socially, and males, who learn more asocially [75,76]. Although these considerations
remain speculative, this theoretical framework could help future studies to investigate the constraints
leading to the formation of matrilineal ‘cultures’ as opposed to group cultures [161], ultimately
questioning culture as a group-level behaviour [160]. s
R. Soc. Open Sci. 10: 230219 Ethics. Our research procedures were evaluated and approved by the Ethics Committee of the Zoological Society of
London and the Ministry of Environment and Tourism, Namibia (MET Research/Collecting Permits 1892/2014,
2009/2015, 2147/2016, 2303/2017, RPIV0039 2018/2019) and adhered to the ASAB/ABS Guidelines for the
Treatment of Animals in Behavioural Research and Teaching. Data accessibility. Research data and R scripts are available through the Dryad Digital Repository: Roatti, Vittoria;
Cowlishaw, Guy; Huchard, Elise; Carter, Alecia (2023), Social network inheritance and differentiation in wild
baboons, Dryad, Dataset, https://doi.org/10.5061/dryad.ffbg79cz1 [129]. y
p
g
y
g
The data are provided in the electronic supplementary material [166]. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 provided in the electronic supplementary material [166 The data are provided in the electronic supplementary material [166]. Authors’ contributions. V.R.: conceptualization, data curation, formal analysis, visualization and writing—original draft;
G.C.: funding acquisition, project administration and writing—review and editing; E.H.: funding acquisition, project
administration and writing—review and editing; A.C.: conceptualization, formal analysis, funding acquisition, project
administration, supervision and writing—review and editing. All authors gave final approval for publication and agreed to be held accountable for the work performed therein. Conflict of interest declaration. We declare we have no competing interests. All authors gave final approval for publication and agreed to be held accountable for the work performed therein. Conflict of interest declaration. We declare we have no competing interests. Funding. The collection of the data we present in this study was organized within the Tsaobis Baboon Project, using the
funding obtained from a Leakey Foundation Research Grant and a Templeton World Charity Foundation Grant (grant
no. TWCF0509) awarded to A.C., and a grant from the Agence Nationale de la Reserche 616 (grant nos. ANR ERS-17-
CE02-0008 and 2018–2021) awarded to E.H., with additional funding from Research England. The study was
developed thanks to a Mary Douglas Scholarship (grant no. 179210) awarded to V.R. by the Sigrid Rausing Trust. This paper is a publication of the ZSL Institute of Zoology’s Tsaobis Baboon Project. Contribution ISEM no. 202x-xxx. Acknowledgements. We are grateful to all the field assistants that worked at the Tsaobis Baboon Project between 2014
and 2019 for their invaluable help in data collection. We also thank the Ministry of Environment and Tourism for
research permission in Namibia, the Gobabeb Research and Training Centre for affiliation, the Tsaobis beneficiaries
for permission to work at Tsaobis, the Snyman and Wittreich families, and Johan Venter for permission to work on
their land. 2.
Silk JB, Alberts SC, Altmann J. 2003 Social
bonds of female baboons enhance infant
survival. Science 302, 1231–1234. (doi:10.1126/
science.1088580) 1.
Snyder-Mackler N et al. 2020 Social
determinants of health and survival in humans
and other animals. Science 368, eaax9553.
(doi:10.1126/science.aax9553) 2.
Silk JB, Alberts SC, Altmann J. 2003 Social
bonds of female baboons enhance infant
survival. Science 302, 1231–1234. (doi:10.1126/
science.1088580)
3.
Silk JB, Beehner JC, Bergman TJ, Crockford C,
Engh AL, Moscovice LR, Wittig RM, Seyfarth RM,
Cheney DL. 2009 The benefits of social capital:
close social bonds among female baboons royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 Ilany A, Holekamp KE, Akçay E. 2021 Rank-
dependent social inheritance determines social
network structure in spotted hyenas. Science
373, 348–352. (doi:10.1126/science.abc1966) 25. Kulahci IG, Rubenstein DI, Bugnyar T, Hoppitt
W, Mikus N, Schwab C. 2016 Social networks
predict selective observation and information
spread in ravens. R. Soc. Open Sci. 3, 160256. (doi:10.1098/rsos.160256) 41. de Waal FBM. 1996 Macaque social culture:
development and perpetuation of affiliative
networks. J. Comp. Psychol. 110, 147–154. (doi:10.1037/0735-7036.110.2.147) 9. Menz CS, Carter AJ, Best EC, Freeman NJ, Dwyer
RG, Blomberg SP, Goldizen AW. 2020 Higher
sociability leads to lower reproductive success in
female kangaroos. R. Soc. Open Sci. 7, 200950. (doi:10.1098/rsos.200950) 42. Jarrett JD, Bonnell TR, Young C, Barrett L,
Henzi SP. 2018 Network integration and limits
to social inheritance in vervet monkeys. Proc. R. Soc. B 285, 20172668. (doi:10.1098/
rspb.2017.2668) 26. Webber QMR, Vander Wal E. 2019 Trends and
perspectives on the use of animal social
network analysis in behavioural ecology: a
bibliometric approach. Anim. Behav. 149,
77–87. (doi:10.1016/j.anbehav.2019.01.010) 10. Brakes P et al. 2021 A deepening understanding
of animal culture suggests lessons for
conservation. Proc. R. Soc. B 288, 20202718. (doi:10.1098/rspb.2020.2718) 43. Berman BYCM. 1982 The ontogeny of social
relationships with group companions among
free-ranging infant rhesus monkeys I. Social
networks and differentiation. Anim. Behav. 30,
149–162. 11. Laland KN. 2004 Social learning strategies. Anim. Learn. Behav. 32, 4–14. (doi:10.3758/
bf03196002) 27. Rodrigues MA. 2007 Age and sex-based
differences in social interactions and spacing in
mantled howling monkeys: implications for
juvenile social development. J. Dev. Process. 2,
103–114. 12. Giraldeau L, Valone TJ, Templeton JJ. 2002
Potential disadvantages of using socially
acquired information. Phil. Trans. R. Soc. Lond. B
357, 1559–1566. (doi:10.1098/rstb.2002.1065) 44. de Waal FBM. 1990 Do rhesus mothers suggest
friends to their offspring? Primates 31,
597–600. (doi:10.1007/BF02382543) 28. Liao Z, Sosa S, Wu C, Zhang P. 2018 The
influence of age on wild rhesus macaques’
affiliative social interactions. Am. J. Primatol. 80,
e22733. (doi:10.1002/ajp.22733) 13. McComb K, Moss C, Durant SM, Baker L, Sayialel
S. 2001 Matriarchs as repositories of social
knowledge in African elephants. Science 292,
491–494. (doi:10.1126/science.1057895) 45. Ilany A, Akçay E. 2016 Social inheritance can
explain the structure of animal social networks. Nat. Commun. 7, 12084. (doi:10.1038/
ncomms12084) 29. Krzyszczyk E, Patterson EM, Stanton MA, Mann
J. 2017 The transition to independence: sex
differences in social and behavioural
development of wild bottlenose dolphins. Anim. Behav. 129, 43–59. (doi:10.1016/j.anbehav. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 4. Cheney DL, Silk JB, Seyfarth RM. 2016 Network
connections, dyadic bonds and fitness in wild
female baboons. R. Soc. Open Sci. 3, 160255. (doi:10.1098/rsos.160255) 35. Mesoudi A, Chang L, Dall SRX, Thornton A. 2016
The evolution of individual and cultural
variation in social learning. Trends Ecol. Evol. 31,
215–225. (doi:10.1016/j.tree.2015.12.012) 20. Pinter-Wollman N et al. 2014 The
dynamics of animal social networks:
analytical, conceptual, and theoretical advances. Behav. Ecol. 25, 242–255. (doi:10.1093/beheco/
art047) 5. Silk JB, Beehner JC, Bergman TJ, Crockford C,
Engh AL, Moscovice LR, Wittig RM, Seyfarth RM,
Cheney DL. 2010 Strong and consistent social
bonds enhance the longevity of female
baboons. Curr. Biol. 20, 1359–1361. (doi:10. 1016/j.cub.2010.05.067) 36. Jones JH. 2011 Primates and the evolution of
long, slow life histories. Curr. Biol. 21,
R708–R717. (doi:10.1016/j.cub.2011.08.025) 21. Croft DP, James R, Krause J. 2008 Exploring
animal social networks. Princeton, NJ: Princeton
University Press. 37. Penndorf J, Aplin L. 2020 Environmental and
life history factors, but not age, influence social
learning about food: a meta-analysis. Anim. Behav. 167, 161–176. (doi:10.1016/j.anbehav. 2020.07.001) 6. Archie EA, Tung J, Clark M, Altmann J, Alberts
SC. 2014 Social affiliation matters: both same-
sex and opposite-sex relationships predict
survival in wild female baboons. Proc. R. Soc. B
281, 20141261. (doi:10.1098/rspb.2014.1261) 22. Atton N, Hoppitt W, Webster MM, Galef BG,
Laland KN. 2012 Information flow through
threespine stickleback networks without social
transmission. Proc. R. Soc. B 279, 4272–4278. (doi:10.1098/rspb.2012.1462) 38. Cheney DL, Seyfarth RM. 2007 Baboon
metaphysics, the evolution of a social mind. Chicago, IL: The University of Chicago Press. d from https://royalsocietypublishing.org/ on 30 May 2023 23. Aplin LM, Farine DR, Morand-Ferron J, Sheldon
BC. 2012 Social networks predict patch discovery
in a wild population of songbirds. Proc. R. Soc. B 279, 4199–4205. (doi:10.1098/rspb. 2012.1591) 7. Ellis S, Franks DW, Nattrass S, Cant MA, Weiss
MN, Giles D, Balcomb KC, Croft DP. 2017
Mortality risk and social network position in
resident killer whales: sex differences and the
importance of resource abundance. Proc. R. Soc. B 284, 20171313. (doi:10.1098/rspb.2017.1313) 39. Cheney DL. 1977 The acquisition of rank and
the development of reciprocal alliances among
free-ranging immature baboons. Behav. Ecol. Sociobiol. 2, 303–318. (doi:10.1007/
BF00299742) 24. Kulahci IG, Ghazanfar AA, Rubenstein DI. 2018
Knowledgeable lemurs become more central in
social networks. Curr. Biol. 28, 1306–1310.e2. (doi:10.1016/j.cub.2018.02.079) 40. Owens NW. 1975 Social play behaviour in free-
living baboons, Papio anubis. Anim. Behav. 23,
387–408. (doi:10.1016/0003-3472(75)90087-1) 8. References 1. Snyder-Mackler N et al. 2020 Social
determinants of health and survival in humans
and other animals. Science 368, eaax9553. (doi:10.1126/science.aax9553) 1. Snyder-Mackler N et al. 2020 Social
determinants of health and survival in humans
and other animals. Science 368, eaax9553. (doi:10.1126/science.aax9553) 2. Silk JB, Alberts SC, Altmann J. 2003 Social
bonds of female baboons enhance infant
survival. Science 302, 1231–1234. (doi:10.1126/
science.1088580) enhance offspring survival. Proc. R. Soc. B 276,
3099–3104. (doi:10.1098/rspb.2009.0681) 19. Carter AJ, Torrents Ticó M, Cowlishaw G. 2016
Sequential phenotypic constraints on social
information use in wild baboons. eLife 5,
e13125. (doi:10.7554/eLife.13125)
20. Pinter-Wollman N et al. 2014 The
dynamics of animal social networks:
analytical, conceptual, and theoretical advances. Behav. Ecol. 25, 242–255. (doi:10.1093/beheco/
art047) enhance offspring survival. Proc. R. Soc. B 276,
3099–3104. (doi:10.1098/rspb.2009.0681) 19. Carter AJ, Torrents Ticó M, Cowlishaw G. 2016
Sequential phenotypic constraints on social
information use in wild baboons. eLife 5,
e13125. (doi:10.7554/eLife.13125) 34. Noble DWA, Byrne RW, Whiting MJ. 2014 Age-
dependent social learning in a lizard. Biol. Lett. 10, 20140430. (doi:10.1098/rsbl.2014.0430) 23 royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 80. 80. Tsaobis Baboon Project. 2019 See http://
tsaobisbaboonproject.org/ (accessed 16 May
2022). 52. 52. Govindarajulu P, Hunte W, Vermeer LA, Horrocks
JA. 1993 The ontogeny of social play in a feral
troop of vervet monkeys (Cercopithecus aethiops
sabaeus): the function of early play. Int. J. Primatol. 14, 701–719. (doi:10.1007/
BF02192187) 67. Bernstein IS, Judge PG, Ruehlmann TE. 1993 Sex
differences in adolescent rhesus monkey
(Macaca mulatta) behavior. Am. J. Primatol. 31,
197–210. (doi:10.1002/ajp.1350310305) 81. Cowlishaw G, Davies JG. 1997 Flora of the Pro-
Namib Desert Swakop River Catchment,
Namibia: community classification and
implications for desert vegetation sampling. J. Arid Environ. 36, 271–290. (doi:10.1006/jare. 1996.0203) 53. Suomi SJ. 2005 Mother-infant attachment, peer
relationships, and the development of social
networks in rhesus monkeys. Hum. Dev. 48,
67–79. (doi:10.1159/000083216) 68. Cheney DL. 1978 Interactions of immature male
and female baboons with adult females. Anim. Behav. 26, 389–408. (doi:10.1016/0003-
3472(78)90057-X) 82. Huchard E, Charpentier MJ, Marshall H, King AJ,
Knapp LA, Cowlishaw G. 2013 Paternal effects
on access to resources in a promiscuous primate
society. Behav. Ecol. 24, 229–236. (doi:10.1093/
beheco/ars158) 54. Cords M, Sheehan MJ, Ekernas LS. 2010 Sex and
age differences in juvenile social priorities in
female philopatric, nondespotic blue monkeys. Am. J. Primatol. 72, 193–205. (doi:10.1002/ajp. 20768) 69. Nakamichi M. 1996 Proximity relationships
within a birth cohort of immature Japanese
monkeys (Macaca fuscata) in a free-ranging
group during the first four years of life. Am. J. Primatol. 40, 315–325. (doi:10.1002/
(SICI)1098-2345(1996)40:4<315::AID-AJP2>3.0. CO;2-0) 83. Altmann J, Altmann S, Hausfater G. 1981
Physical maturation and age estimates of yellow
baboons, Papio cynocephalus, in Amboseli
National Park, Kenya. Am. J. Primatol. 1,
389–399. (doi:10.1002/ajp.1350010404) 55. Lutz MC, Ratsimbazafy J, Judge PG. 2019 Use of
social network models to understand play
partner choice strategies in three primate
species. Primates 60, 247–260. (doi:10.1007/
s10329-018-00708-7) 70. Lonsdorf EV, Anderson KE, Stantonc MA,
Shenderb M, Heintz MR, Goodall J, Murray CM. 2014 Boys will be boys: sex differences in wild
infant chimpanzee social interactions. Anim. Behav. 88, 79–83. (doi:10.1038/jid.2014.371) 84. Alberts SC, Altmann J. 1995 Preparation and
activation: determinants of age at reproductive
maturity in male baboons. Behav. Ecol. Sociobiol. 36, 397–406. (doi:10.1007/
BF00177335) 56. Handen CE, Rodman PS. 1980 Social
development of bonnet macaques from six
months to three years of age: a longitudinal
study. Primates 21, 350–356. (doi:10.1007/
BF02390465) 71. Fruteau C, Voelkl B, van Damme E, Noë R. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 2009
Supply and demand determine the market
value of food providers in wild vervet monkeys. Proc. Natl Acad. Sci. USA 106, 12 007–12 012. (doi:10.1073/pnas.0812280106) 85. Altmann J, Alberts SC. 2003 Variability in
reproductive success viewed from a life-history
perspective in baboons. Am. J. Hum. Biol. 15,
401–409. (doi:10.1002/ajhb.10157) 57. Lonsdorf EV. 2017 Sex differences in primate
behavioral development. J. Neurosci. Res. 95,
213–221. (doi:10.1002/jnr.23862) 72. Marshall H, Carter AJ, Ashford A, Rowcliffe JM,
Cowlishaw G. 2015 Social effects on foraging
behavior and success depend on local
environmental conditions. Ecol. Evol. 5,
475–492. (doi:10.1002/ece3.137710.1002/
ece3.1377) 86. Cheney DL et al. 2004 Factors affecting
reproduction and mortality among baboons in
the Okavango Delta, Botswana. Int. J. Primatol. 25, 401–428. (doi:10.1023/B:IJOP.0000019159. 75573.13) 58. Amici F, Kulik L, Langos D, Widdig A. 2019
Growing into adulthood—a review on sex
differences in the development of sociality
across macaques. Behav. Ecol. Sociobiol. 73, 18. (doi:10.1007/s00265-018-2623-2) 87. CyberTracker Conservation. 2022 See https://
cybertrackerwiki.org/ (accessed 16 May 2022). 73. Hill DA. 1999 Effects of provisioning on the
social behaviour of Japanese and rhesus
macaques: implications for socioecology. Primates 40, 187–198. (doi:10.1007/
BF02557710) 59. Toda K, Ryu H, Furuichi T. 2021 Age and sex
differences in juvenile bonobos in party
associations with their mothers at Wamba. Primates 62, 19–27. (doi:10.1007/s10329-020-
00853-y) 88. Lehmann J, Ross C. 2011 Baboon (Papio anubis)
social complexity—a network approach. Am. J. Primatol. 73, 775–789. (doi:10.1002/ajp.20967) 89. Castles M, Heinsohn R, Marshall HH, Lee AEG,
Cowlishaw G, Carter AJ. 2014 Social networks
created with different techniques are not
comparable. Anim. Behav. 96, 59–67. (doi:10. 1016/j.anbehav.2014.07.023) 74. Kulik L, Langos D, Widdig A. 2016 Mothers
make a difference: mothers develop weaker
bonds with immature sons than daughters. PLoS ONE 11, e0154845. (doi:10.1371/journal. pone.0154845) 60. Barale CL, Rubenstein DI, Beehner JC. 2015
Juvenile social relationships reflect adult patterns
of behavior in wild geladas. Am. J. Primatol. 77,
1086–1096. (doi:10.1002/ajp.22443) 90. Altmann J. 1974 Observational study of
behavior: sampling methods. Behaviour 49,
227–267. 61. Lee P, Mayagoitia L, Mondragón-Ceballos R,
Chiappa P. 2010 Sex differences in learning the
allocation of social grooming in infant
stumptailed macaques. Behaviour 147,
1073–1099. (doi:10.1163/000579510X505436) 75. Reader SM, Laland KN. 2001 Primate
innovation: sex, age and social rank differences. Int. J. Primatol. 22, 787–805. (doi:10.1023/
A:1012069500899) 91. Carter KD, Seddon JM, Frère CH, Carter JK,
Goldizen AW. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 2017.04.011) 46. Altmann J. 1980 Baboon mothers and infants,
1st edn. Chicago, IL: The University of Chicago
Press. 14. Marcoux M, Rendell L, Whitehead H. 2007
Indications of fitness differences among
vocal clans of sperm whales. Behav. Ecol. Sociobiol. 61, 1093–1098. (doi:10.1007/s00265-
006-0342-6) 47. de Lima VCC, Ferreira RG. 2021 Social network
changes during the development of immature
capuchin monkeys (Sapajus spp.). Primates 62,
801–815. (doi:10.1007/s10329-021-00918-6) 30. Boeving ER, Rodrigues MA, Nelson EL. 2020
Network analysis as a tool to understand social
development in spider monkeys. Am. J. Primatol. 82, e23181. (doi:10.1002/ajp.23182) 15. Whiten A, van de Waal E. 2018 The pervasive
role of social learning in primate lifetime
development. Behav. Ecol. Sociobiol. 72, 80. (doi:10.1007/s00265-018-2489-3) 48. Resende BDD, Izar P, Ottoni EB. 2004 Social play
and spatial tolerance in tufted capuchin
monkeys (Cebus apella). Revista de Etologia 6,
55–61. 31. Fedurek P, Lehmann J. 2017 The effect of
excluding juveniles on apparent adult olive
baboons (Papio anubis) social networks. PLoS
ONE 12, e0173146. (doi:10.1371/journal.pone. 0173146) 16. Alberts SC. 2019 Social influences on survival
and reproduction: insights from a long-term
study of wild baboons. J. Anim. Ecol. 88,
47–66. (doi:10.1111/1365-2656.12887) 49. Pereira ME. 1988 Effects of age and sex on
intra-group spacing behaviour in juvenile
savannah baboons, Papio cynocephalus
cynocephalus. Anim. Behav. 36, 184–204. (doi:10.1016/S0003-3472(88)80262-8) 32. Carter AJ, Marshall HH, Heinsohn R, Cowlishaw
G. 2014 Personality predicts the propensity for
social learning in a wild primate. PeerJ 2, e283. (doi:10.7717/peerj.283) 17. Kulahci IG, Quinn JL. 2019 Dynamic
relationships between information transmission
and social connections. Trends Ecol. Evol. 34,
545–554. (doi:10.1016/j.tree.2019.02.007) 50. Cheney DL. 1978 The play partners of immature
baboons. Anim. Behav. 26, 1038–1050. (doi:10. 1016/0003-3472(78)90093-3) 33. Thornton A, Malapert A. 2009 Experimental
evidence for social transmission of food
acquisition techniques in wild meerkats. Anim. Behav. 78, 255–264. (doi:10.1016/j.anbehav. 2009.04.021) 18. van Boekholt B, van de Waal E, Sterck EHM. 2021 Organized to learn: the influence of social
structure on social learning opportunities in a
group. iScience 24, 102117. (doi:10.1016/j.isci. 2021.102117) 51. Berman CM. 1982 The ontogeny of social
relationships with group companions among
free-ranging infant rhesus monkeys II. Sociobiol. 61, 197–204. (doi:10.1007/s00265-
006-0250-9) 66. Kulik L, Amici F, Langos D, Widdig A. 2015 Sex
differences in the development of social
relationships in rhesus macaques (Macaca
mulatta). Int. J. Primatol. 36, 353–376. (doi:10. 1007/s10764-015-9826-4) Differentiation and attractiveness. Anim. Behav. 30, 163–170. (doi:10.1016/S0003-
3472(82)80251-0) 24 royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 2013 Fission–fusion dynamics in
wild giraffes may be driven by kinship, spatial
overlap and individual social preferences. Anim. Behav. 85, 385–394. (doi:10.1016/j.anbehav. 2012.11.011) 76. Carter AJ, Lee AEG, Marshall HH, Ticó MT,
Cowlishaw G. 2015 Phenotypic assortment in
wild primate networks: implications for the
dissemination of information. R. Soc. Open Sci. 2, 140444. (doi:10.1098/rsos.140444) 62. Fairbanks LA, McGuire MT. 1985 Relationships of
vervet mothers with sons and daughters from one
through three years of age. Anim. Behav. 33,
40–50. (doi:10.1016/S0003-3472(85)80118-4) 92. Best EC, Seddon JM, Dwyer RG, Goldizen AW. 2013 Social preference influences female
community structure in a population of wild
eastern grey kangaroos. Anim. Behav. 86,
1031–1040. (doi:10.1016/j.anbehav.2013. 09.008) 63. Lonsdorf EV, Markham AC, Heintz MR, Anderson
KE, Ciuk DJ, Goodall J, Murray CM. 2014 Sex
differences in wild chimpanzee behavior emerge
during infancy. PLoS ONE 9, e99099. (doi:10. 1371/journal.pone.0099099) 77. Barrett L, Halliday J, Henzi SP. 2006 The ecology
of motherhood: the structuring of lactation costs
by chacma baboons. J. Anim. Ecol. 75, 875–886. (doi:10.1111/j.1365-2656.2006.01105.x) (
j
)
78. Silk JB, Altmann J, Alberts SC. 2006 Social
relationships among adult female baboons
(Papio cynocephalus) I. Variation in the strength
of social bonds. Behav. Ecol. Sociobiol. 61,
183–195. (doi:10.1007/s00265-006-0249-2)
79. Silk JB, Alberts SC, Altmann J. 2006 Social
relationships among adult female baboons
(Papio cynocephalus) II. Variation in the quality
and stability of social bonds. Behav. Ecol. 78. Silk JB, Altmann J, Alberts SC. 2006 Social
relationships among adult female baboons
(Papio cynocephalus) I. Variation in the strength
of social bonds. Behav. Ecol. Sociobiol. 61,
183–195. (doi:10.1007/s00265-006-0249-2) 64. Rodrigues MA. 2014 Emergence of sex-
segregated behavior and association patterns in
juvenile spider monkeys. Neotrop. Primates 21,
183–188. (doi:10.1896/044.021.0204) 93. Alberts SC, Fitzpatrick CL. 2012 Paternal care
and the evolution of exaggerated sexual
swellings in primates. Behav. Ecol. 23,
699–706. (doi:10.1093/beheco/ars052) 65. Young GH, Coelho AM, Bramblett CA. 1982 The
development of grooming, sociosexual behavior,
play and aggression in captive baboons in their
first two years. Primates 23, 511–519. (doi:10. 1007/BF02373962) 94. Altmann J, Altmann SA, Hausfater G. 1978
Primate infant’s effects on mother’s future
reproduction. Science 201, 1028–1030. (doi:10. 1126/science.98844) 79. Silk JB, Alberts SC, Altmann J. 2006 Social
relationships among adult female baboons
(Papio cynocephalus) II. Variation in the quality
and stability of social bonds. Behav. Ecol. asnipe. Methods Ecol. Evol. 4, 1187–1194. (doi:10.1111/2041-210X.12121) coalitionary support for grooming in
experimentally induced conflicts. Curr. Biol. 25,
3011–3016. (doi:10.1016/j.cub.2015.10.016) 95. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219
25 72, 130. (doi:10.1007/s00265-018-
2547-x) 130. van Noordwijk MA, van Schaik CP. 2005
Development of ecological competence in
Sumatran orangutans. Am. J. Phys. Anthropol. 127, 79–94. (doi:10.1002/ajpa.10426) 101. Blaszczyk MB. 2018 Consistency in social
network position over changing environments in
a seasonally breeding primate. Behav. Ecol. Sociobiol. 72, 11. (doi:10.1007/s00265-017-
2425-y) 115. Chowdhury MZI, Turin TC. 2020 Variable
selection strategies and its importance in clinical
prediction modelling. Fam. Med. Community
Health 8, e000262. (doi:10.1136/fmch-2019-
000262) 131. Bateson P. 1994 The dynamics of parent-
offspring relationships in mammals. Tree 9,
399–403. (doi:10.1016/0169-5347(94)90066-3) 132. Tsai YJJ, Mann J. 2013 Dispersal, philopatry, and
the role of fission-fusion dynamics in bottlenose
dolphins. Mar. Mammal Sci. 29, 261–279. (doi:10.1111/j.1748-7692.2011.00559.x) 102. Kawazoe T, Sosa S. 2019 Social networks predict
immigration success in wild Japanese
macaques. Primates 60, 213–222. (doi:10.1007/
s10329-018-0702-7) 116. Tredennick AT, Hooker G, Ellner SP, Adler PB. 2021 A practical guide to selecting models for
exploration, inference, and prediction in
ecology. Ecology 102, e03336. (doi:10.1002/
ecy.3336) 133. Borgeaud C, Sosa S, Sueur C, Bshary R. 2017
The influence of demographic variation on social
network stability in wild vervet monkeys. Anim. Behav. 134, 155–165. (doi:10.1016/j.anbehav. 2017.09.028) 103. Turner JW, Bills PS, Holekamp KE. 2018
Ontogenetic change in determinants of social
network position in the spotted hyena. Behav. Ecol. Sociobiol. 72, 10. (doi:10.1007/s00265-
017-2426-x) 117. Stanton MA, Mann J. 2012 Early social networks
predict survival in wild bottlenose dolphins. PLoS ONE 7, e47508. (doi:10.1371/journal.pone. 0047508) 134. Shizuka D, Johnson AE. 2020 How demographic
processes shape animal social networks. Behav. Ecol. 31, 1–11. (doi:10.1093/beheco/arz083) 118. Lehmann J, Majolo B, McFarland R. 2016 The
effects of social network position on the survival of
wild Barbary macaques, Macaca sylvanus. Behav. Ecol. 27, 20–28. (doi:10.1093/beheco/arv169) 104. Farine DR, Whitehead H. 2015 Constructing,
conducting and interpreting animal social
network analysis. J. Anim. Ecol. 84, 1144–1163. (doi:10.1111/1365-2656.12418) 135. Förster S, Cords M. 2005 Socialization of infant
blue monkeys (Cercopithecus mitis stuhlmanni):
allomaternal interactions and sex differences. Behaviour 142, 869–896. (doi:10.1163/
1568539055010138) 105. Croft DP, Madden JR, Franks DW, James R. 2011
Hypothesis testing in animal social networks. Trends Ecol. Evol. 26, 502–507. (doi:10.1016/j. tree.2011.05.012) 119. Thomas R, Lello J, Medeiros R, Pollard A,
Seward A, Vafidis J, Vaughan I. 2015 Data
analysis with R statistical software: a guidebook
for scientists. Eco-Explore. See http://www.eco-
explore.co.uk. 106. Brent LJN, Heilbronner SR, Horvath JE,
Gonzalez-Martinez J, Ruiz-Lambides A,
Robinson AG, Skene JHP, Platt ML. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219
25 125. Douma JC, Weedon JT. 2019 Analysing
continuous proportions in ecology and
evolution: a practical introduction to
beta and Dirichlet regression. Methods Ecol. Evol. 10, 1412–1430. (doi:10.1111/2041-
210X.13234) 110. Wooddell LJ, Kaburu SSK, Dettmer AM. 2020
Dominance rank predicts social network position
across developmental stages in rhesus monkeys. Am. J. Primatol. 82, e23024. (doi:10.1002/
ajp.23024) 96. de Vries HH. 1998 Finding a dominance order
most consistent with a linear hierarchy: a new
procedure and review. Anim. Behav. 55,
827–843. (doi:10.1006/anbe.1997.0708) 111. Berman CM. 1992 Immature siblings and
mother-infant relationships among free-ranging
rhesus monkeys on Cayo Santiago. Anim. Behav. 44, 247–258. (doi:10.1016/0003-
3472(92)90031-4) 126. Albery GF, Morris A, Morris S, Pemberton JM,
Clutton-Brock TH, Nussey DH, Firth JA. 2021
Multiple spatial behaviours govern social
network positions in a wild ungulate. Ecol. Lett. 24, 676–686. (doi:10.1111/ele.13684) 97. Silk J, Cheney D, Seyfarth R. 1999 The structure
of social relationships among female savanna
baboons in Moremi reserve, Botswana. Behaviour 136, 679–703. (doi:10.1163/
156853999501522) 112. Charpentier MJE, Huchard E, Widdig A, Gimenez
O, Sallé B, Kappeler P, Renoult JP. 2012
Distribution of affiliative behavior across kin
classes and their fitness consequences in
Mandrills. Ethology 118, 1198–1207. (doi:10. 1111/eth.12026) 127. Sosa S, Sueur C, Puga-Gonzalez I. 2021 Network
measures in animal social network analysis:
their strengths, limits, interpretations and uses. Methods Ecol. Evol. 12, 10–21. (doi:10.1111/
2041-210X.13366) 98. Huchard E, Cowlishaw G. 2011 Female–female
aggression around mating: an extra cost of sociality
in a multimale primate society. Behav. Ecol. 22,
1003–1011. (doi:10.1093/beheco/arr083) 128. Wey T, Blumstein DT, Shen W, Jordán F. 2008
Social network analysis of animal behaviour: a
promising tool for the study of sociality. Anim. Behav. 75, 333–344. (doi:10.1016/j.anbehav. 2007.06.020) 113. Harrison XA, Donaldson L, Correa-Cano ME,
Evans J, Fisher DN, Goodwin CED, Robinson BS,
Hodgson DJ, Inger R. 2018 A brief introduction
to mixed effects modelling and multi-model
inference in ecology. PeerJ 6, e4794. (doi:10. 7717/peerj.4794) 99. R Core Team. 2022 R: a language and
environment for statistical computing. Vienna,
Austria: R Foundation for Statistical Computing. See https://www.r-project.org/. 129. Roatti V, Cowlishaw G, Huchard E, Carter AJ. 2023 Social network inheritance and
differentiation in wild baboons. Dryad Digital
Repository. (doi:10.5061/dryad.ffbg79cz1) 100. Hobson EA, Avery ML, Wright TF. 2013 An
analytical framework for quantifying and testing
patterns of temporal dynamics in social
networks. Anim. Behav. 85, 83–96. (doi:10. 1016/j.anbehav.2012.10.010) 114. Maestripieri D. 2018 Maternal influences on
primate social development. Behav. Ecol. Sociobiol. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 Dezeure J, Dagorrette J, Baniel A, Carter AJ,
Cowlishaw G, Marshall HH, Martina C, Raby CL,
Huchard E. 2021 Developmental transitions in
body color in chacma baboon infants:
implications to estimate age and developmental
pace. Am. J. Phys. Anthropol. 174, 89–102. (doi:10.1002/ajpa.24118) 25 royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 158. Yu Y, Xiang ZF, Yao H, Grueter CC, Li M. 2013
Female snub-nosed monkeys exchange
grooming for sex and infant handling. PLoS ONE
8, e74822. (doi:10.1371/journal.pone.0074822) 141. Siracusa ER, Higham JP, Snyder-Mackler N, Brent
LJN. 2022 Social ageing: exploring the drivers of
late-life changes in social behaviour in
mammals. Biol. Lett. 18, 20210643. (doi:10. 1098/rsbl.2021.0643) 150. Stanton MA, Gibson QA, Mann J. 2011 When
mum’s away: a study of mother and calf ego
networks during separations in wild bottlenose
dolphins (Tursiops sp.). Anim. Behav. 82,
405–412. (doi:10.1016/j.anbehav.2011.05.026) 159. Laland K, Janik V. 2006 The animal cultures
debate. Trends Ecol. Evol. 21, 542–547. (doi:10. 1016/j.tree.2006.06.005)
ublishing. 142. Rosati AG, Hagberg L, Enigk DK, Otali E, Emery
Thompson M, Muller MN, Wrangham RW,
Machanda ZP. 2020 Social selectivity in aging
wild chimpanzees. Science 370, 473–476. (doi:10.1126/science.aaz9129) 160. Schuppli C, van Schaik CP. 2019 Animal cultures:
how we’ve only seen the tip of the iceberg. Evol. Hum. Sci. 1, e2. (doi:10.1017/ehs.2019.1) 151. Jack K, Schoof V, Isbell L. 2009 What traits
promote male parallel dispersal in primates? Behaviour 146, 701–726. (doi:10.1163/
156853908X399086) 161. van de Waal E, Renevey N, Favre CM, Bshary R. 2010 Selective attention to philopatric models
causes directed social learning in wild vervet
monkeys. Proc. R. Soc. B 277, 2105–2111. (doi:10.1098/rspb.2009.2260) 143. Weiss MN et al. 2021 Age and sex influence
social interactions, but not associations, within a
killer whale pod. Proc. R. Soc. B 288, 20210617. (doi:10.1098/rspb.2021.0617) 152. Silk JB, Alberts SC, Altmann J. 2004 Patterns of
coalition formation by adult female baboons in
Amboseli, Kenya. Anim. Behav. 67, 573–582. (doi:10.1016/j.anbehav.2003.07.001) 144. Shimada M, Sueur C. 2018 Social play among
juvenile wild Japanese macaques (Macaca
fuscata) strengthens their social bonds. Am. J. Primatol. 80, e22728. (doi:10.1002/ajp. 22728) 162. van de Waal E, Krützen M, Hula J, Goudet J,
Bshary R. 2012 Similarity in food cleaning
techniques within matrilines in wild vervet
monkeys. PLoS ONE 7, e35694. (doi:10.1371/
journal.pone.0035694) 153. Kitchen DM, Cheney DL, Seyfarth RM. 2005
Contextual factors meditating contests between
male chacma baboons in Botswana: effects of
food, friends and females. Int. J. Primatol. 26,
105–125. (doi:10.1007/s10764-005-0725-y) 145. Dunbar RIM. 2010 The social role of touch in
humans and primates: behavioural function and
neurobiological mechanisms. Neurosci. Biobehav. Rev. 34, 260–268. (doi:10.1016/j.neubiorev. 2008.07.001) 163. van de Waal E, Bshary R, Whiten A. 2014 Wild
vervet monkey infants acquire the food-processing
variants of their mothers. Anim. Behav. 90,
41–45. royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219
25 2013 Genetic
origins of social networks in rhesus macaques. Sci. Rep. 3, 1042. (doi:10.1038/srep01042) 136. Gibson QA, Mann J. 2008 The size, composition
and function of wild bottlenose dolphin
(Tursiops sp.) mother–calf groups in Shark Bay,
Australia. Anim. Behav. 76, 389–405. (doi:10. 1016/j.anbehav.2008.01.022) 120. Szumilas M. 2010 Explaining odds ratios. J. Can. Acad. Child Adolesc. Psychiatry 19, 3. 121. Brambor T, Clark WR, Golder M. 2006
Understanding interaction models: improving
empirical analyses. Polit. Anal. 14, 63–82. (doi:10.1093/pan/mpi014) 137. Silk JB. 1999 Why are infants so attractive
to others? The form and function of
infant handling in bonnet macaques. Anim. Behav. 57, 1021–1032. (doi:10.1006/anbe.1998. 1065) 107. Sosa S. 2016 The Influence of gender, age,
matriline and hierarchical rank on individual
social position, role and interactional patterns in
Macaca sylvanus at ‘La Forêt des Singes’: a
multilevel social network approach. Front. Psychol. 7, 529. (doi:10.3389/fpsyg.2016.00529) 122. Bauer DJ, Curran PJ. 2005 Probing interactions
in fixed and multilevel regression: inferential
and graphical techniques. Multivar. Behav. Res. 40, 373–400. (doi:10.1207/
s15327906mbr4003_5) 138. Dunayer ES, Berman CM. 2018 Infant handling
among primates. Int. J. Comp. Psychol. 31,
1–32. (doi:10.46867/ijcp.2018.31.02.06) 108. Hoppitt WJE, Farine DR. 2018 Association
indices for quantifying social relationships: how
to deal with missing observations of individuals
or groups. Anim. Behav. 136, 227–238. (doi:10. 1016/j.anbehav.2017.08.029) 123. Farine DR. 2017 A guide to null models for
animal social network analysis. Methods Ecol. Evol. 8, 1309–1320. (doi:10.1111/2041-210X. 12772) 139. Rapaport LG, Brown GR. 2008 Social influences
on foraging behavior in young nonhuman
primates: learning what, where, and how to
eat. Evol. Anthropol. Issues News Rev. 17,
189–201. (doi:10.1002/evan.20180) 124. Farine DR. 2013 Animal social network inference
and permutations for ecologists in R using 109. Borgeaud C, Bshary R. 2015 Wild vervet
monkeys trade tolerance and specific 140. Pereira ME. 1989 Agonistic interactions of
juvenile savanna baboons: II. Agonistic support
and rank acquisition. Ethology 80, 152–171. (doi:10.1111/j.1439-0310.1989.tb00736.x) 149. Silk JB, Beehner JC, Bergman TJ, Crockford C,
Engh AL, Moscovice LR, Wittig RM, Seyfarth RM,
Cheney DL. 2010 Female chacma baboons form
strong, equitable, and enduring social bonds. Behav. Ecol. Sociobiol. 64, 1733–1747. (doi:10. 1007/s00265-010-0986-0) 157. Gumert MD. 2007 Payment for sex in a macaque
mating market. Anim. Behav. 74, 1655–1667. (doi:10.1016/j.anbehav.2007.03.009) 26 royalsocietypublishing.org/journal/rsos
R. Soc. Open Sci. 10: 230219 (doi:10.1016/j.anbehav.2014.01.015) Downloaded from https://royalsocietypublishing.org/ on 3 154. Widdig A, Langos D, Kulik L. 2016 Sex
differences in kin bias at maturation: male
rhesus macaques prefer paternal kin prior to
natal dispersal. Am. J. Primatol. 78, 78–91. (doi:10.1002/ajp.22401) 164. Tanaka I. 1995 Matrilineal distribution of louse
egg-handling techniques during grooming in
free-ranging Japanese macaques. Am. J. Phys. Anthropol. 98, 197–201. (doi:10.1002/ajpa. 1330980208) 146. Nakamichi M. 1989 Sex differences in social
development during the first 4 years in a free-
ranging group of Japanese monkeys, Macaca
fuscata. Anim. Behav. 38, 737–748. (doi:10. 1016/S0003-3472(89)80106-X) 155. Gunst N, Leca JB, Vasey PL. 2013 Development
of sexual and socio-sexual behaviours in free-
ranging juvenile male Japanese macaques,
Macaca fuscata. Behaviour 150, 1225–1254. (doi:10.1163/1568539X-00003088) Downloaded from https://royalsocietypub 165. Tanaka I. 1998 Social diffusion of modified louse
egg-handling techniques during grooming in
free-ranging Japanese macaques. Anim. Behav. 56, 1229–1236. (doi:10.1006/anbe.1998.0891) 147. Altmann J. 1979 Age cohorts as paternal
sibships. Behav. Ecol. Sociobiol. 6, 161–164. (doi:10.1007/BF00292563) 156. Huchard E, Courtiol A, Benavides JA, Knapp LA,
Raymond M, Cowlishaw G. 2009 Can fertility
signals lead to quality signals? Insights from the
evolution of primate sexual swellings. Proc. R. Soc. B 276, 1889–1897. (doi:10.1098/
rspb.2008.1923) 166. Roatti V, Cowlishaw G, Huchard E, Carter AJ. 2023 Social network inheritance and
differentiation in wild baboons. Figshare. (doi:10.6084/m9.figshare.c.6631226) 148. Smith K, Alberts SC, Altmann J. 2003 Wild
female baboons bias their social behaviour
towards paternal half-sisters. Proc. R. Soc. Lond. B 270, 503–510. (doi:10.1098/rspb.2002.2277)
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Ultralow transmission loss in inhibited-coupling guiding hollow fibers
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Optica
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1. INTRODUCTION Since its first proposal [1], hollow-core photonic crystal fiber
(HC-PCF) has proved to be a remarkable platform for our under-
standing on how light is confined in such microstructured wave-
guides, and an outstanding host for gas–laser applications [2,3]. For the case of IC guidance, the condition of a PBG is no longer
required. In analogy with quasi-bound and bound state in a con-
tinuum (QB-BIC), first proposed by Van Neumann and Wigner in
1929 within the context of quantum mechanics [6], a core guided
mode and cladding modes can have the same effective index and
yet propagate without strong interaction. Though QB-BIC re-
mains an uncommon scheme of trapping waves, it is universal,
as shown in a recent review on the subject matter [7]. In the optical
fiber form, QB-BIC works as follows: a core guided mode (often
leaky mode) cannot, or is strongly inhibited to, channel out
through the cladding by a strong reduction in the coupling between
the cladding mode continuum and a core mode. In other words,
the dot product between the field of the core mode jφcorei and the
cladding mode jφcladi is strongly reduced (i.e., hφcladjΔn2j
φcorei →0, with Δn being the photonic structure transverse
index profile). This can be done either by having little spatial in-
tersection between the fields of jφcladi and jφcorei or by having a
strong mismatch in their respective transverse spatial phases. guides, and an outstanding host for gas laser applications [2,3]. In HC-PCF, a core-guided mode is prevented from coupling
to the cladding by either photonic bandgap (PBG) [1] or inhib-
ited-coupling (IC) [4]. In PBG guiding fibers, the coupling of the
core mode to the cladding is disallowed by microengineering the
cladding structure such that its modal spectrum is void from any
propagation modes at the core guided-mode effective index-
frequency space, neff −ω (i.e., fjφcladg Ø, with φclad being
the field wave function of the propagating mode in the cladding
structure) [1]. The lowest transmission loss of 1.2 dB/km at
1620 nm achieved with this type of HC-PCF 12 years ago is
set not by confinement loss (CL) but by surface-roughness
scattering loss (SSL) [5]. The latter scales with the amount of
the optical overlap of the guided light with the silica core contour
(typically in the range of 1%–0.1%), and increases with wave-
length shortening following ∼λ−3 scaling law. Research Article Research Article Vol. 4, No. 2 / February 2017 / Optica
209 209 Ultralow transmission loss in inhibited-coupling
guiding hollow fibers Ultralow transmission loss in inhibited-coupling
guiding hollow fibers 1GPPMM Group, Xlim Research Institute, CNRS UMR 7252, University of Limoges, Limoges, France
2GLOphotonics S.A.S, 1 avenue d’Ester, Ester Technopôle, Limoges, France
3Commissariat à l’Energie Atomique et aux Energies Alternatives, Centre d’Etudes Scientifiques et Techniques d’Aquitaine,
33116 Le Barp Cedex, France 4Department of Engineering “Enzo Ferrari,” University of Modena and Reggio Emilia, I-41125 Modena, Italy
5e-mail: benoit.debord@xlim.fr *Corresponding author: f.benabid@xlim.fr Received 21 September 2016; revised 22 December 2016; accepted 2 January 2017 (Doc. ID 275015); published 8 February 2017 Received 21 September 2016; revised 22 December 2016; accepted 2 January 2017 (Doc. ID 275015); publ Received 21 September 2016; revised 22 December 2016; accepted 2 January 2017 (Doc. ID 275015); published 8 February 2017 Attenuation in photonic bandgap guiding hollow-core photonic crystal fiber (HC-PCF) has not beaten the funda-
mental silica Rayleigh scattering limit (SRSL) of conventional step-index fibers due to strong core-cladding optical
overlap, surface roughness at the silica cladding struts, and the presence of interface modes. Hope has been revived
recently by the introduction of hypocycloid core contour (i.e., negative curvature) in inhibited-coupling guiding HC-
PCF. We report on several fibers with a hypocycloid core contour and a cladding structure made of a single ring from a
tubular amorphous lattice, including one with a record transmission loss of 7.7 dB/km at ∼750 nm (only a factor ∼2
above the SRSL) and a second with an ultrabroad fundamental band with loss in the range of 10–20 dB/km, spanning
from 600 to 1200 nm. The reduction in confinement loss makes these fibers serious contenders for light transmission
below the SRSL in the UV–VIS–NIR spectral range and could find application in high-energy pulse laser beam
delivery or gas-based coherent and nonlinear optics. © 2017 Optical Society of America OCIS codes: (060.2280) Fiber design and fabrication; (060.5295) Photonic crystal fibers; (060.2270) Fiber characterization. https://doi.org/10.1364/OPTICA.4.000209 drawing smaller-pitch PBG HC-PCF with comparable core–
surface uniformity to those operating at longer wavelengths. 2334-2536/17/020209-09 Journal © 2017 Optical Society of America Ultralow transmission loss in inhibited-coupling
guiding hollow fibers
B. DEBORD,1,2,5 A. AMSANPALLY,1 M. CHAFER,1,2 A. BAZ,1 M. MAUREL,1,2 J. M. BLONDY,1
E. HUGONNOT,3 F. SCOL,3 L. VINCETTI,4 F. GÉRÔME,1,2 AND F. BENABID1,2,*
1GPPMM Group, Xlim Research Institute, CNRS UMR 7252, University of Limoges, Limoges, France
2GLOphotonics S.A.S, 1 avenue d’Ester, Ester Technopôle, Limoges, France
3Commissariat à l’Energie Atomique et aux Energies Alternatives, Centre d’Etudes Scientifiques et Techniques d’Aquitaine,
33116 Le Barp Cedex, France
4Department of Engineering “Enzo Ferrari,” University of Modena and Reggio Emilia, I-41125 Modena, Italy
5e-mail: benoit.debord@xlim.fr OCIS codes: (060.2280) Fiber design and fabrication; (060.5295) Photonic crystal fibers; (060.2270) Fiber ch 2. FIBER PARAMETER DESIGN The left panel of Fig. 1(a) shows an example of the SR-TL
HC-PCF that we are exploring. To understand and determine
the design parameters for fiber optimal guidance, we first examine
the nature of its cladding modes. For this purpose, one can con-
sider the SR-TL HC-PCF as an air defect (i.e., core) surrounded
by one ring of an amorphous lattice of isolated tubes (i.e., clad-
ding) [21,22]. In order to calculate the modal spectrum of such an
amorphous lattice, we exploit the fact that we operate in the large
pitch regime [8,23]. Under this regime, the photonic structure is
primarily governed by the modal properties of the lattice
single unit rather than their arrangement in the lattice [23]. Consequently, the modal spectrum of the SR-TL HC-PCF clad-
ding can be determined by considering an infinite lattice of any
symmetry arrangement [see Supplement 1 for the numerical cor-
roboration (Section 1) and its entailed physical background
(Section 2)]. Here, we take a triangular arrangement to calculate
the modal spectrum of the SR-TL HC-PCF cladding [see central
and right panels of Fig. 1(a)]. g
p
g
Figure 1(b) maps the photonic structure propagation modes in
the space neff −F for a lattice of isolated glass tubes of refractive
index ng 1.45 and with representative radius Rt 7 μm,
thickness t 400 nm, and gap between two adjacent tubes
δ 4 μm (corresponding to a lattice pitch of 18 μm, which is
much larger than any of our operating wavelengths). Here, F
2t∕λ
ffiffiffiffiffiffiffiffiffiffiffiffi
n2g −1
q
is a normalized frequency relative to the tube
thickness and refractive index of the glass and air. The plot shows
the density of photonic states (DOPS) normalized to that of the
vacuum [24] (see Supplement 1 for calculation details). The
DOPS diagram shows no PBG regions in the mapped neff −F
space, but a quasi-continuum of propagating modes. The nature
of these modes is revealed in Fig. 1(c) by showing the transverse
profile of their field magnitude for two frequencies (F 0.8 and
F 1.2) at two representative sets of neff. Within the first set
[from 1.2 to 1.3; see the shaded light-red rectangle of Fig. 1(c)],
the modes are all located in the silica tube as expected and are
identifiable in the DOPS diagram by their steep dispersion curves
(close to vertical). 2. FIBER PARAMETER DESIGN The second set of neff crosses the vacuum-line
(i.e., neff 1) and ranges from 1.04 to 0.99 [see the dotted blue
rectangle of Fig. 1(c)]. Within this neff range, the modal content
exhibits, in addition to the just-discussed silica modes, modes re-
siding in air having flat dispersion curves (close to horizontal). Furthermore, and except for F ≈1, air-residing modes seem to
have little interaction with the silica modes as shown by the dis-
tinguishable profile of their dispersion curves. This is in contrast
with the strong hybridization between the air and silica modes at
F ≈1 as shown by the “anti-crossing shape” in the DOPS. This DOPS modal spectral structure, and especially the non-
hybridization of modes from two different materials like air
and silica, can be explained by considering the transverse wave g y
These features for IC guidance are also fulfilled in a tubular lat-
tice, which consists of an arrangement of isolated thin glass tubes
[17], and its fiber form exhibits a hypocycloid core contour [13]. More crucially, when the cladding is reduced to a single ring of
an aperiodic lattice of such tubes, one can fabricate a fiber with a
core contour that is completely void of any connecting nodes
(as proposed in [18]), which strongly favors IC [4,13]. As a matter
of fact, because of the very weak HE11 core-mode field overlap with
the cladding in IC guiding fibers, low confinement loss can be ex-
pectedwith only one claddingring. This isexperimentally illustrated
by the results obtained with a hollow-core fiber having a single ring
of this cladding amorphous lattice [13,14]. In these works, the
fiber cladding tubes were relatively thick (>1 μm), and the achieved
transmission loss was as low as 34 dB/km in a 3–4 μm spectral range. More recent works achieved 30 dB/km at 1 μm using tube thickness
of 830 nm [19] and broad fundamental band with thinner tubes
(down to thickness of 360 nm) [20]. Here, by optimizing such a tubular cladding and operating
with a much thinner glass tube than has previously been achieved,
we report on the fabrication of several ultralow-loss single-ring
tubular lattice HC-PCFs (SR-TL HC-PCF) guiding in the
UV–VIS and NIR. 1. INTRODUCTION Furthermore, all
reported PBG HC-PCFs operating below 800 nm have transmis-
sion loss higher than 100 dB/km because of the difficulty of In a previous paper [4], the design parameters for optimal IC
guidance in air–silica HC-PCF such as Kagome lattice cladding 2334-2536/17/020209-09 Journal © 2017 Optical Society of America Research Article Vol. 4, No. 2 / February 2017 / Optica 210 have been laid down. It has been shown that IC guidance is
enhanced by having a glass microstructure exhibiting a very thin
and elongated glass–membrane network with a minimum num-
ber of sharp bends or nodes, and by operating in the large-pitch
regime or high normalized frequency (i.e., the lattice pitch is
larger than the optical wavelength λ) [8]. These structural features
ensure modes with strong localization in the thin silica web and
with a very fast transverse field oscillation (i.e., high azimuthal-
like number). Furthermore, unlike with PBG, the criterion
hφcladjΔn2jφcorei →0 implies a CL with a strong dependence
on the core-mode profile, and thus the core contour. This led
to the seminal introduction of hypocycloid core-contour design
(i.e., negative curvature) in 2010 [9,10], and which resulted in a
dramatic reduction in transmission loss, as illustrated in the
17 dB/km at 1 μm achieved with Kagome lattice HC-PCF
and its record optical power handling [11,12]. This in turn
has triggered a wide effort to design hollow-core fibers with neg-
ative curvature [13,14]. In a hypocycloid core-contour fiber, the
core has a contour exhibiting a set of alternating negative curva-
ture cups with inner radius Rin, and a field overlap integral
between the core mode and the highly oscillating silica core-sur-
round mode (cladding mode) that is strongly reduced compared
to the previous circular or hexagonal shape in Kagome HC-PCF
[8]. Three reasons explain such a reduction. First, the HE11 mode
field spatial overlap with silica at Rin is reduced to the tangent
sections of the inner cups of the hypocycloid contour. Second,
the larger perimeter of the hypocycloid contour compared to a
circle with radius Rin results in a higher azimuthal-like number
in the silica core-surround modes, and hence stronger transverse
phase mismatch with the core mode. Finally, the spatial overlap
between the core mode with connecting nodes—which support
low azimuthal number modes—is strongly reduced [10,15,16]. 1. INTRODUCTION We count a second fiber that exhibits an ultrabroad fundamental
band with loss range of 10–20 dB/km over one octave spanning
from 600 to 1200 nm. Both fibers present a near single-
modedness, with ∼20 dB extinction between HE11 fundamental
core mode and the strongest first higher-order mode (HOM), and
exhibit bend loss of 0.03 dB/turn for a 30 cm bend diameter at
750 nm. Finally, the results indicate that the SSL is currently the
limiting factor for wavelengths shorter than 1 μm. 2. FIBER PARAMETER DESIGN The vertical axes are the lattice mode
effective indices at F 0.8 and F 1.2. The displayed profiles correspond to modes with effective index above (dashed red box) and crossing (dotted
blue box) the vacuum line. The label on the top left of the mode profile corresponds to the nature of the mode (HE or EH). The numbers at the bottom of
the mode profile correspond, respectively, to the mode effective index and to the indices of the azimuthal and radial components of the transverse wave
number, respectively (m, l). the fiber air core modes in the same manner as with those of
the cladding tube. Consequently, the fiber transmission will
exhibit band edges at F ≈l, corresponding to phase matching
with cladding modes having m nil or close to zero. This transverse
phase-matching
condition
can
be
written
in
the
form
λl−1 2t∕l −1
ffiffiffiffiffiffiffiffiffiffiffiffi
n2g −1
q
, which is used in some of the current
literature as a defining feature of fibers guiding via anti-resonance
reflecting optical waveguide (ARROW) [23,27]. In Supplement 1
(Section 3), we provide some distinctive properties to differentiate
between PBG, IC, and ARROW, and discuss why the latter is not
appropriate in comprehensibly describing the guidance in fibers
such as SR-TL HC-PCF. Conversely, in each band, the fiber con-
finement loss αCL is set by the quantity hφcladjΔn2jφcorei, through
αCL ∝jhφcladjΔn2jφcoreij2 [28], which quantifies the strength of
the power coupling between the core and cladding modes [4]. Similar to coupled mode theory results [29,30], hφcladjΔn2jφcorei
scales with the ratio between the normal modes’ transverse phase
mismatch and their field spatial overlap. In Supplement 1
(Section 5) we show that αCL ∝DO∕gm for the fiber-core
HE11, with DO being the optical overlap of HE11 with the clad-
ding silica dielectric and gm being a positive and increasing
function of m. vector components of each mode. Here, the field profile and
dispersion of each mode far from an anti-crossing region can
be approximated to HEml (i.e., electric field direction is azimu-
thal) or EHml (i.e., electric field direction is radial) given in [25]
for the silica modes, and to those given by Marcatili and
Schmeltzer for the air modes [26]. The suffixes m and l are
the indices of the azimuthal and radial components of the trans-
verse wavenumber, respectively. 2. FIBER PARAMETER DESIGN Among the fabricated fibers, we count one
with a record transmission loss of 7.7 dB/km at ∼750 nm, which
is only 4 dB above the Rayleigh scattering fundamental limit in
silica, and observed guidance down to 220 nm. The loss figure of
7.7 dB/km is lower than ∼12 dB∕km, which is the PBG pro-
jected loss at 750 nm. It is also more than one order of magnitude
less than any reported PBG HC-PCF guiding below 800 nm. Vol. 4, No. 2 / February 2017 / Optica
211 Vol. 4, No. 2 / February 2017 / Optica
211 211 Fig. 1. (a) Schematic of HC-PCF with a single-ring tubular amorphous lattice (i), section of a tubular lattice in a triangular arrangement (ii), and the
details of its unit cell (iii). δ is the inter-tube gap distance, Rt is the tube radius, and t is the tube ring thickness. (b) Density of photonic state (DOPS) of an
infinite tubular lattice in triangular arrangement. The DOPS value is normalized to that of the vacuum. (c) Transverse profiles of the electric field
magnitude of the photonic state (modes) for F 0.8 (fundamental band) and F 1.2 (first higher-order band). The vertical axes are the lattice mode
effective indices at F 0.8 and F 1.2. The displayed profiles correspond to modes with effective index above (dashed red box) and crossing (dotted
blue box) the vacuum line. The label on the top left of the mode profile corresponds to the nature of the mode (HE or EH). The numbers at the bottom of
the mode profile correspond, respectively, to the mode effective index and to the indices of the azimuthal and radial components of the transverse wave
number, respectively (m, l). y
p Fig. 1. (a) Schematic of HC-PCF with a single-ring tubular amorphous lattice (i), section of a tubular lattice in a triangular arrangement (ii), and the
details of its unit cell (iii). δ is the inter-tube gap distance, Rt is the tube radius, and t is the tube ring thickness. (b) Density of photonic state (DOPS) of an
infinite tubular lattice in triangular arrangement. The DOPS value is normalized to that of the vacuum. (c) Transverse profiles of the electric field
magnitude of the photonic state (modes) for F 0.8 (fundamental band) and F 1.2 (first higher-order band). 2. FIBER PARAMETER DESIGN When the silica modes are lon-
gitudinally phase-matched with an air mode (i.e., they have equal
neff), and considering our case of wavelength much smaller than
the radius of the tube, m and l are related via the following
identity (see Supplement 1, Section 2): m
2 Rt
2
l −1 π
t
2
≈2π∕λ2n2g −1:
(1) (1) Furthermore, the silica and air modes strongly hybridize when
they are also transversely phase-matched. For the case of low-azi-
muthal-number air mode such as HE11, this occurs when the
silica mode azimuthal number is nil or close to zero [17]. This
means that the radial number takes the identity l −1π∕t ≈
2π∕λ
ffiffiffiffiffiffiffiffiffiffiffiffi
n2g −1
q
or F ≈l −1 (see Supplement 1, Section 2). As shown in the DOPS diagram near the vacuum line, the
just-shown relations split the lattice modal spectrum into radial
number indexed bands located between F ≈l −1 and F ≈l. Each band is populated by silica modes characterized with a fixed
l and azimuthal number given by Eq. (1). These transverse wave-
number indices are also defining parameters for the transmission
spectral structure and confinement loss of a HC-PCF whose clad-
ding is made up of such a tubular amorphous lattice. Indeed,
Eq. (1) and the transverse phase-matching condition hold for Furthermore, the silica and air modes strongly hybridize when
they are also transversely phase-matched. For the case of low-azi-
muthal-number air mode such as HE11, this occurs when the
silica mode azimuthal number is nil or close to zero [17]. This
means that the radial number takes the identity l −1π∕t ≈
2π∕λ
ffiffiffiffiffiffiffiffiffiffiffiffi
n2g −1
q
or F ≈l −1 (see Supplement 1, Section 2). A
h
h
DOPS d
h
l
h Furthermore, the silica and air modes strongly hybridize when
they are also transversely phase-matched. For the case of low-azi-
muthal-number air mode such as HE11, this occurs when the
silica mode azimuthal number is nil or close to zero [17]. This
means that the radial number takes the identity l −1π∕t ≈
2π∕λ
ffiffiffiffiffiffiffiffiffiffiffiffi
n2g −1
q
or F ≈l −1 (see Supplement 1, Section 2). As shown in the DOPS diagram near the vacuum line, the
just-shown relations split the lattice modal spectrum into radial
number indexed bands located between F ≈l −1 and F ≈l. 2. FIBER PARAMETER DESIGN Figure 2(a) illustrates the effect of increasing δ on the cladding
modal spectrum by the evolution of the electric field magnitude of
a representative silica mode [EH9;2 at wavelength of 700 nm for
t 400 nm (i.e., F 1.2) and tube radius of Rt 7 μm]. The
results show that increasing δ from 2 to 8 μm results in a decrease of
the mode neff from ∼1.00062 to 0.99993 and stronger light con-
finement in the silica tube, as shown in the 1D profile along a gap
line joining the center of two adjacent tubes [z line in Fig. 2(a),
bottom panel]. This trend favors larger inter-tube gap for IC;
however, this would make sense only if the fiber core mode profile
is not altered and kept its zero-order Bessel profile. Figure 2(b)
shows the evolution of the HE11 core mode of a single-ring tube
lattice fiber with eight tubes when the gap is increased. The results
show that increasing δ from 2 to 8 μm leads to an increase of the
mode-field radius relative to the fiber core radius Rc by almost 6%,
which entails a stronger optical spatial overlap of the mode with the
silica. The bottom panel of Fig. 2(b) clearly shows the increase with
δ of field magnitude along the gap line, indicative of the stronger
spread of the core mode in the cladding. Equally, we investigated
the effect of δ on the core mode leakage by calculating the power
flux along the fiber radial and transverse directions using the
Poynting vector (see Supplement 1, Section 4). The results clearly
demonstrate that the transverse power leakage of the HE11 core
mode at the gaps strongly increases with increasing δ, and enhances
by up to 30 dB when δ is increased from 2 to 8 μm for
λ 530 nm. The results also show that the primary power leakage g
p
The top of Fig. 3 summarizes the effect of N and δ on the fiber
confinement loss. Figure 3(a) shows the CL over a representative
spectrum for different N and for fibers with fixed t 400 nm,
Rt 8 μm, and δ 5 μm. The loss is expressed as αCLR4c so as
to only keep the effect of N on the transmission loss trend with
increasing N independent from the associated fiber core radius
change. 2. FIBER PARAMETER DESIGN (a) Field amplitude transverse profile of a unit cell of tubular lattice (top left). First row shows its evolution with inter-tube gap δ when varied
from 2 to 8 μm, and second row shows 1D profile along the gap line (labeled “z”) between the center of two adjacent tubes. (b) Field amplitude transverse
profile of a fiber core with tubular amorphous lattice cladding (bottom right schematics) and its evolution with δ (third row). MFR/RC is the ratio
between the mode-field radius (MFR) and the fiber core inner radius Rc. 1D profile of the fiber core mode along a cladding inter-tube line (labeled “z”). Here, the wavelength of the modes is 700 nm. Fig. 2. (a) Field amplitude transverse profile of a unit cell of tubular lattice (top left). First row shows its evolution with inter-tube gap δ when varied
from 2 to 8 μm, and second row shows 1D profile along the gap line (labeled “z”) between the center of two adjacent tubes. (b) Field amplitude transverse
profile of a fiber core with tubular amorphous lattice cladding (bottom right schematics) and its evolution with δ (third row). MFR/RC is the ratio
between the mode-field radius (MFR) and the fiber core inner radius Rc. 1D profile of the fiber core mode along a cladding inter-tube line (labeled “z”). Here, the wavelength of the modes is 700 nm. tradeoff between these parameters, we start by finding out
the optimal δ, then the number N, and finally the fiber core
radius. channels are the touching nodes between the tube and the outer
silica jacket for δ < 4 μm, and the inter-tube gap for δ > 6 μm. Consequently, and given the stronger impact of the core radius on
the confinement loss (αCL ∝R−4
c [31]) compared to that induced
by the observed cladding modal change, the optimal δ will be a
tradeoff between a value that is sufficiently small to avoid a core
mode leakage being too high, and sufficiently large to avoid a cou-
pling between the tubes being too strong or the formation of con-
necting nodes (which support low-azimuthal-number modes)
during the fabrication process. 2. FIBER PARAMETER DESIGN Each band is populated by silica modes characterized with a fixed
l and azimuthal number given by Eq. (1). These transverse wave-
number indices are also defining parameters for the transmission
spectral structure and confinement loss of a HC-PCF whose clad-
ding is made up of such a tubular amorphous lattice. Indeed,
Eq. (1) and the transverse phase-matching condition hold for As shown in the DOPS diagram near the vacuum line, the
just-shown relations split the lattice modal spectrum into radial
number indexed bands located between F ≈l −1 and F ≈l. Each band is populated by silica modes characterized with a fixed
l and azimuthal number given by Eq. (1). These transverse wave-
number indices are also defining parameters for the transmission
spectral structure and confinement loss of a HC-PCF whose clad-
ding is made up of such a tubular amorphous lattice. Indeed,
Eq. (1) and the transverse phase-matching condition hold for Furthermore, from Eq. (1), it is easy to note that increasing m
can be achieved by either increasing the silica tube radius or
increasing t∕λ, which is consistent with the results reported in
[9,10,31]. However, because Rt is interconnected with the
fiber core radius Rc, δ, and the number of tubes in the
cladding ring N through the identity Rt Rc sinπ∕N −δ∕2
1 −sinπ∕N−1, its increase can have reverse impact on both
the fiber CL and modal content. In order to find the best Fig. 2. (a) Field amplitude transverse profile of a unit cell of tubular lattice (top left). First row shows its evolution with inter-tube gap δ when varied
from 2 to 8 μm, and second row shows 1D profile along the gap line (labeled “z”) between the center of two adjacent tubes. (b) Field amplitude transverse
profile of a fiber core with tubular amorphous lattice cladding (bottom right schematics) and its evolution with δ (third row). MFR/RC is the ratio
between the mode-field radius (MFR) and the fiber core inner radius Rc. 1D profile of the fiber core mode along a cladding inter-tube line (labeled “z”). Here, the wavelength of the modes is 700 nm. Research Article
Vol. 4, No. 2 / February 2017 / Optica
212 Research Article
Vol. 4, No. 2 / February 2017 / Optica
212 Vol. 4, No. 2 / February 2017 / Optica
212 212 Fig. 2. 3. EXPERIMENTAL RESULTS Figure 4(a) shows the micrographs of four fabricated SR-TL
HC-PCFs along with their physical properties. The experimental
fibers have been fabricated using the stack-and-draw technique. Here, we endeavored to vary δ while keeping Rt, t, and Rc as
constant as possible so as to find out the optimal value from
the standpoint of fabrication and transmission performance. The table summarizing these values for the different fibers shows
that δ spans from 2.7 μm (Fiber #4, pink-colored micrograph
frame) to 8 μm (Fiber #1, green-colored micrograph frame). Within this δ span, the core radius Rc ranges from 13.75 to
18.5 μm, corresponding to a maximum relative difference be-
tween the different fiber core sizes of less than 35%. Similarly,
the cladding tube radius Rt and t range from 5.1 μm to
6.15 μm and from 415 nm to 580 nm, respectively, with a maxi-
mum relative difference less than 20% and 40%, respectively. Figure 4(b) shows the four fibers’ measured loss spectra versus
λ∕t. As expected, for the measured wavelength range of 400–
1750 nm and tube thickness of 415–580 nm, the spectra show
three transmission bands corresponding to the fundamental band
(λ∕t > 2), first-order band (1 < λ∕t < 2), and second-order
band (λ∕t < 1). The different fiber-loss curves in the fundamen-
tal and first-order bands clearly demonstrate a dramatic reduction Fig. 3. (a) CL for different N. (b) Ratio between TE01 and HE11 loss. (c) Loss spectra versus δ. (d) Loss and optical overlap of HE11 with the
cladding silica dielectric (DO) spectra for different t values (200, 400,
and 500 nm). Fig. 4. (a) SEM pictures of four fabricated HC tubular lattice
fibers with corresponding geometrical parameters (δ, Rt, t, Rc)
(b) Experimental evolution of the loss spectrum with the gap between
the tubes. proportionally with N; the fiber single-modedness, which is
inversely proportional to N; and bend loss sensitivity, which
favors smaller fiber core size and thus smaller N. We found
N 8 to be a good compromise for a sufficiently low CL
and a modal content dominated by the HE11 mode. Figure 3(c)
shows the CL evolution with δ at two representative wavelengths
of
a
SR-TL
HC-PCF
with
N 8,
t 400 nm,
and
Rt 8 μm. The results show that the inter-tube gap range of
2–6 μm is suitable for low-loss guidance. 2. FIBER PARAMETER DESIGN The results show that, for the same core radius, the
CL decreases with increasing N. However, the decrease rate of
the CL with N drops as N gets larger. For example, choosing
a wavelength of 540 nm, αCLR4c decreases by a factor of 3 when
N is changed from 5 to 6. However, it decreases by less than 20%
when the tube number is increased from 8 to 9. This is due to the
resonance between the core mode and the air modes of the tubes
[15,17]. On the other hand, the power ratio of the higher-order
core modes TE01 and HE11, which is an indicator of how well the
fiber can operate in a single-mode manner, increases with decreas-
ing N, thus favoring a smaller number of tubes for single-mode
operation [see Fig. 3(b)]. Consequently, the optimal N is the
result
of
a
tradeoff
between
CL
decrease,
which
scales Research Article Vol. 4, No. 2 / February 2017 / Optica
213 213 Fig. 3. (a) CL for different N. (b) Ratio between TE01 and HE11 loss. (c) Loss spectra versus δ. (d) Loss and optical overlap of HE11 with the
cladding silica dielectric (DO) spectra for different t values (200, 400,
and 500 nm). 3. EXPERIMENTAL RESULTS In order to address this question, we considered
the simulation of several SR-TL HC-PCFs with their tubes ex-
hibiting corrugated surfaces to mimic a surface roughness along
the azimuthal direction (see Supplement 1, Section 7 for more
details). The results show that CL changes little for wavelengths
larger than 800 nm but increases for shorter wavelengths, consis-
tent with the observed higher transmission loss at short wave-
lengths in the early IC guiding hypocycloid core-contour HC-
PCF [9–11,15,33]. For the second-order band, the trend of the loss decrease with
δ is observed only for δ range of 5–8 μm. For smaller inter-tube
gaps corresponding to tube thicknesses t smaller than 500 nm, the
loss increases with δ decrease (e.g., orange curve compared to the
blue curve). Furthermore, a comparison of the rate at which
the loss decreases with the gap shortening shows that the loss
in the fundamental band decreases at a larger rate than that in
the first-order band. As a matter of fact, it plateaus down to
15 dB/km for δ range of 3.7–2.7 μm (see pink and orange curves). Finally, because the attenuation due to the surface roughness scat-
tering is not taken into account in the calculated CL, the mea-
sured loss spectra don’t necessarily decrease with decreasing
wavelength. This scattering loss stems from the fiber core surface
roughness due to frozen capillary waves during the fiber draw [5]
and takes the form of αSSLλ ς · F ccλ · λ−3 [5,32], with ς
being a constant related to the surface roughness root-mean-
square height and F ccλ being the core mode overlap with
the core contour. In the case of IC guiding HC-PCF, the Now that δ value impact on the transmission loss of fabricated
fibers is demonstrated, we use δ ∼2.5 μm as a parameter target
and undertake two fiber-fabrication campaigns with different
aims. The first aim consists of the fabrication of fibers with
the thinnest tubes possible so as to have the broadest fundamental
band while having loss figures as low as the surface-roughness-
induced transmission loss could permit. The second aim is to have
the lowest loss figure possible in the NIR–VIS spectral range. This
was undertaken in an iterative process of several fiber draws. As
mentioned previously, having a too-thin cladding tube (typically ig. 5. SEM pictures of (a) Fiber #5 and (b) Fiber #6. 3. EXPERIMENTAL RESULTS Based on these design
results, we calculated the CL spectra for different tube thicknesses. Figure 3(d) shows the spectra of CL (top) and DO (bottom)
at different tube thicknesses (t 200, 400, and 500 nm) for
a SR-TL HC-PCF with N 8, Rt 8 μm, and δ 2.5 μm. Within the explored wavelength range of 400–1750 nm, the t
200 nm fiber exhibits one large transmission band corresponding
to the fundamental band with a minimum loss of ∼1 dB∕km at
wavelengths near 600 nm. For the t 400 nm SR-TL HC-PCF,
the spectrum exhibits two transmission bands separated by a high-
loss band centered around 800 nm. Here, the lowest-loss figure is
0.5 dB/km and occurs at wavelengths near 500 nm in the first
higher-order transmission band. For t 500 nm SR-TL HC-
PCF, we observe a spectrum with the three transmission bands
and lowest loss of 0.3 dB/km occurring in the second higher-order
band around 480 nm. Below, we explore these results and scaling
laws in the fabrication of several fibers with N 8. The DO
spectra show the same resonance structure as CL as expected
by IC guidance, and values in the range of 10−5–10−6. The mini-
mum obtainable loss can be reduced by further thinning the tube
thickness and operating in the shorter wavelength as the silica
cladding modes get more localized with F increase. Fig. 4. (a) SEM pictures of four fabricated HC tubular lattice
fibers with corresponding geometrical parameters (δ, Rt, t, Rc). (b) Experimental evolution of the loss spectrum with the gap between
the tubes. Finally, the consistency of the observed CL slope with wave-
length between fibers with different t and the scaling law m ∝1∕t
is noteworthy (see Supplement 1, Section 6). Research Article Vol. 4, No. 2 / February 2017 / Optica 214 of the optical attenuation with δ decrease, from near 1 dB/m
loss level for δ 8 μm (green curve) down to only a few tens
of dB/km for δ 2.7 μm. confinement loss dependence on the core mode overlap with
the cladding raises the question of whether the surface roughness
affects the CL. 3. EXPERIMENTAL RESULTS Transmission curves along the two fiber pieces recorded during cut-back measurement for (c)(i)
iber #5 and (d)(i) Fiber #6. Measured attenuation spectra (black curves) and calculated CL (dotted blue curves) for (c)(ii) Fiber #5 and (d)(ii) Fiber #6. iber #5 reaches a record of 7.7 dB/km, and Fiber #6 exhibits a loss in the range 10–20 dB/km over one octave [(d)(ii)]. The blanked data around 1 μm in
d)(ii) are due to the stronger supercontinuum power at 1064 nm. Bottom: Measured transmission of a 2 m long piece of (e) Fiber #5 and (f) Fiber #6,
with purple-filled curve highlighting UV/DUV guidance. Fig. 5. SEM pictures of (a) Fiber #5 and (b) Fiber #6. Transmission curves along the two fiber pieces recorded during cut-back measurement for (c)(i)
Fiber #5 and (d)(i) Fiber #6. Measured attenuation spectra (black curves) and calculated CL (dotted blue curves) for (c)(ii) Fiber #5 and (d)(ii) Fiber #6. Fiber #5 reaches a record of 7.7 dB/km, and Fiber #6 exhibits a loss in the range 10–20 dB/km over one octave [(d)(ii)]. The blanked data around 1 μm in
(d)(ii) are due to the stronger supercontinuum power at 1064 nm. Bottom: Measured transmission of a 2 m long piece of (e) Fiber #5 and (f) Fiber #6,
with purple-filled curve highlighting UV/DUV guidance. Research Article 215 Vol. 4, No. 2 / February 2017 / Optica
21 In addition to the just-shown low transmission loss, these
fibers present a low bending sensitivity of less than 0.05 dB/turn
for a 30 cm bend diameter over the whole transmission band, and
reach 0.03 dB/turn at 750 nm for Fiber #5 (see Supplement 1). The modal content has also been characterized using spectral and
spatial (S2) imaging techniques [34,35]. Figure 6(a) shows the
typical S2 modal content and the evolution with the group delay
of the interference signal Fourier transform for fiber length of
L 5 m and L 15 m from Fiber #6. The S2 imaging system
comprises an InGaAs camera and tunable laser source with a tun-
ing range of 1010–1070 nm and minimum step size of 40 pm. At
the output of the fiber, a telescope is used to collect the light re-
corded through the camera, which is triggered directly from the
laser and controlled by a PC. 3. EXPERIMENTAL RESULTS For 5 m long fiber, the LP11 mode
presents a multi-path interference (MPI) of 21.4 dB, which cor-
responds to a quasi-single-mode operation and increases to
22.4 dB for a length of 15 m. <250 nm) poses fabrication challenges in keeping the circular
shape of the tubes and also increases the surface scattering through
larger surface roughness that results from an enhanced surface
capillary wave during the draw process. Consequently, reaching
the lowest loss in the shortest wavelength will be a compromise
between CL (i.e., design limited) and SSL (i.e., fabrication lim-
ited). The results of this fabrication campaign are summarized in
Fig. 5. The source used is a supercontinuum which consists of a
piece of a nonlinear PCF and a nanosecond pulsed microchip laser
providing an average spectral power around 100 mW with a sta-
bility <1%. The fiber under test was coiled into a 60 cm bend
curvature and the fiber input ends were prepared using a com-
mercial cleaver. For each transmission measurement, 10 fiber
cleaves were done, demonstrating an error measurement under
the resolution of the optical spectrum analyzer (Ando AQ
6315A), i.e., 0.1 dB. The white light source was coupled into
the fiber using commercial coupling lenses to match the
mode-field diameter between the fiber and the laser source, result-
ing in the present case in a coupling efficiency >90%. The figure
shows the loss and transmission spectra of two fibers. The corre-
sponding scanning electron microscopy (SEM) pictures of the
fabricated fibers are shown in Figs. 5(a) and 5(b). The first
one (Fiber #5) exhibits an average δ value of 2.5 μm, t
545 nm, and Rc 20.5 μm. Its measured loss spectrum (cut
back between 293 and 10 m long pieces) shown in Fig. 5(c)(ii)
highlights ultralow loss in the first-order band with an absolute
record transmission loss, for a HC-PCF of 7.7 dB/km at 750 nm. This loss figure is only about a factor of 2 larger than the silica
Rayleigh scattering limit shown by the grey filled curve. g
The fibers’ polarization-maintaining properties were explored
by launching a laser beam into the fiber from a linearly polarized
laser emitting at 1030 nm and a half-wave plate for polarization
control. 3. EXPERIMENTAL RESULTS The transmitted beam is then passed through a polarizing
beam splitter (PBS), and each of its two output beams is recorded
by a power meter and a camera imaging its reconstructed near-
field beam profile. Figure 6(b) shows the evolution of the polari-
zation extinction ratio (PER) with the fiber length (Fiber #6 is
used). The inset shows the fiber-transmitted laser power from
each of the two output ports of the PBS in function with the
half-wave plate angle. The deduced PER was recorded for differ-
ent fiber lengths. The results show a maximum PER of 15.5 dB
achieved with a fiber length of 16 m, and an evolution with the
fiber length which comprises a decrease in PER as fiber length
decreases for length shorter than 12 m, and then an enhancement
in PER as the fiber length increases for length longer than 12 m. The second design (Fiber #6) presents thinner tubes
(t 227 nm), thus shifting the fundamental band blue edge
to a wavelength as short as 515 nm [Fig. 5(d)(i)]. It exhibits
an average δ value of 3 μm and Rc 20 μm. An ultralarge
low-loss window is demonstrated over one octave ranging
from 600 to 1200 nm, with loss between 10 and 21 dB/km
[Fig. 5(d)(ii)] (cut back between 180 and 13 m long pieces). This is, to our knowledge, the first time that a HC-PCF combines
such a large bandwidth with such a low transmission loss. The
measured transmission spectra of 2 m long sections from the
two fibers are plotted in Figs. 5(e) and 5(f). Both fibers exhibit
guidance in the UV domain. Remarkably, Fiber #5 shows three
UV transmission bands spanning down to 220 nm (purple filling
color). Reliable measurements of loss spectra for wavelengths
shorter than 350 nm were prevented due to the limited dynamics
of the photospectrometer. Fig. 6. (a) Group delay curves of the HOM content propagating in
Fiber #6 for 5 and 15 m long pieces. The reconstructed mode field pro-
files are indicated with the corresponding MPI. (b) Evolution of the PER
versus the fiber length. p
p
Figures 5(c) and 5(d) also show the theoretical CL loss spectra. The CL is calculated numerically for the case of fibers with no
surface roughness. 4. CONCLUSION AND DISCUSSION 31, 3574–3576 (2006). 9. Y. Wang, F. Couny, P. J. Roberts, and F. Benabid, “Low loss broadband
transmission in optimized core-shape Kagome hollow-core PCF,” in
Conference on Lasers and Electro-Optics (IEEE, 2010), paper CPDB4. 10. Y. Y. Wang, N. V. Wheeler, F. Couny, P. J. Roberts, and F. Benabid,
“Low
loss
broadband
transmission
in
hypocycloid-core
Kagome
hollow-core photonic crystal fiber,” Opt. Lett. 36, 669–671 (2011). 11. B. Debord, M. Alharbi, T. Bradley, C. Fourcade-Dutin, Y. Y. Wang, L. Vincetti, F. Gérôme, and F. Benabid, “Hypocycloid-shaped hollow-core
photonic crystal fiber, part I: arc curvature effect on confinement loss,”
Opt. Express 21, 28597–28608 (2013). 12. B. Debord, M. Alharbi, L. Vincetti, A. Husakou, C. Fourcade-Dutin, C. Hoenninger, E. Mottay, F. Gerome, and F. Benabid, “Multi-meter fi-
ber-delivery and pulse self-compression of milli-Joule femtosecond laser
and fiber-aided laser-micromachining,” Opt. Express 22, 10735–10746
(2014). 13. A. D. Pryamikov, A. S. Biriukov, A. F. Kosolapov, V. G. Plotnichenko,
S. L. Semjonov, and E. M. Dianov, “Demonstration of a waveguide re-
gime for a silica hollow-core microstructured optical fiber with a negative
curvature of the core boundary in the spectral region > 3.5 μm,” Opt. Express 19, 1441–1448 (2011). 14. F. Yu, W. J. Wadsworth, and J. C. Knight, “Low loss silica hollow core
fibers for 3–4 μm spectral region,” Opt. Express 20, 11153–11158
(2012). 15. T. D. Bradley, Y. Wang, M. Alharbi, B. Debord, C. Fourcade-Dutin, B. Beaudou, F. Gerome, and F. Benabid, “Optical properties of low loss
(70 dB/km) hypocycloid-core Kagome hollow core photonic crystal fiber
for Rb and Cs based optical applications,” J. Lightwave Technol. 31,
2752–2755 (2013). 16. F. Benabid, F. Gerome, B. Debord, and M. Alharbi, “Kagome PC fiber
goes to extremes for ultrashort-pulse lasers,” Laser Focus World 50,
29–34 (2014). 17. L. Vincetti and V. Setti, “Waveguiding mechanism in tube lattice fibers,”
Opt. Express 18, 23133–23146 (2010). 18. A. N. Kolyadin, A. F. Kosolapov, A. D. Pryamikov, A. S. Biriukov, V. G. Plotnichenko, and E. M. Dianov, “Light transmission in negative curva-
ture hollow core fiber in extremely high material loss region,” Opt. Express 21, 9514–9519 (2013). Funding. Agence
Nationale
de
la
Recherche
(ANR)
(Photosynth); Air Force Office of Scientific Research (AFOSR)
(HOFGAS);
Σ_LIM
Labex
Chaire,
Maturation,
region
Limousin. 19. M. Michieletto, J. K. Lyngsø, C. Jakobsen, J. Lægsgaard, O. Bang, and
T. T. Alkeskjold, “Hollow-core fibers for high power pulse delivery,” Opt. Express 24, 7103–7119 (2016). 20. J. R. REFERENCES 1. T. A. Birks, P. J. Roberts, P. St.J. Russell, D. M. Atkin, and T. J. Shepherd,
“Full
2-D
photonic
bandgaps
in
silica/air
structures,”
Electron. Lett. 31, 1941–1943 (1995). 2. F. Benabid and P. J. Roberts, “Linear and nonlinear optical properties of
hollow core photonic crystal fiber,” J. Mod. Opt. 58, 87–124 (2011). 3. P. St.J. Russell, P. Holzer, W. Chang, A. Abdolvand, and J. C. Travers,
“Hollow-core photonic crystal fibres for gas-based nonlinear optics,” Nat. Photonics 8, 278–286 (2014). 4. F. Couny, F. Benabid, P. J. Roberts, P. S. Light, and M. G. Raymer,
“Generation and photonic guidance of multi-octave optical-frequency
combs,” Science 318, 1118–1121 (2007). 5. P. Roberts, F. Couny, H. Sabert, B. Mangan, D. Williams, L. Farr, M. Mason, A. Tomlinson, T. Birks, J. Knight, and P. St.J. Russell,
“Ultimate low loss of hollow-core photonic crystal fibres,” Opt. Express
13, 236–244 (2005). 4. CONCLUSION AND DISCUSSION 6. J. von
Neumann
and
E. Wigner,
“Über
merkwürdige
diskrete
Eigenwerte,” Phys. Z. 30, 465–467 (1929). In conclusion, we fabricated several SR-TL HC-PCFs guiding in
the NIR–VIS–UV spectral range and showing ultraloss. The
fiber cladding physical properties were adjusted for optimum con-
finement loss and close-to-unity modal content using the IC for-
malism. Among the fabricated fibers we listed, one fiber had a
record loss of 7.7 dB/km at 750 nm, and a second SR-TL
HC-PCF combined an octave-wide transmission band with trans-
mission loss in the range of 10–20 dB/km. We have shown that
the transmission loss is limited by CL for a wavelength longer
than ∼1 μm, indicating that improving the loss at these spectral
ranges and with core diameters comparable to the ones reported
here would require a different cladding design with stronger IC. Nested tube lattice HC-PCFs [36,37] are excellent candidates for
this purpose, as their cladding modes exhibit larger azimuthal
number than the reported tubular lattice for the same neff −ω. The challenge with such a nested tubular lattice is the difficulty
in controlling the different tube thicknesses during the fabrication
process and keeping them the same and constant throughout the
draw. On the other hand, for shorter wavelengths (typically less
than 800 nm), the fiber transmission performance is no longer
limited by the fiber design but by the surface roughness. The lat-
ter affects the propagation loss of SR-TL HC-PCFs via both SSL
and CL. Improving the transmission loss for a wavelength shorter
than 800 nm would require reducing the surface roughness dur-
ing the draw, and the cladding lattice design of any hypocycloid
core-contour HC-PCF will play no significant role until the trans-
mission loss reaches the level of 1 dB/km or below. The reported
results represent an important tool in improving the loss in IC
guiding HC-PCF. Given the much lower optical overlap with
the core surround and the larger transverse dimensions of this
type of fiber compared to PBG fibers, these results could revive
the prospect of developing optical fibers with transmission loss
much lower than the silica Rayleigh scattering limit if the surface
roughness is reduced. 7. C. W. Hsu, B. Zhen, A. D. Stone, J. D. Joannopoulos, and M. Soljačić,
“Bound states in the continuum,” Nat. Rev. Mater. 1, 16048 (2016). 8. F. Couny, F. Benabid, and P. S. Light, “Large-pitch Kagome-structured
hollow-core photonic crystal fiber,” Opt. Lett. Research Article 216 Vol. 4, No. 2 / February 2017 / Optica
2 It is noteworthy that the profiles of the crossed-polarized beams
show that, for an input polarization corresponding to a maximum
PER, the mode of the dominant polarization orientation is mainly
that of HE11 while the one with the crossed polarization shows a
profile of that of the LP11 family [inset of Fig. 6(b)]. We believe
that this PER evolution with the fiber length and the fact that the
mode of the crossed-polarized beams is that of LP11 results from
the back-coupling to the core of the light scattering off the core
inner surface. Indeed, by virtue of stronger optical overlap of the
higher-order core modes with the core contour, the light that is
coupled back from the core-surround surface is likely to couple to
higher-order modes rather than the HE11. See Supplement 1 for supporting content. 3. EXPERIMENTAL RESULTS Comparing αCLλ with the measured loss
shows that, for Fiber #5, the loss is dominated by the CL in the
range of 1300–1750 nm, which is in the range of 80–45 dB/km. However, for the first and second higher-order bands of Fiber #5,
the discrepancy between the measured loss and CL is much
higher, indicative of stronger contribution of SSL. Here, the
CL for a surface-roughness-free HC-PCF reaches a minimum
of ∼1 dB∕km in the first band and ∼0.1 dB∕km in the second
band. This trend is also observed for Fiber #6, where the CL
dominates for wavelengths larger than 1000 nm, and the
surface-roughness-induced SSL and CL increase become the
dominant transmission loss for shorter wavelengths (typically less
than 800 nm). Fig. 6. (a) Group delay curves of the HOM content propagating in
Fiber #6 for 5 and 15 m long pieces. The reconstructed mode field pro-
files are indicated with the corresponding MPI. (b) Evolution of the PER
versus the fiber length. Research Article 4. CONCLUSION AND DISCUSSION Hayes, S. R. Sandoghchi, T. D. Bradley, Z. Liu, R. Slavik, M. A. Gouveia, N. V. Wheeler, G. T. Jasion, Y. Chen, E. Numkam-Fokoua,
M. N. Petrovich, D. J. Richardson, and F. Poletti, “Antiresonant hollow
core fiber with octave spanning bandwidth for short haul data commu-
nications,” in Optical Fiber Communication Conference Postdeadline
Papers (Optical Society of America, 2016), paper Th5A.3. Acknowledgment. The authors thank the PLATINOM
platform for help with the fiber fabrication. 21. H. Kawamura, “Statistics of two-dimensional amorphous lattice,” Prog. Theor. Phys. 70, 352–365 (1983). Research Article 217 Vol. 4, No. 2 / February 2017 / Optica 30. W.-P. Huang, “Coupled-mode theory for optical waveguides: an over-
view,” J. Opt. Soc. Am. A 11, 963–983 (1994). 22. M. Rechtsman, A. Szameit, F. Dreisow, M. Heinrich, R. Keil, S. Nolte,
and M. Segev, “Amorphous photonic lattices: band gaps, effective mass,
and suppressed transport,” Phys. Rev. Lett. 106, 193904 (2011). p
(
)
31. L. Vincetti, “Empirical formulas for calculating loss in hollow core tube
lattice fibers,” Opt. Express 24, 10313–10325 (2016). 23. N. M. Litchinitser, A. K. Abeeluck, C. Headley, and B. J. Eggleton,
“Antiresonant reflecting photonic crystal optical waveguides,” Opt. Lett. 27, 1592–1594 (2002). 32. F. Poletti, “Nested antiresonant nodeless hollow core fiber,” Opt. Express
22, 23807 (2014). 24. J. M. Pottage, D. M. Bird, T. D. Hedley, T. A. Birks, J. C. Knight, P. St.J. Russell, and P. J. Roberts, “Robust photonic band gaps for hollow core
guidance in PCF made from high index glass,” Opt. Express 11,
2854–2861 (2003). 33. M. Alharbi, T. Bradley, B. Debord, C. Fourcade-Dutin, D. Ghosh, L. Vincetti, F. Gérôme, and F. Benabid, “Hypocycloid-shaped hollow-core
photonic crystal fiber, part II: cladding effect on confinement and bend
loss,” Opt. Express 21, 28609–28616 (2013). 34. J. W. Nicholson, A. D. Yablon, S. Ramachandran, and S. Ghalmi,
“Spatially and spectrally resolved imaging of modal content in large-
mode-area fibers,” Opt. Express 16, 7233–7243 (2008). 25. M. M. Z. Kharadly and J. E. Lewis, “Properties of dielectric-tube
waveguides,” Proc. Inst. Electr. Eng. 116, 214–224 (1969). 26. E. A. J. Marcatili and R. A. Schmeltzer, “Hollow metallic and dielectric
waveguides for long distance optical transmission and lasers,” Bell
Syst. Tech. J. 43, 1783–1809 (1964). 35. P. Calvet, “Mise en forme spatiale dans une fibre optique microstructurée
pour la réalisation d’amplificateurs lasers tout fibrés pour les pilotes des
lasers de puissance,” Ph.D. 4. CONCLUSION AND DISCUSSION dissertation (Université des Sciences et
Technologies de Lille, 2014). 27. M. A. Duguay, Y. Kokubun, T. L. Koch, and L. Pfeiffer, “Antiresonant
reflecting optical waveguides in SiO2‐Si multilayer structures,” Appl. Phys. Lett. 49, 13–15 (1986). 36. W. Belardi and J. C. Knight, “Hollow antiresonant fibers with reduced
attenuation,” Opt. Lett. 39, 1853–1856 (2014). 28. D. Marcuse, “Investigation of coupling between a fiber and an infinite
slab,” J. Lightwave Technol. 7, 122–130 (1989). 37. M. S. Habib, O. Bang, and M. Bache, “Low-loss hollow-core silica
fibers with adjacent nested anti-resonant tubes,” Opt. Express 23,
17394–17406 (2015). 29. H. Kogelnik, “An introduction to integrated optics,” IEEE Trans. Microw. Theory Tech. 23, 2–16 (1975).
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Critical Role of the Fusion Protein Cytoplasmic Tail Sequence in Parainfluenza Virus Assembly
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Abstract Interactions between viral glycoproteins, matrix protein and nucleocapsid sustain assembly of parainfluenza viruses at the
plasma membrane. Although the protein interactions required for virion formation are considered to be highly specific,
virions lacking envelope glycoprotein(s) can be produced, thus the molecular interactions driving viral assembly and
production are still unclear. Sendai virus (SeV) and human parainfluenza virus type 1 (hPIV1) are highly similar in structure,
however, the cytoplasmic tail sequences of the envelope glycoproteins (HN and F) are relatively less conserved. To unveil
the specific role of the envelope glycoproteins in viral assembly, we created chimeric SeVs whose HN (rSeVhHN) or HN and F
(rSeVh(HN+F)) were replaced with those of hPIV1. rSeVhHN grew as efficiently as wt SeV or hPIV1, suggesting that the
sequence difference in HN does not have a significant impact on SeV replication and virion production. In sharp contrast,
the growth of rSeVh(HN+F) was significantly impaired compared to rSeVhHN. rSeVh(HN+Fstail) which expresses a chimeric
hPIV1 F with the SeV cytoplasmic tail sequence grew similar to wt SeV or rSeVhHN. Further analysis indicated that the F
cytoplasmic tail plays a critical role in cell surface expression/accumulation of HN and F, as well as NP and M association at
the plasma membrane. Trafficking of nucelocapsids in infected cells was not significantly affected by the origin of F,
suggesting that F cytoplasmic tail is not involved in intracellular movement. These results demonstrate the role of the F
cytoplasmic tail in accumulation of structural components at the plasma membrane assembly sites. Editor: Krzysztof Pyrc, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Poland Received October 31, 2012; Accepted March 7, 2013; Published April 12, 2013 Copyright: 2013 Stone, Takimoto. 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. supported by National Institutes of Health grant R01AI081779. The funders had no role in study design, data collection and analysis
eparation of the manuscript. nding: This work was supported by National Institutes of Health grant R01AI081779. The funders had no role in study design, data c
cision to publish, or preparation of the manuscript. Competing Interests: Toru Takimoto is a PLOS ONE editorial board member. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing
data and materials. Abstract * E-mail: toru_takimoto@urmc.rochester.edu in vivo and when purified singly the M protein self-assembles into
ordered structures [10,11]. A recent study using cryoelectron
tomography showed that M dimers assemble into psudotetrameric
arrays in the virions [9]. Co-expression of M and NP results in the
production of virus-like particles (VLP) containing vRNP-like
structures [12]. In SeV, with temperature sensitive M protein, the
absence of M protein at non-permissive temperatures inhibits viral
assembly [13,14,15]. Other studies with cells persistently infected
with SeV, which expressed an unstable M protein showed a
correlation with reduced virion formation [16]. Raychel Stone, Toru Takimoto* ology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America epartment of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, United Introduction Sendai virus (SeV), the prototypical parainfluenza virus is
composed of six major structural proteins: hemagglutinin-neur-
aminidase (HN), fusion (F), matrix (M), nucleocapsid (NP),
phospho (P) and large (L) proteins. The two surface glycoproteins,
HN and F, are responsible for attachment and fusion, and the M
protein acts as a scaffold that bridges interactions between the viral
envelope proteins and viral nucleocapsid (vRNP) that is composed
of genomic RNA encapsidated with NP and associated with the
polymerase P-L complex [1,2]. The assembly process of parain-
fluenza virus involves multiple viral components with coordinated
localizations. These components include the viral glycoproteins,
which are transported to the plasma membrane through the
exocytic pathway [3,4,5], and other viral proteins, such as the
vRNPs, which utilize the recycling endosome pathway to reach the
cell surface [6]. Role of recycling endosomes in virus assembly has
also been suggested in some negative strand RNA viruses, such as
respiratory syncytial virus (RSV) and influenza A virus [7]. M
proteins are likely to be transported to the plasma membrane in
part by an association with envelope glycoproteins [8]. In virions,
the M protein is found underneath the envelope and interacts with
both envelope glycoproteins and vRNPs [9]. This would suggest
that the M protein acts as an organizer of viral components to
concentrate the proteins at a defined budding site at the plasma
membrane [3]. M protein binds lipid membranes both in vitro and The role of envelope glycoproteins in virus assembly is less clear,
although a specific interaction between the glycoprotein cytoplas-
mic tails and the M protein of parainfluenza viruses has been
thought to be important in the assembly and budding processes. In
the case of parainfluenza virus 5 (PIV5), M and glycoproteins co-
localize unless the cytoplasmic tail of HN or the cytoplasmic tails
of both HN and F have been truncated [17,18,19]. SeV M
becomes raft-associated only when co-expressed with the glyco-
proteins, which intrinsically sort to raft membranes [4]. These
results suggest that M and glycoproteins assemble at specific
locations on plasma membranes through interactions between M
and the glycoprotein cytoplasmic tails. However, the contribution
of HN and F in virus budding and virion formation is likely to
differ between viruses. Previous studies showed that SeV HN was
superfluous for virion budding [20,21,22,23,24]. Citation: Stone R, Takimoto T (2013) Critical Role of the Fusion Protein Cytoplasmic Tail Sequence in Parainfluenza Virus Assembly. PLoS ONE 8(4): e61281.
doi:10.1371/journal.pone.0061281 Critical Role of the Fusion Protein Cytoplasmic Tail
Sequence in Parainfluenza Virus Assembly Raychel Stone, Toru Takimoto* Introduction In contrast, lack
of SeV F reduced production of virions from infected cells, 1 April 2013 | Volume 8 | Issue 4 | e61281 PLOS ONE | www.plosone.org April 2013 | Volume 8 | Issue 4 | e61281 Role of F Cytoplasmic Tail in Virus Assembly highlighting differences in the role of glycoproteins in virion
formation
and
release
[24]. Experiments
involving
use
of
recombinant SeV (rSeV) generated to express F and HN
glycoproteins containing various truncations or amino acid
substitutions in the cytoplasmic tail domains showed that loss of
the
cytoplasmic
tail
of
F
protein
severely
reduced
virion
production [25]. Other paramyxoviruses have also been shown
to require the F protein cytoplasmic tail. Deletion of the
cytoplasmic tail of the RSV F protein resulted in a failure to
assemble RSV proteins into virus-like filaments, causing a
reduction of viral titers by up to 1,000 fold [26,27]. Similarly,
mumps virus also requires the F protein cytoplasmic tail [28], and
the cytoplamic tail of F with full length G protein was shown to be
required for assembly in Hendra virus infections [29]. However,
studies of PIV5 suggest a marked defect in virus budding and
release when the HN cytoplasmic tail was deleted, while F
cytoplasmic tail was dispensable for normal viral budding [19]. These studies highlight the role of glycoprotein cytoplasmic tails in
virion production, but it is unclear how the HN or F cytoplasmic
tails individually contribute to virus assembly and production and
the contribution differs among paramxyoviruses. which is the optimum temperature for hPIV1 growth due to HN
heat stability [34]. Infectious virus at each time point was
quantitated. The rSeVhHN and rSeVh(HN+Fstail) grew to similar
titers as wt SeV and hPIV1, but the rSeVh(HN+F) showed delayed
growth and an overall decrease in infectious virus yield (Fig. 2A). Consistent with the virus growth curve, the rSeVh(HN+F)
exhibited a small plaque phenotype compared with rSeVhHN in
a plaque assay (Fig. 2B). Plaque size of rSeVh(HN+Fstail) was
apparently larger than rSeVh(HN+F), although it is still smaller
than rSeVhHN. These data suggest that the difference in the HN
cytoplasmic tails between SeV and hPIV1 has little effect on SeV
production. In contrast, F cytoplasmic tail plays an important role
in SeV growth and spread. To further characterize viral replication and production from
cells, we determined the amounts of virions produced from
infected cells, as well as viral protein levels in infected cells (Fig. Limited plasma membrane localization of HN and F in
rSeVh(HN+F)-infected cells We next visualized the localization of HN or F in the cytoplasm
and at the plasma membrane in infected cells by IF. A549 cells
were infected with the recombinant viruses and either permeabi-
lized
to
observe
intracellular
protein
localization
or
non-
permeabilized to detect cell surface localization. The cell surface
staining of F in rSeVh(HN+F)-infected cells showed very limited F
at the plasma membrane compared with the F punctae staining
visualized in hPIV1- or rSeVh(HN+Fstail)-infected cells (Fig. 4A). This shows that, in rSeVh(HN+F)-infected cells, F did not
accumulate at the cell surface. Interestingly, the results for HN
protein localization resembled that of F (Fig. 4B), suggesting that
the HN in rSeVh(HN+F)-infected cells is unable to accumulate
stably at the cell surface. Rescue and characterization of rSeVhHN, rSeVh(HN+F)
and rSeVh(HN+Fstail) SeV and hPIV1 are highly homologous, and the overall
homology of the glycoproteins is high (68% for F and 72% for
HN). However, the amino acids in the cytoplasmic domains of F
and HN are not well conserved (28% for F and 23% for HN,
Fig. 1A) [30,31]. The HN cytoplasmic tails of SeV and hPIV1
contain the an identical SYWST sequence in the middle of the tail
domain, which we previously showed to be required for specific
incorporation of HN to SeV [32]. The cytoplasmic tail of SeV and
hPIV1 F protein also shares the TYTLE sequence, which may
play a similar role as HN for specific incorporation into virions
[32]. To determine the role and requirement of cytoplasmic tail
sequences in the glycoproteins for assembly and virion formation,
we created chimeric SeVs which contain hPIV1 HN (rSeVhHN)
or both HN and F (rSeVh(HN+F)), in place of the SeV HN, or HN
and
F
genes,
respectively. In
addition,
rSeVh(HN+Fstail),
containing a chimeric F composed of external and transmembrane
domains from hPIV1 and the cytoplasmic tail (shown in Fig. 1A)
from SeV, was created (Fig. 1B). We did not attempt to rescue
rSeV containing only replacement of hPIV1 F because our
previous study indicated that SeV HN is unable to promote fusion
induced by hPIV1 F [33]. The HN and F regions of the
recombinant viruses were sequenced and the presence of the
designed HN and F genes was confirmed (data not shown). The
expression of the specific viral proteins was confirmed by
immunofluorescent (IF) assay of the cells infected with the viruses
using monoclonal antibodies specific or cross-reactive to hPIV1 or
SeV HN and F proteins (Fig. 1C). Introduction 3). As with the virus growth curve and plaque phenotypes, virion
production in cells infected with rSeVh(HN+F) (Fig. 3A, lane 3)
was significantly lower than wt SeV (lane 1) or rSeVhHN (lane 2). The result was confirmed with three independent assays (Fig. 3C). rSeVh(HN+Fstail) which contains the cytoplasmic tail of SeV F
was
produced
in
a
greater
quantity
than
rSeVh(HN+F),
highlighting the critical role of the F cytoplasmic tail in virus
assembly
and
release. Cells
infected
with
rSeVhHN,
rSeVh(HN+F) and rSeVh(HN+Fstail) produced similar levels of
NP to wt SeV, confirming the same level of viral infection. However, in contrast to NP, HN and F levels in rSeVh(HN+F)-
infected
cell
lysates
was
significantly
less
than
rSeVhHN. Expression of hPIV1 F from rSeVh(HN+F) is expected to be less
than that of SeV F because of the long non-coding region of the
hPIV1 F gene [35]. Level of hPIV1 F in cell lysate was significantly
higher in rSeVh(HN+Fstail)-infected cells (Fig. 3B, lanes 3 and 4). Similarly, more hPIV1 HN was detected in rSeVh(HN+Fstail)-
infected cells than in that of rSeVh(HN+F)-infected cells. Because
rSeVh(HN+F) and rSeVh(HN+Fstail) differ only in the sequence
of the F cytoplasmic tail, the result indicates a major contribution
of the F cytoplasmic tail sequence for accumulation of both
envelope
glycoproteins. Our
data
also
suggest
that
stable
expression/accumulation of the envelope glycoproteins is required
for efficient virion production from infected cells. In this study, we determined the role and specificity of the
glycoprotein cytoplasmic tail sequences in virus assembly using
closely related SeV and human parainfluenza virus type 1 (hPIV1). We
rescued
and
characterized
various
recombinant
SeVs
containing hPIV1 HN or both HN and F. Our data indicate the
critical role of the F cytoplasmic tail in accumulation of viral
envelope proteins and vRNP at the plasma membrane, which is
essential for efficient virion assembly and release. Limited plasma membrane localization of M and NP
proteins in rSeVh(HN+F)-infected cells Progeny virions are formed at the plasma membrane budding
sites, and specific interactions between structural components,
vRNP, M and envelope glycoproteins are considered to be
essential. Since surface glycoproteins were limited in cells infected Next, we compared the growth kinetics of the chimeric viruses
to wt SeV and hPIV1 in LLC-MK2 cells. Cells infected at an MOI
of 0.01 were cultured in the presence of trypsin for 72 h at 34uC, April 2013 | Volume 8 | Issue 4 | e61281 PLOS ONE | www.plosone.org 2 Role of F Cytoplasmic Tail in Virus Assembly Figure 1. Rescue and characterization of rSeVhHN, rSeVh(HN+F) and rSeVh(HN+Fstail). (A) Aligned amino acid sequences of the F and HN
cytoplasmic tails of hPIV1 and SeV. Conserved amino acids are highlighted in red. (B) Schematic diagram of rSeV genomes compared with wt SeV and
hPIV1. With rSeVhHN, the SeV HN gene was replaced with that of hPIV1. In rSeVh(HN+F), both HN and F genes of SeV were replaced with those of
hPIV1. For rSeVh(HN+Fstail), the entire cytoplasmic tail portion of hPIV1 F shown in Figure 1A was replaced with that of SeV F. (C) Expression of HN
and F from rSeVs. IF analysis of A549 cells infected with wt SeV, hPIV1 or rSeV. Origins of HN or F were confirmed using cross reactive (cr) or specific
(sp) mAb for NP (cr M52), HN (SeV-sp S16, hPIV1-sp P24), F (SeV-sp M38, cr P38) or M (hPIV1-sp P3) [12,36,41,42,43]. doi:10.1371/journal.pone.0061281.g001 Figure 1. Rescue and characterization of rSeVhHN, rSeVh(HN+F) and rSeVh(HN+Fstail). (A) Aligned amino acid sequences of the F and HN
cytoplasmic tails of hPIV1 and SeV. Conserved amino acids are highlighted in red. (B) Schematic diagram of rSeV genomes compared with wt SeV and
hPIV1. With rSeVhHN, the SeV HN gene was replaced with that of hPIV1. In rSeVh(HN+F), both HN and F genes of SeV were replaced with those of
hPIV1. For rSeVh(HN+Fstail), the entire cytoplasmic tail portion of hPIV1 F shown in Figure 1A was replaced with that of SeV F. (C) Expression of HN
and F from rSeVs. IF analysis of A549 cells infected with wt SeV, hPIV1 or rSeV. Origins of HN or F were confirmed using cross reactive (cr) or specific
(sp) mAb for NP (cr M52), HN (SeV-sp S16, hPIV1-sp P24), F (SeV-sp M38, cr P38) or M (hPIV1-sp P3) [12,36,41,42,43]. Limited plasma membrane localization of M and NP
proteins in rSeVh(HN+F)-infected cells doi:10.1371/journal.pone.0061281.g001 of the xy plane were analyzed and reconstructed to allow a lateral
view of the cell (Fig. 5). In cells infected with wt SeV or hPIV1,
accumulation of both M and NP was clearly detected. Similarly,
rSeVhHN showed similar patterns of M and NP localization,
suggesting that the difference in HN sequence between SeV and
hPIV1 does not have a significant impact on the accumulation of
M and NP in infected cells. As seen with the HN and F surface
staining in rSeVh(HN+F)-infected cells, the localizations of NP
and M below the plasma membrane were limited, signifying that
the viral protein interactions necessary for assembly and virion
formation are suboptimal in rSeVh(HN+F)-infected cells. Addi-
tionally, the NP and M in rSeVh(HN+F)-infected cells formed
large aggregates within the cytoplasm, which differs from the more
evenly distributed pattern observed with the other recombinant
viruses. These results suggest that lack of envelope glycoproteins at
the plasma membrane also affects the localization of vRNP and M. with rSeVh(HN+F), we next determined whether M and vRNP
were accumulated below the plasma membrane in rSeVh(HN+F)-
infected cells. A549 were infected with wt or recombinant viruses,
the cell surface was biotinylated and processed for IF analysis using
anti-SeV NP (Texas Red) or SeV M (FITC) Ab and streptavidin
conjugated to Cy5 for cell surface staining. Using a confocal
microscope, 3D reconstructions from deconvoluted z-stack images Figure 2. Virus growth kinetics and plaque formation of rSeVs. (A) Multi-step growth curve of the viruses in LLC-MK2 cells. Cells were
infected with wt or chimeric viruses at MOI 0.01 and incubated at 34uC. Aliquots of infected cell supernatants were collected at indicated times
after infection and viral titers of supernatants were determined in LLC-
MK2 cells. (B) Plaque formation of the wt and rSeVs. LLC-MK2 cells were
infected with SeV, rSeVhHN, rSeVh(HN+F), rSeVh(HN+Fstail) or hPIV1
and cultured at 34uC with medium containing agarose. Plaques were
identified using crystal violet staining. doi:10.1371/journal.pone.0061281.g002 Discussion In this study, we determined the role of glycoproteins in virus
assembly and formation in the context of an infection by taking
advantage of the low amino acid homology of HN and F
cytoplasmic tail sequences between SeV and hPIV1, which share
only 23 and 28% identity, respectively. Our data indicate that 1)
the difference in the HN cytoplasmic tail sequence does not affect
SeV growth and virion formation, 2) the F cytoplasmic tail
sequence plays a major role in accumulation of both HN and F at
the cell surface, and 3) lack of a specific F cytoplasmic sequence
results in a reduction in the accumulation of M and vRNP at the
plasma membrane and overall virion production. These findings
highlight the critical role of F cytoplasmic tail in virus assembly
and suggest that specific interactions between F cytoplasmic tail
and M are a key factor for recruiting all viral structural
components to plasma membrane budding sites. p
p
g
The important role of SeV F in virus budding is consistent with
our previous study showing that expression of SeV F, but not HN
by itself induced VLP formation [36]. The major contribution of
F, but not HN in SeV virion production was also suggested in a
previous study using siRNA to knock down HN or F protein
expression [24]. Comparison of virion production from cells
infected with rSeVh(HN+F) and rSeVh(HN+Fstail) clearly indi-
cates that the SeV F cytoplasmic tail includes a critical domain
necessary for the formation of progeny virions (Fig. 2). Replace-
ment of the hPIV1 F cytoplasmic tail with that of SeV significantly
increased HN and F cell surface accumulation (Fig. 4) and virion
production (Fig. 3), suggesting that a specific interaction between F
cytoplasmic tail and M plays a major role in virus assembly and
budding site formation. Although SeV and hPIV1 F share the
TYTLE sequence in the cytoplasmic tail (Fig. 1A), other parts of
the sequence are likely to be responsible for the lack of hPIV1 F
accumulation at the surface of rSeVh(HN+F)-infected cells. F
proteins located at the cell surface could be internalized through
lack of a specific interaction with M, and due to the specific HN-F
interaction, HN may also be internalized in rSeVh(HN+F)-
infected cells resulting in reduced accumulation of both HN and
F proteins at the cell surface. Lack of envelope protein accumulation does not affect
vRNP trafficking in the cytoplasm (A) Virio
production from LLC-MK2 cells infected with SeV (lane 1), rSeVhH
(lane 2), rSeVh(HN+F) (lane 3), or rSeVh(HN+Fstail) (lane 4). Cells we
i f
t d t
MOI
f 1
d l b l d
ith [35S] M t/C
f
16 h L b l immunoprecipitated using anti-SeV or hPIV1 HN, F or NP antibodies. (C) Amounts of NP in released virions (A) were quantified and shown as
the average of three independent experiments with standard
deviations. doi:10.1371/journal.pone.0061281.g003 of rSeVLeGFP (Videos S1 and S2). The average velocities of
LeGFPs
which
traveled
greater
than
2 mm
in
length
in
rSeVh(HN+F)LeGFP-infected
cells
were
0.24–0.73 mm/sec,
which is similar to the kinetics observed for rSeVLeGFP [6]. The movement of these vRNPs was indicative of movement along
microtubules, similar to that of rSeVLeGFP. These results
suggest
that
trafficking
of
vRNPs
in
the
cytoplasm
of
rSeVh(HN+F)LeGFP-infected cells is unaffected, however, the
vRNPs are recycled back into the cytoplasm due to a deficiency in
the accumulation of M and envelope glycoproteins at the plasma
membrane. Lack of envelope protein accumulation does not affect
vRNP trafficking in the cytoplasm In our previous study, we rescued a rSeV with eGFP fused to
the L protein (rSeVLeGFP), and used live cell digital video
microscopy to visualize movement of vRNP [6]. Using this virus,
we showed that vRNP transport is microtubule dependent and
involves the recycling endosome pathway. To determine if the
reduced vRNP accumulation at the plasma membrane and
formation of large vRNP
aggregates in the cytoplasm
of
rSeVh(HN+F)-infected cells were due to inefficient vRNP trans-
port, we rescued a rSeVh(HN+F)LeGFP, which expresses L fused
to eGFP in the background of rSeVh(HN+F). The accumulation
of vRNP-M complexes and vRNP movement were compared
between rSeVLeGFP and rSeVh(HN+F)LeGFP at different times
after infection. At 12 h p.i., vRNP-M complexes had already
formed in the cytoplasm of the rSeVh(HN+F)LeGFP-infected
cells, unlike rSeVLeGFP nucleocapsid, which exhibited a primar-
ily punctate appearance with some larger areas of accumulated
vRNP (Fig. 6). In spite of the large vRNP accumulations, small
punctae were also present in the rSeVh(HN+F)LeGFP-infected
cells and the movement of these nucleocapsids was similar to that Figure 2. Virus growth kinetics and plaque formation of rSeVs. (A) Multi-step growth curve of the viruses in LLC-MK2 cells. Cells were
infected with wt or chimeric viruses at MOI 0.01 and incubated at 34uC. Aliquots of infected cell supernatants were collected at indicated times
after infection and viral titers of supernatants were determined in LLC-
MK2 cells. (B) Plaque formation of the wt and rSeVs. LLC-MK2 cells were
infected with SeV, rSeVhHN, rSeVh(HN+F), rSeVh(HN+Fstail) or hPIV1
and cultured at 34uC with medium containing agarose. Plaques were
identified using crystal violet staining. doi:10.1371/journal.pone.0061281.g002 Figure 2. Virus growth kinetics and plaque formation of rSeVs. (A) Multi-step growth curve of the viruses in LLC-MK2 cells. Cells were
infected with wt or chimeric viruses at MOI 0.01 and incubated at 34uC. Aliquots of infected cell supernatants were collected at indicated times
after infection and viral titers of supernatants were determined in LLC-
MK2 cells. (B) Plaque formation of the wt and rSeVs. LLC-MK2 cells were
infected with SeV, rSeVhHN, rSeVh(HN+F), rSeVh(HN+Fstail) or hPIV1
and cultured at 34uC with medium containing agarose. Plaques were
identified using crystal violet staining. doi:10.1371/journal.pone.0061281.g002 April 2013 | Volume 8 | Issue 4 | e61281 3 PLOS ONE | www.plosone.org Role of F Cytoplasmic Tail in Virus Assembly Figure 3. Virus production from infected cells. Discussion Alternatively, it is also possible that
efficient translocation of F to the plasma membrane through the
exocytic pathway may require a stable interaction with a specific
viral M protein. Figure 3. Virus production from infected cells. (A) Virion
production from LLC-MK2 cells infected with SeV (lane 1), rSeVhHN
(lane 2), rSeVh(HN+F) (lane 3), or rSeVh(HN+Fstail) (lane 4). Cells were
infected at a MOI of 1 and labeled with [35S] Met/Cys for 16 h. Labeled
progeny virions released from the cells were purified and analyzed by
SDS-PAGE. (B) Viral proteins produced in infected cells. HN, F and NP
proteins in [35S]-labeled cell lysates as described in (A) were In sharp contrast, rSeVhHN which expresses hPIV1 HN and
other components of SeV, replicated and assembled virions as
efficiently as wt SeV. SeV and hPIV1 HN cytoplasmic tails share
no identity except the SYWST sequence (Fig. 1A), which we
previously identified to be required for specific incorporation of April 2013 | Volume 8 | Issue 4 | e61281 PLOS ONE | www.plosone.org April 2013 | Volume 8 | Issue 4 | e61281 4 Role of F Cytoplasmic Tail in Virus Assembly Figure 4. Surface and subcellular localization of HN and F proteins. A549 cells were infected with SeV, hPIV1 or rSeVs and incubated for 16 h
at 34uC. Cells were then fixed and treated with mAbs against SeV or hPIV1 F (A) or HN (B) without (upper panels) or with (lower panels)
permeabilization. Anti-mouse IgG-Texas Red was used as secondary. doi:10.1371/journal.pone.0061281.g004 Figure 4. Surface and subcellular localization of HN and F proteins. A549 cells were infected with SeV, hPIV1 or rSeVs and incubated for 16 h
at 34uC. Cells were then fixed and treated with mAbs against SeV or hPIV1 F (A) or HN (B) without (upper panels) or with (lower panels)
permeabilization. Anti-mouse IgG-Texas Red was used as secondary. doi:10.1371/journal.pone.0061281.g004 HN and SeV F from cDNAs induced membrane fusion [33]. However, in rSeVh(HN+F)-infected cells, reduction in both HN
and F surface expression suggests that SeV M-hPIV1 HN
interaction may not be sufficient to retain hPIV1 HN at the
surface. In other words, HN may be able to accumulate at the
surface through interactions with F, which can be accumulated at
the surface through the interaction of its cytoplasmic tail and M. In HN proteins into progeny SeV [32]. Discussion It is possible that the common
SYWST sequence is responsible for the interaction with both SeV
and hPIV1 M proteins. Our previous data support this possibility
since chimeric Newcastle disease virus HN containing the
cytoplasmic SYWST sequence was incorporated into progeny
SeV virions [32]. The hPIV1 HN and SeV F are considered to
function coordinately for infection, because expression of hPIV1 HN proteins into progeny SeV [32]. It is possible that the common
SYWST sequence is responsible for the interaction with both SeV
and hPIV1 M proteins. Our previous data support this possibility
since chimeric Newcastle disease virus HN containing the
cytoplasmic SYWST sequence was incorporated into progeny
SeV virions [32]. The hPIV1 HN and SeV F are considered to
function coordinately for infection, because expression of hPIV1 Figure 5. M and NP localization below the plasma membrane of infected cells. A549 cells were infected with the indicated viruses for 16 h
at 34uC. Cells were processed for IF using anti-SeV NP mAb or M rabbit serum. Top 3 panels represent localization of M and NP. Merged images are
deconvoluted z-stack images of the xy-plane created using an Olympus FV1000 confocal microcope. Bottom 3 panels show plasma membrane, M
and NP and are z-stack reconstructions of the ROIs indicated in the whole cell images by white rectangles in the merged images. doi:10.1371/journal.pone.0061281.g005 Figure 5. M and NP localization below the plasma membrane of infected cells. A549 cells were infected with the indicated viruses for 16 h
at 34uC. Cells were processed for IF using anti-SeV NP mAb or M rabbit serum. Top 3 panels represent localization of M and NP. Merged images are
deconvoluted z-stack images of the xy-plane created using an Olympus FV1000 confocal microcope. Bottom 3 panels show plasma membrane, M
and NP and are z-stack reconstructions of the ROIs indicated in the whole cell images by white rectangles in the merged images. doi:10.1371/journal.pone.0061281.g005 April 2013 | Volume 8 | Issue 4 | e61281 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 5 Role of F Cytoplasmic Tail in Virus Assembly Figure 6. Accumulation of vRNP in rSeVh(HN+F)LeGFP-infected
cells. A549 infected with rSeVLeGFP or rSeVh(HN+F)LeGFP were fixed
at the indicated hours after infection, and processed for IF to detect M
protein (red). doi:10.1371/journal.pone.0061281.g006 described in this study, it is also possible that F cytoplasmic domain
contributes to vRNP association to the matrix array. cDNA synthesis and cloning The
helical vRNP were also detected in association with the matrix
arrays, which is consistent with the idea that M protein is a key
organizer of virus assembly. Although vRNP association to the
glycoprotein tails in the membrane lacking matrix arrays was not Cell and viruses Therefore, vRNP accumulation at
assembly sites may require a stable interaction with M at the
plasma membrane, which could be provided by specific interac-
tion of M with the F cytoplasmic tail. It is not known if F
cytoplasmic tail directly interacts with vRNP at the plasma
membrane. A recent cryoelectron tomography study of Newcastle
disease virus (NDV) showed that the glycoproteins were anchored
in the gaps between repeating units in the matrix array [9]. The
helical vRNP were also detected in association with the matrix
arrays, which is consistent with the idea that M protein is a key
organizer of virus assembly. Although vRNP association to the
glycoprotein tails in the membrane lacking matrix arrays was not Although our data and previous studies support a major role for
F in virus assembly and budding, contribution of the envelope
glycoprotein cytoplasmic tails in virus budding seems to differ
between paramyxoviruses. In the case of parainfluenza virus 5
(PIV5), truncation of F cytoplasmic tail affects budding less than
that of truncations in HN cytoplasmic tail in reducing viral
budding and production [17,19]. IF study of the cells infected with
HN
cytoplasmic
tail-truncated
viruses
showed
HN
and
F
distributed all across the cell surface, unlike wt PIV5-infected
cells where HN and F formed highly localized patches on the cell
surface, suggesting that reduced budding could be due to
redistribution of the membrane glycoproteins at the surface [17]. Materials and Methods Figure 6. Accumulation of vRNP in rSeVh(HN+F)LeGFP-infected
cells. A549 infected with rSeVLeGFP or rSeVh(HN+F)LeGFP were fixed
at the indicated hours after infection, and processed for IF to detect M
protein (red). doi:10.1371/journal.pone.0061281.g006 Discussion Overall, characterization of the recombinant SeVs containing
envelope glycoproteins from hPIV1 highlights the importance of
the F cytoplasmic tail in recruitment/accumulation of viral
structural components at assembly sites on the infected cell
membrane. Clearly, our study indicates that the F cytoplasmic
domain critical for virion production is not shared between SeV
and hPIV1. It is predicted that lack of a specific interaction
between
the
F
cytoplasmic
tail
with
M causes
inefficient
recruitment and enrichment of structural components at budding
sites, which is essential for virion assembly and production. Rescue of rSeVhHN, rSeVh(HN+F), rSeVh(HN+Fstail), and
rSeVh(HN+F)LeGFP The recombinant SeVs were rescued as described previously
[35]. Briefly,
HeLa
T4+
cells
infected
with
vTF7.3
were
transfected
with
2
mg
of
pSeVhHN,
pSeVh(HN+F),
pSeVh(HN+Fstail), or pSeVh(HN+F)LeGFP together with sup-
porting plasmids pTF1SeVNP (1mg), pTF1SeVP (1 mg), and
pTF1SeVL (0.1 mg) by Lipofectamine 2000 (Invitrogen). After
36 h incubation in DMEM supplemented with 0.15% bovine
serum albumin plus araC (40 mg/ml), the cells were treated with
trypsin and overlayed onto LLC-MK2 cells and cultured in
DMEM containing trypsin. The rescued viruses were plaque
purified in LLC-MK2 cells, and the stock viruses were grown in
LLC-MK2 cells. cDNA synthesis and cloning y
g
The full genome cDNA of rSeV (pSeV(E), strain Enders) [39]
was mutated at the non-coding regions between M and F, F and
HN, and HN and L to include unique restriction sites for FseI,
NotI, and AscI, respectively. The SeV HN and F genes in pSeV(E)
were replaced with those of hPIV1 HN and F cDNAs produced by
PCR using primers containing appropriate restriction sites. The
SeV cDNAs containing the hPIV1 HN gene, and both hPIV1 HN
and F, were designated as pSeVhHN and pSeVh(HN+F). The
hPIV1 F cDNA encoding the SeV cytoplasmic tail sequence was
constructed using PCR for gene splicing by overlap extension [40]. pSeVh(HN+Fstail) was produced by replacing the hPIV1 F gene
in pSeVh(HN+F) with that of the chimeric F cDNA. The
pSeVh(HN+F)LeGFP was constructed by replacing the XhoI/KpnI
fragment of pSeVh(HN+F) with that of pSeVLeGFP, which
included the L gene tagged with eGFP [6]. g y
p
[
]
It is well established that viral M protein is the key driver of
virus assembly and budding. M protein links together the major
structural components of the virus through interaction with vRNP
and
envelope
glycoproteins
via
their
cytoplasmic
tails. In
rSeVh(HN+F)-infected cells, in addition to HN and F, M and
vRNP accumulation at the plasma membrane was reduced (Fig. 5). IF analysis of cells infected with rSeVh(HN+F) showed extensive
localization of M with vRNP in the cytoplasm as large aggregates. This lack of accumulation of vRNP at the plasma membrane is
unlikely due to a defect in vRNP trafficking to the plasma
membrane, because movement of vRNP in rSeVh(HN+F)LeGFP-
infected cells seems to be as efficient as rSeVLeGFP (Videos S1
and S2). M and vRNP showed extensive co-localization at the
plasma membrane of cells infected with SeV, hPIV1, rSeVhHN,
and rSeVh(HN+Fstail), but not with rSeVh(HN+F) (Fig. 5). M and
vRNP co-localize mainly in large clusters in the cytoplasm in
rSeVh(HN+F)-infected cells. Therefore, vRNP accumulation at
assembly sites may require a stable interaction with M at the
plasma membrane, which could be provided by specific interac-
tion of M with the F cytoplasmic tail. It is not known if F
cytoplasmic tail directly interacts with vRNP at the plasma
membrane. A recent cryoelectron tomography study of Newcastle
disease virus (NDV) showed that the glycoproteins were anchored
in the gaps between repeating units in the matrix array [9]. Cell and viruses LLC-MK2, A549, HeLa and HeLa T4+ [37] cells were cultured
in Dulbecco’s modified Eagle’s medium (DMEM) with 8% fetal
calf serum (FCS). SeV (strain Enders), hPIV1 (strain C-35) and the
recombinant SeVs: rSeVhHN, rSeVh(HN+F), rSeVh(HN+Fstail),
rSeVLeGFP [6], and rSeVh(HN+F)LeGFP were grown in LLC-
MK2 cells in DMEM supplemented with acetylated trypsin
(2 mg/ml). Recombinant vaccinia virus vTF7.3, which expresses
T7 polymerase [38] was grown in HeLa cells. this case, a specific F cytoplasmic tail and M interaction is the
major factor for accumulation of envelope proteins at the budding
site, which is likely to be required for efficient budding and virion
production. production. Although our data and previous studies support a major role for
F in virus assembly and budding, contribution of the envelope
glycoprotein cytoplasmic tails in virus budding seems to differ
between paramyxoviruses. In the case of parainfluenza virus 5
(PIV5), truncation of F cytoplasmic tail affects budding less than
that of truncations in HN cytoplasmic tail in reducing viral
budding and production [17,19]. IF study of the cells infected with
HN
cytoplasmic
tail-truncated
viruses
showed
HN
and
F
distributed all across the cell surface, unlike wt PIV5-infected
cells where HN and F formed highly localized patches on the cell
surface, suggesting that reduced budding could be due to
redistribution of the membrane glycoproteins at the surface [17]. It is well established that viral M protein is the key driver of
virus assembly and budding. M protein links together the major
structural components of the virus through interaction with vRNP
and
envelope
glycoproteins
via
their
cytoplasmic
tails. In
rSeVh(HN+F)-infected cells, in addition to HN and F, M and
vRNP accumulation at the plasma membrane was reduced (Fig. 5). IF analysis of cells infected with rSeVh(HN+F) showed extensive
localization of M with vRNP in the cytoplasm as large aggregates. This lack of accumulation of vRNP at the plasma membrane is
unlikely due to a defect in vRNP trafficking to the plasma
membrane, because movement of vRNP in rSeVh(HN+F)LeGFP-
infected cells seems to be as efficient as rSeVLeGFP (Videos S1
and S2). M and vRNP showed extensive co-localization at the
plasma membrane of cells infected with SeV, hPIV1, rSeVhHN,
and rSeVh(HN+Fstail), but not with rSeVh(HN+F) (Fig. 5). M and
vRNP co-localize mainly in large clusters in the cytoplasm in
rSeVh(HN+F)-infected cells. Live cell trafficking of vRNP HeLa cells in a DT35 dish (Bioptechs) were infected with
rSeVLeGFP or rSeVh(HN+F)LeGFP for 18 h, then the move-
ment of LeGFP was recorded using a Leica DMIRB inverted
fluorescence microscope equipped with Image-Pro Plus software
(Mediacybernetics) while maintaining the cells at 37uC on a DTC3
temperature-controlled stage. The video image data were analyzed
using NIH ImageJ software. References 11. Hewitt JA, Nermut MV (1977) A morphological study of the M-protein of
Sendai virus. J Gen Virol 34: 127–136. 1. Takimoto T, Portner A (2004) Molecular mechanism of paramyxovirus
budding. Virus Res 106: 133–145. 1. Takimoto T, Portner A (2004) Molecular mechanism of paramyxovirus
budding. Virus Res 106: 133–145. 2. Coronel EC, Takimoto T, Murti KG, Varich N, Portner A (2001) Nucleocapsid
incorporation into parainfluenza virus is regulated by specific interaction with
matrix protein. J Virol 75: 1117–1123. 12. Coronel EC, Murti KG, Takimoto T, Portner A (1999) Human parainfluenza
virus type 1 matrix and nucleoprotein genes transiently expressed in mammalian
cells induce the release of virus-like particles containing nucleocapsid-like
structures. J Virol 73: 7035–7038. p
3. Lamb RA, Parks GD (2007) Paramyxoviridae: the viruses and their replication. In:
Knipe DM, Howley PM, editors. Fields Virology. 5th edition ed. Philadelphia,
PA: Lippincott Williams & Wilkins. pp. 1449–1496. 13. Kondo T, Yoshida T, Miura N, Nakanishi M (1993) Temperature-sensitive
phenotype of a mutant Sendai virus strain is caused by its insufficient
accumulation of the M protein. J Biol Chem 268: 21924–21930. 4. Ali A, Nayak DP (2000) Assembly of Sendai virus: M protein interacts with F
and HN proteins and with the cytoplasmic tail and transmembrane domain of F
protein. Virology 276: 289–303. 14. Yoshida T, Hamaguchi M, Naruse H, Nagai Y (1982) Persistent infection by a
temperature-sensitive mutant isolated from a Sendai virus (HVJ) carrier culture:
its initiation and maintenance without aid of defective interfering particles. Virology 120: 329–339. 5. Sanderson CM, McQueen NL, Nayak DP (1993) Sendai virus assembly: M
protein binds to viral glycoproteins in transit through the secretory pathway. J Virol 67: 651–663. 15. Yoshida T, Nagai Y, Maeno K, Iinuma M, Hamaguchi M, et al. (1979) Studies
on the role of M protein in virus assembly using a ts mutant of HVJ (Sendai
virus). Virology 92: 139–154. J
6. Chambers R, Takimoto T (2010) Trafficking of Sendai virus nucleocapsids is
mediated by intracellular vesicles. PLoS One 5: e10994. 16. Roux L, Waldvogel FA (1982) Instability of the viral M protein in BHK-21 cells
persistently infected with Sendai virus. Cell 28: 293–302. 7. Bruce EA, Stuart A, McCaffrey MW, Digard P (2012) Role of the Rab11
pathway in negative-strand virus assembly. Biochem Soc Trans 40: 1409–1415. 17. Acknowledgments We thank Allen Portner (St. Jude Children’s Research Hospital) for
providing antibodies and Leslie MacDonald for technical assistance. For
the microscopy data, we would like to thank Linda Callahan for the use of
the URMC Confocal and Conventional Microscopy Core and Brian Ward
for the use of his Leica DMIRB inverted light microscope. We thank Allen Portner (St. Jude Children’s Research Hospital) for
providing antibodies and Leslie MacDonald for technical assistance. For
the microscopy data, we would like to thank Linda Callahan for the use of
the URMC Confocal and Conventional Microscopy Core and Brian Ward
for the use of his Leica DMIRB inverted light microscope. Purification of produced virions and
radioimmunoprecipitation (RIP) p
p
LLC-MK2 cells infected with the viruses at MOI 1 for 16 h
were labeled with [35S] Met/Cys (Perkin Elmer) for an additional
16 h. The supernatants were collected and purified by ultracen-
trifugation over 40% glycerol cushions at 190,0006 g for 2 h at
4uC. The virus pellet was resuspended in Laemmli sample loading
buffer and analyzed by SDS-PAGE. For immunoprecipitation of
viral proteins, labeled cells were lysed with TNE buffer (10 mM
Tris [pH 7.4], 150 mM NaCl, 0.5% NP-40, and 1 mM EDTA)
and incubated with Protein G-Dynabeads pre-incubated with anti-
SeV NP (M52), HN (M2) or F (M38) or hPIV1 NP (P19), HN
(P24) or F (P12) monoclonal antibody (mAb). The samples were
analyzed by SDS-PAGE. Band intensities were quantified with
BioRad Quantity One software and the amount of NP detected in
purified virions was normalized to NP expressed in cell lysates. Immunofluorescence (IF) assays and confocal microscopy Immunofluorescence (IF) assays and confocal microscopy
Localization of viral proteins in cells infected with wt and
recombinant SeVs was determined by IF assay using confocal
microscope. A549 cells were infected with SeV, rSeVhHN,
rSeVh(HN+F), rSeVh(HN+Fstail) or hPIV1 at MOI 0.8. After
16 h, cells were fixed with 4% paraformaldehyde (PFA) for 15 min
and either permeabilized with 0.1% Triton X-100 for 10 min at
room temperature (RT) or left unpermeabilized. HN and F
proteins were detected by reaction with a-hPIV1 HN (P24), a-SeV
HN (M2), a-hPIV1 F (P12) and a-SeV F (M38) followed by goat
anti-mouse
Texas
Red
(Molecular
Probes)
[41,42,43]. For
detection of M and NP, permeabilized cells were reacted with
anti-M rabbit serum followed by anti-rabbit IgG-FITC, and anti-
NP mAb followed by anti-mouse Texas Red. Z-stack reconstruc-
tions of the samples were obtained using an Olympus FV1000
confocal microscope with a 636 oil immersion objective. These
experiments were repeated at least three times and representative Author Contributions Conceived and designed the experiments: RS TT. Performed the
experiments: RS TT. Analyzed the data: RS TT. Contributed reagents/
materials/analysis tools: RS TT. Wrote the paper: RS TT. Conceived and designed the experiments: RS TT. Performed the
experiments: RS TT. Analyzed the data: RS TT. Contributed reagents/
materials/analysis tools: RS TT. Wrote the paper: RS TT. Plaque assay LLC-MK2 cells were infected with the viruses and cultured at
34uC with medium containing acetylated trypsin (2 mg/ml) and
0.9% SeaKem LE agarose (Lonza). Plaques were identified using
crystal violet staining. Virus growth in LLC-MK2 cells Cells in a six-well plate were infected with the recombinant
SeVs at an MOI of 0.01 and incubated at 34uC in 2 ml DMEM
containing 0.15% BSA and acetylated trypsin (2 mg/ml). Culture
medium (200 ml) was harvested every 12 h after infection. Virus
titers in the supernatant were determined by tissue culture
infection in LLC-MK2. April 2013 | Volume 8 | Issue 4 | e61281 PLOS ONE | www.plosone.org 6 Role of F Cytoplasmic Tail in Virus Assembly images are shown in the Figures. Parameters for each experiment,
such as exposure length and laser power, were kept the same by re-
loading the specific parameters from previous experimental files. Supporting Information Video S1
Movement of LeGFP in rSeVLeGFP-infected
A549 cells. Live infected cells were observed using a Leica
DMIRB inverted fluorescence microscope equipped with Image-
Pro Plus software. The movements of LeGFP were recorded while
maintaining the cells at 37uC. (AVI) (
)
infected A549 cells. Live infected cells were observed using a
Leica DMIRB inverted fluorescence microscope equipped with
Image-Pro Plus software. The movements of LeGFP were
recorded while maintaining the cells at 37uC. (AVI) Role of F Cytoplasmic Tail in Virus Assembly 20. Portner A, Marx PA, Kingsbury DW (1974) Isolation and characterization of
Sendai virus temperature-sensitive mutants. J Virol 13: 298–304. 32. Takimoto T, Bousse T, Coronel EC, Scroggs RA, Portner A (1998) Cytoplasmic
domain of Sendai virus HN protein contains a specific sequence required for its
incorporation into virions. J Virol 72: 9747–9754. p
J
21. Portner A, Scroggs RA, Marx PS, Kingsbury DW (1975) A temperature-
sensitive mutant of Sendai virus with an altered hemagglutinin-neuraminidase
polypeptide: consequences for virus assembly and cytopathology. Virology 67:
179–187. 33. Bousse T, Takimoto T, Gorman WL, Takahashi T, Portner A (1994) Regions
on the hemagglutinin-neuraminidase proteins of human parainfluenza virus
type-1 and Sendai virus important for membrane fusion. Virology 204: 506–514. 22. Stricker R, Roux L (1991) The major glycoprotein of Sendai virus is dispensable
for efficient virus particle budding. J Gen Virol 72 (Pt 7): 1703–1707. 34. Takimoto T, Portner A (1994) The human parainfluenza virus type-1 prototypic
strain contains a heat-labile hemagglutinin-neuraminidase protein. Virus Res 32:
85–92. 23. Tuffereau C, Portner A, Roux L (1985) The role of haemagglutinin-
neuraminidase glycoprotein cell surface expression in the survival of Sendai
virus-infected BHK-21 cells. J Gen Virol 66 (Pt 11): 2313–2318. 35. Bousse T, Matrosovich T, Portner A, Kato A, Nagai Y, et al. (2002) The long
noncoding region of the human parainfluenza virus type 1 f gene contributes to
the read-through transcription at the m-f gene junction. J Virol 76: 8244–8251. 24. Gosselin-Grenet AS, Mottet-Osman G, Roux L (2010) Sendai virus particle
production: basic requirements and role of the SYWST motif present in HN
cytoplasmic tail. Virology 405: 439–447. 36. Takimoto T, Murti KG, Bousse T, Scroggs RA, Portner A (2001) Role of matrix
and fusion proteins in budding of Sendai virus. J Virol 75: 11384–11391. y p
gy
25. Fouillot-Coriou N, Roux L (2000) Structure-function analysis of the Sendai virus
F and HN cytoplasmic domain: different role for the two proteins in the
production of virus particle. Virology 270: 464–475. 37. Maddon PJ, Dalgleish AG, McDougal JS, Clapham PR, Weiss RA, et al. (1986)
The T4 gene encodes the AIDS virus receptor and is expressed in the immune
system and the brain. Cell 47: 333–348. p
p
gy
26. Oomens AG, Bevis KP, Wertz GW (2006) The cytoplasmic tail of the human
respiratory syncytial virus F protein plays critical roles in cellular localization of
the F protein and infectious progeny production. References Schmitt AP, He B, Lamb RA (1999) Involvement of the cytoplasmic domain of
the hemagglutinin-neuraminidase protein in assembly of the paramyxovirus
simian virus 5. J Virol 73: 8703–8712. 8. Sanderson CM, Wu HH, Nayak DP (1994) Sendai virus M protein binds
independently to either the F or the HN glycoprotein in vivo. J Virol 68: 69–76. 9. Battisti AJ, Meng G, Winkler DC, McGinnes LW, Plevka P, et al. (2012)
Structure and assembly of a paramyxovirus matrix protein. Proc Natl Acad
Sci U S A 109: 13996–14000. 18. Schmitt AP, Leser GP, Morita E, Sundquist WI, Lamb RA (2005) Evidence for a
new viral late-domain core sequence, FPIV, necessary for budding of a
paramyxovirus. J Virol 79: 2988–2997. 10. Heggeness MH, Smith PR, Choppin PW (1982) In vitro assembly of the
nonglycosylated membrane protein (M) of Sendai virus. Proc Natl Acad Sci U S A
79: 6232–6236. 10. Heggeness MH, Smith PR, Choppin PW (1982) In vitro assembly of the
nonglycosylated membrane protein (M) of Sendai virus. Proc Natl Acad Sci U S A
79: 6232–6236. 19. Waning DL, Schmitt AP, Leser GP, Lamb RA (2002) Roles for the cytoplasmic
tails of the fusion and hemagglutinin-neuraminidase proteins in budding of the
paramyxovirus simian virus 5. J Virol 76: 9284–9297. 7 PLOS ONE | www.plosone.org April 2013 | Volume 8 | Issue 4 | e61281 April 2013 | Volume 8 | Issue 4 | e61281 Role of F Cytoplasmic Tail in Virus Assembly Role of F Cytoplasmic Tail in Virus Assembly Role of F Cytoplasmic Tail in Virus Assembly J Virol 80: 10465–10477. 38. Fuerst TR, Niles EG, Studier FW, Moss B (1986) Eukaryotic transient-
expression system based on recombinant vaccinia virus that synthesizes
bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 83: 8122–8126. p
p
g
y p
J
27. Shaikh FY, Cox RG, Lifland AW, Hotard AL, Williams JV, et al. (2012) A
critical phenylalanine residue in the respiratory syncytial virus fusion protein
cytoplasmic tail mediates assembly of internal viral proteins into viral filaments
and particles. MBio 3. 39. Zhan X, Slobod KS, Krishnamurthy S, Luque LE, Takimoto T, et al. (2008)
Sendai virus recombinant vaccine expressing hPIV-3 HN or F elicits protective
immunity and combines with a second recombinant to prevent hPIV-1, hPIV-3
and RSV infections. Vaccine 26: 3480–3488. p
28. Li M, Schmitt PT, Li Z, McCrory TS, He B, et al. (2009) Mumps virus matrix,
fusion, and nucleocapsid proteins cooperate for efficient production of virus-like
particles. J Virol 83: 7261–7272. 40. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid
genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77: 61–68. p
J
29. Popa A, Pager CT, Dutch RE (2011) C-terminal tyrosine residues modulate the
fusion activity of the Hendra virus fusion protein. Biochemistry 50: 945–952. 41. Portner A (1981) The HN glycoprotein of Sendai virus: analysis of site(s)
involved in hemagglutinating and neuraminidase activities. Virology 115: 375–
384. 30. Merson JR, Hull RA, Estes MK, Kasel JA (1988) Molecular cloning and
sequence determination of the fusion protein gene of human parainfluenza virus
type 1. Virology 167: 97–105. 42. Portner A, Scroggs RA, Metzger DW (1987) Distinct functions of antigenic sites
of the HN glycoprotein of Sendai virus. Virology 158: 61–68. 31. Gorman WL, Gill DS, Scroggs RA, Portner A (1990) The hemagglutinin-
neuraminidase glycoproteins of human parainfluenza virus type 1 and Sendai
virus have high structure-function similarity with limited antigenic cross-
reactivity. Virology 175: 211–221. 43. Gorman WL, Pridgen C, Portner A (1991) Glycosylation of the hemagglutinin-
neuraminidase glycoprotein of human parainfluenza virus type 1 affects its
functional but not its antigenic properties. Virology 183: 83–90. PLOS ONE | www.plosone.org April 2013 | Volume 8 | Issue 4 | e61281 PLOS ONE | www.plosone.org 8
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Polonica in „Registrum Equitum Sancti Sepulchri” (1561-1848)
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Nasza Przeszłość
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cc-by-sa
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OLGIERD LENCZEWSKI, Warszawa
https://orcid.org/0000-0001-6567-328X
leniec1@wp.pl OLGIERD LENCZEWSKI, Warszawa
https://orcid.org/0000-0001-6567-328X
leniec1@wp.pl OLGIERD LENCZEWSKI, Warszawa
https://orcid.org/0000-0001-6567-328X
leniec1@wp.pl KEYWORDS: Registrum Equitum Sancti Sepulchri, Michele Piccirillo, Custody of
the Holy Land, Knights of the Holy Sepulchre, Knights of Jerusalem. POLONICA IN THE REGISTRUM EQUITUM
SANCTI SEPULCHRI (1561-1848) ABSTRACT: The Registrum Equitum Sancti Sepulchri is a valuable source for
presenting the contacts of Poles with the Holy Land in the years 1561-1848. On the
pages of this book, you can find information about approximately 1,590 people who
took part in the fitting ceremony for knights of the Holy Sepulchre in Jerusalem. Fifteen of them refer to the Polish Order of the Holy Sepulchre. To demonstrate their
presence in the book, one should take into account the historical and geographical
location of Poland at that time. Index equites can be inconsistent. Generally,
information about the nominees includes: noble titles, public or ecclesiastical
positions, family affiliation, origin. It happens, however, that data on knights has been
reduced to a minimum. Before the liber began to be maintained, notes were made
about the people knighted. It is estimated that, in the years 1348-1560, 817 knights
were fitted. However, it is not known how many people more precisely enlisted under
the banner of the Knights of the Holy Sepulchre until 1560, because these notes were
confiscated by the Turkish authorities. Although, on the basis of available sources,
it is possible to reconstruct the list created during this period. Based on the registrum
equitum, it is impossible to determine how many citizens of the Commonwealth
received the title eques Sancti Sepulchri, because the Franciscan monks did not
register every knighthood. This is evidenced by the examples contained in this article. It should be noted that the title eques hierosolymitanus was used not only by the
knights of the Holy Sepulchre. This title was also claimed by other orders: the Knights
of Malta, the Knights of Jerusalem from the Order of Saints Maurice and Lazarus,
or the Knights of the Order of the Golden Fleece. The author of the article did not
make use of the entire transcription made by Father Michele Piccirillo, OFM in the
book Registrum Equitum SSmi Sepulchri D.N.J.C. (1561-1848), but the photocopies
of the originals included in this publication, because he noticed that the above edition
does not meet editorial criteria, and contains many inaccuracies. However, the
publisher should be thanked for publishing the said register together with photocopies
of the manuscript. OLGIERD LE
36 136 OLGIERD LENCZEWSKI In this essay I have undertaken to discuss the polonics contained in the
Registrum Equitum Sancti Sepulchri (1561-1848). 1 C. de O d r i o z o l a y G r i m a u d, Libro de ora de la sagrada orden militar
Jerosalimitana del Santo Sepulcro de N. p. Jesús Cristo, containing the names of the
knights of that Order created by RR. Guardians of the Holy Sepulchre, Custos of the
Holy Land, of the Order of Minors of St. Francis, from the year 1561 to 1848 with
reference to the archives of the convent of San Salvador in Jerusalem and other
historical dates followed of an appendix with supporting notes, Zaragoza 1900.
Ś g
yg
(
) p
4 H. G a p s k i, Wstępny wykaz polskich rycerzów Grobu Naszego w Jerozolimie
(up to and including the nineteenth century), in: Zsuprectwo w Polsce Zakonu
Rycerzskiego Grobu Boże w Jerozolimie, ed. U. P r z y m u s, J. K a m i ń s k i,
Katowice 2008, pp. 136-137. y
y
(
) p
3 N. G o l i c h o w s k i, Palestyna, czyli pokłosie zebrane z różnych autorów i własne
spostrzeżenia, [b.m.d.w.], manuscript in the Archives of the Bernardine Province
in Kraków, RGP-k-94, vol. 2, pp. 225-226; idem, Ze Ziemi Świętej: polscy rycerze
Grobu Pańskiego, „Tygodnik Katolicki”, 23 (1892), p. 179. 1 C. de O d r i o z o l a y G r i m a u d, Libro de ora de la sagrada orden militar
Jerosalimitana del Santo Sepulcro de N. p. Jesús Cristo, containing the names of the
knights of that Order created by RR. Guardians of the Holy Sepulchre, Custos of the
Holy Land, of the Order of Minors of St. Francis, from the year 1561 to 1848 with
reference to the archives of the convent of San Salvador in Jerusalem and other
historical dates followed of an appendix with supporting notes, Zaragoza 1900.
2 Kawalerowie Grobu Św. Polacy, „Krzyż”, 23 (1867), p. 186.
3 N. G o l i c h o w s k i, Palestyna, czyli pokłosie zebrane z różnych autorów i własne
spostrzeżenia, [b.m.d.w.], manuscript in the Archives of the Bernardine Province
in Kraków, RGP-k-94, vol. 2, pp. 225-226; idem, Ze Ziemi Świętej: polscy rycerze
Grobu Pańskiego, „Tygodnik Katolicki”, 23 (1892), p. 179.
4 H. G a p s k i, Wstępny wykaz polskich rycerzów Grobu Naszego w Jerozolimie
(up to and including the nineteenth century), in: Zsuprectwo w Polsce Zakonu
Rycerzskiego Grobu Boże w Jerozolimie, ed. U. P r z y m u s, J. K a m i ń s k i,
Katowice 2008, pp. 136-137. f
f
pp
pp
g
2 Kawalerowie Grobu Św. Polacy, „Krzyż”, 23 (1867), p. 186. POLONICA IN THE REGISTRUM EQUITUM
SANCTI SEPULCHRI (1561-1848) So far, this issue has
been examined by three Poles: Fr Jukundin Bielak OFM, Fr Norbert
Golichowski OFM, and Henryk Gapski, referring to the work of Charles
de Odriozola y Grimaud.1 I noticed that this needs to be queried. again The aim of this article is to revise the state of research on polonics
contained in the book of the Knights of the Holy Sepulchre (1561-1848). This is due to the fact that the above-mentioned researchers, in addition
to the names noted with the annotation: polonus or indicating the
Polish origin of the knight, gave surnames unrelated to the former
Commonwealth. Importantly, they did not establish the identity of all of
the knights. Therefore, it is necessary to examine this problem closer. g
y
The beginnings of Polish research on these polonica date back to
the second half of the 19th century. Most likely the first Polish list was
published in the magazine Krzyż.2 It contained the names of four knights
from the lands of the former Polish-Lithuanian Commonwealth. It was
likely prepared by Father Jukundyn Bielak, OFM. Another list of its
citizens was noted by Father Norbert Golichowski, OFM. He published
this list in his diary and in the Tygodnik Katolicki weekly.3 It has 21
Polish surnames. After Golichowski’s research, the aforementioned
book was not used methodically until the twenty-first century (której? Odriozoli?). Recently, the search for Polish traces in the registrum was
continued by Henryk Gapski.4 His query showed that 18 knights had been
placed in the liber. Materials concerning milites Sancti Sepulchri also POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… 137 appear in herbariums (Paprocki’s,5 Niesiecki’s,6 Boniecki’s,7 Uruski’s,8
Dachnowski’s,9 Dworzaczek Files10), in an essay by Jan S. Bystron,
in the notes of Rev.11 Jan Fijałek,12 and in the works of Stanisław
A. Korwin13 and Jerzy Pertek.14 appear in herbariums (Paprocki’s,5 Niesiecki’s,6 Boniecki’s,7 Uruski’s,8
Dachnowski’s,9 Dworzaczek Files10), in an essay by Jan S. Bystron,
in the notes of Rev.11 Jan Fijałek,12 and in the works of Stanisław
A. Korwin13 and Jerzy Pertek.14 In 2006, owing to the efforts of Fr Michele Piccirillo OFM,
the manuscript Registrum Equitum Sancti Sepulchri (1561-1848) was
reissued, containing a list of the knights of the Holy Sepulchre.15 This
manuscript is in the Historical Archives of the Custody of the Holy
Land. It consists of two parts: A (1561-1831) and B (1831-1848).16
The first one is incomplete. 5 B. P a p r o c k i, Herby rycerstwa polskiego, ed. K. J. T u r o w s k i, Warszawa 1988.
6 K. N i e s i e c k i, Herbarz Polski, ed. J. N. B o b r o w i c z, 1-10, Lipsk 1839-1845.
7 A. B o n i e c k i, Herbarz Polski, vol. 1-16, Warszawa 1899-1913.
8 P. U r u s k i, Rodzina. Herbarz szlachty polskiej, vol. 1-16, Warszawa 1904-1938.
9 J. K. D a c h n o w s k i, Herbarz szlachty Prus Królewskich z XVII w., Kórnik 1995.
10 http://teki.bkpan.poznan.pl/index_monografie.html [access: 19.10.2022].
11J. P. B y s t r o ń, Polacy w Ziemi Świętej, Syrji i Egypt 1147-1914, Kraków 1930.
12 Notaty ks. Jana Fijałka: „Pątnictwo polskie. Pielgrzymki do Ziemi Świętej” XI-XVIII w,
manuscript in the PAU and PAN library in Krakow, 5313. 13 S. A. K o r w i n, Stosunki Polish z Ziemi Świętą, Warszawa 1958.
14 14 J. P e r t e k, Polacy na morzach i oceanach, vol. 1, Poznań 1981. 5 B. P a p r o c k i, Herby rycerstwa polskiego, ed. K. J. T u r o w s k i, Warszawa 1988.
6 K. N i e s i e c k i, Herbarz Polski, ed. J. N. B o b r o w i c z, 1-10, Lipsk 1839-1845.
7 A. B o n i e c k i, Herbarz Polski, vol. 1-16, Warszawa 1899-1913.
8 P. U r u s k i, Rodzina. Herbarz szlachty polskiej, vol. 1-16, Warszawa 1904-1938.
9 J. K. D a c h n o w s k i, Herbarz szlachty Prus Królewskich z XVII w., Kórnik 1995.
10 http://teki.bkpan.poznan.pl/index_monografie.html [access: 19.10.2022].
11J. P. B y s t r o ń, Polacy w Ziemi Świętej, Syrji i Egypt 1147-1914, Kraków 1930.
12 Notaty ks. Jana Fijałka: „Pątnictwo polskie. Pielgrzymki do Ziemi Świętej” XI-XVIII w,
manuscript in the PAU and PAN library in Krakow, 5313.
13 S. A. K o r w i n, Stosunki Polish z Ziemi Świętą, Warszawa 1958.
14 J. P e r t e k, Polacy na morzach i oceanach, vol. 1, Poznań 1981.
15 Registrum Equitum SSmi Sepulchri D.N.J.C. (1561-1848). Monoscritti dell’Archivo
storico della Custodia di Terra Santa a Gerusalemme, ed. M. P i c c i r i l l o, Studium
Biblicum Franciscanum Collectio Maior 46, Jerusalem-Milano 2006.
16 L. L e m m e n s, Collectanea ex Archivo Hierosolymitano deprompta, ed. G. G o -
l u b o v i c h, Quaracchi 1933, p. 253; The Historical Archive of the Custody of the
Holy Land (1230-1970), vol. 2, ed. A. M a i a r e l l i, Milan 2012, p. 450.
17 Registrum Equitum..., pp. 3-17. The number of knights of the Holy Sepulchre in
this period was approximately 125.
18 Ibidem, p. 5.
19 Ibidem, p. 15. 15 Registrum Equitum SSmi Sepulchri D.N.J.C. (1561-1848). Monoscritti dell’Archivo
storico della Custodia di Terra Santa a Gerusalemme, ed. M. P i c c i r i l l o, Studium
Biblicum Franciscanum Collectio Maior 46, Jerusalem-Milano 2006.
16 L. L e m m e n s, Collectanea ex Archivo Hierosolymitano deprompta, ed. G. G o -
l u b o v i c h, Quaracchi 1933, p. 253; The Historical Archive of the Custody of the
Holy Land (1230-1970) vol 2 ed A M a i a r e l l i Milan 2012 p 450 15 Registrum Equitum SSmi Sepulchri D.N.J.C. (1561-1848). Monoscritti dell’Archivo
storico della Custodia di Terra Santa a Gerusalemme, ed. M. P i c c i r i l l o, Studium
Biblicum Franciscanum Collectio Maior 46, Jerusalem-Milano 2006. Biblicum Franciscanum Collectio Maior 46, Jerusalem-Milano 2006.
16 L. L e m m e n s, Collectanea ex Archivo Hierosolymitano deprompta, ed. G. G o -
l u b o v i c h, Quaracchi 1933, p. 253; The Historical Archive of the Custody of the
Holy Land (1230-1970), vol. 2, ed. A. M a i a r e l l i, Milan 2012, p. 450. 19 Ibidem, p. 15. 18 Ibidem, p. 5. 17 Registrum Equitum..., pp. 3-17. The number of knights of the Holy Sepulchre in
this period was approximately 125.
18 Ibid
5 POLONICA IN THE REGISTRUM EQUITUM
SANCTI SEPULCHRI (1561-1848) It contains deficiencies, which cover the
years 1563-1572, 1575, 1577-1581, 1589, 1591-1596.17 These blanks
are explained in this section of the registry: Illi Milites qui creati fuerunt
in tempore regiminis Admodum Redi Pros Fris Bonifacii de Ragusio
Guard.i S. Montis Sion, annotati Fuerunt in alio libro, qui tempore
belli Regni Cipri combustus fuit a Turcis cum omnibus aliis libris in
quibus descripti erant alii Milites creati ab aliis Guardianis Sacri
Montis Sion eius Antecessoribus18 and multi alii milites tempore istius
Guard.i creati fuerunt, sed propter Turcharum persequutionis non
fuerunt notati in ordinario libro et ideo cognitio de his non habetur.19 16 L. L e m m e n s, Collectanea ex Archivo Hierosolymitano deprompta, ed. G. G o -
l u b o v i c h, Quaracchi 1933, p. 253; The Historical Archive of the Custody of the
Holy Land (1230-1970), vol. 2, ed. A. M a i a r e l l i, Milan 2012, p. 450. y
(
)
p
17 Registrum Equitum..., pp. 3-17. The number of knights of the Holy Sepulchre in
this period was approximately 125. 18 Ibid
5 19 Ibidem, p. 15. 138 OLGIERD LENCZEWSKI In addition, there are other deficiencies in the years: 1652, 1676-1678,
1684, 1689, 1698, 1717, 1739, 1747-1748, 1751-1752, 1757, 1759-1760,
1768, 1770-1771, 1774-1775, 1779, 1781, 1784-1788, 1791-1793,
1798-1799, the reasons for which are unknown. In addition, there are other deficiencies in the years: 1652, 1676-1678,
1684, 1689, 1698, 1717, 1739, 1747-1748, 1751-1752, 1757, 1759-1760,
1768, 1770-1771, 1774-1775, 1779, 1781, 1784-1788, 1791-1793,
1798-1799, the reasons for which are unknown. The register was created by the Italian Franciscans. It records
information about 1,590 people who obtained the rank of a Knight
of Jerusalem.20 In the information about the appointed knights, in addition
to the name and surname, the following data was included: noble title,
public or church position, family affiliation, origin.21 It is impossible
to precisely verify the nationality of each knight if one does not take
into account the historical and geographical location of the given
country at the given time.22 In the article I discuss the territories of the
former Commonwealth (Royal Prussia, Lithuania, Ukraine and Belarus),
i.e. the lands within the reach of the Crown until 1795. This is due to
the fact that I am raising the issue of polonics contained in the book of
the Knights of the Holy Sepulchre, covering the years of 1561-1848. 20 Indice onomastico, in: Registrum Equitum..., pp. 420-437. Parma citing the work of
Jean-Pierre de Gennes (Les chevaliers du Saint Sépulchre de Jérusalem, French 2004)
reports that, at that time, 1,671 people were appointed knights of the Holy Sepulchre,
T. P a r m a, Rytíři, dámy a poutníci: Dějiny a současnost Rytířského řádu Božího
hrobu jeruzalémského a jeho působení v českých zemích, Olomouc 2020, p. 109.
21 O
ti i d
i
R
i t
E
it
419 20 Indice onomastico, in: Registrum Equitum..., pp. 420-437. Parma citing the work of
Jean-Pierre de Gennes (Les chevaliers du Saint Sépulchre de Jérusalem, French 2004)
reports that, at that time, 1,671 people were appointed knights of the Holy Sepulchre,
T. P a r m a, Rytíři, dámy a poutníci: Dějiny a současnost Rytířského řádu Božího
hrobu jeruzalémského a jeho působení v českých zemích, Olomouc 2020, p. 109.
21 Onomastic index, in: Registrum Equitum..., p. 419.
22 Ibidem.
23 M. P i c c i r i l l o, Introduzione, in: Registrum Equitum..., p. VIII. Of which in the
14th century the title of miles was given to only 20 people.
24 Vol. P a r m a, op. cit., p. 109. Unfortunately, their names were not included, as de
Gennes compiled only a personal roster of French cavaliers. I obtained this information
courtesy of Dr. Tomáš Parma (e-mail dated November 11, 2022).
25 B. P a p r o c k i, op. cit., p. 953; H. L u l e w i c z, Sołtan Aleksandrowicz, PSB, 40,
Warszawa-Kraków 2000-2001, pp. 341-342; H. G a p s k i, op. cit., p. 136; M. L u -
b c z y ń s k i, J. P i e l a s, Szydłowiecki Krzysztof, PSB, 49, Warszawa-Kraków 2014, y
(
)
25 B. P a p r o c k i, op. cit., p. 953; H. L u l e w i c z, Sołtan Aleksandrowicz, PSB, 40,
Warszawa-Kraków 2000-2001, pp. 341-342; H. G a p s k i, op. cit., p. 136; M. L u -
b c z y ń s k i, J. P i e l a s, Szydłowiecki Krzysztof, PSB, 49, Warszawa-Kraków 2014, 23 M. P i c c i r i l l o, Introduzione, in: Registrum Equitum..., p. VIII. Of which in
14th century the title of miles was given to only 20 people.
24 23 M. P i c c i r i l l o, Introduzione, in: Registrum Equitum..., p. VIII. Of which in the
14th century the title of miles was given to only 20 people.
24 Vol. P a r m a, op. cit., p. 109. Unfortunately, their names were not included, as de
Gennes compiled only a personal roster of French cavaliers. I obtained this information
courtesy of Dr. Tomáš Parma (e-mail dated November 11, 2022).
25 B. P a p r o c k i, op. cit., p. 953; H. L u l e w i c z, Sołtan Aleksandrowicz, PSB, 40,
Warszawa-Kraków 2000-2001, pp. 341-342; H. G a p s k i, op. cit., p. 136; M. L u -
b c z y ń s k i, J. P i e l a s, Szydłowiecki Krzysztof, PSB, 49, Warszawa-Kraków 2014, 24 Vol. P a r m a, op. cit., p. 109. Unfortunately, their names were not included, as
Gennes compiled only a personal roster of French cavaliers. I obtained this informa
courtesy of Dr. Tomáš Parma (e-mail dated November 11, 2022). j
j
p
ý
,
, p
omastic index, in: Registrum Equitum..., p. 419. 30 Registrum A, k. 2, photocopy in: Registrum Equitum…, p. 4. Jan Pudłowski (d. 1614),
Sochaczew army (1585-1603) see: H. K o w a l s k a, Pudłowski Melchior, PSB, 29,
Wrocław-Warszawa-Krakow-Gdańsk-Łódź 1986, p. 344. The data included in brackets
was obtained thanks to the courtesy of Andrew Haratym, M.A. p. 551; K. B a c z k o w s ki, Mikołaj Rozembarski, PSB, 32, Wrocław-Warszawa-
Kraków 1991, p. 384.
26 Bogusław X of Pomerania was included in this list only because he was related to the
Jagiellonian dynasty (he married the daughter of King Casimir Jagiellon, Princess Anna).
27 S. B a r ą c z, Żywoty sławnych Ormian w Polsce, Lwów 1856, p. 349; Notaty
ks. Jana Fijałka, k. 145, 180; H. B a r y c z, „Z zagadnień podróżnictwa polskiego
w dawnych wiekach”, Przegląd Współczesny, 10 (1938), p. 72; W. P o c i e c h a,
Królowa Bona (1494-1557): czasy i ludzie odrodzenia, vol. 2, Poznań 1949, p. 49;
J. P e r t e k, op. cit, p. 297; Chronografia albo Dziejopis Żywiecki, ed. P. G r o d z i s k i,
I. D w o r n i c k a, Żywiec 1987, p. 574; J. K u r t y k a, Latyfundium tęczyńskie: dobra
i właściciele (XIV-XVII w.), Kraków 1999, pp. 179-180, 183; O. L e n c z e w s k i,
Wybrane opisy Ziemi Świętej w relacji polskich pielgrzymów od XV do XVIII w.,
UKSW Warszawa 2017, mps, p. 44-46. 28 De Gennes estimates that 20 Poles were recorded in the register, but does not
mention their names, as he was only interested in French personalities, see footnote 24.
29 Registrum A (1561-1831), k. 1, photocopy in: Registrum Equitum…, p. 3. Přemek
z Víckova (1535-1584), nephew of the Olomouc bishop, see: T. P a r m a, op. cit.,
p. 303. p. 551; K. B a c z k o w s ki, Mikołaj Rozembarski, PSB, 32, Wrocław-Warszawa-
Kraków 1991, p. 384.
26 Bogusław X of Pomerania was included in this list only because he was related to the
Jagiellonian dynasty (he married the daughter of King Casimir Jagiellon, Princess Anna).
27 S. B a r ą c z, Żywoty sławnych Ormian w Polsce, Lwów 1856, p. 349; Notaty
ks. Jana Fijałka, k. 145, 180; H. B a r y c z, „Z zagadnień podróżnictwa polskiego
w dawnych wiekach”, Przegląd Współczesny, 10 (1938), p. 72; W. P o c i e c h a,
Królowa Bona (1494-1557): czasy i ludzie odrodzenia, vol. 2, Poznań 1949, p. 49;
J. P e r t e k, op. cit, p. 297; Chronografia albo Dziejopis Żywiecki, ed. P. G r o d z i s k i,
I. D w o r n i c k a, Żywiec 1987, p. 574; J. K u r t y k a, Latyfundium tęczyńskie: dobra
i właściciele (XIV-XVII w.), Kraków 1999, pp. 179-180, 183; O. L e n c z e w s k i,
Wybrane opisy Ziemi Świętej w relacji polskich pielgrzymów od XV do XVIII w.,
UKSW Warszawa 2017, mps, p. 44-46.
28 De Gennes estimates that 20 Poles were recorded in the register, but does not
mention their names, as he was only interested in French personalities, see footnote 24.
29 Registrum A (1561-1831), k. 1, photocopy in: Registrum Equitum…, p. 3. Přemek
z Víckova (1535-1584), nephew of the Olomouc bishop, see: T. P a r m a, op. cit.,
p. 303.
30 Registrum A, k. 2, photocopy in: Registrum Equitum…, p. 4. Jan Pudłowski (d. 1614),
Sochaczew army (1585-1603) see: H. K o w a l s k a, Pudłowski Melchior, PSB, 29,
Wrocław-Warszawa-Krakow-Gdańsk-Łódź 1986, p. 344. The data included in brackets
was obtained thanks to the courtesy of Andrew Haratym, M.A. 51; K. B a c z k o w s ki, Mikołaj Rozembarski, PSB, 32, Wrocław-Warszawa-
ków 1991, p. 384. mention their names, as he was only interested in French personalities, see footnote 24.
29 Registrum A (1561-1831), k. 1, photocopy in: Registrum Equitum…, p. 3. Přemek
z Víckova (1535-1584), nephew of the Olomouc bishop, see: T. P a r m a, op. cit.,
p. 303. POLONICA IN THE REGISTRUM EQUITUM
SANCTI SEPULCHRI (1561-1848) Before the register was created, notes on candidates were written
down. It is estimated that, in the years 1348-1496, 653 people obtained
the rank of miles, and in the years 1500-156023 – 164, including five
Poles.24 On the basis of surviving testimonies, it is known that this title
was also adopted by other Poles at that time, e.g. Jan Łaski (ca. 1450),
Mikołaj Jordan from Zakliczyn (second half of the 15th century),
Aleksander Sołtan (ca. 1468), Krzysztof Szydłowiecki (ca. 1492) and
Mikołaj Rozembarski (ca. 1495).25 Later records of nominated knights POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… 139 until 1560 have not survived to our times, because they were burned
by the Turkish authorities ca. 1570. Based on other materials, it is possible
to reconstruct the list of people who obtained the title of knight of the
Holy Sepulchre. It was initially estimated that the above group included
the following Poles, i.e. Bogusław X (1497), Eberhardt Ferber (1497),
Marcin Wartan (first half of the 16th century), Krzysztof Nuchorski (1509),
Andrzej Łaski (1511), Mikołaj Wolski (1511), Stanisław Włoszek
from Tęczyn (1513), Jan from Tęczyn (1513), Janusz Latalski (1513),
Jan Tarnowski known as ‘Ciężki’ (1513),26 Przecław Lanckoroński
(1513), Kasper Maciejowski (ca. 1514), Piotr Stano (ca. 1514), Hieronim
Jarosławski (1517), Jan Amor Tarnowski (1517), Hieronim Łaski
(ca. 1518), Marcin Broniewski (1518), Stanisław Łaski (1520), Andrzej
from Żywiec (1521), Jan Gostyński (1529), Jan Kościelecki (1532), Kilian
from Latoszyn (1534), Stanisław from Tęczyn and Batorz (ca. 1535),
Jan Firlej (ca. 1535), Andrzej from Tęczyn, known as Pępek (ca. 1535).27 Until now, it was assumed that in the register from 1561 to 18
one can find mentions of 26 citizens of the Commonwealth:28 – 1561, 17 Augustii, Primislaus de Vickoua;29 – 1561, 17 Augustii, Primislaus de Vickoua;29 – 1576, 24 Augusti, Iohannis/Iohannes Pudloschii Polonus;30 – 1576, 24 Augusti, Iohannis/Iohannes Pudloschii Polo 30 Registrum A, k. 2, photocopy in: Registrum Equitum…, p. 4. Jan Pudłowski (d. 1614),
Sochaczew army (1585-1603) see: H. K o w a l s k a, Pudłowski Melchior, PSB, 29,
Wrocław-Warszawa-Krakow-Gdańsk-Łódź 1986, p. 344. The data included in brackets
was obtained thanks to the courtesy of Andrew Haratym, M.A. 38 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8.
39 Ibidem. POLONICA IN THE REGISTRUM EQUITUM
SANCTI SEPULCHRI (1561-1848) OLGIERD LENCZEWSKI 140 – 1582, 29 Octobris Bernardus Bruntalskii Baro Marcomannus
<de> Wirben;31 – 1582, 29 Octobris Bernardus Bruntalskii Baro Marcomannus
<de> Wirben;31 – 1583, Nicolaus Christophorus Radzuul [should be: Radzivil] Dux
Olic[a]e et in[N]esvisch Comes in Schidlonie [should be:
Schidloviec?] ac Mhir Sacri Rom[ani] Imper[ii] Princeps
Archimarschalcus Ducatus Lituaniae;32 – 1583, Abraham Burgo Grauius et Baro a Donaminor D[omi]nus
Incraschen;33 – 1583, Abraham Burgo Grauius et Baro a Donaminor D[omi]nus
Incraschen;33 – 1583, Georgius Koss Prutenus;34
35 – 1583, Michael Konarski;35 – 1583, Petrus Bulina [should be: Bylina] Polonus;36 – 1583, Andreas Skorulski Littuanus;37 – 1583, Colonellus Melchior Custii Eques S[an]ct[a]e Ecclesi[a]e
Amanus Cantonsiluanie;38
1583 R d l h
Phiff
L
i
39 – 1583, Colonellus Melchior Custii Eques S[an]ct[a]e Ecclesi[a]e
Amanus Cantonsiluanie;38 – 1583, Rudolphus Phifferro, Lucernensis;39 31 Registrum A, k. 2, photocopy in: Registrum Equitum…, p. 6. Bernard Bruntálský
from Vrbno (d. 1613), see: T. P a r m a, op. cit., p. 304. Annotations regarding the
origin of Bruntáls by Fr Bertrand Zimolong, OFM, see: idem Schlesische Pilger im
Hl. Lande 1561-1695, Breslau 1938, p. 266, ftn. 56. 31 Registrum A, k. 2, photocopy in: Registrum Equitum…, p. 6. Bernard Bruntálský
from Vrbno (d. 1613), see: T. P a r m a, op. cit., p. 304. Annotations regarding the
origin of Bruntáls by Fr Bertrand Zimolong, OFM, see: idem Schlesische Pilger im
Hl. Lande 1561-1695, Breslau 1938, p. 266, ftn. 56. 32 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Mikołaj Krzysztof
Radziwiłł „Sierotka” (1549-1616), prince, diarist, see: T. S ł a b c z y ń s k i, Słownik
polskich podróżników i odkrywców, Warszawa 2017, p. 330. 32 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Mikołaj Krzysztof
Radziwiłł „Sierotka” (1549-1616), prince, diarist, see: T. S ł a b c z y ń s k i, Słownik
polskich podróżników i odkrywców, Warszawa 2017, p. 330. 33 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Abraham III from
Donín (1561-1613), companion of the peregrination of M. K. Radziwiłł to the Holy
Land, see: M. K. R a d z i w i ł ł „Sierotka”, Podróż do Ziemi Świętej. Syrii i Egiptu
1581-1584, by L. K u k u l s k i, Warszawa 1962, pp. 66-67; T. P a r m a, op. cit., p. 304. 34 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. 31 Registrum A, k. 2, photocopy in: Registrum Equitum…, p. 6. Bernard Bruntálský
from Vrbno (d. 1613), see: T. P a r m a, op. cit., p. 304. Annotations regarding the
origin of Bruntáls by Fr Bertrand Zimolong, OFM, see: idem Schlesische Pilger im
Hl. Lande 1561-1695, Breslau 1938, p. 266, ftn. 56.
32 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Mikołaj Krzysztof
Radziwiłł „Sierotka” (1549-1616), prince, diarist, see: T. S ł a b c z y ń s k i, Słownik
polskich podróżników i odkrywców, Warszawa 2017, p. 330.
33 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Abraham III from
Donín (1561-1613), companion of the peregrination of M. K. Radziwiłł to the Holy
Land, see: M. K. R a d z i w i ł ł „Sierotka”, Podróż do Ziemi Świętej. Syrii i Egiptu
1581-1584, by L. K u k u l s k i, Warszawa 1962, pp. 66-67; T. P a r m a, op. cit., p. 304.
34 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Jerzy Kos, companion of
the peregrination of M. K. Radziwiłł to the Holy Land, see: M. K. R a d z i w i ł ł
„Sierotka”, op. cit., pp. 66-67.
35 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Michał Konarski
(1557-1613), Pomeranian Voivode, companion of the peregrination of M. K. Radziwiłł to
the Holy Land, see: M. K. R a d z i w i ł ł „Sierotka”, op. cit. pp. 66-67; F. M i n c e r,
Konarski Michał, PSB, 13, Wrocław-Warszawa-Kraków 1967-1968, pp. 467-468.
36 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Piotr Bylina (d. 1603),
companion of the peregrination of M. K. Radziwiłł to the Holy Land, see: M. K. R a d z i w i łł
„Sierotka”, op. cit., pp. 66-67; A. B o n i e c k i, op. cit., vol. 2, Warszawa 1900, p. 285.
37 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Andrzej Skorulski
(d. 1637), Kaunas Marshal, companion of the peregrination of M. K. Radziwiłł to the
Holy Land, see: M. K. R a d z i w i ł ł „Sierotka”, op. cit., pp. 66-67; H. L u l e w i c z,
Skorulski Andrzej, PSB, 38, Warszawa 1997-1998, p. 269.
38 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8.
39 Ibidem. 35 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Michał Konarski
(1557-1613), Pomeranian Voivode, companion of the peregrination of M. K. Radziwiłł to
the Holy Land, see: M. K. R a d z i w i ł ł „Sierotka”, op. cit. pp. 66-67; F. M i n c e r,
Konarski Michał, PSB, 13, Wrocław-Warszawa-Kraków 1967-1968, pp. 467-468. Skorulski Andrzej, PSB, 38, Warszawa 1997-1998, p. 269. 34 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Jerzy Kos, companion of
the peregrination of M. K. Radziwiłł to the Holy Land, see: M. K. R a d z i w i ł ł
„Sierotka”, op. cit., pp. 66-67. 33 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Abraham III from
Donín (1561-1613), companion of the peregrination of M. K. Radziwiłł to the Holy
Land, see: M. K. R a d z i w i ł ł „Sierotka”, Podróż do Ziemi Świętej. Syrii i Egiptu
1581-1584, by L. K u k u l s k i, Warszawa 1962, pp. 66-67; T. P a r m a, op. cit., p. 304. 32 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Mikołaj Krzysztof
Radziwiłł „Sierotka” (1549-1616), prince, diarist, see: T. S ł a b c z y ń s k i, Słownik
polskich podróżników i odkrywców, Warszawa 2017, p. 330. 40 Ibidem.
41 Registrum A, k. 5v, photocopy in: Registrum Equitum…, p. 20. Wratlisav of Donína
in Lemberk (d. 1606), owner of Lemberk Castle, Maltese knight. According to
Golichowski, he is a burgrave from Lviv, see: the same, Palestyna, p. 225; Ze Ziemi
Świętej, p. 179. In Odriozoli y Grimauda, a remark appeared next to his name:
Lithuanus. Lembergensis, see: idem, op. cit., p. 9. 43 Registrum A, k. 10, photocopy in: Registrum Equitum…, p. 38. Jan Drahatemski
(d. 1615), painter, see, Navis peregrinorum: ein Pilgerverzeichnis aus Jerusalem
von 1561 bis 1695: mit Angaben über Pilger aus Deutschland, England, Frankreich,
Italien und den Niederlanden, sowie aus anderen europäischen und aussereuropäischen
Ländern, ed. B. Z i m o l o n g, Köln 1938, p. 32. Zdaniem Odriozoli y Grimauda to:
Joannes Brahatemsky Palatinus Dioecesis Vilnensis, see: idem, op. cit., p. 12. 46 Registrum A, k. 24, photocopy in: Registrum Equitum…, p. 94. Jean de la Cron
(d. 1665), military man, see: T. P a r m a, op. cit., p. 306. Odriozola y Grimaud
described him as: Polonus Dioecesiae Vilnius, see: idem, op. cit., p. 19. 44 Registrum A, k. 10, photocopy in: Registrum Equitum…, p. 38. Teofil Szemberg
(d. ca. 1640), royal secretary, diplomat, see: M. N a g i e l s k i, Szemberg Teofil, PSB, 48,
Warszawa-Kraków 2012-2013, pp. 127-128. 40 Ibidem.
41 Registrum A, k. 5v, photocopy in: Registrum Equitum…, p. 20. Wratlisav of Donína
in Lemberk (d. 1606), owner of Lemberk Castle, Maltese knight. According to
Golichowski, he is a burgrave from Lviv, see: the same, Palestyna, p. 225; Ze Ziemi
Świętej, p. 179. In Odriozoli y Grimauda, a remark appeared next to his name:
Lithuanus. Lembergensis, see: idem, op. cit., p. 9.
42 Registrum A, k. 7, photocopy in: Registrum Equitum…, p. 26. His identity could not
be established.
43 Registrum A, k. 10, photocopy in: Registrum Equitum…, p. 38. Jan Drahatemski
(d. 1615), painter, see, Navis peregrinorum: ein Pilgerverzeichnis aus Jerusalem
von 1561 bis 1695: mit Angaben über Pilger aus Deutschland, England, Frankreich,
Italien und den Niederlanden, sowie aus anderen europäischen und aussereuropäischen
Ländern, ed. B. Z i m o l o n g, Köln 1938, p. 32. Zdaniem Odriozoli y Grimauda to:
Joannes Brahatemsky Palatinus Dioecesis Vilnensis, see: idem, op. cit., p. 12.
44 Registrum A, k. 10, photocopy in: Registrum Equitum…, p. 38. Teofil Szemberg
(d. ca. 1640), royal secretary, diplomat, see: M. N a g i e l s k i, Szemberg Teofil, PSB, 48,
Warszawa-Kraków 2012-2013, pp. 127-128.
45 Registrum A, k. 11v, photocopy in: Registrum Equitum…, p. 44. His identity could
not be established.
46 Registrum A, k. 24, photocopy in: Registrum Equitum…, p. 94. Jean de la Cron
(d. 1665), military man, see: T. P a r m a, op. cit., p. 306. Odriozola y Grimaud
described him as: Polonus Dioecesiae Vilnius, see: idem, op. cit., p. 19. 45 Registrum A, k. 11v, photocopy in: Registrum Equitum…, p. 44. His identity could
not be established. 42 Registrum A, k. 7, photocopy in: Registrum Equitum…, p. 26. His identity could not
be established. POLONICA IN THE REGISTRUM EQUITUM
SANCTI SEPULCHRI (1561-1848) Jerzy Kos, companion of
the peregrination of M. K. Radziwiłł to the Holy Land, see: M. K. R a d z i w i ł ł
„Sierotka”, op. cit., pp. 66-67. 35 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Michał Konarski
(1557-1613), Pomeranian Voivode, companion of the peregrination of M. K. Radziwiłł to
the Holy Land, see: M. K. R a d z i w i ł ł „Sierotka”, op. cit. pp. 66-67; F. M i n c e r,
Konarski Michał, PSB, 13, Wrocław-Warszawa-Kraków 1967-1968, pp. 467-468. 36 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Piotr Bylina (d. 1603),
companion of the peregrination of M. K. Radziwiłł to the Holy Land, see: M. K. R a d z i w i łł
„Sierotka”, op. cit., pp. 66-67; A. B o n i e c k i, op. cit., vol. 2, Warszawa 1900, p. 285. 37 Registrum A, k. 2v, photocopy in: Registrum Equitum…, p. 8. Andrzej Skorulski
(d. 1637), Kaunas Marshal, companion of the peregrination of M. K. Radziwiłł to the
Holy Land, see: M. K. R a d z i w i ł ł „Sierotka”, op. cit., pp. 66-67; H. L u l e w i c z,
Sk
l ki A d
j PSB 38 Wars a a 1997 1998 p 269 POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… 47 Registrum A, k. 29, photocopy in: Registrum Equitum…, p. 114. Franciszek
Kazimierz Wysocki (d. 1681), Sublime Porte deputy, a cub-bearer from Sochaczew
(from 1661), see: J. P. Ł ą t k a, Słownik Polaków w Imperium Osmańskim i w Republice
Turcji, ed. 2, revised, Kraków 2015, p. 452. The data included in brackets was obtained
thanks to the courtesy of Andrew Haratym M.A. 48 Registrum A, k. 30, photocopy in: Registrum Equitum…, p. 118. Franciszek Meniński
(1623-1698), orientalist and lexicographer, see: T. S ł a b c z y ń s k i, op. cit., pp. 270-271.
49 Registrum A, k. 67, photocopy in: Registrum Equitum…, p. 270. Herakliusz
Lisowski (1734-1809), Uniate Archbishop of Polotsk, see: L. Ż y t k o w i c z,
Lisowski Józef, PSB,17, Wrocław-Warszawa-Kraków 1972, pp. 473-474. 53 Preceded by the word Polonus in the margin. 52 Registrum A, k. 72, photocopy in: Registrum Equitum…, p. 290. Antonín Prokresch
(1795-1876), Austrian General, see: T. P a r m a, op. cit., p. 311. 51 Preceded by the word Austriacus in the margin. 47 Registrum A, k. 29, photocopy in: Registrum Equitum…, p. 114. Franciszek
Kazimierz Wysocki (d. 1681), Sublime Porte deputy, a cub-bearer from Sochaczew
(from 1661), see: J. P. Ł ą t k a, Słownik Polaków w Imperium Osmańskim i w Republice
Turcji, ed. 2, revised, Kraków 2015, p. 452. The data included in brackets was obtained
thanks to the courtesy of Andrew Haratym M.A.
48 Registrum A, k. 30, photocopy in: Registrum Equitum…, p. 118. Franciszek Meniński
(1623-1698), orientalist and lexicographer, see: T. S ł a b c z y ń s k i, op. cit., pp. 270-271.
49 Registrum A, k. 67, photocopy in: Registrum Equitum…, p. 270. Herakliusz
Lisowski (1734-1809), Uniate Archbishop of Polotsk, see: L. Ż y t k o w i c z,
Lisowski Józef, PSB,17, Wrocław-Warszawa-Kraków 1972, pp. 473-474.
50 Registrum A, k. 71, photocopy in: Registrum Equitum…, p. 286. Emeryk Stefan
Leonard Plater (1799-1864), Commander of the Cross of the Holy Sepulchre, see:
S. K o n a r s k i, Platerowie, Buenos Aires-Paris 1967, p. 167.
51 Preceded by the word Austriacus in the margin.
52 Registrum A, k. 72, photocopy in: Registrum Equitum…, p. 290. Antonín Prokresch
(1795-1876), Austrian General, see: T. P a r m a, op. cit., p. 311.
53 Preceded by the word Polonus in the margin. 50 Registrum A, k. 71, photocopy in: Registrum Equitum…, p. 286. Emeryk Stefan
Leonard Plater (1799-1864), Commander of the Cross of the Holy Sepulchre, see:
S. K o n a r s k i, Platerowie, Buenos Aires-Paris 1967, p. 167. POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… 141 – 1583, Mauritius Phiffer, Lucernensis;40 – 1602, 17 Augusti, Vratislaus Burgraff et Baro de Dona in Lemberg;41 – 1606, 7 septembris, Andreas Lind Suecus Polonus;42 – 1614, 30 Martii, Joannis Drahatemsky de Palatino Boemus
Catt[oli]cae Civitatis Pilsnae;43 – 1613, 6 novembris, Theophilus Szemberg Polonus;44 – 1618, septembris, Ioannis Bialcouius Sacerdos Polonus Di[o]ecesis
Vilnensis in Lithuania;45 – 1650, 27 septembris, Ill[ustrissi]mus Dominus D[ominus]Ioannes
seu Jean de la Coron Gubernator Civitatis pilsenensis D[omi]n[u]s
in Poritsch etc. creatus Fuit Eques SS.mi Sepulchri in absentia,
suscipiente pro eo habitum Equestris Militiae Rev[erendo] P[at]re
Electo Zwinner Strict[ioris] Obs[ervanti]ae Prov[inci]ae Boemiae
Concionatore Terraeq[ue] S[anct]ae Discreto eius Procuratore;46 q
– 1667, 24 Novem[bris], Illustrissimus et Excell[entissi]mus
D[omi]nus D[omi]nus Casimirus Wysocky Pincerna Sockaviensis
[sic!] Sacrae Regie Maiestatis Poloniae Sec[re]t[a]rius et ad
praesens apud Portam Ottomanicam legatus Creatus fuit Eques
SSmi Sepulchri suscipiente pro eo habitum Equestris Militiae 44 Registrum A, k. 10, photocopy in: Registrum Equitum…, p. 38. Teofil Szemberg
(d. ca. 1640), royal secretary, diplomat, see: M. N a g i e l s k i, Szemberg Teofil, PSB, 48,
Warszawa-Kraków 2012-2013, pp. 127-128. 45 Registrum A, k. 11v, photocopy in: Registrum Equitum…, p. 44. His identity could
not be established. 46 Registrum A, k. 24, photocopy in: Registrum Equitum…, p. 94. Jean de la Cron
(d. 1665), military man, see: T. P a r m a, op. cit., p. 306. Odriozola y Grimaud
described him as: Polonus Dioecesiae Vilnius, see: idem, op. cit., p. 19. 142 OLGIERD LENCZEWSKI in absen[tia] R.P.F. Dominico Lardizaval Ord[inis] Min[or]
Obs[ervanti]ae Prov[inci]ae Beticae Terrae Sanctae Proc[urato]re
generale eiq[ue] Procuratore;47 – 1669, 20 iun[ius], Illustrissimus Dominus D[ominus] Franciscus
de Mesgnienski [sic!] Sacrae Caesareae Maiestatis Consiliarus,
et Orientalium linguarum supremus Interpraes [sic!] Creatus fuit
Eques S. Smi Sepulc[hri] ipse personaliter comparens postulans,
et recipiens habitum;48 – 1805, 4 februarii, Creatus fuit Eques Excellentissimus et
Reverendissimus D[ominus] Archiepiscopus Heraclius Lisovski
Di[o]ecesis Polocensi[s] ex Polonia Russia per Procuratorem,
qui fuit R.P. Clemens Perez Minoris Observ[an]tiae Prov[incia]e
Castell[an]ae, necessaria requisita produxit;49 – 1827, 7 aprilis, Ill[ustrissi]mus Stephanus de Plater Polonus, cujus
Procurator fuit Fr. Leonard Plater (1799-1864), Commander of the Cross of the Holy Sepulchre,
S. K o n a r s k i, Platerowie, Buenos Aires-Paris 1967, p. 167. 59 It should be noted that the term Lemberg was used to describe the cities Lviv and
Lwówek, see: Orbis latinus oder der wichtigsten lateinischen Orts- und Ländernamen,
by T. G. J. G r a e s s e, F. Benedict, Berlin 1909, p. 179. 54 Registrum B, k. 17, photocopy in: Registrum Equitum…, p. 332. Adam Sołtan (1792-1863),
landowner, received the Order of the Holy Sepulchre from the Pope, see: Z. S u d o l s k i,
Adam Ludwik Michał Sołtan, PSB, 40, Warszawa-Kraków 2001, pp. 344-345.
55 Preceded by the word Theutonicus in the margin.
56 Registrum B (1831-1847), k. 17, photocopy in Registrum Equitum…, p. 332. Count
Edmund Zichy de Vázsonykői (1811-1894), politician, see: https://hu.wikipedia.org/
wiki/Zichy_Edmund [access: 27.03.2020].
57 Registrum B, k. 34, photocopy in: Registrum Equitum…, p. 366. So far, it has not
been possible to precisely determine the personal data of this knight. Importantly,
we have different versions of reading this surname. These variants are shown in the
table below. Only Horain’s variations are excluded from this list: Ladislao Chodkiemenz,
see: Cz. H o r a i n, Na tropie Polaków w Ziemi Świętej. Rejestr pielgrzymów i po-
dróżników polskich do Ziemi Świętej przybyłych w latach 1845-1921; 1931-1947,
by M. K ł a k u s, Katowice-Toulon 2022, p. 98. 54 Registrum B, k. 17, photocopy in: Registrum Equitum…, p. 332. Adam Sołtan (1792-1863),
landowner, received the Order of the Holy Sepulchre from the Pope, see: Z. S u d o l s k i,
Adam Ludwik Michał Sołtan, PSB, 40, Warszawa-Kraków 2001, pp. 344-345.
55 P
d d b th
d Th
t
i
i th
i 58 T. P a r m a, op. cit., p. 304. POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… Emanuel Bajon Vice Procurator G[e]n[era]lis
Terrae Sanctae;50 – 1829, 23 aprilis, Ill[ustrissi]mus D[omi]nus Antonius Prokesch
Austriacus51 ex Nobili familia Austriaca S[acrae] M[aiestatis]
I[mperialis] R[egiae] A[postolicae] in Exercitu Dux et Diversorum
Ordinum Eques;52 – 1842, 6 aprilis, Illu[strissi]mus Dominus D[o]minus Adam Soleland
[sic!] Polonus,53 per Procurat[orem];54 – 1842, 6 aprilis, Illu[strissi]mus Dominus D[o]minus Adam Soleland
[sic!] Polonus,53 per Procurat[orem];54 50 Registrum A, k. 71, photocopy in: Registrum Equitum…, p. 286. Emeryk Stefan
Leonard Plater (1799-1864), Commander of the Cross of the Holy Sepulchre, see:
S. K o n a r s k i, Platerowie, Buenos Aires-Paris 1967, p. 167. POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… 143 – 1842, 9 Maii, Edmundus Comes Zichy de VasonRő [should be:
Vá(z)sonykő(i)] Theutonicus55 Suae Maiestatis Sacratissimae
Imperatoris Austriae Regni Hungariae et Bohemiae Camerarius;56 – 1847, 13 decembris, Illustrissimus ac Nobil[li]mus Vir Ladislaus
Codenieiviz Comes Polonus, creatus fuit Eques SS. Sepulcri
D.N.I.C personaliter.57 As can been seen, the notes about them are more or less precise: – eight surnames contain the note Polonus; – three surnames provide areas that belonged to the Commonwealth
at that time (Lituaniae/Littuanus, Diecesis Polocensi); – one surname contains information about the office held by this
knight in Poland: pincerna sockaviensis; – three surnames do not have further data on the origin of the knight:
Michael Konarski, Franciscus de Mesgnienski; Abraham Burgo
Grauius. Parma considers the last of the knights to be a Czech;58 – ten surnames refer to areas that were not within the reach of the
Commonwealth (Boemus, Austriacus, Theutonicus, Cantonsiluanie,
Lemberg,59 Lucernensis, Wirben, Vickoua). I have shown that the pages of the book of the knights of the Holy
Sepulchre contain records of 15 citizens of the former Commonwealth. The
following table depicts that no such conclusion had been reached to date.60 144 OLGIERD LENCZEWSKI Table 1. 60 Surnames in which there is no national annotation refer to citizens from the lands of
the former Commonwealth. The notes with footnotes do not originate from the book
of the knights of the Holy Sepulchre. 61 Spis Polaków, którzy odwiedzili Ziemię Świętą, „Krzyż”, 22 (1867), p. 178. 62 H. F r e y t a g, Preuβische Jerusalempilger von 14. bis 16. Jahrhundert, „Archiv
für Kultur-Geschichte”, 3 (1905), p. 150; J. Pogonowski, Pamiątka po Feliksie
Odrowążu Pieniążku, „Miesięcznik Heraldyczny”, 6 (1930), p. 125; idem, Szabla-
batorówka Feliksa Odrowąża Pieniążka, „Broń i Barwa”, 4 (1935), p. 89; F. M i n c e r,
Koene-Jaski Jerzy, PSB, 13, Wrocław-Warszawa-Kraków 1967-1968, p. 256;
H. G a p s k i, op. cit., p. 137; O. L e n c z e w s k i, Wybrane opisy…, pp. 47-52; Idem,
Polonica w księdze pielgrzymów jerozolimskich – Navis peregrinorum (1561-1695),
„Nasza Przeszłość”, 132 (2019), p. 98. 67 A. W e j n e r t, Kawalerowe złotéj ostrogi w Polsce do XIX w., Warszawa 1879;
T. Sz u l c, Eques auratus w dawnej Rzeczypospolitej, „Acta Universitatis Lodzensis:
Folia Iuridica”, 38 (1988), p. 76; H. L u l e w i c z, Sapieha Mikołaj z Kodnia h. Lis,
PSB, 35, Warszawa-Kraków 1994, p. 69; A. B o g u c k i, Polskie nazwy rycerstwa
w średniowieczu: przyczynki do historii ustroju społecznego, Włocławek 2001, p. 95. 63 N. G o l i c h o w s k i, Palestyna, p. 226; idem, Ze Ziemi Świętej, pp. 179-180.
64 S
l i
G
b
ł
i ji” 1 (1988)
24 66 K. N i e s i e c k i, op. cit., vol. 2, by J. N. B o b r o w i c z, Leipzig 1839, p. 173;
idem, vol. 3, p. 46; idem, vol. 6, p. 29; idem, vol. 7, p. 128; idem, vol. 8, p. 412; Idem,
vol. 10, p. 85. 64 S. K l i m, Rycerze Bożego Grobu, „Perła Misji”, 15 (1988), p. 24.
65 67 A. W e j n e r t, Kawalerowe złotéj ostrogi w Polsce do XIX w., Warszawa 1879;
T. Sz u l c, Eques auratus w dawnej Rzeczypospolitej, „Acta Universitatis Lodzensis:
Folia Iuridica”, 38 (1988), p. 76; H. L u l e w i c z, Sapieha Mikołaj z Kodnia h. Lis,
PSB, 35, Warszawa-Kraków 1994, p. 69; A. B o g u c k i, Polskie nazwy rycerstwa
w średniowieczu: przyczynki do historii ustroju społecznego, Włocławek 2001, p. 95.
68 H. K o w a l s k a, Pieniążek Prokop, PSB, 26, Wrocław-Warszawa-Krakow-Gdańsk
1981, p. 107; B. P a p r o c k i, op. cit., p. 412; M. N a g i e l s k i, Sapieha Krzysztof
h. Lis, PSB, 35, Warszawa-Kraków 1994, pp. 68-69. 68 H. K o w a l s k a, Pieniążek Prokop, PSB, 26, Wrocław-Warszawa-Krakow-Gdańsk
1981, p. 107; B. P a p r o c k i, op. cit., p. 412; M. N a g i e l s k i, Sapieha Krzysztof
h. Lis, PSB, 35, Warszawa-Kraków 1994, pp. 68-69. 65 F. S i a r c z y ń s k i, Obraz wieku reignowania Zygmunta III, króla polskiego i szwe-
dzkiego, zawiera opis osób życie pod jego władowania, p. 1, Lwów 1858, p. 63.
66 65 F. S i a r c z y ń s k i, Obraz wieku reignowania Zygmunta III, króla polskiego i szwe-
dzkiego, zawiera opis osób życie pod jego władowania, p. 1, Lwów 1858, p. 63.
66 K. N i e s i e c k i, op. cit., vol. 2, by J. N. B o b r o w i c z, Leipzig 1839, p. 173; POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… List of Polish Knights of the Holy Sepulchre
according to previous research
Bielak
Golichowski
Gapski
Lenczewski
Jan Pudolski
(Pudłowski)
Jan Pudłowski
Jan Pudłowski
Jan Pudłowski
Piotr Bulina
Piotr Bulina
Piotr Bylina
Piotr Bylina
Andrzej Lind
Andrzej Lind
Andrzej Lind
Kazimierz Wisoski
(Wysocki)
Franciszek Kazimierz
Wysocki
Franciszek Kazimierz
Wysocki
Franciszek Kazimierz
Wysocki
Franciszek Meniński
Franciszek Mericuski
Franciszek Meniński
Jean Drahotemski vel
Brahatemski, Czech
Count Wladyslaw
Chodkiewicz
Ladislas Codeikievitz
Count Wladyslaw
Chodkiewicz
Bernard Bruntalski,
Silesian
Michał Konarski
Michał Konarski
Michał Konarski
Jerzy Kos
Jerzy Kos
Jerzy Kos
Andrzej Skorulski
Andrzej Skorulski
Andrzej Skorulski
Przemysław
from Wicko, Silesian
Primislas Vickova,
Silesian
Jan Białkowius
Jean Biolcavis
Jan Białkowius Table 1. List of Polish Knights of the Holy Sepulchre
according to previous research POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… 145 Jean Coron,
Czech
Herakliusz Lisowski
Herakljusz Lissowski
Herakliusz Lisowski
Antoni Prokresh,
Austrian
[Edmund] Zichy,
Austrian
Mikołaj Krzysztof
Radziwiłł
Mikołaj Krzysztof
Radziwiłł
Mikołaj Krzysztof
Radziwiłł
Abraham Burgo,
Silesian
Wratysław,
Silesian
Teofil Szemberg
Teofil Szemberg
Count Adam Soldand
(Sołtan?)61
Count Adam
Solelenad
Adam Sołtan
Stefan Plater
Stefan Plater
Melchior Kusti,
Swiss
Rudolf Phiffer,
Swiss
Maurycy Phiffer,
Swiss 146 OLGIERD LENCZEWSKI The register does not include:62 Jerzy Koene-Jaski (1561), Feliks
Odrowąż-Pieniążek (1563), Maurycy Paweł Henik (1585), Hieronim
Strzała (1599), Joannes Baptista Sappi (1613). After 1848, information
about knights is stored at the headquarters of the Jerusalem Patriarchate.63 g
q
During the pontificate of Pius XII (1939-1958), the requirements for
the appointment of the knights of the Holy Sepulchre were reverted,
but their framework was modernised. According to the modernised
version, it was necessary to: preserve and popularise faith in the Holy
Land, support the development of missions in the Latin Patriarchate in
Jerusalem, as well as charity, cultural and social works.64 y
Not only the knights of the Holy Sepulchre held the rank of equites
hierosolimitanum. The above title was also claimed by other orders. Some point out that this title was also received during a stay in Malta,
where knighthoods were also awarded.65 Heraldic Fr Kasper Niesiecki SJ
describes them as Knights of Jerusalem.66 This title was also used by
the Knights of the Order of the Golden Fleece67 and the Jerusalem
Knights of the Order of Saints Maurice and Lazarus.68 62 H. F r e y t a g, Preuβische Jerusalempilger von 14. bis 16. Jahrhundert, „Archiv
für Kultur-Geschichte”, 3 (1905), p. 150; J. Pogonowski, Pamiątka po Feliksie
Odrowążu Pieniążku, „Miesięcznik Heraldyczny”, 6 (1930), p. 71 H. J. B r e u n i n g v o n B u c h e n b a c h, Orientalische Reyß Deß Edlen unnd
Besten Hanß Jacob Breüning von und zu Buochenbach so er selb ander in der Tuerckey
under deß Tuerckischen Sultans Jurisdiction und Gebiet so wol in Europa als Asia unnd
Africa ohn einig Cuchtum oder FreyGleit benantlich in Griechen Land Egypten Arabien
Palestina das Heylige Gelobte Land und Syrien nicht ohne sondere grosse Gefahr vor
dieser Zeit verrichtet. Alles in Fuenff underschiedliche Meerfahrten disponiert und
abgetheylet auch was in einer jeden derselben von tag zu tag fuergangen ordentlich vom
Authore selbsten verzeichnet: darinn ein jede abgesonderte Materi under ihr eigen
Capitel oder Titul gebracht und mit schönen Kupfferstuecken gezieret. Mit angehenckter
Summarischer Computation aller Meylen..., Straßburg 1612, p. 241.
72 69 O. L e n c z e w s k i, Wybrane opisy..., pp. 96-97; idem, Polonica w księdze...,
pp. 105-106.
70 Idem, Polonica w księdze..., p. 106.
71 H. J. B r e u n i n g v o n B u c h e n b a c h, Orientalische Reyß Deß Edlen unnd
Besten Hanß Jacob Breüning von und zu Buochenbach so er selb ander in der Tuerckey
under deß Tuerckischen Sultans Jurisdiction und Gebiet so wol in Europa als Asia unnd
Africa ohn einig Cuchtum oder FreyGleit benantlich in Griechen Land Egypten Arabien
Palestina das Heylige Gelobte Land und Syrien nicht ohne sondere grosse Gefahr vor
dieser Zeit verrichtet. Alles in Fuenff underschiedliche Meerfahrten disponiert und
abgetheylet auch was in einer jeden derselben von tag zu tag fuergangen ordentlich vom
Authore selbsten verzeichnet: darinn ein jede abgesonderte Materi under ihr eigen
Capitel oder Titul gebracht und mit schönen Kupfferstuecken gezieret. Mit angehenckter
Summarischer Computation aller Meylen..., Straßburg 1612, p. 241.
72 Chronografia albo..., p. 342.
73 Dziennik podróży Jana Tarnowskiego do Ziemi Świętej z 1518 r., trans. R. S a w a,
ed. D. C h e m p e r e k, „Odrodzenie i Reformacja”, 49 (2005), p. 191.
74 K. M i g o ń, Podróże Ślązaków na Wschód, „Kwartalnik Opolski”, 2 (1966), p. 35;
T. Parma, op. cit., p. 302. 69 O. L e n c z e w s k i, Wybrane opisy..., pp. 96-97; idem, Polonica w księdze...,
pp. 105-106.
0 74 K. M i g o ń, Podróże Ślązaków na Wschód, „Kwartalnik Opolski”, 2 (1966), p. 35;
T. Parma, op. cit., p. 302. POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… 125; idem, Szabla-
batorówka Feliksa Odrowąża Pieniążka, „Broń i Barwa”, 4 (1935), p. 89; F. M i n c e r,
Koene-Jaski Jerzy, PSB, 13, Wrocław-Warszawa-Kraków 1967-1968, p. 256;
H. G a p s k i, op. cit., p. 137; O. L e n c z e w s k i, Wybrane opisy…, pp. 47-52; Idem,
Polonica w księdze pielgrzymów jerozolimskich – Navis peregrinorum (1561-1695),
„Nasza Przeszłość”, 132 (2019), p. 98. Ś 65 F. S i a r c z y ń s k i, Obraz wieku reignowania Zygmunta III, króla polskiego i szwe-
dzkiego, zawiera opis osób życie pod jego władowania, p. 1, Lwów 1858, p. 63. 66 K. N i e s i e c k i, op. cit., vol. 2, by J. N. B o b r o w i c z, Leipzig 1839, p. 173;
idem, vol. 3, p. 46; idem, vol. 6, p. 29; idem, vol. 7, p. 128; idem, vol. 8, p. 412; Idem,
vol. 10, p. 85. ed. D. C h e m p e r e k, „Odrodzenie i Reformacja”, 49 (2005), p. 191.
4
Ś 73 Dziennik podróży Jana Tarnowskiego do Ziemi Świętej z 1518 r., trans. R. S a w a,
ed. D. C h e m p e r e k, „Odrodzenie i Reformacja”, 49 (2005), p. 191. 3 Dziennik podróży Jana Tarnowskiego do Ziemi Świętej z 1518 r., trans. R. S a w g
f
p
73 Dziennik podróży Jana Tarnowskiego do Ziemi Świętej z 1518 r., trans. R. S a w a,
ed. D. C h e m p e r e k, „Odrodzenie i Reformacja”, 49 (2005), p. 191.
74 K M i g o ń Podróże Ślązaków na Wschód
Kwartalnik Opolski” 2 (1966) p 35; Dziennik podróży Jana Tarnowskiego do Ziemi Świętej z 1518 r., trans. R. S a w a,
ed. D. C h e m p e r e k, „Odrodzenie i Reformacja”, 49 (2005), p. 191.
74 K. M i g o ń, Podróże Ślązaków na Wschód, „Kwartalnik Opolski”, 2 (1966), p. 35;
T. Parma, op. cit., p. 302. 72 Chronografia albo..., p. 342. POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… 147 It is assumed that decorating a coat of arms, commemorative medal
or tombstone with the Jerusalem cross indicates obtaining in Jerusalem
the rank of eques auratus Hierosolymitanus Sancti Sepulchri.69 De facto,
the above emblem does not determine whether a person received this
title in Jerusalem or not.70 According to a German pilgrim from the
second half of the 16th century, Hans Jakob Breuning von Buchenbach,
only those who visited Jerusalem had the right to place the Jerusalem
cross in their coat of arms.71 A later testimony from 1710, states that
a certain pilgrim showed signs that he had been in Jerusalem with the
Jerusalem coats of arms on his right hand [...], expressed as if beautifully
drawn in ink, so that it could not be obliterated in any way.72 Initially, the rank of a knight of the Holy Sepulchre could only be
obtained by nobles. Therefore, before the fitting ceremony began,
in order to verify the candidate’s origins, the following questions were
asked: Of what status are you, are you of noble birth, or are you of
parents of a noble family? Do you have sufficient means to lead an
honest life and exercise the rank of a knight without commerce or
crafts?73 This principle was not always adhered to. An example is
knighting the of Jan Rindfleisch, a merchant from Wrocław (1481),
in Jerusalem.74 In addition, the knight was required to defend and
liberate the Holy Church and its servants from persecution by infidels,
and, if necessary, act against them personally or to send out trained men, OLGIERD LENCZEWSKI 148 as well as to build and consolidate peace among Christians.75 Those who
were appointed eques Sancti Sepulchri, after obtaining permission from
the guardian, could introduce two more knights of the Order of the Holy
Sepulchre into the ranks.76 Hence the Knight William de Boldensel in
1336 post missam feci duos milites nobiles supra sepulchrum gladios
accingendo et alia observando, quae in professione militaris ordinis fieri
consueverunt.77 Emperors and popes were also authorised to confer
the rank of miles. In Martin Ketzel’s account of Augsburg (1476) we can
read that the Saxon duke Albrecht established 72 knights.78 The above
power of attorney was also received by the procurator of the Minorites on
Mount Zion, brother Jan of Prussia. 75 F. F. von T r o i l o, Orientalische Reise Beschreibung, Dresden 1733, p. 299.
76 J. K a m a n n, Die Pilgerfahrten Nürnberger Bürger nach Jerusalem im. 15 Jahrhundert,
“Mitteilungen des Vereins für Geschichte der Stadt Nürnberg”, 2 (1880), pp. 109-110.
77 Itinerarium Guilielmi de Boldensele, ed. C. L. G r o t e f e n d, „Zeitschrift des
Historischen Vereins für Niedersachsen”, (1855), p. 267.
78 R. R ö h r i c h t, Deutsche Pilgerreisen nach dem Heiligen Lande, Innsbruck 1900,
p. 155.
79 Ibid., p. 193; A. M a ń k o w s k i, Pielgrzymki Pomorzan w wiekach średnich,
Pelpin 1935, p. 6; H. F r e y t a g, op. cit., p. 142;
80 M. P i c c i r i l l o, op. cit., in: Registrum Equitum…, p. VII.
81 Ibidem.
82 G. R o t h, „Grób Święty” w Zgorzelcu, w: Kult św. Jakuba Apostoła Większego
w Europie Środkowo-Wschodniej, ed. R. K n a p i ń s k i, Lublin 2002, p. 277. 82 G. R o t h, „Grób Święty” w Zgorzelcu, w: Kult św. Jakuba Apostoła Większego
w Europie Środkowo-Wschodniej, ed. R. K n a p i ń s k i, Lublin 2002, p. 277. p
p
y
g
p
80 M. P i c c i r i l l o, op. cit., in: Registrum Equitum…, p. VII. 79 Ibid., p. 193; A. M a ń k o w s k i, Pielgrzymki Pomorzan w wiekach średn
Pelpin 1935, p. 6; H. F r e y t a g, op. cit., p. 142; 83 Canon Pietro Casola’ Pilgrimage to Jerusalem in the Year 1494, trans. from Italian
by M. Margaret N e w e t t, Manchester 1907, p. 264.
84 J. K a m a n n, op. cit., p. 110.
85 J. A. K o ś c i e l n a, Pomorzanie w Ziemi Świętej (do końca XVI w.). Rekonesans
badawczy, „Stargardia”, 10 (2015), p. 139.
86 F. M i n c e r, Konarski Michał, p. 468.
87 Registrum Equitum..., pp. 159, 171.
88 Ibid., p. 161. The above sum likely constituted a deposit for the renovation of the
Church of the Holy Sepulchre in Jerusalem. In many cases, candidates were exempted
from the fee because of their services to the Catholic Church, see: ibidem, pp. 179-187.
89 Ibid., p. 195.
90 Registrum A, k. 12v, photocopy in: Registrum Equitum…, p. 48.
91 Ibidem, k. 72, photocopy in: Registrum Equitum…, p. 290. 91 Ibidem, k. 72, photocopy in: Registrum Equitum…, p. 290. 90 Registrum A, k. 12v, photocopy in: Registrum Equitum…, p. 48. POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… During his long stay in the Holy
Land (1479-1499), he presided over the ceremonies of knighthood of the
Holy Sepulchre. As part of this undertaking, he collected a certain fee
for the maintenance of the holy places. During one of the investitures,
he awarded the title of miles hierosolymitanus (1497) to Pomeranian ruler
Bogusław X and his 26-person entourage, including Eberhard Ferber
of Gdańsk.79 The privilege of appointing equites was also granted to
imperial envoys: in dicto Sancto Sepulchro forno facti Cavalieri aurati
sette pellegrini da uno legato imperiale, con grandissima solennità,
devozione et riverentia.80 The adjective “aurati” used in the above-cited
quotation indicates that super Sanctissimum Domini Sepulchrum fuit
cingulo militari insignitus atque solemniter decorates.81 q
There is a known case of awarding the title of knight of Jerusalem
to a person who committed an act of violence. We are speaking of the
mayor of Görlitz, George Emerich, who likely went to Jerusalem as
penance for this act, where he received absolution and was bestowed
the rank of eques (1465).82 POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… anon Pietro Casola’ Pilgrimage to Jerusalem in the Year 1494, trans. from Italian
M. Margaret N e w e t t, Manchester 1907, p. 264. 89 Ibid., p. 195. by M. Margaret N e w e t t, Manchester 1907, p. 264.
84 J. K a m a n n, op. cit., p. 110.
85 J. A. K o ś c i e l n a, Pomorzanie w Ziemi Świętej (do końca XVI w.). Rekonesans
badawczy, „Stargardia”, 10 (2015), p. 139.
86 F. M i n c e r, Konarski Michał, p. 468.
87 Registrum Equitum..., pp. 159, 171.
88 Ibid., p. 161. The above sum likely constituted a deposit for the renovation of the
Church of the Holy Sepulchre in Jerusalem. In many cases, candidates were exempted
from the fee because of their services to the Catholic Church, see: ibidem, pp. 179-187.
89 Ibid p 195 92 Ibidem, k. 67, photocopy in: Registrum Equitum…, p. 270.
93 F-R. de C h a t e a u b r i a n d, Opis podróży z Paryża do Jerozolimy, by P. H e r t z,
Warszawa 1980, p. 327.
94 Ibidem, p. 453, ftn. 23.
95 Ibidem, p. 327.
96 L’archivo storico..., p. 449.
97 Travelogue..., pp. 191-192. POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… 149 There were instances when the fitting ceremony took place in the
absence of the secretary, who should approve the rank granted by
issuing the appropriate patent. It was then necessary to find a deputy
who issued the newly elected privilegia, certifying the obtaining of the
title of eques Sancti Sepulchri. In 1494, due to the lack of a scribe,
Father Pietro da Casola himself wrote out certificates for ten pilgrims for
their appointment as knights of the Holy Sepulchre; in accordance with
the recommendation given by the guardian, he wrote several letters
confirming that these pilgrims had taken part in the fitting ceremony.83 g
p g
p
g
y
In the 16th century, the requirement of noble origin of candidates
for miles Sancti Sepulchri lost its significance.84 From that moment
onward, anybody could receive the rank of a knight, even heretics.85
A likely example is the inclusion of Protestant Michał Konarski (1583)
among the Order of the Holy Sepulchre.86 In the 17th century, the
ceremony itself acquired a new framework. Before proceeding with the
fitting, the candidate had to receive a positive opinion ex consensus/ex
plenu R.R.P.P. Discretorii Instante87 and fuit ei donata summa 100
zecchinorum.88 Post requisita debite examinata was appointed a knight of
the Holy Sepulchre.89 In fact, as can be seen from the entries in the book,
not everyone adhered to the above principle. It should be noted that, starting from 1619, many people obtained
the title of knight of the Holy Sepulchre per procuratorem.90 In this
way, e.g. the eminent French Egyptologist Jean-François Champollion
and the Italian archaeologist Ippolito Rosellini became knights.91 Sometimes the Franciscans themselves knighted those who had
rendered great service to the Catholic faith. One of them was a French OLGIERD LENCZEWSKI 150 pilgrim and diplomat from the first half of the 19th century, François-
René de Chateaubriand. 96 L’archivo storico..., p. 449. POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… This pilgrim received the rank of equitis
Sancti Sepulchri on 12 September 1806.92 The eminent French thinker
modestly wrote of himself that he had rendered little merit [...] to the
faith.93 These merits are, among others, the work entitled Genius of
Christianity (1802).94 It is worth noting that the proposition to take
part in the ceremony came from the Minorites – they wanted to grant
me an honour that I did not demand and that I did not deserve”,
stressed Chateaubriand.95 The above-described character of the ceremonial lasted until 1847,
when Pope Pius IX, by virtue of the Nulla celebrior bull, awarded the
Patriarch of Jerusalem the right to establish equites.96 Accounts of the knighthood ceremony in Jerusalem were included
in many memoirs of sojourns to the Holy Land. Below is a description
of the ceremony written down by the aforementioned Jan Amor
Tarnowski:97 The method of fitting the knights of the Holy Sepulchre, as well as
the rule that applies to the knights of the said Sepulchre: The knight who is to be fitted, after a prior confession, listens to the
Mass, receives communion during its celebration, and is then led into
the vestibule of the Holy Sepulchre, and asked by the father guardian:
– What are you asking for? He answers on his knees: – I ask you to fit me
as a knight of the Holy Sepulchre of our Lord Jesus Christ. The guardian
then asks him: – Of what status are you, are you of noble birth, or are
you of parents of a noble family? He asks him a third time: – Do you
have sufficient means to lead an honest life and exercise the rank of
a knight without trade or craftsmanship? The knight then answers:
– I have sufficient resources by the grace of God. POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… Question: – Are you
willing with your heart and mouth to swear that you wish to keep the
oaths which oblige you to do so, and which are as follows: to hear
Mass when granted the opportunity, to offer up temporal goods and POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… 151 your life in a general war against the infidels, or to go to war
personally, to neglect your own interests, as far as possible, to defend
against persecutors the Holy Church of God and its servants,
completely avoid unjust wars, shameful services, tournaments for
profit, duels, except for in the needs of war, and in particular to
consolidate peace and harmony among Christians, to enlarge the
Christian community, to protect widows and orphans, to avoid evil
oaths, poverty, blasphemy, usury, rape, sacrilege, murder, drunkenness,
places and persons unequal in condition, avoid sins of the flesh like
pestilence, and appear in every way before God and man? Are you
willing and ready with your heart and mouth to declare this, to do
this, and to maintain this? He replies, – I'm ready. Then they enter the
Holy Sepulchre, the knight with a sword drawn from its sheath. Then
the guardian performs ceremonies and blessings on the sword and the
knight, while the knight holds the naked sword. After completing them,
the knight places his foot on the Holy Sepulchre, and the guardian
puts a spur on him, fits his sword, and wraps a golden belt around him,
placing a gold chain around his neck. The then knight rests his head
on the Holy Sepulchre, and the guardian touches his neck three times
with the sword drawn from its sheath, saying: – I render you a knight
in the name of the Father and of the Son and of the Holy Spirit, and
also in the name of the Blessed Virgin Mary – and kisses the knight on
both cheeks. Next, he leads the knight out of the Sepulchre, chanting
the Te Deum laudamus, and singing, and leads him to the chapel of
the Blessed Virgin Mary, where our Lord Jesus Christ appeared for
the first time after his resurrection to his Mother, the Blessed Virgin
Mary. After the singing is over, the guard embraces the knight, kisses
him, and all the ceremonies associated with the fitting end here. POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… 153 Manuscript sources Golichowski N., Palestyna, czyli pokłosie zebrane z różnych autorów i własne
spostrzeżenia, manuscript at the Provincial Archive of Bernardine Monks
in Krakow, RGP-k-94, vol. 2. Ś Golichowski N., Palestyna, czyli pokłosie zebrane z różnych autorów i własne
spostrzeżenia, manuscript at the Provincial Archive of Bernardine Monks
in Krakow, RGP-k-94, vol. 2. Ś Notaty ks. Jana Fijałka: „Pątnictwo polskie. Pielgrzymki do Ziemi Świętej”
XI-XVIII w., manuscript at Library of the Polish Academy of Arts and
Sciences and the Polish Academy of Sciences in Krakow, 5313. Conclusions Until my research, the number of Poles recorded in the discussed
book oscillated between four and 21 names. I proved that 15 Poles
enlisted under the banner of the knights of the Holy Sepulchre in the
years 1561-1847. The reason for these discrepancies should be
explained as follows: 1. Bielak likely had problems with deciphering the manuscript of
the book, so he noted only four surnames, 1. Bielak likely had problems with deciphering the manuscript of
the book, so he noted only four surnames, OLGIERD LENCZEWSKI 152 2. some researchers (Golichowski and Gapski) included in their
lists knights unrelated to the area of the former Commonwealth
(Switzerland, Silesia, Bohemia and Austria). One of them,
Bruntalski, was considered its citizen because he had a Slavic-
sounding surname, 3. when creating his list, Gapski referred, inter alia, to the work of
Odriozoli y Grimaud, in which knights from Silesia, Bohemia and
Austria were referred to as Poloni. However, he was unacquainted
with the lists prepared by Bielak and Golichowski. In addition, the differences between my research on the book and
those of my predecessors are based on the fact that: 1. the lists drawn up by Bielak, Golichowski and Gapski were
limited to providing the surname of the knight. Therefore, we do
not know that Franciszek Kazimierz Wysocki and Adam Sołtan
obtained the rank of eques per procura, 2. apart from the registrum Equitum, they did not refer to the liber
Navis peregirnorum (1561-1695), in which we find mention of
a knight of the Holy Sepulchre (1613) from the territories of the
former Polish-Lithuanian Commonwealth, 3. I managed to establish the identity of the knight named “Soleland”
and indicate that it is still difficult to verify three names: “Linda”,
“Bialcouiusa” and “Codenieiviza”. Importantly, my predecessors
did not pay attention to these issues, 4. the aforementioned researchers did not indicate what areas were
within the reach of the former Commonwealth of Poland. OLGIERD LENCZEWSKI OLGIERD LENCZEWSKI Printed sources Breuning von Buchenbach H. J., Orientalische Reyß Deß Edlen unnd Besten
Hanß Jacob Breüning von und zu Buochenbach so er selb ander in der
Tuerckey under deß Tuerckischen Sultans Jurisdiction und Gebiet so wol
in Europa als Asia unnd Africa ohn einig Cuchtum oder FreyGleit benantlich
in Griechen Land Egypten Arabien Palestina das Heylige Gelobte Land und
Syrien nicht ohne sondere grosse Gefahr vor dieser Zeit verrichtet. Alles in
Fuenff underschiedliche Meerfahrten disponiert und abgetheylet auch was
in einer jeden derselben von tag zu tag fuergangen ordentlich vom Authore
selbsten verzeichnet: darinn ein jede abgesonderte Materi under ihr eigen
Capitel oder Titul gebracht und mit schönen Kupfferstuecken gezieret. Mit
angehenckter Summarischer Computation aller Meylen…, Straßburg 1612. Canon Pietro Casola’ Pilgrimage to Jerusalem in the Year 1494, trans. from
Italian by M. Margaret Newett, Manchester 1907. Chateaubriand F-R. de, Opis podróży z Paryża do Jerozolimy, by P. Hertz,
Warszawa 1980. Chronografia albo Dziejopis Żywiecki, ed. S. Grodziski, I. Dwornicka,
Żywiec 1987. Ś Dziennik podróży Jana Tarnowskiego do Ziemi Świętej z 1518 r., trans. R. Sawa,
by D. Chemperek, „Odrodzenie i Reformacja”, 49 (2005), pp. 181-194. Itinerarium Guilielmi de Boldensele, ed. C. L. Grotefend, „Zeitschrift des
Historischen Vereins für Niedersachsen”, (1855), pp. 236-286. Navis peregrinorum: ein Pilgerverzeichnis aus Jerusalem von 1561 bis 1695:
mit Angaben über Pilger aus Deutschland, England, Frankreich, Italien und
den Niederlanden, sowie aus anderen europäischen und aussereuropäischen
Ländern, ed. B. Zimolong, Köln 1938. Ś Radziwiłł M. K. „Sierotka”, Podróż do Ziemi Świętej. Syrii i Egiptu 1581-1584,
by L. Kukulski, Warszawa 1962. Registrum Equitum SSmi Sepulchri D.N.J.C. (1561-1848). Monoscritti dell’Archivo
storico della Custodia di Terra Santa a Gerusalemme, ed. M. Piccirillo,
Studium Biblicum Franciscanum Collectio Maior 46, Jerusalem-Milano 2006. Registrum Equitum SSmi Sepulchri D.N.J.C. (1561 1848). Monoscritti dell Archivo
storico della Custodia di Terra Santa a Gerusalemme, ed. M. Piccirillo,
Studium Biblicum Franciscanum Collectio Maior 46, Jerusalem-Milano 2006. Troilo von F. F., Orientalische Reise Beschreibung, Dresden 1733. 154 Studies Baczkowski K., Mikołaj Rozembarski, PSB, 32, Wrocław-Warszawa-Kraków
1991, pp. 384-387. Barącz S., Żywoty sławnych Ormian w Polsce, Lwów 1856. Barycz H., Z zagadnień podróżnictwa polskiego w dawnych wiekach,
„Przegląd Współczesny”, 10 (1938), pp. 66-84. Bogucki A., Polskie nazwy rycerstwa w średniowieczu: przyczynki do historii
ustroju społecznego, Włocławek 2001. j
p
g
Boniecki A., Herbarz Polski, vol. 1-16, Warszawa 1899-1913. Ś Horain Cz., Na tropie Polaków w Ziemi Świętej. Rejestr pielgrzymów i po-
dróżników polskich do Ziemi Świętej przybyłych w latach 1845-1921;
1931-1947, by M. Kłakus, Katowice-Toulon 2022. Ś Bystroń J. S., Polacy w Ziemi Świętej, Syrji i Egipcie 1147-1914, Kraków 1930. Dachnowski J K Herbarz szlachty Prus Królewskich z XVII w Kórnik 1995 Bystroń J. S., Polacy w Ziemi Świętej, Syrji i Egipcie 1147-1914, Kraków 1930. Dachnowski J. K., Herbarz szlachty Prus Królewskich z XVII w., Kórnik 1995 Gapski H., Wstępny wykaz polskich rycerzy Grobu Bożego w Jerozolimie
(do XIX w. włącznie), in: Zwierzchnictwo w Polsce Zakonu Rycerskiego
Grobu Bożego w Jerozolimie, red. U. Przymus, J. Kamiński, Katowice 2008,
pp. 136-137. Gennes J-P. de, Les chevaliers du Saint Sépulchre de Jérusalem, French 2004. Ś Golichowski N., Ze Ziemi Świętej: polscy rycerze Grobu Pańskiego,
„Tygodnik Katolicki”, 23 (1892), pp. 179-180. Freytag H., Preuβische Jerusalempilger von 14. bis 16. Jahrhundert, „Archiv
für Kultur-Geschichte“, 3 (1905), pp. 129-154. Indice onomastico, w: Registrum Equitum…, pp. 419-437. Kamann J., Die Pilgerfahrten Nürnberger Bürger nach Jerusalem im. 15
Jahrhundert, „Mitteilungen des Vereins für Geschichte der Stadt Nürnberg“, 2 (1880),
pp. 78-163. Kaniewska I., Latalski Janusz, PSB, 16, Wrocław-Warszawa-Kraków 1971,
pp. 563-565. pp
Kawalerowie Grobu Św. Polacy, „Krzyż”, 23 (1867), pp. 186. Klim S., Rycerze Grobu Bożego, „Perła Misji”, 15 (1988), pp. 23-24. Konarski S., Platerowie, Buenos Aires-Paris 1967. Ś Korwin S. A., Stosunki Polski z Ziemią Świętą, Warszawa 1958. Kościelna J. A., Pomorzanie w Ziemi Świętej (do końca XVI w.). Rekonesans
badawczy, „Stargardia”, 10 (2015), pp. 131-148. Kotarski H., Lanckoroński Przecław, PSB, 16, Wrocław-Warszawa-Kraków
1971, pp. 449-450. Kowalska H., Pieniążek Prokop, PSB, 26, Wrocław-Warszawa-Kraków-
Gdańsk 1981, pp. 106-108. Taż, Pudłowski Melchior, PSB, 29, Wrocław-Warszawa-Kraków-Gdańsk-
Łódź 1986, pp. 342-344. POLONICA IN THE REGISTRUM EQUITUM SANCTI SEPULCHRI… 155 Kurtyka J., Latyfundium tęczyńskie: dobra i właściciele (XIV-XVII w.),
Kraków 1999. Kurtyka J., Latyfundium tęczyńskie: dobra i właściciele (XIV-XVII w.),
Kraków 1999. L’archivo storico della Custodia di Terra Santa (1230-1970), vol. 2,
ed. A. Maiarelli, Milano 2012. Lemmens L., Collectanea ex Archivo Hierosolymitano deprompta,
ed. G. Golubovich, Quaracchi 1933. Studies Lenczewski O., Polonica w księdze pielgrzymów jerozolimskich – Navis
peregrinorum (1561-1695), „Nasza Przeszłość”, 132 (2019), pp. 95-111. p
g
(
) pp
Lubczyński M., Pielas J., Szydłowiecki Krzysztof, PSB, 49, Warszawa-
Kraków 2014, pp. 551-566. Lulewicz H., Sapieha Mikołaj z Kodniu h. Lis, PSB, 35, Warszawa-Kraków
1994, pp. 116-120. Idem, Skorulski Andrzej, PSB, 38, Warszawa 1997-1998, pp. 269-270. Idem, Sołtan Aleksandrowicz, PSB, 40, Warszawa-Kraków 2000-2001,
pp. 341-343. Łątka J. S., Słownik Polaków w Imperium Osmańskim i w Republice Turcji,
ed. 2, revised, Kraków 2015. Manikowska H., Jerozolima-Rzym-Compostela. Wielkie pielgrzymowanie
u schyłku średniowiecza, Wrocław 2008.f Mańkowski A., Pielgrzymki Pomorzanw wiekach średnich, Pelpin 1935. Ś Migoń K., Podróże Ślązaków na Wschód, „Kwartalnik Opolski”, 2 (1966),
pp. 31-49. Mincer F., Koene-Jaski Jerzy, PSB, 13, Wrocław-Warszawa-Kraków 1967-1968,
p. 256. Idem, Konarski Michał, PSB, 13, Wrocław-Warszawa-Kraków 1967-1968,
pp. 467-469. Nagielski M., Sapieha Krzysztof h. Lis, PSB, 35, Warszawa-Kraków 1994,
pp. 68-70. Idem, Szemberg Teofil, PSB, 48, Warszawa-Kraków 2012-2013, pp. 127-130. Niesiecki K., Herbarz Polski, ed. J. N. Bobrowicz, vol. 1-10, Lipsk 1839-1845. Odriozola y Grimaud Carlos de, Libro de ora de la sagrada orden militar
Jerosalimitana del Santo Sepulcro de N. S. Jesucristo, que contiene los
nombres de los caballeros de dicha Orden creados por los RR. PP. Guardianes del Santo Sepulcro, Custodios de Tierra Santa, de la Orden de
Menores de San Francisco, desde el año 1561 á 1848 con referencia á los
archivos del convento de San Salvador de Jerusalén y otros dates
históricos seguido de un apéndice con notas justificativas, Zaragoza 1900. g
p
j
f
g
Orbis latinus oder der wichtigsten lateinischen Orts- und Ländernamen,
by T. G. J. Graesse, F. Benedict, Berlin 1909. y
Paprocki B., Herby rycerstwa polskiego, ed. K. J. Turowski, Warszawa 198 Paprocki B., Herby rycerstwa polskiego, ed. K. J. Turowski, Warszawa 1988. Parma T., Rytíři, dámy a poutníci: Dějiny a současnost Rytířského řádu Božího
hrobu jeruzalémského a jeho působení v českých zemích, Olomouc 2020. Parma T., Rytíři, dámy a poutníci: Dějiny a současnost Rytířského řádu Božího
hrobu jeruzalémského a jeho působení v českých zemích, Olomouc 2020. 156 OLGIERD LENCZEWSKI Parola P. L., Registrum Equitum Ssmi Domini Nostri Jesu Christi (1561-1848),
„Città del Vaticano”, (2011), pp. 29-32. Pertek J., Polacy na morzach i oceanach, vol. 1, Poznań 1981. Piccirillo M., Introduzione, in: Registrum Equitum…, pp. VII-IX. Pociecha W., Królowa Bona (1494-1557): czasy i ludzie odrodzenia, vol. 2,
Poznań 1949. Studies Pogonowski J., Pamiątka po Feliksie Odrowążu Pieniążku, „Miesięcznik
Heraldyczny”, 6 (1930), pp. 124-125. Idem, Szabla-batorówka Feliksa Odrowąża Pieniążka, „Broń i Barwa”, 4 (1935),
pp. 89-90. Röhricht R., Deutsche Pilgerreisen nach dem Heiligen Lande, Innsbruck 1900
Ś Röhricht R., Deutsche Pilgerreisen nach dem Heiligen Lande, Innsbruck 1900. Roth G., „Grób Święty” w Zgorzelcu, in: Kult św. Jakuba Apostoła Większego
w Europie Środkowo-Wschodniej, ed. R. Knapiński, Lublin 2002, pp. 269-285. Siarczyński F Obraz wieku panowania Zygmunta III króla polskiego i szwedz Roth G., „Grób Święty” w Zgorzelcu, in: Kult św. Jakuba Apostoła Większego
w Europie Środkowo-Wschodniej, ed. R. Knapiński, Lublin 2002, pp. 269-285. Siarczyński F., Obraz wieku panowania Zygmunta III, króla polskiego i szwedz-
kiego, zawierający opis osób żyjących pod jego panowaniem, p. 1, Lwów 1858. Słabczyński T., Słownik polskich podróżników i odkrywców, Warszawa 2017. Spis Polaków, którzy odwiedzili Ziemię Świętą, „Krzyż”, 22 (1867), p. 178. Sudolski Z., Adam Ludwik Michał Sołtan, PSB, 40, Warszawa-Kraków 2001,
pp. 344-346. Szulc T., Eques auratus w dawnej Rzeczypospolitej, „Acta Universitatis
Lodzensis: Folia Iuridica”, 38 (1988), pp. 59-96. (
) pp
Uruski S., Rodzina. Herbarz szlachty polskiej, vol. 1-16, Warszawa 1904-1938. (
) pp
Uruski S., Rodzina. Herbarz szlachty polskiej, vol. 1-16, Warszawa 1904-1938. y p
j
Wejnert A., Kawalerowe złotéj ostrogi w Polsce do XIX w., Warszawa 1879. Zimolong B., Schlesische Pilger im Hl. Lande 1561-1695, Breslau 1938. Wejnert A., Kawalerowe złotéj ostrogi w Polsce do XIX w., Warszawa 1879. Zimolong B., Schlesische Pilger im Hl. Lande 1561-1695, Breslau 1938. Żytkowicz L., Lisowski Józef, PSB,17, Wrocław-Warszawa-Kraków 1972,
pp. 473-474. Żytkowicz L., Lisowski Józef, PSB,17, Wrocław-Warszawa-Kraków 1972,
pp. 473-474. Online sources https://hu.wikipedia.org/wiki/Zichy_Edmund [access: 27.03.2020]. http://teki.bkpan.poznan.pl/index_monografie.html [access: 19.10.2022]. OLGIERD LENCZEWSKI – theologian, deals with the analysis of Old
Polish peregrinations to the Holy Land and the reception of the Holy Land in
geographical and travel writing.
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https://openalex.org/W2726018660
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https://journals.eco-vector.com/uroved/article/download/6772/5404
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Russian
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The infertility overcoming in men with retrograde ejaculation and anejaculation
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Urologičeskie vedomosti
| 2,017
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cc-by
| 3,067
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УДК 616.69-008.3 + 616.697
DOI: 10.17816/uroved7210-15 УДК 616.69-008.3 + 616.697
DOI: 10.17816/uroved7210-15 УДК 616.69-008.3 + 616.697
DOI: 10.17816/uroved7210-15 Received: 11.04.2017 Accepted: 12.06.2017 In this descriptive retrospective, single-center study, we analyzed the local database of 82 men treated for infertility
due to retrograde ejaculation and anejaculation in the International Centre for Reproductive Medicine. Spermatozoa for
IVF/ICSI cycles were retrieved from post-ejaculatory urine and by aspiration biopsies (PESA/TESA). An algorithm for
diagnosis and treatment of infertile men with retrograde ejaculation and anejaculation has been suggested. Keywords: retrograde ejaculation; aspermia; IVF/ICSI; male infertility. Keywords: retrograde ejaculation; aspermia; IVF/ICSI; male infertility. ВВЕДЕНИЕ расте 50–59, 60–69 и 70–80 лет соответственно. Эти нарушения семяизвержения снижали качество
жизни каждому второму опрошенному мужчи-
не [1], уменьшали степень сексуального удовлет-
ворения их партнерш [2], а также нередко служи-
ли поводом для обращения к врачу, в том числе
и в связи с невозможностью добиться наступления
беременности. За последние годы были получены
и систематизированы знания о работе двух веду-
щих и взаимосвязанных механизмов семяизвер- По данным Европейской ассоциации урологов,
нарушения семяизвержения — ретроградная эяку-
ляция (РЭ) и анэякуляция (аспермия) в структуре
обращений за медицинской помощью по поводу
бесплодия в браке составляют менее 1 % случаев. В то же время в исследовании MSAM-7 снижение
объема эякулята или его полное отсутствие в те-
чение месяца, предшествующего анкетированию,
отметили 30,1, 54,9 и 74,3 % респондентов в воз- © И.А. Корнеев 1, 2, Р.Д. Зассеев 2 1 ФГБОУ ВО «Первый Санкт-Петербургский государственный медицинский университет имени акаде-
ми ка И.П. Павлова» Минздрава России, Санкт-Петербург;
2 АО «Международный центр репродуктивной медицины», Санкт-Петербург 1 ФГБОУ ВО «Первый Санкт-Петербургский государственный медицинский университет имени акаде-
ми ка И.П. Павлова» Минздрава России, Санкт-Петербург; р
р ур
2 АО «Международный центр репродуктивной медицины», Санкт-Петербург Для цитирования: Корнеев И.А. , Зассеев Р.Д.. Преодоление бесплодия у мужчин с ретроградной эякуляцией и анэякуляцией //
Урологические ведомости. – 2017. – Т. 7. – № 2. – С. 10–15. doi: 10.17816/uroved7210-15 Статья принята к печати: 12.06.2017 Дата поступления: 11.04.2017 Представлены данные о 82 мужчинах (средний возраст — 35 ± 15 лет), получивших лечение по поводу бес-
плодия, обусловленного ретроградной эякуляцией и анэякуляцией в Международном центре репродуктивной
медицины. Сперматозоиды, пригодные для использования в программах вспомогательных репродуктивных
технологий (ЭКО/ИКСИ), были получены из посткоитальной мочи или хирургическим путем при чрескожных
аспирационных биопсиях органов мошонки — ПЕСА/ТЕСА. Предложен алгоритм диагностической и лечебной
тактики у бесплодных мужчин с ретроградной эякуляцией и анэякуляцией. Ключевые слова: ретроградная эякуляция; аспермия; ЭКО/ИКСИ; мужское бесплодие. © I.A. Korneyev 1, 2, R.D. Zasseev 2 1 FSBEI HE “Academician I.P. Pavlov First St Petersburg State Medical University” of the Ministry of Healthcare
of Russia, Saint Petersburg, Russia; g
2 International Center for Reproductive Medicine, Saint Petersburg, Russia For citation: Korneyev IA, Zasseev RD. The infertility overcoming in men with retrograde ejaculation and anejaculation. Urologicheskie
vedomosti. 2017;7(2):10-15. doi: 10.17816/uroved7210-15 ОРИГИНАЛЬНЫЕ СТАТЬИ ОРИГИНАЛЬНЫЕ СТАТЬИ 10 УРОЛОГИЧЕСКИЕ ВЕДОМОСТИ 2017 Том 7 № 2 ISSN 2225-9074 ОРИГИНАЛЬНЫЕ СТАТЬИ 11 жения — эмиссии и экспульсии, а также описаны
патофизиологические причины РЭ и анэякуляции
(АЭ). Это способствовало пониманию необходи-
мости проведения и предложению методологии
выбора диагностических и лечебных мероприятий
для успешной диагностики, дифференциальной
диагностики и коррекции приводящих к беспло-
дию расстройств семяизвержения. Современное
развитие и широкое применение вспомогательных
репродуктивных технологий (ВРТ) — ЭКО, ИКСИ,
позволяющих с высокой вероятностью добиться
наступления беременности при наличии небольшо-
го числа сперматозоидов, диктуют необходимость
внесения корректив в методологию обследования
и лечения таких пациентов. Сравнительно неболь-
шое число работ, посвященных решению проблемы
бесплодия у мужчин с нарушениями семяизверже-
ния [3, 4], послужило предпосылкой к настоящему
исследованию. скожные аспирационные биопсии придатков яичек
(ПЕСА) или яичек (ТЕСА по методике, предложен-
ной L. Levineet, E. Lisek (1998) [5], в модификации). При этом придаток яичка и яичко фиксировали
рукой, захватывали кожу мошонки и растягивали
ее рукой над яичком и головкой придатка. Прида-
ток удерживали между большим и указательным
пальцами, в то время как другой рукой через кожу
вводили иглу 21g (в придаток) или 19g (в яичко)
перфузионной системы «Бабочка». Систему при-
соединяли к шприцу 20 мл и, выдвинув поршень,
создавали в ней отрицательное давление. Трубку
системы перекрывали зажимом, а иглу перемеща-
ли внутри придатка яичка или яичка до появления
желтоватой жидкости в просвете системы. После
этого иглу извлекали, снимали зажим и промыва-
ли систему 1 мл среды HAM F-10, забуференной
22 мМ HEPES. Бригада эмбриологов проводила ми-
кроскопию полученного материала, анализировала
количество, подвижность и морфологию спермато-
зоидов и делала вывод о возможности их использо-
вания в программе вспомогательной репродукции
и криоконсервации. МАТЕРИАЛЫ И МЕТОДЫ
ИССЛЕДОВАНИЯ С 1998 по 2013 г. мы наблюдали 82 мужчин
(средний возраст — 35 ± 15 лет), состоящих в бес-
плодных браках и имеющих нарушения семяиз-
вержения: РЭ и АЭ у 54 (66 %) и 28 (34 %) соответ-
ственно. Чаще всего ретроградное семяизвержение
у мужчин развивалось на фоне сахарного диабета,
реже — после эндоскопических оперативных вме-
шательств на шейке мочевого пузыря. У подавля-
ющего большинства больных причиной аспермии
было травматическое повреждение спинного мозга,
кроме того, мы наблюдали несколько случаев пси-
хогенной аспермии, обусловленной необходимо-
стью получить эякулят в условиях медицинского
центра ВРТ для проведения ЭКО, и двух пациентов
с первичной идиопатической аспермией. РЕЗУЛЬТАТЫ У 48 (89 %) мужчин с ретроградной эякуля-
цией в посткоитальной моче были обнаружены
и использованы в программах ВРТ — экстракор-
поральном оплодотворении (ЭКО) с интрацито-
плазматическим введением сперматозоидов в яй-
цеклетку (ИКСИ) — жизнеспособные подвижные
сперматозоиды. У 6 (11 %) мужчин количественные
и качественные характеристики сперматозоидов
посткоитальной мочи были признаны неудовлет-
ворительными. С целью получения сперматозои-
дов для проведения ЭКО/ИКСИ им потребовалось
выполнение оперативных вмешательств — ПЕСА
и ТЕСА, при этом у всех мужчин были получены
сперматозоиды, пригодные для ВРТ, частота насту-
пления беременности на перенос после проведения
ЭКО/ИКСИ составила 52 %. У пациентов с ретроградным семяизвержени-
ем получение сперматозоидов для программ ВРТ
проводили из посткоитальной мочи. С целью под-
готовки к этому мужчинам предлагали принять
препарат, приводящий к ощелачиванию мочи (би-
карбонат натрия вечером и утром перед получе-
нием эякулята), просили собрать первую порцию
мочи непосредственно после появления оргазма
при мастурбации и незамедлительно передать ее
в лабораторию ВРТ. Всем пациентам с аспермией, обратившимся
за медицинской помощью в связи с проблемой бес-
плодия в браке, с целью получения сперматозоидов
и применения их в программах ВРТ (ЭКО/ИКСИ)
производили хирургические вмешательства —
ПЕСА и ТЕСА. У всех мужчин были получены
сперматозоиды, пригодные для ВРТ, частота насту-
пления беременности на перенос после проведения
ЭКО/ИКСИ составила 50 %. У мужчин с АЭ и отсутствием в посткоиталь-
ной моче сперматозоидов, пригодных для ВРТ,
применяли оперативные вмешательства — чре- УРОЛОГИЧЕСКИЕ ВЕДОМОСТИ УРОЛОГИЧЕСКИЕ ВЕДОМОСТИ 2017 Том 7 № 2 ISSN 2225-9074 ОРИГИНАЛЬНЫЕ СТАТЬИ 12 Послеоперационный период у всех мужчин
протекал благополучно, в первые сутки после
вмешательства им было рекомендовано ноше-
ние суспензория, а при появлении болей — при-
менение нестероидных противовоспалительных
средств per os. Жалобы на небольшой дискомфорт
и слабовыраженные болевые ощущения в области
мошонки прекращались спустя 24–48 часов. Под ко-
жей мошонки у некоторых пациентов были замет-
ны небольшие кровоизлияния, однако обычно они
бесследно исчезали в течение недели и не требова-
ли лечебных мероприятий. экспульсии способствует РЭ, представляющей со-
бой частичный или полный ретроградный выход
семенной жидкости в полость мочевого пузыря. Следует отметить, что у некоторых мужчин при-
чиной азооспермии может быть обструкция эяку-
ляторных протоков: поствоспалительная, пост-
травматическая (в том числе и послеоперационная)
или идиопатическая [8]. У них, как и у мужчин с РЭ,
может наблюдаться низкий объем эякулята, пре-
имущественно состоящий из секрета предстатель-
ной железы. Это состояние редко сопровождается
замедлением семяизвержения, и его следует отли-
чать от РЭ. Для восстановления проходимости се-
мявыносящих путей таким пациентам может быть
применена трансуретральная резекция семявыно-
сящих протоков. Таким образом, у пациентов, име-
ющих схожие клинические проявления нарушений
семяизвержения, можно выявить три различных
патофизиологических механизма, требующих про-
ведения дифференциальной диагностики. ОБСУЖДЕНИЕ Согласно современным представлениям эякуля-
торный рефлекс активируется при взаимодействии
центральной и периферической нервной систем. Спинальный эякуляторный центр при половом воз-
буждении получает сигналы от супраспинальных
зон, а также афферентные сигналы чувствительных
нервов половых органов. Координация импульсов,
поступающих со стороны симпатической, парасим-
патической и соматической систем, а также форми-
рование сигнала в гипоталамусе и преоптической
зоне головного мозга приводит к семяизвержению,
в котором можно выделить две фазы — эмиссию
и экспульсию. Во время эмиссии в уретру выде-
ляется секрет предстательной железы и семенных
пузырьков, а также происходит сокращение шейки
мочевого пузыря, препятствующее ретроградно-
му забросу компонентов эякулята [6]. Растяжение
простатического отдела мочеиспускательного кана-
ла секретом половых желез при эмиссии служит пу-
сковым механизмом второй фазы эякуляции — экс-
пульсии, непроизвольных ритмичных сокращений
поперечно-полосатой мускулатуры тазового дна,
и одновременное расслабление мочевого сфинкте-
ра. Это способствует толчкообразному выделению
эякулята из наружного отверстия мочеиспуска-
тельного канала [7]. Обследование мужчин с нарушениями эякуля-
ции следует начать с тщательного сбора анамнеза,
обращая внимание на свидетельства способности
иметь антеградное семяизвержение, в том числе
ночные поллюции достижение оргазма, в различ-
ных условиях. Одним из симптомов, о которых сле-
дует расспросить пациентов, является появление
мутной примеси в посткоитальной моче. Необхо-
димо также уточнить историю применения лекар-
ственных препаратов, перенесенных лечебных про-
цедур и оперативных вмешательств, учесть наличие
сопутствующих заболеваний — гипогонадизма, де-
прессии, неврологических расстройств. Признаки
этих заболеваний могут быть заметны и при физи-
кальном обследовании. При выявлении признаков нарушений углевод-
ного обмена и эндокринных расстройств показана
соответствующая лабораторная диагностика. О на-
личии РЭ можно судить по результатам исследова-
ния посткоитальной мочи [9]: обнаружение в ней
сперматозоидов и фруктозы позволяет подтвердить
наличие РЭ, а также предоставляет возможность
подготовить сперматозоиды для последующего ис-
пользования с применением ВРТ [10]. Выявление
расширения семенных пузырьков при трансрек-
тальном ультразвуковом сканировании свидетель-
ствует об обструкции эякуляторных протоков. Изучение физиологии семяизвержения позво-
лило получить представление о патологических
механизмах анэякуляции и РЭ, которые часто на-
блюдаются при нейрогенных расстройствах и на-
рушении анатомической целостности органов
мочеполовой системы и нервных волокон. При от-
сутствии секрета половых желез в задней уретре
из-за нарушения фазы эмиссии невозможен запуск
экспульсии, что клинически проявляется как АЭ. Неполное закрытие шейки мочевого пузыря в фазу Для преодоления проблемы бесплодия при РЭ
необходимо восстановить способность мужчин
к антеградной эякуляции и рассчитывать на на- УРОЛОГИЧЕСКИЕ ВЕДОМОСТИ 2017 Том 7 № 2 2017 Том 7 № 2 ISSN 2225-9074 ОРИГИНАЛЬНЫЕ СТАТЬИ 13 Препарат
Класс
Дозировка
Эффективность,%
Побочные эффекты
Псевдоэфедрин
Бромфенинамин
Имипрамин
Мидодрин
Эфедрин
Альфа-адреномиметик
Анигистаминное
средство
Трициклический
антидепрессант
Альфа-адреномиметик
Непрямой
альфа-адреномиметик
60 мг 4 р/д или
120 мг 2 р/д
16–24 мг/д
26 мг 2 р/д
15 мг/д
50–100 мг/д
30
38
65
62
20
Повышение артериального давле-
ния, боль в животе, тошнота, рвота
Антихолинэргические
Антихолинэргические,
нарушения сердечного ритма
Головная боль, сухость во рту
Тахикардия, повышение артериаль-
ного давления, тошнота, головная
боль
Таблица 1
Препараты, применявшиеся для фармакотерапии при ретроградной эякуляции (по А. Kamischke и Е. Nieschlag, 2002) функции афферентных или эфферентных нервных
волокон семенных пузырьков, семявыносящих
протоков, шейки мочевого пузыря и задней уре-
тры [13]. ступление естественного зачатия. В тех случаях,
когда это представляется возможным, нарушаю-
щий семяизвержение препарат следует отменить
или заменить его на препарат, принадлежащий
к другой фармакологической группе. А. Kamischke
и Е. Nieschlag в 2002 г. систематизировали представ-
ления о фармакотерапии, которую можно предло-
жить пациентам с РЭ с целью восстановления анте-
градного семяизвержения [11] (табл. 1). ступление естественного зачатия. В тех случаях,
когда это представляется возможным, нарушаю-
щий семяизвержение препарат следует отменить
или заменить его на препарат, принадлежащий
к другой фармакологической группе. А. Kamischke
и Е. Nieschlag в 2002 г. систематизировали представ-
ления о фармакотерапии, которую можно предло-
жить пациентам с РЭ с целью восстановления анте-
градного семяизвержения [11] (табл. 1). р
У многих пациентов с АЭ сперматозоиды, при-
годные для ВРТ, могут быть обнаружены в эяку-
ляте, полученном при использовании пенильной
вибростимуляции и электроэякуляции [14, 15]. Вибростимуляцию целесообразно применять у па-
циентов с сохраненной дугой рефлекса, состоящей
из чувствительных афферентных волокон полово-
го члена и эфферентных симпатических нервов,
а также участков спинного мозга на уровне их
входа (S2-S4) и выхода (Т10-Т12). В связи с этим
оптимальными кандидатами для проведения ви-
бростимуляции являются мужчины с поражением
спинного мозга выше сегмента Т10, у 88 % из них
она может привести к получению сперматозоидов,
в то время как в остальных случаях успех проце-
дуры не превышает 15 % [16]. Электроэякуляция
выступает альтернативой пенильной вибрости-
муляции, применяется по аналогичным показа-
ниям и служит высокоэффективной методикой,
позволяющей успешно получить сперматозоиды
у 97–100 % мужчин с анэякуляцией вследствие по-
вреждения спинного мозга [17]. Для преодоления проблемы бесплодия у муж-
чин с РЭ в программах ВРТ можно использовать
сперматозоиды, полученные из посткоитальной
мочи. УРОЛОГИЧЕСКИЕ ВЕДОМОСТИ Предварительно (за 12 часов и за 2 часа пе-
ред получением эякулята) пациенту рекомендуют
принять 50 мг бикарбоната натрия с целью още-
лачивания мочи и минимизации ее токсического
воздействия на сперматозоиды. Непосредственно
перед семяизвержением больной должен помо-
читься [12]. Посткоитальную мочу путем мочеиспу-
скания или катетеризации собирают в стерильный
контейнер и после добавления специального рас-
твора, позволяющего сохранить жизнеспособность
сперматозоидов, направляют в лабораторию ВРТ. При получении большого числа сперматозоидов
они могут быть использованы для внутриматоч-
ных инсеминаций, если концентрация и подвиж-
ность сперматозоидов невысоки, более вероятно
применение экстракорпорального оплодотворения
(ЭКО) и инъекции сперматозоидов в цитоплазму
яйцеклетки (ИКСИ). Отсутствие жизнеспособных сперматозоидов
в посткоитальной моче и анэякуляция являются
показанием для применения методов их хирурги-
ческой экстракции. Количество и качество спер-
матозоидов, полученных при операции, в боль-
шинстве случаев не только позволяют применить
их для оплодотворения яйцеклетки в условиях ла-
боратории ВРТ непосредственно после получения,
но и дают возможность произвести криоконсерва- Наиболее частыми причинами нарушений эмис-
сии являются сахарный диабет и повреждение
спинного мозга, кроме того, они могут быть след-
ствием воздействия целого ряда различных, в том
числе и ятрогенных, факторов, приводящих к дис- ЛИТЕРАТУРА 1. Rosen R, Altwein J, Boyle P, et al. Lower urinary tract symptoms
and male sexual dysfunction: the multinational survey of the aging
male (MSAM-7). Eur Urol. 2003;44(6):637-649. doi: 10.1016/j. eururo.2003.08.015. цию и хранение для использования в последующих
попытках проведения ЭКО [18]. На основании данных, представленных в лите-
ратуре, принимая во внимание необходимость ис-
пользования лекарственных препаратов в соответ-
ствии с инструкциями к их применению и риски
развития побочных эффектов, а также опираясь
на собственный опыт применения хирургических
методов экстракции сперматозоидов, мы предлага-
ем алгоритм диагностической и лечебной тактики
у бесплодных мужчин с РЭ и АЭ (рис. 1). УРОЛОГИЧЕСКИЕ ВЕДОМОСТИ УРОЛОГИЧЕСКИЕ ВЕДОМОСТИ 2017 Том 7 № 2 ISSN 2225-9074 ОРИГИНАЛЬНЫЕ СТАТЬИ 14 14
ОРИГИНАЛЬНЫЕ СТАТЬИ
Рис. 1. Алгоритм диагностической и лечебной тактики у бесплодных мужчин с ретроградной эякуляцией и анэякуля-
цией (ВРТ — вспомогательные репродуктивные технологии, ПЕСА — чрескожная аспирационная биопсия придатка яичка,
ТЕСА — чрескожная аспирационная биопсии яичка)
Отсутствие
эякулята
Ретроградная
эякуляция
Появление анте-
градной эякуляции
Сохранение ретро-
градной эякуляции
Получение сперма-
тозоидов из посткои-
тальной мочи
Получены
сперматозоиды
хорошего качества
Не получены
сперматозоиды
хорошего качества
Ощелачивание
мочи
Получены
сперматозоиды
Получены
сперматозоиды
Не получены
сперматозоиды
Не получены
сперматозоиды
Сперма донора. Усыновление
Сперма донора. Усыновление
Фармакокоррекция
ПЕСА/ТЕСА
ПЕСА/ТЕСА
ВРТ
ВРТ
ВРТ
Отсутствие
эмиссии
Анализ
посткоитальной мочи Отсутствие
эякулята Анализ посткоитальной мочи Отсутствие
эмиссии Ретроградная
эякуляция ПЕСА/ТЕСА Фармакокоррекция Получены
сперматозоиды Не получены
сперматозоиды Сохранение ретро-
градной эякуляции Появление анте-
градной эякуляции Ощелачивание
мочи Сперма донора. Усыновление ВРТ Получение сперма-
тозоидов из посткои-
тальной мочи Не получены
сперматозоиды
хорошего качества Получены
сперматозоиды
хорошего качества ВРТ ПЕСА/ТЕСА Получены
сперматозоиды Не получены
сперматозоиды Сперма донора. Усыновление Рис. 1. Алгоритм диагностической и лечебной тактики у бесплодных мужчин с ретроградной эякуляцией и анэякуля-
цией (ВРТ — вспомогательные репродуктивные технологии, ПЕСА — чрескожная аспирационная биопсия придатка яичка,
ТЕСА — чрескожная аспирационная биопсии яичка) Таким образом, на современном этапе специали-
сты обладают достаточно разнообразным арсеналом
лечебных подходов, позволяющих рассчитывать
на успешную подготовку к программам преодоления
бесплодия в браке у мужчин с АЭ и РЭ. цию и хранение для использования в последующих
попытках проведения ЭКО [18]. УРОЛОГИЧЕСКИЕ ВЕДОМОСТИ 2017 Том 7 № 2 ISSN 2225-9074 ОРИГИНАЛЬНЫЕ СТАТЬИ 15 11. Coogan CL, Hejase MJ, Wahle GR, et al. Nerve sparing post-
chemotherapy retroperitoneal lymph node dissection for advanced
testicular cancer. J Urol. 1996;156:1656-1658. 2. Calabro RS, Polimeni G, Ciurleo R, et al. Neurogenic ejaculatory dis-
orders: focus on current and future treatments. Recent Pat CNS Drug
Discov. 2011;6(3):205-221. doi: 10.2174/157488911796958011. 3. Rowland D, McMahon CG, Abdo C, et al. Disorders of orgasm
and ejaculation in men. J Sex Med. 2010;7(4):1668-1686. doi: 10.1111/j.1743-6109.2010.01782.x. 12. Arafa M, El Tabie O. Medical treatment of retrograde ejaculation
in diabetic patients: a hope for spontaneous pregnancy. J Sex
Med. 2008;5(1):194-198. doi: 10.1111/j.1743-6109.2007. 00456.x. 4. Betocchi C, Verze P, Palumbo F, et al. Ejaculatory disorders: patho-
physiology and managemen. Nat Clin Pract Urol. 2008;5(2):93-103. doi: 10.1038/ncpuro1016. 13. Wein AJ, Kavoussi LR, Novick AC, et al. Campbell-Walsh Urol-
ogy. 10th ed. Chapter 22: Surgical Management of Male Infertility. Philadelphia PA: Elsevier Saunders; 2012. 5. Barazani Y, Stahl P, Nagler H, Stember D. Management of ejacula-
tory disorders in infertile men. Asian J Androl. 2012;14(4):525-9. doi: 10.1038/aja.2012.29. 14. Ohl DA, Quallich SA, Sønksen J, et al. Anejaculation and retro-
grade ejaculation. Urol Clin North Am. 2008;35(2):211-220. doi: 10.1016/j.ucl.2008.01.014. 6. Hershlag A, Schiff SF, DeCherney AH. Retrograde ejaculation. Hum Reprod. 1991;6:255-258. 15. Brackett NL, Ibrahim E, Iremashvili V, et al. Treatment for ejacula-
tory dysfunction in men with spinal cord injury: an 18-year single
center experience. J Urol. 2010;183(6):2304-2308. doi: 10.1016/j. juro.2010.02.018. 7. Polakoski KL, Syner FN, Zaneveld LJ. Biochemistry of human semi-
nal plasma. In: Human semen and fertility regulation in men. E. S. E.,
Hafez, E. D., C.V. Mosby Co St. Louis, Mosby; 1976. P. 133-143. 16. Ohl DA, Quallich SA, Sonksen J, et al. Anejaculation: an elec-
trifying approach. Semin Reprod Med. 2009;27(2):179-185. doi: 10.1055/s-0029-1202307. 8. Ozgok Y, Tan MO, Kilciler M, et al. Diagnosis and treat-
ment of ejaculatory duct obstruction in male infertilit. Eur Urol. 2001;39(1):24-29. 17. Brackett NL, Lynne CM, Ibrahim E, et al. Treatment of infertility in
men with spinal cord injury. Nat Rev Urol. 2010;7(30):162-172. doi: 10.1038/nrurol.2010.7. 9. Revenig L, Leung A, Hsiao W. Ejaculatory physiology and patho-
physiology: assessment and treatment in male infertility. Transl
Androl Urol. 2014;3(1):41-49. 18. Levine LA, Lisek EW. Successful sperm retrieval by per cu-
taneous epididymal and testicular sperm aspiration. J Urol. 1998;159(2):437-440. doi: 10.1016/s0022-5347(01)63943-0. 10. Colpi G, Weidner W, Jungwirth A, et al. УРОЛОГИЧЕСКИЕ ВЕДОМОСТИ EAU guidelines on ejacu-
latory dysfunction. Eur Urol. 2004;46(5):555-558. doi: 10.1016/j. eururo.2004.07.020. Information about the authors: Сведения об авторах: Игорь Алексеевич Корнеев — д-р мед. наук, профессор
кафедры урологии. ФГБОУ ВО «Первый Санкт-
Петербургский государственный медицинский университет
имени академика И.П. Павлова» Минздрава России; врач-
уролог, Международный центр репродуктивной медицины. Санкт-Петербург. E-mail: iakorneyev@yandex.ru. Igor A. Korneyev — doctor of medical science, professor. Department of Urology. Academician I.P. Pavlov First
St Petersburg State Medical University of the Ministry of
Healthcare of the Russian Federation; urologist, International
Center for Reproductive Medicine. Saint Petersburg, Russia. E-mail: iakorneyev@yandex.ru. Руслан Дзамболатович Зассеев — врач-уролог,
Международный центр репродуктивной медицины, Санкт-
Петербург. E-mail: r.zasseev@gmail.com. Ruslan D. Zasseev — urologist, International Center
for Reproductive Medicine. Saint Petersburg, Russia. E-mail: r.zasseev@gmail.com. 2017 Том 7 № 2 ISSN 2225-9074
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https://openalex.org/W4289202888
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https://www.ej-eng.org/index.php/ejeng/article/download/2841/1268
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English
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Relationship between Different Anaerobic Digestion Parameters in a Pig-dung Aided Water Hyacinth Digestion Process
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European journal of engineering and technology research
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cc-by
| 3,004
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Relationship between Different Anaerobic
Digestion Parameters in a Pig-dung Aided
Water Hyacinth Digestion Process Ochuko M. Ojo, Josiah O. Babatola, and Taiwo O. Olabanji activities, as well as clogging irrigation channels [6]. The
conditions that allow WH to thrive cannot be easily controlled
hence it is difficult to eradicate this noxious plant [7]. Several
studies have attempted to utilize this problematic plant for the
production of biogas [1], [5], [8]–[13]. Animal dungs contain
nutrients which are detrimental to public health, but which
can be used as biomass for fuel generation [14]. Pigs produce
a huge amount of dungs that could be harmful to the
environment if not properly managed [15]. Manure nutrients
(total nitrogen and total phosphorus) found in Pig dung (PD)
are considered the main source of environmental pollution
[16]. In Nigeria and sub-Saharan Africa in general, pig
production has increased in great measure as a result of
increase in the animal protein needs of the people caused by
rapid increase in population [17]. There is a continuous need
to properly harness these two constituents of environmental
pollution. Abstract — This study is aimed at assessing the relationship
between different anaerobic digestion parameters (biogas
quality, retention time, pH, temperature, biogas pressure,
volume of biogas produced and cumulative volume of gas
produced) in a Pig-dung (PD) aided Water Hyacinth (WH)
digestion process in order to maximize biogas yield in terms of
quantity and quality. 25 - litre capacity plastic prototype
digesters were used in the study and eleven (11) mix ratios of PD
and WH were prepared namely: 10 WH: 0 PD, 9 WH: 2 PD, 8
WH: 2 PD, 7 WH: 3 PD, 6 WH: 4 PD, 5 WH: 5 PD, 4 WH: 6 PD,
3 WH: 7 PD, 2 WH: 8 PD, 1 WH: 9 PD and 0 WH: 10 PD. The
digestion process was evaluated for a retention period of 40 days. A bivariate Pearson correlation analysis was carried out to
examine the relationship between the quality of gas produced
and other variables. The results revealed that daily gas
production yields greatly improved in the co-digestion runs with
mix 3 WH: 7 PD recording the highest maximum daily yield of
9.5 L with a cumulative gas volume of 140 L. For this mix, the
methane content of the gas produced increased from 5.8% on
day 4 to 69.2% on day 20. European Journal of Engineering and Technology Research
ISSN: 2736-576X European Journal of Engineering and Technology Research
ISSN: 2736-576X DOI: http://dx.doi.org/10.24018/ejeng.2022.7.4.2841
Submitted on June 15, 2022.
Published on July 31, 2022.
O. M. Ojo, Federal University of Technology, Akure, Nigeria.
(e-mail: omojo@futa.edu.ng) Relationship between Different Anaerobic
Digestion Parameters in a Pig-dung Aided
Water Hyacinth Digestion Process The least quantity and quality of gas
was produced by mix 10 WH: 0 PD with a maximum daily yield
2.34 L and a cumulative gas yield of 32.18 L. The digestion of all
the mixes occurred within a mesophilic temperature range of
28.2 to 31.4 0C and an increase in temperature within the
digestion resulted in an increase in the quality of gas produced. The gas pressure ranged from 1 bar to 3.324 bars with an
increase in gas pressure leading to a corresponding increase in
volume of gas produced. The pH of the substrates ranged from
6.1 to 8.4 with the values low at the start of the digestion process
and gradually increasing to its maximum at the end of the
digestion process. The results revealed a very strong, positive
and significant association between the quality of the biogas
produced and other digestion parameters. Co-digestion may be referred to as the anaerobic digestion
(AD) of multiple biodegradable substrates (feedstocks) in an
AD system [6]. The ultimate goal of co-digestion is to
improve the biogas yield from an AD process. Several
parameters are used the evaluate the effectiveness of an AD
process they include biogas quality, retention time, hydrogen
ion concentration, temperature, biogas pressure, volume of
biogas produced and cumulative volume of gas produced. The aim of this study is to assess the relationship between
different anaerobic digestion in order to maximize biogas
yield in terms of quantity and quality. Keywords — Anaerobic digestion, biogas quality, correlation
analysis, relationship. A. Digestion Process 25-litre capacity plastic prototype digesters, with a
thickness of 2 mm, height of 400 mm and width of 320 mm
were used in the study. The substrates used in this research
are WH and PD. Fresh WH were harvested from a private
pond in Akure, Ondo State, while PD were collected from the
animal farm of the Federal University of Technology, Akure. WH were chopped into small sizes ranging from 1 cm to
2 cm. Both feedstocks were appropriately weighed. Eleven
(11) co-digestion mix ratios of WH to PD were evaluated as
follows: 10 WH: 0 PD, 9 WH: 2 PD, 8 WH: 2 PD, 7 WH: 3
PD, 6 WH: 4 PD, 5 WH: 5 PD. 4 WH: 6 PD, 3 WH:7 PD, 2
WH: 8 PD, 1 WH: 9 PD, 0 WH: 10 PD. J. O. Babatola, Federal University of Technology, Akure, Nigeria.
(e-mail: jobabatola@futa.edu.ng)
T. O. Olabanji, Federal University of Technology, Akure, Nigeria.
(e-mail: oreoluwataiwo27@gmail.com). B. Data Analysis The pH of the substrates ranged from 6.1 to 8.4 with the
values low at the start of the digestion process and gradually
increasing to its maximum at the end of the digestion process. The results revealed a weak and negative association between
the gas quality and the hydrogen ion concentration of the
different mix substrates as shown in Fig. 2. However, the
association was not statistically significant for the PD-aided
WH digestion. This may be due to the microbial composition
of the mix substrates. The PD mix recorded very high
microbial count before and after digestion. A bivariate Pearson correlation analysis was carried out to
examine the relationship between the quality of gas and the
other variables. The null hypothesis (H0) and alternative hypothesis (H1) of
the significance test for correlation were expressed in the
following ways for the two-tailed test. H0: ρ = 0 (the correlation coefficient is 0; there is no
association) H0: ρ = 0 (the correlation coefficient is 0; there is no
association) H1: ρ ≠ 0 (the correlation coefficient is not 0; a non-zero
correlation could exist. B. Correlation between Gas Quality and Retention Time The association between the gas quality and retention time
for the different mixes was not statistically significant. The
results revealed a negative and weak association between the
quality of gas produced and the retention time for the PD–
aided mix as shown in Fig. 1. This is due to the fact that the
quality of the gas produced by the evaluated mixes followed
a unimodal path increasing from the beginning of the
digestion process to a peak and then decreasing gradually till
the end of the retention time. This behavior resulted from the
batch feeding method employed in this research described by
[19]. The hydrogen ion concentration was derived from the pH
using equation 1. 𝑝𝐻= −𝑙𝑜𝑔10[𝐻+]
(1) (1) A rotameter flowmeter of model LZM-4T with a capacity
of 0.1-1L/Min equipped with a measuring tube was used for
the gas flow measurements while a manometer was used to
measure the pressure of the gas. The quality of the gas
produced was determined using a locally fabricated
potassium hydroxide (KOH) solution biogas quality
determination apparatus as specified by [18]. C. Correlation between the Gas Quality and the pH and
Hydrogen Ion Concentration C. Correlation between the Gas Quality and the pH and
Hydrogen Ion Concentration D. Correlation between Biogas Quality and Temperature The digestion of all the mixes occurred within a mesophilic
temperature range of 28.2 to 31.4 °C and an increase in
temperature within the digestion resulted in an increase in the
quality of gas produced. The mesophilic temperature range is
ideal for AD as this temperature range adequately supports
microbial activities within the digester [11], [20]. The
correlation between temperature and biogas quality was
statistically significant at 1% which means that the
temperature within the digester clearly affects the quality of
the gas produced with up to 99 % confidence level. Fig. 3
shows that the association between the temperature and gas
quality was positive implying that an increase in temperature
within the digestion leads to an increase in the quality of gas
produced. I. INTRODUCTION The main conventional source of energy in Nigeria today
is fossil fuel [1]. Unfortunately, fossil fuels are non-
renewable and eventually, their deposits will be exhausted
[2]. Biogas is a renewable energy that can be exploited to
meet the ever-increasing energy demands of man (especially
in developing countries like Nigeria) without depleting the
environment [3]. Water hyacinth (WH) are very problematic plants that
cover water bodies, hindering the penetration of the sun’s
rays and thereby reducing the photosynthetic processes of
submerged plants [4], [5]. WH cause severe environmental
and economic problems by impeding navigation and fishing The digestion process was evaluated for a retention period
of 40 days. The temperature within the digester was measured J. O. Babatola, Federal University of Technology, Akure, Nigeria. (e-mail: jobabatola@futa.edu.ng)
T. O. Olabanji, Federal University of Technology, Akure, Nigeria. (e-mail: oreoluwataiwo27@gmail.com). Vol 7 | Issue 4 | July 2022
10 Vol 7 | Issue 4 | July 2022
10 Vol 7 | Issue 4 | July 2022
10 European Journal of Engineering and Technology Research
ISSN: 2736-576X European Journal of Engineering and Technology Research
ISSN: 2736-576X using a mercury thermometer calibrated in degree centigrade
while the pH was determined using a pH meter. B. Correlation between Gas Quality and Retention Time European Journal of Engineering and Technology Research
ISSN: 2736-576X F. Correlation between Biogas Quality (%CH4) and
Daily Gas Volume Fig. 6. Association between biogas quality (%CH4) and cumulative volume
of gas produced for PD - aided WH digestion. The results revealed a very strong and positive association
between the daily volume and quality of the gas produced by
PD-aided WH digestion mixes as shown in Fig. 5. The
association was statistically significant at 1%. This implies
that an increase in the volume of gas produced leads to a
corresponding increase in the quality of the gas. E. Correlation between Biogas Quality (%CH4) and Gas
Pressure The results revealed that WH single-substrate digestion
produced a cumulative gas volume of 32.18 L while PD
single-substrate digestion produced a cumulative gas volume
of 94.47 L. This could be linked to the nature of organics
present in the two substrates [21]. Mix 3 WH: 7 PD recorded
the cumulative gas volume of 140 L. For this mix, the
methane content of the gas produced increased from 5.8% on
day 4 to 69.2% on day 20. The least cumulative volume of
gas was produced by mix 10 WH: 0 PD with a cumulative gas
yield of 32.18 L. The results revealed that the relationship
between the gas quality and the cumulative gas produced for
the different mixes was not statistically significant as shown
in Fig. 6. This is due to the fact that the digestion was a batch-
fed process with the cumulative volume of gas reaching its
ultimate value at the end of the 40-days retention time. The gas pressure ranged from 1 bar to 3.324 bars with an
increase in gas pressure leading to a corresponding increase
in volume of gas produced. A strong and positive association
between the pressure of the produced gas and its quality was
observed for the different mixes of PD aided WH digestion,
as shown in Fig. 4. The association was significant at 1%. This implies that the more the pressure of the gas, the more
the quality of the gas would be. Fig. 4. Association between biogas quality (%CH4) and Biogas Pressure for
PD -aided WH digestion. Fig. 6. Association between biogas quality (%CH4) and cumulative volume
of gas produced for PD - aided WH digestion. Fig. 4. Association between biogas quality (%CH4) and Biogas Pressure for
PD -aided WH digestion. A. Daily and Cumulative Gas Production A. Daily and Cumulative Gas Production Daily gas production yields greatly improved in the co-
digestion runs. For the PD - aided WH digestion, mix 3 WH:
7 PD recorded the highest daily yield of 9.5 L on the 20th day,
starting its gas production on the 3rd day and ending on the
34th day. The least daily yield of gas was produced by mix
10 WH: 0 PD with a maximum daily yield 2.34 L. The results
also revealed that WH single-substrate digestion produced a
cumulative gas volume of 32.18 L while PD single-substrate
digestion produced a cumulative gas volume of 94.47 L. Fig. 1. Association between biogas quality (%CH4) and retention time for
PD - aided WH digestion. Fig. 2. Association between biogas quality (%CH4) and Hydrogen ion
concentration for PD-aided WH digestion. Fig. 2. Association between biogas quality (%CH4) and Hydrogen ion
concentration for PD-aided WH digestion. Fig. 2. Association between biogas quality (%CH4) and Hydrogen ion
concentration for PD-aided WH digestion. Fig. 2. Association between biogas quality (%CH4) and Hydrogen ion
concentration for PD-aided WH digestion. Fig. 1. Association between biogas quality (%CH4) and retention time for
PD - aided WH digestion. Vol 7 | Issue 4 | July 2022
1 DOI: http://dx.doi.org/10.24018/ejeng.2022.7.4.2841 European Journal of Engineering and Technology Research
ISSN: 2736-576X Fig. 3. Association between biogas quality (%CH4) and Temperature for
PD - aided WH digestion. Fig. 5. Association between biogas quality (%CH4) and Volume of gas
produced for PD-aided WH digestion. G. Correlation between Biogas Quality (%CH4) and
Cumulative Gas Volume Fig. 5. Association between biogas quality (%CH4) and Volume of gas
produced for PD-aided WH digestion. Fig. 3. Association between biogas quality (%CH4) and Temperature for
PD - aided WH digestion. E. Correlation between Biogas Quality (%CH4) and Gas
Pressure REFERENCES [1]
Ojo O.M. Biogas Production through Anaerobic Co-Digestion of
Water Hyacinth and Poultry Manure. Nigerian Research Journal of
Engineering and Environmental Sciences. 2021;6(2):735–740. [2]
Akinola A.O. Renewable Energy Development for Sustainable
National Growth. 2016 SEET Annual Conference Proceedings, FUTA,
Nigeria, pp. 123–130, 2016. [3]
Roopnarain A., Adeleke R. Current Status, Hurdles and Future
Prospects of Biogas Digestion Technology in Africa. Renew. Sust. Energ. Rev. 2017;67:1162–1179. [4]
Perna C., Burrows D. Improved dissolved oxygen status following
removal of exotic weed in important fish habitat lagoons of the tropical
Burdekin River floodplain, Australia, Mar. Pollution Bulletin,
2005;51:138–148. [5]
Ojo O.M., Babatola J.O., Adesina A.A., Akinola A.O., Lafe O. Synergistic Effect of co-digesting different mix ratios of Water
Hyacinth and Cow-dung for Biogas production. FUTA Journal of
Engineering and Engineering Technology, Nigeria, 2018;12(1):54–59. Engineering and Engineering Technology, Nigeria, 2018;12(1):54 [6]
Ojo O.M., Babatola J.O. Appraisal of Cumulative Volume of Biogas
Produced from Water Hyacinth and Selected Animal Dungs Co-
Digestion Mixes. Journal of Civil Engineering and Urbanism, 2021;11
(5):58–64. [7]
Howard G.W., Matindi, S.W. Les espèces étrangères envahissantes
dans les zones humides de l’Afrique, UICN, GISP, RAMSAR, 15 pp. 2003. [8]
Katima J.H.Y. Production of biogas from water hyacinth: Effect of
substrate concentration, particle size and incubation period. Tanzania
Journal of Science, 2001;27(1):107–119. [9]
Patil J.H., Molayan A.R., Bhargav S., Sowmya S.R. Anaerobic co-
digestion of water hyacinth with primary sludge, Research Journal of
Chemical Science, 2011;1(3):72–77. ( )
[10] Ojo O.M. Biomethanation of Water Hyacinth and Selected Animal
Dungs for Biogas Production. Ph.D Thesis in the Department of Civil
Engineering, The Federal University of Technology, Akure; 2017. [11] Ojo O.M., Babatola J.O., Akinola A.O. Regression Analysis of Biogas
Production from the Co-Digestion of Water Hyacinth and Pig Dung. FUOYE
Journal
of
Engineering
and
Technology,
Nigeria. 2018;3(2):141–145. [12] Ojo O.M., Babatola J.O. Association between Biogas Quality and
Digester Temperature for Selected Animal Dung-Aided Water
Hyacinth Digestion Mixes. Journal of Applied Science and
Environmental Management, 2020;24(6):966-959. [13] Adelodun A.A., Olajire T.M., Ojo O.M. Biogas Generation from Co-
Digestion Waste Systems: The Role of Water Hyacinth; IntechOpen,
2022, pp. 1–20. [14] Sillar B. Dung by preference: The choice of fuel as an example of how
Andean pottery production is embedded within wider technical, social
and economic practices. Archaeometry. 2000;42:43–60. and economic practices. Archaeometry. 2000;42:43–60. [15] Okoli C.G., Edo F.A., Ogbuewu I.P., Nwajiobi I.J., Enemor V.H.A. Okoli I.C. IV. CONCLUSION This study has affirmed that there exist a very strong,
positive and significant association between the quantity and
quality of biogas produced and other digestion parameters. The yield of biogas from anerobic co-digestion of different
feedstocks can be enhanced if the digestion parameters are
closely monitored. DOI: http://dx.doi.org/10.24018/ejeng.2022.7.4.2841 Vol 7 | Issue 4 | July 2022
12 European Journal of Engineering and Technology Research
ISSN: 2736-576X European Journal of Engineering and Technology Research
ISSN: 2736-576X [20] Wang H., Lehtomaki A., Tovanen K., Puhakka J. Impact of crop
species on bacteria community structure during anaerobic co-digestion
of crops and cow manure. Bioresource Technology, 2009;100:2311–
2315. ACKNOWLEDGMENT The authors would like to appreciate the Federal University
of Technology, Akure for creating an enabling environment
to carry out this research. [21] Ojo, O.M. Daily and Cumulative Biogas Yields from Selected Animal
Dungs. In: Ayeni, A.O., Sanni, S.E., Oranusi, S.U. (eds) Bioenergy and
Biochemical Processing Technologies. Green Energy and Technology. Springer, Cham, pp. 37–44, 2022. https://doi.org/10.1007/978-3-030-
96721-5_4. CONFLICT OF INTEREST The authors declare that they do not have any conflict of
interest. DOI: http://dx.doi.org/10.24018/ejeng.2022.7.4.2841 European Journal of Engineering and Technology Research
ISSN: 2736-576X REFERENCES Biochemical values of pig dung collected from different
locations in Imo state, southeastern Nigeria. Asian J. Biol. Sci.,
2019;12:470–476. [16] Izmaylov A., Briukhanov A., Shalavina E., Vasilev E. Pig Manure
Management: A Methodology for Environmentally Friendly Decision-
Making. Animals, 2022;12:747. https://doi.org/10.3390/ani12060747. [17] Iregbu G.U., Kubkomawa I.H., Okoli C.G., Ogundu E.C., Uchegbu
M.C., Okoli I.C. Environmental concerns of pig waste production and
its potentials as biofuel source. J. Anim. Vet. Sci., 2014;1:17–24. [18] Abdel-Hadi M.A., Abd El-Azeem S.A.M. Effect of heating, mixing
and digester type on biogas production from buffalo dung. Misr
Journal of Agricultural Engineering, 2008;25(4):1454–1477. [19] Yu H.Q, Fang H.H.P. Acidogenesis of dairy wastewater at various pH
levels. Water Science Technology, 2002;43(10):201–206. Vol 7 | Issue 4 | July 2022
13 DOI: http://dx.doi.org/10.24018/ejeng.2022.7.4.2841
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Seeking menstrual products: a qualitative exploration of the unmet menstrual needs of individuals experiencing homelessness in New York City
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© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Seeking menstrual products: a qualitative
exploration of the unmet menstrual needs
of individuals experiencing homelessness
in New York City Caitlin Gruer1* , Kim Hopper1, Rachel Clark Smith1, Erin Kelly1, Andrew Maroko2 and Marni Sommer1 Abstract 168th Street, Room 537, New York, NY 10032,
USA USA
Full list of author information is available at the end of the article Full list of author information is available at the end of the article Full list of author information is available at the end of the article *Correspondence: Caitlin.gruer@columbia.edu
1 Department of Sociomedical Sciences, Mailman School of Public Health,
Columbia University, 722 W. 168th Street, Room 537, New York, NY 10032,
USA Gruer et al. Reprod Health (2021) 18:77
https://doi.org/10.1186/s12978-021-01133-8 Gruer et al. Reprod Health (2021) 18:77
https://doi.org/10.1186/s12978-021-01133-8 Open Access Abstract Background: There has been increasing recognition that certain vulnerable populations in the United States of
America struggle to meet their menstruation-related needs, including people experiencing homelessness. Media and
policy attention on this subject has focused on the provision of free menstrual products to vulnerable populations,
including a New York City legislative bill, which guarantees access to menstrual products for Department of Homeless
Services shelter residents (Intros 1123-A). Methods: This qualitative study explored the challenges people experiencing homelessness in New York City face in
accessing menstrual products. Data collection was conducted from June to August 2019 and included: Semi-struc-
tured key informant interviews with staff from relevant government agencies and homeless service providers (n = 15),
and semi-structured in-depth interviews with individuals with experience living on the street and in shelters (n = 22). Data were analysed using thematic analysis. Results: Key themes that emerged included: (1) insufficient and inconsistent access to menstrual products; (2) sys-
temic challenges to providing menstrual products; and (3) creative solutions to promote access to menstrual prod-
ucts. Both shelter- and street-living individuals reported significant barriers to accessing menstrual products. While
both populations struggle, those in shelters were more likely to be able to purchase menstrual products or access free
products at their shelter, while those living on the streets were more likely to have to resort to panhandling, theft, or
using makeshift materials in place of menstrual products. Across both populations, individuals described barriers to
accessing free products at shelters and service providers, primarily due to distribution systems that rely on gatekeep-
ers to provide a few pads or tampons at a time, sometimes of inadequate quality and only upon request. Shelters and
service providers also described challenges providing these products, including inconsistent supply. Conclusion: These findings highlight the critical importance of expanding and improving initiatives seeking to
provide access to menstrual products for vulnerable populations. Despite policy level efforts to support menstrual
product access, individuals experiencing homelessness in New York City, whether living in shelters or on the street, are
often not able to access the menstrual products that they need to manage their monthly menstrual flow. *Correspondence: Caitlin.gruer@columbia.edu *Correspondence: Caitlin.gruer@columbia.edu
1 Department of Sociomedical Sciences, Mailman School of Public Health,
Columbia University, 722 W. Plain English summary struggle to meet their menstruation-related needs [10,
11]. For instance, people experiencing homelessness
face unique challenges to managing their menstruation
whether living in shelters or on the street [12, 13], such
as lack of access to menstrual products [14–16], difficulty
maintaining personal hygiene [17], menstrual shame and
stigma [18, 19], and gynaecological issues [20]. This paper
seeks to add further nuance to this picture of unmet need
by exploring how those experiencing homelessness in
New York City (NYC) manage their periods, and how—
short of re-housing—their needs could be more effec-
tively addressed. We focus in particular on difficulties
related to securing menstrual products. g
y
In recent years, the media and policymakers have drawn
attention to the challenges that people experiencing
homelessness in the United States face managing their
menstruation. There are a growing number of initiatives
focused on providing menstrual products (pads, tam-
pons, etc.), including a bill passed in New York City
requiring homeless shelters to provide these items to cli-
ents. This study explores the menstruation experiences
of people with experience living in shelters or on the
streets of New York City. Qualitative data was gathered
through interviews with 22 individuals with experience
of homelessness, and 15 staff from relevant government
agencies and service providers. Data were analysed using
thematic analysis. Three main themes were found: chal-
lenges individuals experiencing homelessness faced
accessing menstrual products; difficulties shelters, and
service providers experienced providing access to these
products; and recommendations for improvements. All participants described difficulty obtaining sufficient
menstrual products to manage their monthly period. People living in shelters could more often purchase the
products themselves, while those living on the streets
sometimes had to resort to theft, panhandling or make-
shift alternatives. Participants also described challenges
accessing free products at shelters and service providers,
because of the distribution methods, while shelter and
service provider staff reported difficulties maintaining
consistent stock. Recommendations include: increasing
free product distribution in locations open twenty-four
hours a day; training shelter and service provider staff
to improve their comfort providing these services; and
rethinking distribution strategies. Our findings empha-
size the importance of initiatives that provide access to
these essential items for vulnerable populations. NYC has the largest population of people experiencing
homelessness in the USA. © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gruer et al. Reprod Health (2021) 18:77 Page 2 of 11 Keywords: Menstruation, Homelessness, New York City, Menstrual product Plain English summary In 2019, an average of 63,839
people slept in NYC homeless shelters every night [21],
and a point-in-time count estimated that at minimum
an additional 3,588 individuals were living on the streets
[22]. The latter is likely much higher due to difficulties in
locating and estimating this population [23]. The number
of women in the shelter system has continued to increase
over the last 18 years [21, 22]. Additionally, and likely an
underestimate, the Department of Housing and Urban
Development’s (HUD) 2018 point-in-time estimate of
homelessness suggested that 0.7% of those experiencing
homelessness identify as transgender or gender-non-
conforming [24]. All told, the number of people men-
struating while experiencing homelessness in NYC is
significant. i
Existing evidence suggests that this population encoun-
ters substantial difficulties related to menstruation,
including acquiring menstrual products, accessing bath-
rooms, and maintaining basic personal hygiene [14, 19]. These challenges are compounded by the stigma still
attached to menstruation, marking it as something to be
hidden from view and shielded from others [25]. Soci-
etal norms stipulate that its management be discreet
and secret. For people experiencing homelessness, this
is particularly challenging: they often lack access to the
resources required to avoid leaks and prevent menstrual
accidents, thus reducing their chances of maintaining
their appearance and blending in with the general pub-
lic. Perversely, this ability to “pass” as someone conven-
tionally housed plays a vital role in their ability to access
precisely
those
resources—commercial
bathrooms,
employment opportunities—that would better equip
them to manage their menstruation [26, 27]. Background Menstruation has gained global attention in the past
decade as a critical public health and human rights
issue [1–3]. This includes growing evidence document-
ing what those who menstruate need in order to man-
age their monthly periods comfortably and confidently:
access to safe, private toilets with adequate water sup-
ply, bathing and laundering spaces, menstrual materials
and supplies (e.g. menstrual products, underwear), and
quality information and education [4–9]. While atten-
tion initially focused on low-income countries, there
has been increasing recognition that certain vulnerable
populations in the United States of America (USA) also Access to menstrual products has received much atten-
tion from the media and policymakers. In 2016, NYC Gruer et al. Reprod Health (2021) 18:77 Gruer et al. Reprod Health (2021) 18:77 Page 3 of 11 The sample for the IDIs included people with experi-
ence living on the street and/or in shelters ages 18 years
and older. To capture a diversity of menstruation-related
issues, we sought to interview individuals across a range
of age categories including: 18–25; 26–35; 36–45; and 46
and above. IDI participants (n = 22) were identified from
among those seeking services at homeless service provid-
ers using convenience sampling. These service providers
provided clients with information about the study via
announcements, posted flyers, and sign-up sheets at the
check-in desk. Interested clients identified themselves to
the service provider who in turn introduced them to the
research team. IDI participants received a small incentive
for their participation ($10 or $15 subway card). passed a legislative bill which guarantees access to men-
strual products for Department of Homeless Services
shelter residents (Intros 1123-A). Referred to as the NYC
Menstrual Policy for the purposes of this paper, Intros
1123-A requires city-run and city-funded shelters to
provide tampons and pads to clients. It applies to fam-
ily shelters, single adult shelters, HIV/AIDS shelters,
domestic violence shelters, youth detention facilities, and
congregate care facilities for youth waiting for placement
with a foster care agency [28]. While some shelters pro-
vided these products already, the bill sought to codify this
service, ensuring that all shelter residents have a source
of these necessities. Methods We conducted a qualitative study that sought to under-
stand the menstruation experiences of people living on
the streets and in shelters in NYC. The research methods
included: (1) key informant interviews (KII) conducted
with staff from relevant government agencies and home-
less service providers; and (2) in-depth interviews (IDI)
with people experiencing homelessness while menstru-
ating. This paper will explore findings from the KIIs and
IDIs related to menstrual products; other findings from
the KIIs and IDIs are described elsewhere [27].h The study received Internal Review Board (IRB)
approval from the Columbia University Institutional
Review Board. Based on respondent preference, KIIs were conducted
in person or online. The interviews ranged from 30 to
60 min, were conducted in a confidential setting, and
were digitally recorded unless otherwise requested by the
interviewee. We used a semi-structured interview guide
tailored to the organization’s work and mission, its role
in providing support to people experiencing homeless-
ness, the specific needs and challenges encountered while
providing this support (related to menstruation and more
broadly), and any recommendations for how these expe-
riences could be improved.fi Data collection
d KIIs and IDIs were conducted in NYC by two-person
research teams during the period of June–August 2019. The research team consisted of the Principal Investigator
(PI), a graduate research assistant (RA), and a member of
the PI’s staff research team. All interviewers were females
and had conducted work or research with individuals
experiencing homelessness in the past. Before starting
data collection, the team conducted reflexivity exercises
to consider our positionality and the power dynamics in
the exchange, as well as discussing own experiences men-
struating while living in NYC. The larger research team
also included a Co-investigator (Co-I) with significant
experience in research, practice and policy related to
homelessness. All participants provided informed con-
sent prior to the start of each interview. Background This bill was the first of its kind in
the USA, though since its passing, other cities and states,
such as Los Angeles, Chicago, and New York State, have
instituted similar policies [10].h All participants presented as female; however, we did
not explicitly ask about gender identification. The age of
the participants ranged from 18 to 62, and all participants
had menstruated while experiencing homelessness. All
participants had lived in a shelter, and a segment (n = 7)
of the participants had experienced both street and shel-
ter living. This study was conducted in NYC between May and
August 2019 in collaboration with the Coalition for the
Homeless, one of the oldest advocacy and direct service
organizations in NYC. The Coalition provided invalu-
able insights into the predicament of those facing home-
lessness in NYC today, and facilitated the connection to
individuals experiencing homelessness and to service
providers throughout the city. Sample and recruitmenth The sample for the KIIs included a range of govern-
ment and service provider actors (male and female) from
agencies and organizations providing services to people
experiencing homelessness. They included both front-
line providers, and those with relevant oversight or man-
agement roles. Key informants (n = 15) were sampled
purposively to ensure that participants represented agen-
cies and organizations conversant with the issue, exist-
ing challenges, and/or recommendations for possible
improvements to the current situation. This included rel-
evant government agencies, food pantries, youth shelters
and street outreach providers. IDIs were conducted at the offices of the collaborat-
ing service providers in a confidential space where the
conversation could not be overheard and were digi-
tally recorded with the consent of the participant. The Gruer et al. Reprod Health (2021) 18:77 Gruer et al. Reprod Health (2021) 18:77 Page 4 of 11 interviews were conducted using a semi-structured inter-
view guide that explored the participant’s current and
recent housing status and personal history, before delv-
ing into the participant’s experience managing their men-
struation while experiencing homelessness. This included
questions around the facilities and alternate locations
used as bathrooms, bathing and laundering; access to
menstrual products; and their recommendations for
improving the menstrual experiences of people experi-
encing homelessness. interviews were conducted using a semi-structured inter-
view guide that explored the participant’s current and
recent housing status and personal history, before delv-
ing into the participant’s experience managing their men-
struation while experiencing homelessness. This included
questions around the facilities and alternate locations
used as bathrooms, bathing and laundering; access to
menstrual products; and their recommendations for
improving the menstrual experiences of people experi-
encing homelessness. to other necessities were common. For example, secur-
ing menstrual products could mean going without
medications or more expensive food items. Others
reported having to rely on makeshift solutions when
they were unable to afford products: I would run out of money and I ended up doing
things rather than [buying them]… like, cutting up
old t-shirts and stuff like that.—IDI 15 Respondents living in shelters also mentioned shelter-
based distributions, though less frequently. Depending
on the shelter, the distribution method varied: mass
distributions on designated supply days; requests to a
caseworker, shelter staff, or the check-in desk; or, rarely,
unencumbered access to a centrally located repository. Sample and recruitmenth A few participants reported that such distributions
made their lives easier by reducing their financial bur-
den and ensuring that they could access products more
regularly. Consistently, though, participants described
the availability of products as uneven and insufficient. Some reported a complete absence of menstrual prod-
ucts at their shelter. Data analysis
I
i Interviews were transcribed and analysed by two mem-
bers of the research team, using Malterud’s ‘systematic
text condensation’ method for thematic cross-case analy-
sis [29]. This included the following steps: (1) identifica-
tion of preliminary themes; ((2) creative development
of qualitative codes; (3) condensation of coded text; (4)
synthesis and reconceptualization [29]. The PI reviewed
and helped to revise the final codebook, and the analy-
sis team used Dedoose analytic software to code the data. The themes identified from the data were shared with full
research team for discussion, refinement, and validation. The full team met multiple times to discuss the themes
identified, the commonalities and differences emerging
within the themes, and to discuss and resolve discrepan-
cies between codes. After data analysis was complete, the
final analytical themes were presented to representatives
from the Coalition for the Homeless for discussion and
to affirm the data. The resultant analytical themes are
presented below, along with illustrative excerpts from
interviews. Rationing methods, too, were described, with men-
strual products being distributed by staff members
selectively and only upon request. Participants reported
receiving only a few pads or tampons at a time, necessi-
tating asking for products repeatedly, sometimes multi-
ple times a day. Some shelters permit product requests
only on specific distribution days during a given week or
month; still others require the request be made to a cer-
tain staff member who is only on duty during certain days
or hours. Such restrictive distribution systems meant
that—even where officially “provisioned”—respond-
ents could not rely on shelters as a primary source of
products. Resultsh Three key themes related to the availability and acces-
sibility of menstrual products for people experiencing
homelessness in NYC proved salient throughout the anal-
ysis: (1) insufficient and inconsistent access to menstrual
products; (2) systemic challenges to providing menstrual
products; and (3) creative solutions to promote access to
menstrual products. A number of shelter clients added nuance to such
requests, describing how disclosing their need for men-
strual products elicited shame and humiliation, feelings
heightened if the gatekeeper was stationed in a public
place (e.g., the check-in desk), or male: …the humiliation is that you have to keep going back
to them and asking them, and when you’re asking
for, because they have police and security, so it’s not
private. So you’re asking for it in front of NYPD and
DHS security, and most of those are male staff. —IDI
07 Insufficient and inconsistent access to menstrual products
h
fi d
f
d
h Insufficient and inconsistent access to menstrual products
An overarching finding from respondents with experi-
ence living both in shelters and on the street was the
formidable struggle to access menstrual products
consistently. Those living in shelters most commonly
reported purchasing their own menstrual products,
often relying on discount or dollar stores to minimize
costs. Revenue sources varied, with a majority of partic-
ipants reporting using their own income, depending on
financial support from friends or family members, or
public assistance funds. Trade-offs made with respect Other shelter clients were simply unwilling to disclose
their menstruating status due to social norms and taboos,
preventing them from accessing the free menstrual prod-
ucts even when available. A few respondents reported
experiencing or witnessing harassment, including sexual
harassment, by shelter staff: Gruer et al. Reprod Health (2021) 18:77 Gruer et al. Reprod Health (2021) 18:77 Page 5 of 11 Page 5 of 11 like that. They might have deodorant and soap and
toothpaste, but they don’t have the personal items
that you might need. So, you have to ask for that. And if they have it, they don’t mind. A lot of times
they don’t carry it, though, so you have to go from
place to place to place, and you have to go in pub-
lic restrooms and stuff like that, because that’s not
something that we think about. And you think about
giving people water and food and soap to be clean
and, but you don’t think about the other aspects, like
vitamins, you know, and tampons and this stuff, it’s,
it’s not so easy. —IDI 21 … the women have to go and ask for menstrual
products, and then you have the male staff smiling,
smirking at them… Women feel that they’re being
discriminated against, harassed, sexually harassed
because they’re menstruating —IDI 07 The types and quality of menstrual products provided
by the shelters were also identified as problematic. For
example, some women preferred using tampons but
only pads were available, or vice versa. This meant set-
tling for what was available, or finding another way to
acquire their preferred product. Insufficient and inconsistent access to menstrual products
h
fi d
f
d
h Many complained about
the quality or absorbency of available products, say-
ing that pads provided by their shelter were too small or
too thin to manage what they described as their “heavy
[blood] flow.” This made it necessary to change the prod-
uct much more often, requiring access to safe, clean
spaces for changing—a contingency not readily available
to them [27]. As a result, even when menstrual products
were available at their shelter, some participants opted to
acquire their products elsewhere: Those with experience living on the street also
described similar complaints heard from shelter resi-
dents with respect to procuring preferred products, set-
tling for inferior quality and quantity, and having to deal
with gatekeepers. As a result, some participants who had
lived on the street reported resorting to panhandling for
menstrual products, which posed its own difficulties. As
one woman described: “I get more cigarettes than I could
get tampons [while begging on the subway].” (IDI 16) A
few participants had resorted to pilfered menstrual prod-
ucts while others, like their counterparts in the shelters,
made do with makeshift materials, such as old pieces of
clothing, or wads of toilet paper. I was always like, if I had like, six, five dollars, I’d
go get pads with wings, cause it would just make life
so much easier. And the reason I needed so many of
those from [service provider] was cause they didn’t
work and I had to go to the bathroom every two
three hours instead of every four hours or five. —IDI
13 Although none of our participants self-identified as
trans masculine or gender non-conforming, a number
of key informants noted that such clients often encoun-
ter even more significant barriers to obtaining menstrual
products. As one youth services provider shared: Lastly, a number of participants reported being una-
ware of whether their shelter provided free menstrual
products or not. A few explained that it had never
occurred to them that these products might be avail-
able, when so many other basic items are not, and so they
had never asked. Despite clearly outlining how necessary
menstrual products were for their daily life, the women
did not view them as something they could expect to
receive. Insufficient and inconsistent access to menstrual products
h
fi d
f
d
h Similarly, when questioned, most participants
had not heard of the 2016 law requiring shelters to pro-
vide menstrual products, and were unaware of their legal
right to access products at their shelter. They’re, like, presenting with . . . a gender that
doesn’t match someone who would be having a
period, and so that’s super complicated and embar-
rassing, um, and so I think there’s just, yeah, there’s
so much shame and stigma around it... —KII 05 They’re, like, presenting with . . . a gender that
doesn’t match someone who would be having a
period, and so that’s super complicated and embar-
rassing, um, and so I think there’s just, yeah, there’s
so much shame and stigma around it... —KII 05 As a result, transgender and gender non-conforming
individuals may only be willing or able to access men-
strual products from service providers where they feel
accepted and have developed trusting relationships. Unsurprisingly, participants with experience living on
the street described even greater difficulties accessing
menstrual products, as they lack access to shelter sup-
plies and were less likely to report alternative sources of
funding and support. Most commonly, these participants
relied on service organizations for handouts. However,
not all homeless service providers in NYC distribute
menstrual products, and even when they did, availability
was uncertain. Menstrual products are not viewed as an
essential item in “care packages”: Systemic barriers to providing menstrual products
dd
h d ffi
l
h
h Sometimes they have
tampons, most of the time they don’t have tampons. Most of the time they have, uh, the pads. Yeah. And
uh, you have to just take what they’ve got. —IDI 21 Systemic barriers to providing menstrual products
dd
h d ffi
l
h
h Cause I, in shelter, I’ve been working in
shelter for, since 2010, um, and it was always some-
thing that we just provided to the clients, like we had
to, yeah. —KII 012 Service providers may also face challenges with staff
comfort distributing products—the reciprocal version
of the “gatekeeper” issue mentioned earlier. While our
sample of service provider informants was predomi-
nantly women, a few mused that their male counterparts
might feel uncomfortable discussing menstruation or feel
embarrassed when providing these supplies to women
and transgender clients. The fear of stock outs appeared to intersect with the
distribution system in many shelters, as gatekeeping staff
monitor the usage and supply of these products and pre-
vent one client from taking the entire supply. Shelter staff
seemed unaware of the potential stress that the rationing
system may cause some clients. I guess like one thing I was thinking about was sort
of the, the diversity and gender diversity of our staff
and, you know, the comfortability of asking for prod-
ucts or talking about problems with male versus
female versus transgender staff…or even, just like
how our, kind of our male staff feel [inaudible] feel
about offering, um, [menstrual products]. —KII 09 I guess like one thing I was thinking about was sort
of the, the diversity and gender diversity of our staff
and, you know, the comfortability of asking for prod-
ucts or talking about problems with male versus
female versus transgender staff…or even, just like
how our, kind of our male staff feel [inaudible] feel
about offering, um, [menstrual products]. —KII 09 Non-shelter service providers faced tougher chal-
lenges because they supply these products at their own
discretion and expense. Most rely on product donations
through arrangements ranging from formal partnerships
with commercial companies to ad hoc donations from
private individuals. While such approaches reduce pro-
curement costs, they also make it harder for service pro-
viders to control the type and quality of products made
available—an eventuality not lost on clients: A staff member’s own personal discomfort with dis-
tributing products may in turn contribute to creating an
uncomfortable environment for clients. No. No no no no no no no no no, there’s no choice. You get what they’ve got. Because most of their stuff
is donated too. Some, you have to get what they’ve
got and utilize what they have. Systemic barriers to providing menstrual products
dd
h d ffi
l
h
h In addition to the difficulties that participants themselves
communicated in accessing menstrual products, shel-
ter staff and service providers described how the system
itself inhibits their ability to consistently provide men-
strual products to their clients. While all the service pro-
viders articulated the importance of menstrual products,
many described not always being able to provide their cli-
ents with regular access. They give you a care package; they don’t necessarily
have personal things like that, like tampons and stuff Shelters are included under the mandate of the
2016 NYC Menstrual Policy, which means that the Gruer et al. Reprod Health (2021) 18:77 Page 6 of 11 Gruer et al. Reprod Health (2021) 18:77 Department of Citywide Administrative Services (DCAS)
must provide a supply of menstrual products sufficient to
meet the needs of their residents. However, shelter staff
reported that the products were not always delivered in
a timely enough manner to ensure a consistent stock. These delays contributed to occasional stock outs and to
fear that the products might run out. In addition, aware-
ness of the NYC Menstrual Policy was uneven among the
shelter staff. Some knew nothing about the policy; others
were uncertain about how it would be implemented over
time. As the director of one shelter shared: have robust donation programs; however, to date, these
products have not been popular with the clients and may
be difficult for the clients to change, clean and store while
living in a shelter or on the street: We’ve been partnering with [menstrual cup com-
pany] for a few years, um, they send us a big box of
[menstrual cups] every 6 months or so. Um, but with
the [menstrual cups] it hasn’t been, it just hasn’t
picked up for the young people and so we’ve haven’t
gotten a shipment in the last six months since we
have a big supply of it still. —KII 10 I didn’t know about the, the menstrual [policy], I
had no idea. Cause I, in shelter, I’ve been working in
shelter for, since 2010, um, and it was always some-
thing that we just provided to the clients, like we had
to, yeah. —KII 012 I didn’t know about the, the menstrual [policy], I
had no idea. Creative solutions to promote access to menstrual
productsh A few participants described how
this system is already in place in some shelters or ser-
vice providers and how this makes accessing products
less fraught: I would recommend that’s really nice is, is what they
do at, it’s an awful place, [shelter], is, is, the having
them available in a way so that…[you] don’t even
have to ask, I mean you’ve got women from all differ-
ent types of backgrounds, some people might, some
people might be shy about it. —IDI 15 I would recommend that’s really nice is, is what they
do at, it’s an awful place, [shelter], is, is, the having
them available in a way so that…[you] don’t even
have to ask, I mean you’ve got women from all differ-
ent types of backgrounds, some people might, some
people might be shy about it. —IDI 15 While some programs are constrained in what they
can currently offer (e.g., SNAP and WIC cannot cover
non-food items), these suggestions illustrate how these
products could be incorporated into existing systems. Two service providers and one participant living in a
shelter suggested a citywide campaign to distribute free
menstrual products in the same manner as NYC con-
doms, which are widely available and easily accessible. The equating of condoms with menstrual products, the
latter an essential gendered item, pointedly raises an
important issue of societal equity. Beyond making products freely available, participants
recommended smaller adjustments to improve the cur-
rent systems. The most common suggestion was that a
larger number of products should be distributed at one
time per client, reducing the need to resupply during one
menstrual period, improving clients’ sense of independ-
ence and dignity, and assuring not being “tied” to a given
provider or location during their period. Plainly, too, this
would increase their ability to seek other services, work
or go about their day. Related, a few participants sug-
gested that products be distributed regularly and rou-
tinely in a shelter instead of only on demand, so that
clients could have consistent access without having to
ask for them. There was also a repeated recommendation
that service providers distribute menstruation-support-
ive supplies along with the menstrual products, such as
soap, towels, feminine wipes, and underwear. Creative solutions to promote access to menstrual
productsh These sup-
plies are already available at some service providers, and
are a much-appreciated resource: A second set of recommendations built on the idea of
increased accessibility, proposing an expanded number
of locations where menstrual products could be made
available, especially ones accessible twenty-four hours
a day. This was particularly important to participants
who had spent time living on the street to ensure that
they could access products during hours when service
providers were closed. Proposed locations varied, but a
common suggestion was public restrooms. As one par-
ticipant experiencing homelessness (IDI 10) described,
“I’d have [menstrual products] for free in the bath-
room…[in] any public bathroom.” Another participant without a stable home recom-
mended a van that would drive around the city to cer-
tain drop-off points, distributing products to those who
need them. Still another participant who had spent
time living on the street recommended the installation
of menstrual product vending machines across the city
where products could be purchased using a credit card,
or accessed by those on public assistance who would
be given a specific card for the machines. Importantly,
such a machine would be accessible 24 hours a day. Par-
ticipants noted that these approaches would not only
improve menstrual product access for people experi-
encing homelessness, but also for residents struggling
to make ends meet and for anyone finding themselves
in urgent need of a menstrual product while in the city. I honestly would make sure that every single shelter,
because they do have the resources, that they would
give any woman, any woman that’s menstruating,
whatever, access to what they need, how many they
need, whenever. Not giving them a limit, because the
human body changes. Um, access to towels, soap,
and everything you can clean yourself —IDI 20 I honestly would make sure that every single shelter,
because they do have the resources, that they would
give any woman, any woman that’s menstruating,
whatever, access to what they need, how many they
need, whenever. Not giving them a limit, because the
human body changes. Um, access to towels, soap,
and everything you can clean yourself —IDI 20 Others recommended making sure more clients knew
that menstrual products were available, given repeated
indications that shelter clients often have no idea these
products were stocked. …letting people know, this item is available to you,
I think would be a huge help because people, again, Creative solutions to promote access to menstrual
productsh The participants experiencing homelessness and those
seeking to meet their needs shared a number of ideas
for improving access to menstrual products. Resound-
ingly, the most common recommendation stressed the
need to increase the availability of free and low-cost
menstrual products throughout the city. To accomplish
this, multiple service providers suggested system-level
changes, such as the inclusion of menstrual products in
public assistance programs such as Supplemental Nutri-
tion Assistance Program (SNAP), Special Supplemental
Nutrition Program for Women, Infants, and Children
(WIC), or Medicare/Medicaid. As one Director of Social
Services suggested: Nor were service providers always able to predict when
donations will arrive, which could lead to stock-outs or
rationing of the products similar to what occurs within
the shelter system: Yeah, I, yeah, I’ve noticed, [stock out of a certain
type of product] doesn’t happen a lot, but it does,
you know, in any social service agency, like, it kind of
fluctuates. —KII 06 Most people who are like, street homeless, they’re
entitled to some type of medical insurance, right? I
think that Advil and um, pads or tampons, what-
ever they want to use, should be part of like, some
type of like, your health coverage. Cause it is, like, A number of key informants also described how well-
intentioned donation efforts, often the most expedient
source for products, did not always align with client pref-
erences. For example, some menstrual cup companies Gruer et al. Reprod Health (2021) 18:77 Page 7 of 11 Gruer et al. Reprod Health (2021) 18:77 health, like, we women, we, I mean, I wish I didn’t
get my period, right, but unfortunately it’s, we have
to, right? So it’s like, if they, if they have Medicaid,
then I think Medicaid should be, you know, hav-
ing like, maybe every month you should get like two
packs of like tampons or be entitled to purchase
two packs of, um, menstruation, or the menstrua-
tion cups should be something that your health
insurance covers, uh, you know? —KII 007 at familiar venues throughout the city. Another centred
on improving the current distribution systems. The
most common recommendation was that products be
made freely available, instead of requiring interaction
with a gatekeeper. Discussionhi The findings of this study uncovered multiple challenges
that people experiencing homelessness, in shelters and
on the street, face accessing menstrual products. Some
differences did emerge: those in shelters were more likely
to have a source of funds to purchase menstrual prod-
ucts or access to free products, while those living on the
streets were more likely to have to resort to panhandling,
theft, or using makeshift materials in place of menstrual
products. Across both populations, while individuals
were grateful that some service providers and shelters
provide free menstrual products, they described many
barriers to accessing these products, primarily due to a
system that allots only a few pads or tampons at a time,
insufficient for an entire menstrual period, and only
upon request. There was a palpable sense of frustration
amongst many respondents that they had to worry about
how to access such a basic necessity. It is important to note that individuals experiencing
homelessness in NYC likely have greater access to men-
strual products than most. Alone among USA cities,
NYC has a court-enforced right to shelter and, while bar-
riers remain, the NYC Menstrual Policy does provide a
channel through which the majority of individuals expe-
riencing homelessness in the city can (in theory) access
menstrual products. In most other locales, there is no
such guaranteed channel. Instead, individuals experi-
encing homelessness must find their own methods of
acquiring these essential items. While NYC has the larg-
est population of people experiencing homelessness in
the country, it only represents a fraction of the estimated
568,000 people in the USA experiencing homelessness on
a given night [31], a number that is likely on the rise due
to the economic fallout of the COVID-19 pandemic. This
issue has been long ignored due in part to the discomfort
and socially sanctioned silence around this issue, with a Put most plainly, participants experiencing homeless-
ness in NYC were not always able to access the menstrual
products that they need to manage their monthly men-
strual flow. Similar challenges have been documented
in other cities across the USA. For example, a series of
interview and focus group discussions with 20 adolescent
women experiencing homelessness in Seattle, Washing-
ton found that the women struggled to access menstrual
products, resulting in theft, or having to go without food
[14]. Creative solutions to promote access to menstrual
productsh Clients could receive a list of
what supplies and materials are available for them, and
where and how to access them. Alternatively, a list could
be publicly posted detailing the supplies that are available
and where they are located. Participants also had a range of suggestions for how
to improve the accessibility of these products within
the shelters and at other service providers. One idea
was for more service providers to stock menstrual
products, making it easier for clients to access products …letting people know, this item is available to you,
I think would be a huge help because people, again, Gruer et al. Reprod Health (2021) 18:77 Page 8 of 11 Gruer et al. Reprod Health (2021) 18:77 absorbency menstrual products may be particularly
important in that they reduce the frequency in which
an individual must find a safe, private place to change
[16]. Given the challenges accessing bathing and laundry
spaces, menstrual stains caused by leaking or inadequate
menstrual products may be harder to resolve, requir-
ing the wearing of clothes with visible menstrual stains
[18, 27]. Beyond the embarrassment and shame that this
may cause, it also impacts an individual’s ability to pass
as someone who is not experiencing homelessness, which
is critical for their ability to work, to move about the city
without harassment, or to access commercial toilets [18,
27]. While access to quality menstrual products is by no
means a solution to these problems, it is a step towards
comprehensively addressing these basic needs. Just as
critical, is ensuring consistent access to toilets and bath-
ing and laundry facilities, as explored in a previous paper
resulting from this study [27]. you’re not just gonna, if I’m always asking you for
something, like you said, and they say no, then it’s
like, it makes me feel like when I really need some-
thing, you’re definitely not gonna have it, so I feel
like, even allowing people to know that these options
are available for you if necessary, you know, if
needed…it would be very helpful, or even like, if you
go into shelter systems, and it’s your first time where
you’ve been there, they’ll be like hey, and by the way,
just in case needed, or even if they put like a little
poster or something, letting people know that these
items are available to you. Creative solutions to promote access to menstrual
productsh —IDI 09 Across all the interviews, the principal recommen-
dation was, as one service provider, who had previ-
ously experienced homelessness, succinctly put: “[what
we need is] just more access, in more spaces, with less
stigma.” —KII 4. Participants shared that even when menstrual prod-
ucts were available through their shelter or other service
providers, there were significant barriers to accessing and
using these products, such as the necessity of disclos-
ing one’s menstruating status to gatekeepers, the lack of
availability of preferred type of menstrual product, and
insufficient quality and quantity of products. Similar
difficulties have been reported elsewhere. For example,
during interviews with 40 women experiencing home-
lessness in Bristol, England, the women described chal-
lenges accessing menstrual products, lack of awareness
about which service providers might supply them, and
embarrassment around having to ask staff for products
[19]. This suggests a gendered discrimination within shel-
ter systems; an occurrence possibly rooted in an internal-
ized misogyny that limits recognition of women’s basic
needs [30]. Discussionhi In Albany, NY, interviews with five women experi-
encing homelessness revealed difficulty obtaining men-
strual products from service providers and food pantries
as they were frequently out of stock [15]. These popula-
tions also face concurrent challenges accessing private,
safe spaces for changing menstrual products, and for
bathing and laundering [16, 19, 27]. Thus, quality, high Gruer et al. Reprod Health (2021) 18:77 Gruer et al. Reprod Health (2021) 18:77 Page 9 of 11 Page 9 of 11 Overwhelmingly, the largest barrier to the sugges-
tions proposed is lack of funding; however, compared to
many city-funded initiatives, these costs would be mini-
mal. We fund what we care about, and to date, address-
ing the menstrual needs of vulnerable populations has
been a low priority. Efforts such as the NYC Menstrual
Policy show positive progress, but have not yet achieved
their goal of ensuring menstrual product access. At the
national level, there has also been some progress. A
growing number of cities and states have passed policies
mandating the distribution of free pads in public schools,
homeless shelters and correctional facilities. Additionally,
during the COVID-19 pandemic, the USA government
classified pads and tampons for the first time as medical
expenses that are eligible for flexible spending accounts
(FSA) and health saving account (HSA) reimbursement
in the Coronavirus Aid, Relief and Economic Securities
(CARES) Act (35). While this will only support certain
segments of the USA population, it is an important rec-
ognition that menstrual products are essential items. pervasive assumption of personal responsibility for men-
struation regardless of personal circumstance. Simply
recognizing the current situation as an untenable state of
affairs is a categorical break with the past. pervasive assumption of personal responsibility for men-
struation regardless of personal circumstance. Simply
recognizing the current situation as an untenable state of
affairs is a categorical break with the past. f
g
p
Our findings from NYC support the benefit of the NYC
Menstrual Policy as they reinforce that many people
experiencing homelessness struggle to access menstrual
products. However, the findings also highlight ways in
which the current system could better meet the needs of
this population. As recommended by the study partici-
pants, menstrual products should be provided through
an open distribution system by removing (or reducing)
the requirement that clients interact with a gatekeeper to
access these products. Limitationsh There are some limitations important to note. First, the
study sample was small, and although it included partici-
pants with experience living on the street and in shelters,
it was predominantly those living in shelters. Similarly,
while our study was open to all individuals with con-
current experiences of being homeless and menstruat-
ing, all of our study participants were cisgender women. It is likely that individuals who menstruate and do not
identify or present as female face increased challenges
to accessing menstrual products, as was also noted by a
number of key informants. A larger sample would have
likely captured a broader range of experiences, challenges
and successes around accessing menstrual products
while experiencing homelessness. Second, our sample
was limited to individuals who were capable of providing
informed consent. As a result, the study excluded indi-
viduals struggling with mental health conditions, who
represent a sizeable portion of the homeless population,
and likely face even more significant barriers to manag-
ing their menstruation. Policies could be considered to promote menstrual
product access for all populations across the city, such as
the wide-scale distribution of menstrual products mod-
elled on the NYC condom campaign suggested by our
participants, or the recent Scottish legislation mandat-
ing free menstrual products in all public spaces and for
those in need [32]. There are a few examples of these
types of broader initiatives appearing around the USA
[33]; however, to date, the only ones that have passed and
been implemented have been small-scale pilots in small,
wealthy localities, where the demand for these products
is likely quite low [34]. All policies or programs that seek to address the
menstrual challenges faced by individuals experienc-
ing homelessness must consider the way that menstrual
stigma may impact the delivery and uptake of services. As mentioned above, systems that require little to no
interaction with a gatekeeper are preferable. Service pro-
viders should consider the potential gendered inequity
inherent in the notion that these resources require a gate-
keeper, especially if other necessities are freely available. Additionally, all shelter and service provider staff might
benefit from sensitization training so that they can better
support their clients and their menstrual needs. Discussionhi There is anecdotal evidence that
open distribution systems are viable as they are already in
use in some shelters and service providers in NYC. Other
small improvements could be made such as increasing
the number of menstrual products provided at one time;
providing a range of types of products (e.g. pads, tam-
pons) and absorbencies; and ensuring the products are
of adequate quality. In addition, expanding the mandate
and associated resources to a broader array of providers
of homeless services could make it easier for individuals
living on the street to access these products. Currently,
it is easier for an individual experiencing homelessness
to obtain soap, a toothbrush, or toothpaste, than it is to
access menstrual products; however, menstrual products
are just as essential. Received: 13 January 2021 Accepted: 2 April 2021 needed to better understand the menstrual experiences
and needs of a broader range of individuals experienc-
ing homelessness, our findings suggest a few actionable
items that could improve menstrual product access in
NYC. These include increasing free product distribu-
tion in locations more widely accessible to individuals
living on the street; training shelter and service pro-
vider staff on menstruation to improve their comfort
around providing these services; and rethinking distri-
bution strategies in shelters and at other service provid-
ers. This study also underscores the critical importance
of expanding efforts that seek to provide access to these
essential items for vulnerable populations across the
country. needed to better understand the menstrual experiences
and needs of a broader range of individuals experienc-
ing homelessness, our findings suggest a few actionable
items that could improve menstrual product access in
NYC. These include increasing free product distribu-
tion in locations more widely accessible to individuals
living on the street; training shelter and service pro-
vider staff on menstruation to improve their comfort
around providing these services; and rethinking distri-
bution strategies in shelters and at other service provid-
ers. This study also underscores the critical importance
of expanding efforts that seek to provide access to these
essential items for vulnerable populations across the
country. Competing interests p
g
The authors declare that they have no competing interests. 18. Mitchell MK, Ramsey LR, Nelson S. The body image of women at a home-
less service group. Gend Issues. 2018;35(1):38–51. Declarations 13. Parrillo A, Feller E. Menstrual hygiene plight of homeless women, a public
health disgrace. R I Med J. 2017;(December):14–5. Available from: http://
www.rimed.org/rimedicaljournal/2017/12/2017-12-14-pov-parrillo.pdf. Abbreviations CARES: Coronavirus Aid, Relief and Economic Securities; DCAS: Department of
Citywide Administrative Services; FSA: Flexible Spending Account; HSA: Health
Spending Account; HUD: Department of Housing and Urban Development;
IDI: In-depth interview; IRB: Institutional Review Board; KII: Key informant inter-
view; NYC: New York City; SNAP: Supplemental Nutrition Assistance Program;
USA: United States of America; WIC: Special Supplemental Nutrition Program
for Women, Infants, and Children. 5. Mason L, Nyothach E, Alexander K, Odhiambo FO, Eleveld A, Vulule J,
et al. “We keep it secret so no one should know”—a qualitative study to
explore young schoolgirls attitudes and experiences with menstruation
in rural Western Kenya. PLoS ONE. 2013;8:e79132. 5. Mason L, Nyothach E, Alexander K, Odhiambo FO, Eleveld A, Vulule J,
et al. “We keep it secret so no one should know”—a qualitative study to
explore young schoolgirls attitudes and experiences with menstruation
in rural Western Kenya. PLoS ONE. 2013;8:e79132. y
6. Hennegan J, Shannon AK, Rubli J, Schwab KJ, Melendez-Torres GJ. Wom-
en’s and girls’ experiences of menstruation in low- and middle-income
countries: a systematic review and qualitative metasynthesis. PLOS Med. 2019;16:1002803. Availability of data and materials The dataset used (all transcripts) and analysed during the current study are
available from the corresponding author on reasonable request. 12. McNamara B. How periods perpetuate homelessness. Teen. 2017. https://
www.teenvogue.com/story/homelessness-and-periods. 12. McNamara B. How periods perpetuate homelessness. Teen. 2017. https://
www.teenvogue.com/story/homelessness-and-periods. Funding g
This study was funded by the Sid and Helaine MHM Faculty Support Fund and
the Osprey Foundation. This study was funded by the Sid and Helaine MHM Faculty Support Fund and
the Osprey Foundation. 11. Herbert AC, Ramirez AM, Lee G, North SJ, Askari MS, West RL, et al. Puberty experiences of low-income girls in the United States: a system-
atic review of qualitative literature from 2000 to 2014. J Adolesc Heal. 2016;60(4):363–79. https://doi.org/10.1016/j.jadohealth.2016.10.008. Acknowledgements 7. Sommer M, Caruso BA, Sahin M, Calderon T, Cavill S. A time for global
action : addressing girls ’ menstrual hygiene management needs in
schools. PLoS Med. 2016. https://doi.org/10.1371/journal.pmed.1001962. We would like to thank the Coalition for the Homeless, the Door, the Midtown
Outreach Project (CUCS) and all of the service providers in NYC and individuals
experiencing homelessness who so generously provided their insights. 8. Scandurra L, Khorn D, Charles T-A, Sommer M. Cambodian boys’ transi-
tions into young adulthood: exploring the influence of societal and
masculinity norms on young men’s health. Cult Heal Sex. 2017;19(7):767. 8. Scandurra L, Khorn D, Charles T-A, Sommer M. Cambodian boys’ transi-
tions into young adulthood: exploring the influence of societal and
masculinity norms on young men’s health. Cult Heal Sex. 2017;19(7):767. Consent for publication 16. Kuhlmann SA, Bergguist PE, Danjoint D, Wall L. Unmet menstrual hygiene
needs among low-income women. Obstet Gynecol. 2019;133(2):238–44. Not applicable. Not applicable. 17. Durfor SL. Personal hygiene self-management of chronically unsheltered
homeless women. University of Wisconsin Milwaukee; 2015. Authors’ contributions Conceived and designed the study: MS, KH, AM, CG. Conducted interviews:
CG, MS, RS. Performed the analyses: CG and EK. Drafted the manuscript: CG
and MS. All authors edited, read and approved the final manuscript. 9. Schmitt M, Clatworthy D, Ogello T, Sommer M, Schmitt ML, Clatworthy D,
et al. Making the case for a female-friendly toilet. Water. 2018;10(9):1193. 9. Schmitt M, Clatworthy D, Ogello T, Sommer M, Schmitt ML, Clatworthy D,
et al. Making the case for a female-friendly toilet. Water. 2018;10(9):1193. 10. BRAWS, University of D.C. David A. Clarke School of Law Legislation Clinic. Periods, poverty, and the need for policy: a report on menstrual inequity
in the United States. Washington DC; 2018. Available from: https://cdn.
ymaws.com/www.law.udc.edu/resource/resmgr/LegClinic/2018_perio
ds_policy_report.pdf. References 1. VanLeeuwen C, Torondel B. Improving menstrual hygiene management
in emergency contexts: literature review of current perspectives. Int J
Womens Health. 2018;10:169–86. 1. VanLeeuwen C, Torondel B. Improving menstrual hygiene management
in emergency contexts: literature review of current perspectives. Int J
Womens Health. 2018;10:169–86. 2. Phillips-Howard PA, Caruso B, Torondel B, Zulaika G, Sahin M, Sommer M. Menstrual hygiene management among adolescent schoolgirls in low-
and middle-income countries: research priorities. Glob Health Action. 2016;9(1):1–7. 2. Phillips-Howard PA, Caruso B, Torondel B, Zulaika G, Sahin M, Sommer M. Menstrual hygiene management among adolescent schoolgirls in low-
and middle-income countries: research priorities. Glob Health Action. 2016;9(1):1–7. 3. MacRae ER, Clasen T, Dasmohapatra M, Caruso BA. “It’s like a burden
on the head”: Redefining adequate menstrual hygiene management
throughout women’s varied life stages in Odisha, India. Federici S, editor. PLoS One. 2019;14(8):e0220114. 3. MacRae ER, Clasen T, Dasmohapatra M, Caruso BA. “It’s like a burden
on the head”: Redefining adequate menstrual hygiene management
throughout women’s varied life stages in Odisha, India. Federici S, editor. PLoS One. 2019;14(8):e0220114. 4. McMahon SA, Winch PJ, Caruso BA, Ogutu EA, Ochari IA, Rheingans
RD. “The girl with her period is the one to hang her head” Reflections
on menstrual management among schoolgirls in rural Kenya. BMC Int
Health Hum Rights. 2011;11(1):7. http://www.biomedcentral.com/1472-
698X/11/7. Conclusion Overall, this study illustrated how individuals experi-
encing homelessness in NYC, whether living in shelters
or on the street, are often not able to access the men-
strual products that they need to manage their monthly
menstrual flow. While the 2016 NYC Menstrual Pol-
icy represented an important step towards ensuring
this population can access menstrual products, as yet
it does not assure consistent and adequate access to
these basic necessities. Although additional research is Gruer et al. Reprod Health (2021) 18:77 Gruer et al. Reprod Health (2021) 18:77 Page 10 of 11 Page 10 of 11 Received: 13 January 2021 Accepted: 2 April 2021 Received: 13 January 2021 Accepted: 2 April 2021 Ethics approval and consent to participate Ethical review and approval was obtained from the Columbia University
Institutional Review Board to allow human participant research. To participate
in the qualitative interview participants provided verbal consent to participate
and be recorded. No respondents declined participation. Participants were
informed that they could decline to answer any question. 14. Ensign J. Reproductive health of homeless adolescent women in Seattle,
Washington, USA Reproductive Health of Homeless Adolescent Women
in Seattle, Washington, USA. Women Health. 2008;0242. in Seattle, Washington, USA. Women Health. 2008;0242. 15. Levit R. Waiting and menstruation: a look at homeless and at-risk
women’s experiences. University of Albany, State University of New York;
2017. Author details
1 19. Vora S. The realities of period poverty: how homelessness shapes
women’s lived experiences of menstruation. In: Bobel C, Winkler IT, Fahs
B, Hasson KA, Kissling EA, Roberts T-A, editors. The Palgrave handbook
of critical menstruation studies. Singapore: Springer Singapore; 2020. p. 31–47. https://doi.org/10.1007/978-981-15-0614-7_4. 1 Department of Sociomedical Sciences, Mailman School of Public Health,
Columbia University, 722 W. 168th Street, Room 537, New York, NY 10032,
USA. 2 Department of Environmental, Occupational, and Geospatial Health
Sciences, CUNY School of Public Health, 55 W. 125th Street, Room 508, New
York, NY 10027, USA. Gruer et al. Reprod Health (2021) 18:77 Page 11 of 11 Gruer et al. Reprod Health (2021) 18:77 20. Wenzel SL, Andersen RM, Gifford DS, Gelberg L. Homeless women ’ s
gynecological symptoms and use of medical care. J Health Care Poor
Underserved. 2019;12(3):323–41. 30. Manne K. Down girl: the logic of misogyny. Oxford: Oxford University
Press; 2017. 31. Henry M, Watt R, Mahathey A, Ouellette J, Sitler A. The 2019 annual home-
less assessment report (AHAR) to Congress. 2020. 21. Routhier G. State of the Homeless 2019. New York City; 2019. https://
www.coalitionforthehomeless.org/wp-content/uploads/2019/04/State
OfThe-Homeless2019.pdf. Accessed 17 Dec 2020. 32. Specia M. Scotland is 1st nation to make period products free. The New
York Times. 2020. https://www.nytimes.com/2020/11/24/world/europe/
scotland-free-period-products.html. Accessed 18 Dec 2020. 2. NYC Department of Homeless Services. NYC Hope 2019 Results. 23. Hopper K, Shinn M, Laska E, Meisner M, Wanderling J. Estimating numbers
of unsheltered homeless people through plant-capture and postcount
survey methods. Am J Public Health. 2008;98(8):1438–42. https://doi.org/
10.2105/AJPH.2005.083600. 33. Barajas J. “Menstrual equity”: L.A. may offer free tampons and pads in
public bathrooms - Los Angeles Times. Los Angeles Times. 2019. https://
www.latimes.com/california/story/2019-12-28/la-moves-to-provide-free-
tampons-and-pads-in-public-restrooms. Accessed 21 Aug 2020. 24. Romero AP, Goldberg SK, Vasquez LA. LGBT people and housing afford-
ability, and homelessness. 2020. https://williamsinstitute.law.ucla.edu/
wp-content/uploads/LGBT-Housing-Apr-2020.pdf. 34. Berkowitz K. A new initiative is trying to make searching for tampons
easier for Highland Park women. It’s part of a national movement. Chicago Tribune. 2019. https://www.chicagotribune.com/suburbs/highl
and-park/ct-hpn-free-tampons-feminine-hygiene-tl-0919-20190912-24sfi
he3pngsjeit6pg4qdbptu-story.html. Accessed 21 Aug 2020. 25. Johnston-Robledo I, Chrisler JC. The menstrual mark: menstruation as
social stigma. Sex Roles. 2013;68:9–18. 26. Goffman E. Stigma: notes on the management of spoiled identity. New
York: Simon & Schuster; 1963. 35. Simmons-Duffin S. Coronavirus Legislation Allows For OTC Drugs : Coro-
navirus Updates : NPR. NPR. 2020. https://www.npr.org/sections/coron
avirus-live-updates/2020/04/01/825490269/save-those-pharmacy-recei
pts-new-payment-flexibility-courtesy-of-covid-rescue-ac. Accessed 20
Nov 2020. 27. Author details
1 Sommer M, Gruer C, Smith RC, Maroko A, Hopper K. Menstruation and
homelessness: challenges faced living in shelters and on the street in
New York City. Health Place. 2020;66:1353–8292. http://creativecommons.
org/licenses/by/4.0/ 28. The New York City Council. File #: Int 1123-2016. 2016. https://legistar.
council.nyc.gov/LegislationDetail.aspx?ID=2637112&GUID=31322AF8-
376A-4D7F-93B2-4243BA5E4181. Accessed 20 Nov 2020 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. 29. Malterud K. Systematic text condensation: a strategy for qualitative analy-
sis. Scand J Public Health. 2012;40(8):795–805. •
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http://oro.open.ac.uk/84128/1/84128.pdf
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English
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Rapid Eocene diversification of spiny plants in subtropical woodlands of central Tibet
|
Nature communications
| 2,022
|
cc-by
| 11,394
|
Open Research Online Policy This document has been downloaded from Open Research Online, The Open University's
repository of research publications. This version is being made available in accordance
with Open Research Online policies available from Open Research Online (ORO) Policies Citation Zhang, Xinwen; Gélin, Uriel; Spicer, Robert A; Wu, Feixiang; Farnsworth, Alexander; Chen,
Peirong; Del Rio, Cédric; Li, Shufeng; Liu, Jia; Huang, Jian; Spicer, Teresa E V; Tomlinson,
Kyle W; Valdes, Paul J; Xu, Xiaoting; Zhang, Shitao; Deng, Tao; Zhou, Zhekun and Su,
Tao (2022). Rapid Eocene diversification of spiny plants in subtropical woodlands of
central Tibet. Nature communications, 13(1), article no. 3787. https://oro.open.ac.uk/84128/ https://creativecommons.org/licenses/by/4.0/ Rapid Eocene diversification of spiny plants in
subtropical woodlands of central Tibet Xinwen Zhang
1,2,12, Uriel Gélin3,12, Robert A. Spicer
1,4, Feixiang Wu5,6, Alexander Farnsworth
7,8,
Peirong Chen1,2, Cédric Del Rio
1,9, Shufeng Li1,2,10, Jia Liu1,10, Jian Huang1,10, Teresa E. V. Spicer1,
Kyle W. Tomlinson
3, Paul J. Valdes
7, Xiaoting Xu1,2, Shitao Zhang11, Tao Deng
5,6, Zhekun Zhou
1,10 &
Tao Su
1,2,10✉ Spinescence is an important functional trait possessed by many plant species for physical
defence against mammalian herbivores. The development of spinescence must have been
closely associated with both biotic and abiotic factors in the geological past, but knowledge of
spinescence evolution suffers from a dearth of fossil records, with most studies focusing on
spatial patterns and spinescence-herbivore interactions in modern ecosystems. Numerous
well-preserved Eocene (~39 Ma) plant fossils exhibiting seven different spine morphologies
discovered recently in the central Tibetan Plateau, combined with molecular phylogenetic
character reconstruction, point not only to the presence of a diversity of spiny plants in
Eocene central Tibet but a rapid diversification of spiny plants in Eurasia around that time. These spiny plants occupied an open woodland landscape, indicated by numerous mega-
fossils and grass phytoliths found in the same deposits, as well as numerical climate and
vegetation modelling. Our study shows that regional aridification and expansion of herbi-
vorous mammals may have driven the diversification of functional spinescence in central
Tibetan woodlands, ~24 million years earlier than similar transformations in Africa. 1 CAS Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, China. 2 University of
Chinese Academy of Sciences, Beijing, China. 3 Center for Integrative Conservation, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences,
Mengla, China. 4 School of Environment, Earth and Ecosystem Sciences, The Open University, Milton Keynes, UK. 5 CAS Key Laboratory of Vertebrate
Evolution and Human Origins, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, Beijing, China. 6 CAS Center for
Excellence in Life and Paleoenvironment, Chinese Academy of Sciences, Beijing, China. 7 School of Geographical Sciences and Cabot Institute, University of
Bristol, Bristol, UK. 8 State Key Laboratory of Tibetan Plateau Earth System, Environment and Resources, Institute of Tibetan Plateau Research, Chinese
Academy of Sciences, Beijing, China. 9 CR2P - Centre de Recherche en Paléontologie – Paris, MNHN - Sorbonne Université - CNRS, Paris, France. 10 Center of
Plant Ecology, Core Botanical Gardens, Chinese Academy of Sciences, Mengla, China. 1 CAS Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, China. 2 University of
Chinese Academy of Sciences, Beijing, China. 3 Center for Integrative Conservation, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences,
Mengla, China. 4 School of Environment, Earth and Ecosystem Sciences, The Open University, Milton Keynes, UK. 5 CAS Key Laboratory of Vertebrate
Evolution and Human Origins, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, Beijing, China. 6 CAS Center for
Excellence in Life and Paleoenvironment, Chinese Academy of Sciences, Beijing, China. 7 School of Geographical Sciences and Cabot Institute, University of
Bristol, Bristol, UK. 8 State Key Laboratory of Tibetan Plateau Earth System, Environment and Resources, Institute of Tibetan Plateau Research, Chinese
Academy of Sciences, Beijing, China. 9 CR2P - Centre de Recherche en Paléontologie – Paris, MNHN - Sorbonne Université - CNRS, Paris, France. 10 Center of
Plant Ecology, Core Botanical Gardens, Chinese Academy of Sciences, Mengla, China. 11 Faculty of Land Resource Engineering, Kunming University of Science
and Technology, Kunming, China. 12These authors contributed equally: Xinwen Zhang, Uriel Gélin. ✉email: sutao@xtbg.org.cn Versions If this document is identified as the Author Accepted Manuscript it is the version after peer
review but before type setting, copy editing or publisher branding ARTICLE TURE COMMUNICATIONS | (2022) 13:3787 | https://doi.org/10.1038/s41467-022-31512-z | www.nature.com/naturecommunications Results
l Geological age. We document a total of 44 spine-bearing fossil
specimens collected from two fossil localities (Fig. 1, Supple-
mentary Fig. 1): the Dayu locality (32° 20′ N, 89° 46′ E), within
the middle member of the Niubao Formation, is considered to be
~39 Ma (Bartonian, early late Eocene) based on radiometric
(U/Pb) dating35,36, while the other site at Xiede (31° 58′ N, 88°
25′ E) we consider age-equivalent as it contains a wide range of
similar fossils in addition to spine-bearing plants. Both localities
are within the central Tibetan Bangong-Nujiang Suture Zone. Spinescence (a general term for the phenomena of spines,
prickles, and thorns on plants) is an important functional trait
shared by numerous plant families worldwide and mainly pro-
vides physical protection against vertebrate herbivores11,12. Sharp
spines can hurt the bodies and mouthparts of herbivores, and
thus restrict their feeding rates13. In general, spine architecture
and spatial distribution are largely influenced by mammal her-
bivory and correlate strongly with the spatial distribution of
megaherbivores13–16. Both prickles and thorns can defend against
herbivores and even climbing mammals17, but whether they have
other functional properties is unclear. Today, spinescence is
common in open habitats with abundant herbivores and rela-
tively arid climates, such as savanna, featuring an open tree
canopy with a grassy understory18–20. Spine morphology. Spine morphology is here divided into
prickles (modified epidermis) and thorns (modified axial stems). According to size and growth pattern, we classify prickles into
two types and thorns into five types. Further details are given in
Supplementary Figs. 2–8, Supplementary Note 1. Prickles. Morphotype I (Fig. 2b) Prickles arranged on stems
alternately. The average length of prickles is 4.9 ± 1.6 mm. The
mean width at the base of each prickle is 5.8 ± 1.2 mm. Each
prickle curves upward at an angle between 45° and 90°. Morphotype II (Fig. 2e) Prickles occur on stems irregularly. The average length of prickles is 3.8 ± 1.2 mm. The mean width at
base of each prickle is 5.2 ± 1.5 mm. The prickles grow almost
perpendicular to the stem. To better understand mechanisms underlying the evolution of
plant spines, it is necessary to explore their occurrence in deep
time. Spiny plants in Africa, evidenced by both plant and mam-
mal phylogenies, underwent a massive radiation during the early
Miocene, particularly in African savannas18, which are char-
acterised as open-canopied ecosystems. Results
l Spiny species have been
linked to the arrival and diversification of bovids from Eurasia
during the Neogene, as well as climatic drying in Africa that
promoted the development of open vegetation18,21. Even though
spiny plants are distributed worldwide, our understanding of
their evolutionary history remains woefully incomplete. Thorns. Morphotype III (Fig. 2h) Thorns grow on stems oppo-
sitely and densely. The average length of thorns is 5.6 ± 2.1 mm. They grow on the stem at an angle of ~90°. Morphotype IV (Fig. 2i) Thorns grow on stems alternately. The
average length of thorns is 4.7 ± 1.7 mm. Each thorn curves
slightly upwards or downwards. g
y
p
Morphotype V (Fig. 2d) Thorns grow on stems alternately. The
average length of thorns is 29.0 ± 14.7 mm. They grow on the
stem at an angle of ~90°. Morphotype VI (Fig. 2j) Thorns grow on stems alternately and
at an acute angle of less than 45°. The average length of thorns is
5.9 ± 1.4 mm. Fossils of plant spines are physical evidence for the presence of
plant spinescence at the place and time the plant was alive;
nevertheless, they have been largely ignored and scarcely reported
compared to other plant organs, such as leaves, fruits, and seeds. Previously, spiny fossils have been largely overlooked in fossil
floras, with a few exceptions such as the Eocene Green River
flora22 and Oligocene Bridge Creek flora23,24 of North America,
and the Miocene Tortonian flora25 of Europe. Overall, their
occurrence in fossil floras is not well documented and the eco-
logical and evolutionary significance of these scattered spiny
fossils have never been explored at continental scale. Morphotype VII (Fig. 2k) Thorns grow on stems in pairs
oppositely. The average length of thorns is 2.6 ± 0.6 mm. They
grow on the stem at an angle of ~90°. Phylogeny of spiny plants. The accumulation curve of spiny
eudicots (Fig. 3a) suggests that the first spiny plant species
emerged during the Paleogene in Eurasia. This strengthens our
findings pointing to an early occurrence of spiny plants in Asia,
although fossil spines are generally under-explored over this
period. The proportion of spiny plants remained low until 40 Ma,
but has risen exponentially since then (Fig. 3a). Rapid Eocene diversification of spiny plants in
subtropical woodlands of central Tibet 11 Faculty of Land Resource Engineering, Kunming University of Science
and Technology, Kunming, China. 12These authors contributed equally: Xinwen Zhang, Uriel Gélin. ✉email: sutao@xtbg.org.cn 1 NATURE COMMUNICATIONS | (2022) 13:3787 | https://doi.org/10.1038/s41467-022-31512-z | www.nature.com/naturecommunications TURE COMMUNICATIONS | (2022) 13:3787 | https://doi.org/10.1038 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z P
l
p
h P
lant functional traits refer to plant characters that impact
plant survival, growth, or reproduction1,2, which in turn
have significant influences on ecosystem processes3,4. Functional traits enable plants to better acquire and retain
resources, promote niche differentiation, and reduce interspecific
competition, while functional trait redundancy maintains the
stability of ecosystem function5–7. Although most studies focus
on
the
role
of
functional
traits
in
structuring
modern
ecosystems8–10, little is known about how these functional traits
evolved in the geological past, particularly under varied envir-
onmental stressors. Plant functional traits are shaped by the
interaction between plants and the surrounding environment,
including faunal interactions, during the long process of evolution
in deep time1; therefore, it is crucial to investigate the evolu-
tionary history of plant functional traits to better understand the
mechanisms moulding them. early diversification history of spiny species in Eurasia. Using
proxy and modelling data, we reconstruct the vegetation, climate
and herbivory that favoured spiny plant evolution in the Eocene
of central Tibet. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z More details are given in Supplementary Tables 1–3. Palaeoclimate and palaeoelevation. We applied the Climate-Leaf
Analysis Multivariate Program (CLAMP) to the Dayu leaf flora
(Supplementary Figures 9, 10), which indicates a climate with
summers that were warm (warm month mean temperatures of
24–30 °C) and dry (summer vapour pressure deficit (VPD.sum)
of 10.2–17.2 hPa), and winters that were cool (cold month mean
0.2–7.2 °C) and moist (VPD.win 2.8–5.8 hPa). Occasional frosts
may have occurred but were not harsh, nor prolonged, and thus
survivable by palms that also occur in the Dayu section. The dry
bulb mean annual temperature of 15.6 °C exceeds, but is close to,
the lower survivable limit for palms (14.2 °C)37. The wet/dry
precipitation ratio is 4.6:1, suggesting a borderline monsoonal
climate. More details are given in Supplementary Tables 1–3. reconstructed from proxies, and CLAMP is known to return dry
bulb temperatures that reflect evapotranspirational cooling in dry
regimes where groundwater is plentiful39. Overall, the modelling
and proxy thermal regimes are similar, pointing out to drying and
cooling climate in central Tibet by the mid-late Eocene
accompanied
by
some
within-valley
increase
in
elevation
(~1 km) since ~47 Ma33. Herbaceous plant diversity. Herbaceous fossil specimens of the
Dayu flora (n = 315), classified as monocots based on their par-
allel veins and gross morphology, account for ~38% of all plant
fossil specimens found at Dayu. Using leaf size, length, and stem
growth form, we were able to classify herbaceous fossils into six
morphotypes (Supplementary Fig. 11). Currently, we cannot
determine if they were terrestrial or aquatic plants due to limited
preservation. However, there are abundant herbaceous phytoliths
in the bounding sediments, representing several mainly non-
aquatic grass subfamilies, including Chloridoideae and Pooideae
(Supplementary Figure 12). The diverse morphology of phytoliths
observed from the fossil-bearing outcrops (Supplementary Fig-
ure 12) indicates a species-rich grass community within an eco-
system that also contained palms and numerous other woody
taxa, but evidence for such a diverse grass component does not
occur in older Tibetan floras, e.g., the middle Eocene Jianglang
flora from central Tibet33 (Supplementary Fig. 13). Both the moist enthalpy and the wet bulb terrestrial lapse rate
approaches for palaeoaltimetry38 gave similar results for the
elevation of the Dayu fossil assemblage: 2.6 ± 1.2 km and
2.7 ± 0.9 km respectively (Supplementary Note 2). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z DY2
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XD
Tibetan Plateau
Mt. Everest
0
500 km
N
DY2
DY1
XDA1
XDB3
Fig. 1 The location of two fossil sites from the early late Eocene of central Tibetan Plateau, China. a Map showing the fossil sites (red stars) from the
middle member of the Niubao Formation, central Tibetan Plateau. The base map was downloaded from Natural Earth (https://www.naturalearthdata.com/). XD, the site in Xiede village; DY, the site in Dayu village. b, c Outcrop drone images of fossil sites near Dayu51 and Xiede villages. d The stratigraphy of
Dayu51 and Xiede sections. The colour of each layer reflects the colour of the rock. 75°E
80°E
20°N
25°N
30°N
35°N
40°N
85°E
90°E
95°E
100°E
105°E
45°N
a
Lhasa
DY
XD
Tibetan Plateau
Mt. Everest
0
500 km
N 40°N 25°N c
XDA1
XDB3 DY2
DY1
b c Fig. 1 The location of two fossil sites from the early late Eocene of central Tibetan Plateau, China. a Map showing the fossil sites (red stars) from the
middle member of the Niubao Formation, central Tibetan Plateau. The base map was downloaded from Natural Earth (https://www.naturalearthdata.com/). XD, the site in Xiede village; DY, the site in Dayu village. b, c Outcrop drone images of fossil sites near Dayu51 and Xiede villages. d The stratigraphy of
Dayu51 and Xiede sections. The colour of each layer reflects the colour of the rock. Palaeoclimate and palaeoelevation. We applied the Climate-Leaf
Analysis Multivariate Program (CLAMP) to the Dayu leaf flora
(Supplementary Figures 9, 10), which indicates a climate with
summers that were warm (warm month mean temperatures of
24–30 °C) and dry (summer vapour pressure deficit (VPD.sum)
of 10.2–17.2 hPa), and winters that were cool (cold month mean
0.2–7.2 °C) and moist (VPD.win 2.8–5.8 hPa). Occasional frosts
may have occurred but were not harsh, nor prolonged, and thus
survivable by palms that also occur in the Dayu section. The dry
bulb mean annual temperature of 15.6 °C exceeds, but is close to,
the lower survivable limit for palms (14.2 °C)37. The wet/dry
precipitation ratio is 4.6:1, suggesting a borderline monsoonal
climate. Results
l For approxi-
mately 20 Myr after their emergence we estimate that spines
occurred in only 2 lineages, but during the late Eocene there was a
four-fold increase from 2 to 8 spiny lineages within the next
10 Myr, closely matching the 7 morphotypes found in our fossil
records. The beginning of this substantial radiation of spiny
plants, estimated according to molecular clock techniques, closely
matches the timing of our fossil spines, reinforcing the hypo-
thesised early emergence of spiny plant diversity in Asia. The
mid-late Eocene, therefore, witnessed a step change in the abiotic
and/or biotic environment of the woody flowering plants in
Eurasia. p
Here we report exceptionally rich assemblages of spiny plant
fossils collected from late Eocene (~39 Ma) sediments in central
Tibet (Fig. 1). The Tibetan region has undergone dramatic evo-
lutionary and climatic change since the collision of the Indian and
Eurasian plates began between 65–55 Ma26–30. Habitat differ-
entiation favoured the diversification and turnover of plant and
mammal species31,32 as central Tibet changed from hosting a
closed subtropical humid lowland forest valley ecosystem during
the middle Eocene33 to the open and dry highland steppe of
today34. These spiny fossils together with phytoliths and plant
megafossils, including those of monocotyledonous herbs and
dicotyledonous woody species, point to an initial opening-up of
the vegetation as early as the late Eocene in central Tibet. Com-
bined with molecular phylogenetic analyses, we document the TURE COMMUNICATIONS | (2022) 13:3787 | https://doi.org/10.1038/s41467-022-31512-z | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | (2022) 13:3787 | https://doi.org/10.1038/s41467-022-31512-z | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z As grass tends not to grow in abundance in shaded
conditions, all these megafossils and phytoliths strongly suggest
the progressive development of a semi-open habitat dominated by
herbaceous plants with trees forming a broken canopy, such as is
seen in modern woodlands40,41. This interpretation is further
supported by experiments using fully coupled ocean-atmosphere-
vegetation climate modelling with Eocene boundary conditions
and a Tibetan Central Valley topography (Supplementary Note 3,
Supplementary Fig. 14). These simulations show early Eocene and diversity. The phytolith assemblage from the ~47 Ma
Jianglang section, where the megafossils are interpreted to
represent a subtropical forest, is dominated by those forms
produced by woody taxa, and these greatly exceed grass forms
(Supplementary Fig. 13, Supplementary Table 4). Within the
lower part of the Dayu section (~39 Ma), phytoliths are still
dominated by those produced by woody plants but bulliform
phytoliths typically produced by grasses become more common
(Supplementary Figure 13). The main fossil-bearing layer higher
in the Dayu section preserves not only the numerous spiny taxa
but also abundant phytoliths where, among those that could be
identified, 66% were produced by grasses and only 34% by woody
plants. As grass tends not to grow in abundance in shaded
conditions, all these megafossils and phytoliths strongly suggest
the progressive development of a semi-open habitat dominated by
herbaceous plants with trees forming a broken canopy, such as is
seen in modern woodlands40,41. This interpretation is further
supported by experiments using fully coupled ocean-atmosphere-
vegetation climate modelling with Eocene boundary conditions
and a Tibetan Central Valley topography (Supplementary Note 3,
Supplementary Fig. 14). These simulations show early Eocene Herbivorous mammal fossils of the Tibetan Plateau. Available
fossil records indicate that the regional diversity of large herbi-
vorous mammals increased substantially from the Paleogene to
the Neogene (Fig. 3a). Large herbivores were abundant in central
Asia after the early Eocene. There are numerous fossil records of
Paleogene herbivorous mammals from the Tibetan Plateau and its
surrounding regions, mainly represented by Brontotheriidae,
Hyracodontidae,
Amynodontidae,
Paraceratheriidae,
and
Anthracotheriidae (Supplementary Data 1), and recently a rhino
(Plesiaceratherium sp., Rhinocerotidae) was found from the early
Miocene strata of the central Tibetan Plateau42. At the broader
scale of central Asian countries, we observed a first peak of
herbivore diversification in the first half of the Eocene (~50 Ma,
Fig. 3a), followed by a second peak between the late Eocene to
early Oligocene (~40–28 Ma; Fig. 3a). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z Numerical climate modelling for central Tibet, with a valley
floor set at 2.5 km bounded by 5 km high East-West trending
mountain ranges, also indicates a winter-wet warm climate within
the valley (a mean annual air surface temperature 23–26 °C, and a
cold month mean surface air temperature of 9–11 °C depending
on location) with progressively more arid conditions towards the
east
where
some
minor
summer
rain
also
is
predicted
(Supplementary Note 3). Note that these model temperatures
are slightly warmer than those reconstructed from CLAMP, but
the nominal model valley floor elevation is lower than that Progression from the older flora to the younger in the Bangor
and Lunpola basins shows an increase in phytolith abundance 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z Fig. 2 Morphotypes of spiny fossils from the upper Eocene Dayu and Xiede sections, central Tibetan Plateau. a Prickles of living species Rubus
alceifolius. b, d, e, h–k, Seven morphotypes of spiny fossils. c Enlargement of b. f Enlargement of h. g Thorns of living species Xylosma racemosum. (For c, f, scale bars = 1 mm; for others, scale bars = 10 mm). Fig. 2 Morphotypes of spiny fossils from the upper Eocene Dayu and Xiede sections, central Tibetan Plateau. a Prickles of living species Rubus
alceifolius. b, d, e, h–k, Seven morphotypes of spiny fossils. c Enlargement of b. f Enlargement of h. g Thorns of living species Xylosma racemosum. (For c, f, scale bars = 1 mm; for others, scale bars = 10 mm). closed forest vegetation transitioning to a more open system by
the middle Eocene. and diversity. The phytolith assemblage from the ~47 Ma
Jianglang section, where the megafossils are interpreted to
represent a subtropical forest, is dominated by those forms
produced by woody taxa, and these greatly exceed grass forms
(Supplementary Fig. 13, Supplementary Table 4). Within the
lower part of the Dayu section (~39 Ma), phytoliths are still
dominated by those produced by woody plants but bulliform
phytoliths typically produced by grasses become more common
(Supplementary Figure 13). The main fossil-bearing layer higher
in the Dayu section preserves not only the numerous spiny taxa
but also abundant phytoliths where, among those that could be
identified, 66% were produced by grasses and only 34% by woody
plants. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z 11) and abundant phytoliths (Fig. 4, Supplementary Fig. 12)
representing the grass family (Poaceae) and attributable to the
subfamilies Chloridoideae, Pooideae, and potentially Bambusoi-
deae point to an open ecosystem46. These well-preserved fossils of
herbaceous plants show that the vegetation could not have been
dense forest, even though the assemblage inevitably will have
been biased towards representing a more tree-rich community
bordering the ancient lake shoreline where water was most
abundant. This is emphasised by the presence in the region of
several amphibious mammals that had a similar niche to modern
hippos47 in African savanna. Away from lake margins, the
relatively seasonally dry environment would support fewer trees
and shrubs and thus more open vegetation. Woody species that
have been found preserved in the Dayu section
include
Koelreuteria lunpolaensis48, Ailanthus maximus49, Cedrelosper-
mum tibeticum50, and several species in Malvaceae and Fabaceae
(Supplementary Figs. 9, 10), which are families commonly found
in open ecosystems, as well as a palm, Sabalites tibetensis51. Our
census of the leaf flora from the same layer at the Dayu site shows
that the modern affinities of 10 fossil taxa in the flora belong to
families/orders that contain spine-bearing species, i.e., Malvaceae,
Rosales, Fabaceae, Ulmaceae, Cannabaceae, Menispermaceae,
Simaroubaceae, Anacardiaceae, Myrtaceae, and Araliaceae (Sup-
plementary Table 5). Notably, the plant diversity was much lower
than in the earlier middle Eocene (~47 Ma) Jianglang subtropical
flora reported recently from the adjacent Bangor (Baingoin)
Basin33. Fig. 3 The lineage accumulation curve of spiny plants in Asia and the
elevational changes of central Tibetan Plateau. a The red line represents a
log plot of lineage accumulation of spiny plants in eudicots in Eurasia during
the Cenozoic. The blue curve represents a log plot of lineage accumulation
of mammalian herbivore species in central Asia during the Cenozoic. Being
log plots, the linear rise is indicative of exponential diversification. Source
data are provided as Source Data files. b South (left) to north (right)
transects of mean elevation changes across Himalaya-Tibet at different
phases in its geological evolution (modified from Su, et al.51). Fig. 3 The lineage accumulation curve of spiny plants in Asia and the
elevational changes of central Tibetan Plateau. a The red line represents a Eocene still belonged to Brontotheriidae, a few species of Hyr-
acodontidae and Lophialetidae emerged, suggesting that these
clades may have helped drive the spiny plant diversification
reflected by our findings. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z Most of the second peak
species belonged to Perissodactyls (~84%, n = 187), mainly from
the Hyracodontidae, Brontotheriidae and Lophialetidae families. While most of the species identified to family level in the late NATURE COMMUNICATIONS | (2022) 13:3787 | https://doi.org/10.1038/s41467-022-31512-z | www.nature.com/naturecommunications 4 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z Among the main changes in the late
Eocene-early Oligocene fauna, the proportion of Artiodactyls
increased to ~43% (n = 124), Rhinocerotidae, Paraceratheriidae
and Lophiomerycidae started to diversify, and finally cervids and
bovids appeared (Supplementary Data 1). In addition, many
hippo-like amphibious species belonging to Anthracotheriidae,
Amynodontidae and Anthracobunidae were present both in the
first half of the Eocene and in the late Eocene–early Oligocene,
with 30 and 36 species respectively. This is consistent with
sedimentological evidence for the presence of water bodies, at
least seasonally, which over time became increasingly brackish to
saline43. Furthermore, these clades included several very large
herbivores that required large amounts of food, both grass and
tree leaves. These megaherbivores have been found since in open
habitats such as savanna44, adding to our interpretation that
increasing herbaceous (predominantly grass) and spiny plants co-
existed in an open canopy semi-wooded environment. Factors promoting the diversification of spiny plants in central
Tibet. The Tibetan Plateau experienced a complex geological
evolution, leading to dramatic palaeoenvironmental changes both
locally and regionally52–54. Although the details of Tibetan oro-
geny are still being resolved, the latest geological and palaeon-
tological studies have revealed that the plateau did not rise as a
single coherent entity55–57. A deep East-West trending valley
existed in what is now the central Tibetan Plateau for more much
of the Paleogene (Fig. 3b), with the valley bottom being at an
elevation of ~1500 m at 47 Ma33 and ~2600–2700 m at ~39 Ma
(Supplementary Note 2), bounded by the Gangdese and the
Tanggula (Qiangtang) highlands with crest heights > 4 km to the
south and north58 respectively. It was not until just prior to the
Neogene that the modern low relief high elevation plateau
formed36,51,59. During the middle Eocene, a warm and humid NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z ARTICLE Age(Ma)
~39Ma
0
2
4
6
60
40
20
Paleogene
Neogene
Spiny plant lineages
Mammal lineages
b
Gangdese
Himalaya
Qiangtang
0
2
4
6
8
Modern
Mid Miocene
Mid-late Eocene
Lunpola
S
N
log(number of lineages)
a
Elevation (km)
Fig. 3 The lineage accumulation curve of spiny plants in Asia and the
elevational changes of central Tibetan Plateau. a The red line represents a
log plot of lineage accumulation of spiny plants in eudicots in Eurasia during
the Cenozoic. The blue curve represents a log plot of lineage accumulation
of mammalian herbivore species in central Asia during the Cenozoic. Being
log plots, the linear rise is indicative of exponential diversification. Source
data are provided as Source Data files. b South (left) to north (right)
transects of mean elevation changes across Himalaya-Tibet at different
phases in its geological evolution (modified from Su, et al.51). Cenozoic floras worldwide. The evolutionary history of spiny
plants is still poorly known largely due to a dearth of fossil
records, which may be because spinescence tends to be most
prevalent in semi-arid to arid environments where fossilisation
potential is normally low18,45. The rich spine assemblages from
Tibet enable us to investigate the morphological diversity of spiny
plants in a highly unusual context. The fossils studied here
represent both prickles and thorns divisible into seven morpho-
types (Fig. 2). Although with only morphological characters it is
difficult to assign unambiguously these fossils to specific taxa,
these spiny fossils show a range of distinct morphological
characters, indicating that a wide variety of spiny species existed
in the same community in central Tibet during the late Eocene. ll
l
f
l
d
h
h
l
d ll Age(Ma)
~39Ma
0
2
4
6
60
40
20
Paleogene
Neogene
Spiny plant lineages
Mammal lineages
b
log(number of lineages)
a b
Gangdese
Himalaya
Qiangtang
0
2
4
6
8
Modern
Mid Miocene
Mid-late Eocene
Lunpola
S
N
Elevation (km) y
g
All plant fossil evidence, together with numerical modelling
(Supplementary Note 3; Supplementary Fig. 14), indicate that
central Tibet hosted a landscape that supported an open habitat,
seemingly in the form of woodlands, during the late Eocene. Woodlands are usually considered to be tree-rich communities
with open canopies and grassy understories40,41. Today, spiny
plants appear to be most abundant in open canopy communities,
where
predation
pressure
from
mammalian
herbivores
is
high11,18. Numerous fossilised monocots (Fig. 4, Supplementary
Fig. NATURE COMMUNICATIONS | (2022) 13:3787 | https://doi.org/10.1038/s41467-022-31512-z | www.nature.com/naturecommunications Discussion
h l The late Eocene diversification of spiny plants and semi-open
woodlands in central Tibet. The rich assemblage of spiny plant
fossils from the late Eocene (~39 Ma) along the Bangong-Nujiang
Suture Zone in central Tibet traces the evolutionary history of
spiny plants in Eurasia back to the Paleogene. These fossils evi-
dence an early diversification of spiny plants in the Tibetan region
contemporaneous with the emergence of open semi-wooded
habitats by the late Eocene, and early in the transition of central
Tibet to full plateau formation which appears to have been almost
complete early in the Oligocene36. Based on current knowledge, this late Eocene flora in central
Tibet bears the richest diversity of spiny plants known among 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z Fig. 4 Typical herbaceous fossils and phytoliths from the Dayu section. a–d Phytoliths in different forms extracted from the Dayu sediments. a True
saddle; b Rondel; c Elongate; d Bulliform. e, f Herbaceous megafossils from Dayu section. e Sympodial branching; f Wide leaf with parallel veins; g Stem
with swollen nodes. For phytolith analysis, we performed three replicate experiments with consistent results. (For a–d, scale bars = 10 µm; for e, f, scale
bars = 10 mm). Fig. 4 Typical herbaceous fossils and phytoliths from the Dayu section. a–d Phytoliths in different forms extracted from the Dayu sediments. a True
saddle; b Rondel; c Elongate; d Bulliform. e, f Herbaceous megafossils from Dayu section. e Sympodial branching; f Wide leaf with parallel veins; g Stem
with swollen nodes. For phytolith analysis, we performed three replicate experiments with consistent results. (For a–d, scale bars = 10 µm; for e, f, scale
bars = 10 mm). airflow evidently penetrated the valley, supporting a humid cli-
mate and subtropical biota33,54,60. The palaeoclimate of the Dayu
flora derived from the CLAMP analysis is similar, but slightly
drier, than that experienced by the older Jianglang flora nearby
(Supplementary Table 1), and is consistent with an early onset of
regional drying that became more pronounced after the end of
the Oligocene43. The reduction in growing season precipitation is
largely a function of the shorter growing season, but the mean
monthly growing season precipitation does indicate slightly drier
conditions for the Dayu assemblage. Eocene, central Tibet was an intermontane lowland bounded by
mountains that seem to have exceeded 4,000 metres59. Despite
the initial influx of moisture, the retreat of the Neotethys during
that period resulted in a reduction in the amount of water vapour
entering the then shallowing valley, which, together with a
growing Himalaya, led to the increasingly dry Neogene environ-
mental conditions across the Tibet and central Asia64. A recent
study demonstrates both drying and cooling occurred in central
Tibet as the surface elevation of the Tibetan Central Valley
increased from ~2.5 km to more than 4 km between 39 Ma and
29 Ma36. Eocene, central Tibet was an intermontane lowland bounded by
mountains that seem to have exceeded 4,000 metres59. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z 15) to those of our fossils and found that only eudicots exhibit
the same spine morphology87 as those of our fossils. We classified fossil spines into
prickles and thorns according to two criteria: prickles originate from the epidermis
of plant organs such as stems, leaves and petioles, whereas thorns are modified
branches and include internal vascular bundles. For fossil specimens, due to the
different structures of prickles and thorns, we observed distinct scars at the
junction between prickles and stems, but these are absent in thorny species. Secondly, thorns always grow from stem nodes and therefore display a regular
phyllotaxy, but prickles tend to be distributed randomly along the internodes. h l
l
f
d For morphological comparison of spinescence among modern species, we
consulted online data sources including JSTOR Global Plants (http://plants.j
stor.org/) and the Chinese Virtual Herbarium (http://www.cvh.org.cn/). Living
species of Rubus alceifolius and Xylosma racemosum were collected from XTBG
and from the wild in the Xishuangbanna region, Yunnan. The plant fossils reported here record the beginnings of
vegetation opening-up under a drying/cooling climate as the
modern Tibetan Plateau began to form from the late Eocene
onwards, and the observed spinescence marks the early develop-
ment of defence mechanisms against large herbivore feeding
pressure in the region. We infer that in the late Eocene climate
changes in central Tibet, driven by the onset of global cooling61
and regional tectonism74, resulted in changes in vegetation that
allowed increased access by large herbivores. This in turn led to
further opening-up of the landscape favouring further diversifica-
tion of herbivorous mammals and spiny plants. This string of
climate/plant/animal interactions long pre-dated the middle
Miocene arrival of herbivorous mammals (especially bovids) in
Africa where similarly linked evolutionary feedback processes
took place some 24 million years later18. Phylogenetic analyses. To investigate the emergence and early diversification of
spiny eudicots, we reconstructed the evolutionary history of spines across species of
woody eudicots represented on the mega-phylogeny of plants from Zanne et al.88
using the make.simmap function in phytools89 and the ape90 and Geiger91
R-libraries (Supplementary Code 1). We coded, as a binary trait, for the presence of
spines in plants found in Eurasia (n = 1590 species) mostly using scans from
https://plants.jstor.org/. We excluded species when the origin appeared ambiguous
between more than one continent. Methods
Thi This conservation of energy means that we can exploit the difference in moist
enthalpy between two locations (Hlow and Hhigh) to determine the height difference
between them, ΔZ, as follows: The Dingqinghu Formation is characterised by grey lacustrine mudstones
together with sandstones and oil shales, with a total thickness of about ~1,000
metres36,42,76. It bears rich records of animals and plants, including two Oligocene
ostracode assemblages: Austrocypis-Cyprinotus-Pelocypris and Ilyocypris-
Limnocythere81,82, fishes including Plesioschizothorax83, and mammals such as
Plesiaceratherium42. ΔZ ¼ ðHlow HhighÞ=g
ð2Þ ð2Þ Thermal lapse rates describe changes in temperature with changing elevation,
and in general (with exception of a temperature inversion) temperature measured
at Earth’s surface declines with increasing height and can be expressed as: Fossils in this study are from two sites in the Niubao Formation (Fig. 1). According to recent radiometric dating throughout the Dayu section, the age of the
plant fossil horizons in the Dayu section (32° 20′ N, 89° 46′ E) is ~39 Ma36
(Bartonian, early late Eocene). For the other site in Xiede (31° 58′ N, 88° 25′ E), the
biota is similar to that at Dayu, and many species are common to both sites, such as
a climbing perch Eoanabas thibetana84, a water strider Aquarius lunpolaensis85,
and plants, e.g., Limnobiophyllum pedunculatum86, Sabalites tibetensis51,
Cedrelospermum tibeticum50, and Ailanthus maximus49. Therefore, we consider
both sites to be age-equivalent, dating from the early late Eocene. Γε ¼ dT=dz
ð3Þ ð3Þ where Γε is the terrestrial lapse rate, dT is the change in temperature, and dz is the
change in height. We used wet bulb lapse rates because these are more reliable than
those based on dry bulb temperatures and are independent of season38. The local
terrestrial wet bulb lapse rate was determined from modelling as in Farnsworth
et al.38 using a best estimate realistic topography. Cedrelospermum tibeticum50, and Ailanthus maximus49. Therefore, we consider
both sites to be age-equivalent, dating from the early late Eocene. Sea level moist enthalpy was derived from model data adjusted to be compatible py
j
p
with CLAMP values using Eocene archived proxy data ranging from northern
India to Svalbard, adjusted for palaeolatitude, following the methodology of Su
et al.33. Wet bulb temperature at mean sea level was also obtained from the
adjusted model data. Further details are given in Supplementary Note 2. Morphological observations. In total, 44 spiny fossils were collected from the
Dayu and Xiede localities. Methods
Thi This research complies with all relevant ethical regulations. The fossil excavation
was permitted by Department of Natural Resources and Department of Science &
Technology in Tibetan Autonomous Region, China. Among the palaeoclimate metrics that CLAMP returns in this calibration are
moist enthalpy and wet bulb mean annual temperature and both can be used to
assess palaeoelevation. The former uses conservation of energy principles, and the
latter exploits the reduction in temperature that occurs as a land surface increases
in height (a terrestrial thermal lapse rate). Both approaches were used to determine
the height at which the Dayu flora existed. Geological setting. Fossils in this study are from the Dayu and Xiede sections,
which are located within the Bangong-Nujiang Suture Zone that is aligned roughly
East-West through what is now the central Tibetan Plateau. The present average
surface elevation of these two fossil sites is about 4700 metres42, and predominantly
vegetated by alpine steppe34 (Supplementary Figure 1). The Bangong-Nujiang Suture Zone hosts thick Cenozoic deposits, the lower
part of which in the adjacent Lunpola and Bangor basins comprise the Paleocene-
Eocene Niubao Formation and the upper part the Oligocene-Miocene Dingqinghu
(Dingqing) Formation75–77. The Niubao Formation, up to 3,000 metres thick36, is
mainly formed of fluviatile red clastic rocks interspersed with greyish green
mudstones and sandstones. Plant fossils have recently been reported from
mudstones in the middle part of the formation near the top of the lower member of
the Niubao Formation, and include, amongst others, Ailanthus maximus49,
Lagokarpos tibetensis78, Illigera eocenica79, and Asclepiadospermum marginatum
and A. ellipticum80. As a parcel of air rises against a mountain front, temperature declines, humidit
rises and potential energy increases, but overall the energy it contains is
conserved93. This energy is called moist static energy (h) and excludes kinetic
energy, which tends to be small except during a hurricane. As a parcel of air rises against a mountain front, temperature declines, humidity
rises and potential energy increases, but overall the energy it contains is p
gy
gy
conserved93. This energy is called moist static energy (h) and excludes kinetic
energy, which tends to be small except during a hurricane. h ¼ H þ Zg
ð1Þ ð1Þ where H is moist enthalpy, Z is height, and g is gravitational acceleration (a
constant 9.81 cm/s2). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z To control for the relatively recent origin and
diversification of spines, we allowed rates to vary through time (parameter Δ = 9),
and constrained evolutionary transitions to preclude reversals from the spiny to
non-spiny state, reflecting the rarity of this evolutionary event, and avoiding
potential bias due to the over-representation of non-spiny lineages among woody
taxa92. Palaeoenvironmental reconstruction using Climate-Leaf Analysis Multivariate
Program (CLAMP). We applied CLAMP (http://clamp.ibcas.ac.cn) to the woody
dicot leaf forms (24 distinct morphotypes in the Dayu flora, Supplementary Figures
9, 10) with the PhysgAsia2 (http://clamp.ibcas.ac.cn/CLAMP_PhysgAsia2.html)
training set and Worldclim2 climate data (http://worldclim.com/version2)
calibration74 to derive palaeoclimate metrics. Scoring of the fossil leaves followed
the CLAMP protocols. The CLAMP scoresheets are given as Supplementary
Tables 2, 3. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z In terrestrial ecosystems, mammalian herbivores influence the
length, density, and distribution of plant spines14,67,68. For
example, the rapid accumulation of spiny plants in Africa since
the middle Miocene closely matched the arrival of bovids18. The
earlier emergence of ruminants, and other extant families of
herbivores in Eurasia may have triggered the early radiation of
spiny plants in Tibet by filtering out the undefended plant species. Mammalian herbivory is a major driver of global vegetation
dynamics69, and large animals are thought to have the capacity to
create
open
ecosystems
by
reducing
woody
biomass70–72. Exclosure experiments conducted in Africa reveal that species-
rich megaherbivore communities can effectively reduce woody
vegetation coverage by 15–95%73. In terrestrial ecosystems, mammalian herbivores influence the
length, density, and distribution of plant spines14,67,68. For
example, the rapid accumulation of spiny plants in Africa since
the middle Miocene closely matched the arrival of bovids18. The
earlier emergence of ruminants, and other extant families of
herbivores in Eurasia may have triggered the early radiation of
spiny plants in Tibet by filtering out the undefended plant species. Central Laboratory in XTBG. Digital photographs of extant and fossil specimens
were measured using ImageJ 1.52a software (http://rsb.info.nih.gov.ig/). The morphological definitions of spines followed Cornelissen2, and Bell and
Bryan87. We compared spine morphologies in living eudicots and monocots
(Supplementary Fig. 15) to those of our fossils and found that only eudicots exhibit
the same spine morphology87 as those of our fossils. We classified fossil spines into
prickles and thorns according to two criteria: prickles originate from the epidermis
of plant organs such as stems, leaves and petioles, whereas thorns are modified
branches and include internal vascular bundles. For fossil specimens, due to the
different structures of prickles and thorns, we observed distinct scars at the
junction between prickles and stems, but these are absent in thorny species. Secondly, thorns always grow from stem nodes and therefore display a regular
phyllotaxy, but prickles tend to be distributed randomly along the internodes. For morphological comparison of spinescence among modern species, we
consulted online data sources including JSTOR Global Plants (http://plants.j
stor.org/) and the Chinese Virtual Herbarium (http://www.cvh.org.cn/). Living
species of Rubus alceifolius and Xylosma racemosum were collected from XTBG
and from the wild in the Xishuangbanna region, Yunnan. The morphological definitions of spines followed Cornelissen2, and Bell and
Bryan87. We compared spine morphologies in living eudicots and monocots
(Supplementary Fig. NATURE COMMUNICATIONS | (2022) 13:3787 | https://doi.org/10.1038/s41467-022-31512-z | www.nature.com/naturecommunicatio NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z Despite
the initial influx of moisture, the retreat of the Neotethys during
that period resulted in a reduction in the amount of water vapour
entering the then shallowing valley, which, together with a
growing Himalaya, led to the increasingly dry Neogene environ-
mental conditions across the Tibet and central Asia64. A recent
study demonstrates both drying and cooling occurred in central
Tibet as the surface elevation of the Tibetan Central Valley
increased from ~2.5 km to more than 4 km between 39 Ma and
29 Ma36. Undoubtedly, palaeoenvironmental changes during the devel-
opment of the Tibetan Plateau stimulated an overturn of plant
taxa and an opening-up of the vegetation as the floor of the
central valley rose in the latter part of the Paleogene and regional
drying took place. These changes may have stimulated herbivore
access, increased herbivory pressure on plants, and drove the
evolution of defensive spines. Moreover, at this time global
climate began its transition from a ‘warm house’ to a ‘cool house’
and
eventually
an
icehouse
condition61. Nevertheless,
the
presence of palm fossils in the Dayu flora indicated that the
coldest month mean temperature and mean annual temperature
in central Tibet at ~39 Ma were not below 5.2 °C and 14.2 °C,
respectively51. Temperature, precipitation and solar radiation are
major abiotic factors that determine vegetation growth, distribu-
tion ranges and dynamic change62. When thermal conditions are
sufficient for vegetation growth, water becomes the main limiting
factor controlling vegetation status63. During the early late Since open habitats are generally able to carry larger groups
of herbivores and these animals have a greater impact on plants
whose
growth
is
also
limited
by
reduced
rainfall
and
temperature, herbivore pressure during this period inevitably
increased, driving the evolution of defensive spines. Numerous
fossils of large herbivorous mammals have been found on the
Tibetan
Plateau
and
adjacent
regions,
evidencing
their
presence in the Paleogene (Supplementary Data 1). Examples
include the late Eocene Brontotheriidae65, Hyracodontidae,
late Oligocene Paraceratheriidae66, as well as the early Miocene
Rhinocerotidae42. Although many species are now extinct,
fossil
records
show
that
herbivorous
mammals
became
increasingly diversified, including an important turnover of
herbivore families, notably the emergence of bovids and
cervids, from the Paleogene to the Neogene (Fig. 3a, the blue
curve). NATURE COMMUNICATIONS | (2022) 13:3787 | https://doi.org/10.1038/s41467-022-31512-z | www.nature.com/naturecommunications 6 ARTICLE Trait-based tests
of coexistence mechanisms. Ecol. Lett. 16, 1294–1306 (2013). 8. Wright, S. J. et al. Functional traits and the growth–mortality trade-off in
tropical trees. Ecology 91, 3664–3674 (2010). 9. Wright, I. J. et al. The worldwide leaf economics spectrum. Nature 428,
821–827 (2004). 10. Ruiz-Jaen, M. C. & Potvin, C. Can we predict carbon stocks in tropical
ecosystems from tree diversity? Comparing species and functional diversity in
a plantation and a natural forest. New Phytol. 189, 978–987 (2011). 11. Grubb, P. J. A positive distrust in simplicity-lessons from plant defences and
from competition among plants and among animals. J. Ecol. 80, 585–610
(1992). Herbaceous plant and vegetation types in the late Eocene. To understand the
vegetation type, we assessed the diversity of grass species in the flora. We studied
megafossils, pollen grains/spores, and phytoliths indicative of grass from the
spiny plant fossil-bearing layer of the Dayu section. Pollen grains and spores
were poorly preserved in Dayu and Xiede sections, preventing us from further
palynological investigation, but phytoliths were relatively well preserved. The
extraction and identification of phytoliths from sediments followed the methods
of Strömberg99 and Lu100. The procedure consisted of treatment with 30%
hydrogen peroxide (H2O2) and cold 15% hydrochloric acid (HCl), followed by
heavy liquid separation using zinc bromide (ZnBr2, density 2.35 g/cm3) and
mounting on a microscope slide with Canada balsam. From each extracted
sample, at least one slide was prepared for phytolith counts and analysis under a
LEICA DM 1000 microscope at 400× magnification. For these samples with
poorly preserved phytoliths somewhat less than 100 diagnostic forms were
counted (Supplementary Table 4). 12. Hanley, M. E., Lamont, B. B., Fairbanks, M. M. & Rafferty, C. M. Plant
structural traits and their role in anti-herbivore defence. Perspect. Plant Ecol. 8, 157–178 (2007). 13. Burns, K. C. Spinescence in the New Zealand flora: parallels with Australia. N. Z. J. Bot. 54, 273–289 (2016). 14. Goheen, J. R., Young, T. P., Keesing, F. & Palmer, T. M. Consequences of
herbivory by native ungulates for the reproduction of a savanna tree. J. Ecol. 95, 129–138 (2007). 15. Goldel, B., Kissling, W. D. & Svenning, J.-C. Geographical variation and
environmental correlates of functional trait distributions in palms (Arecaceae)
across the New World. Bot. J. Linn. Soc. 179, 602–617 (2015). across the New World. Bot. J. Linn. Soc. 179, 602–617 (2015 16. Alves-Silva, E. & Del-Claro, K. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z M2.1aD94. Model boundary conditions (topography, bathymetry, and ice sheet
configurations; at 0.5×0.5° resolution and downscaled to model resolution) for the
Bartonian (~39 Ma) are provided by Getech Plc. Stage-specific solar luminosity was
calculated using the methods of Gough95. Atmospheric CO2 concentrations were
prescribed at 1,120 ppm, consistent with the Phanerozoic CO2 compilation of
Foster et al.96. Each experiment was run for 12,422 model years to allow the surface
and deep ocean to reach equilibrium and achieve a state with no net energy
imbalance at the top of the atmosphere. This is fundamental as ocean circulation
can take many thousands of model years to establish its equilibrium state. Climate
means were calculated from the last 100-years of each simulation. Time-varying
latitude and longitude plate palaeo-rotations were provided for the Dayu location
to allow for accurate comparison within the model. Dayu was located at a
palaeolatitude of 32° 30′ N, 82° 54′ E using the Getech Plc. plate model. Tibetan
orography was constrained to represent high (5 km) Gangdese and Tanggula
mountain systems bounding an East-West trending valley system the floor of
which was set at 2.5 km (Supplementary Fig. 14A). The dynamic vegetation scheme
TRIFFID97,98 (Top-down Representation of Interactive Foliage and Flora Including
Dynamics), which predicts the distribution and properties of global vegetation
based on plant functional types using a competitive, hierarchical formulation
derived from the land-atmosphere climate interactions in the model (TRIFFID was
executed and updated every 10 model days) to predict vegetation types likely to
existed within the valley (Supplementary Fig. 14B). 2. Cornelissen, J. H. C. et al. A handbook of protocols for standardised and easy
measurement of plant functional traits worldwide. Aust. J. Bot. 51, 335–380
(2003). 3. Liu, X. J. & Ma, K. P. Plant functional traits concepts, applications and future
directions. Sci. Sin. Vitae 45, 325–339 (2015). 4. Diaz, S., Cabido, M. & Casanoves, F. Plant functional traits and environmental
filters at a regional scale. J. Veg. Sci. 9, 113–122 (1998). 5. Kraft, N. J. B., Godoy, O. & Levine, J. M. Plant functional traits and the
multidimensional nature of species coexistence. Proc. Natl Acad. Sci. USA 112,
797–802 (2015). 6. Barton, K. E. Tougher and thornier: general patterns in the induction of
physical defence traits. Func. Ecol. 30, 181–187 (2016). p y
7. Adler, P. B., Fajardo, A., Kleinhesselink, A. R. & Kraft, N. J. B. Data availability 25. Lancucka-Srodoniowa, M. Tortonian flora from the “Gdow Bay” in the south
of Poland. Acta Palaeobot. 7, 1–134 (1966). All data analysed in this paper are available as part of the Article and Supplementary
Information. All fossils are deposited at the Paleoecology Collections of Xishuangbanna
Tropical Botanical Garden (XTBG), Chinese Academy of Sciences. Additional data
related to this paper may be requested from the authors. Correspondence and requests
for materials should be addressed to T.S. (sutao@xtbg.org.cn). Source data are provided
with this paper. 26. Yuan, J. et al. Rapid drift of the Tethyan Himalaya terrane before two-stage
India-Asia collision. Natl Sci. Rev. 8, nwaa173 (2021). 26. Yuan, J. et al. Rapid drift of the Tethyan Himalaya terr India-Asia collision. Natl Sci. Rev. 8, nwaa173 (2021). 27. Spicer, R. A. et al. Why the ‘Uplift of the Tibetan Plateau’is a myth. Natl Sci. Rev. 8, nwaa091 (2021). 28. Spicer, R. A. Tibet, the Himalaya, Asian monsoons and biodiversity–In what
ways are they related? Plant Divers. 39, 233–244 (2017). y
y
29. DeCelles, P. G., Kapp, P., Gehrels, G. E. & Ding, L. Paleocene-Eocene foreland
basin evolution in the Himalaya of southern Tibet and Nepal: implications for 29. DeCelles, P. G., Kapp, P., Gehrels, G. E. & Ding, L. Paleocene-Eocene foreland
basin evolution in the Himalaya of southern Tibet and Nepal: implications for
the age of initial India-Asia collision. Tectonics 33, 824–849 (2014). ARTICLE Herbivory causes increases in leaf spinescence
and fluctuating asymmetry as a mechanism of delayed induced resistance in a
tropical savanna tree. Plant Ecol. Evol. 149, 73–80 (2016). tropical savanna tree. Plant Ecol. Evol. 149, 73–80 (2016). 17. Cooper, S. M. & Ginnett, T. F. Spines protect plants against browsing by small
climbing mammals. Oecologia 113, 219–221 (1998). Paleogene herbivorous mammal fossils. We used fossil records of all Artio-
dactyls and Perissodactyls from fossilworks (http://fossilworks.org/) (Supplemen-
tary Data 1). Both are orders including mammals that have similar ecological roles
as modern ungulates. From fossil records, we built a dataset with the first occur-
rence of each species using the maximum date estimate as the species are very likely
older than the first fossil. This allowed us to deduce a cumulative curve of species
(n = 658 species) as an estimate of speciation rate. We focused on the fossil records
from countries including and surrounding the Tibetan Plateau, including China,
India, Afghanistan, Kazakhstan, Kyrgyzstan, Mongolia, Myanmar, Nepal, Pakistan,
Turkmenistan, and Uzbekistan. 18. Charles-Dominique, T. et al. Spiny plants, mammal browsers, and the origin
of African savannas. Proc. Natl Acad. Sci. USA 113, E5572–E5579 (2016). 19. Ratnam, J., Tomlinson, K. W., Rasquinha, D. N. & Sankaran, M. Savannahs of
Asia: antiquity, biogeography, and an uncertain future. Philos. Trans. R. Soc. B. 371, 20150305 (2016). 20. Scholes, R. & Archer, S. Tree-grass interactions in savannas. Annu. Rev. Ecol. Syst. 28, 517–544 (1997). 21. Cerling, T. E. Development of grasslands and savannas in East Africa during
the Neogene. Palaeogeogr. Palaeoclimatol. Palaeoecol. 97, 241–247 (1992). 22. Brown, R. W. Additions to the flora of the Green River formation. U. S. Geol. Surv. Prof. Paper, U. S. Gov. Print. Off. 154-J, 279–292 (1929). Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article. 23. Manchester, S. Oligocene fossil plants of the John Day Formation, Oregon. Or. Geol. 49, 115d–127d (1987). 24. Meyer, H. W. & Manchester, S. R. Oligocene Bridge Creek flora of the John Day
Formation, Oregon (Univ. California Press, 1997). Code availability the age of initial India-Asia collision. Tectonics 33, 824–849 (2 y
Code for phylogeny reconstruction is provided in the Supplementary Information. 30. Royden, L. H., Burchfiel, B. C. & van der Hilst, R. D. The geological evolution
of the Tibetan Plateau. Science 321, 1054–1058 (2008). of the Tibetan Plateau. Science 321, 1054–1058 (2008). Received: 3 November 2021; Accepted: 21 June 2022; Received: 3 November 2021; Accepted: 21 June 2022; Received: 3 November 2021; Accepted: 21 June 2022; 31. Deng, T., Wu, F. X., Zhou, Z. K. & Su, T. Tibetan Plateau: an evolutionary
junction for the history of modern biodiversity. Sci. China Earth Sci. 63,
172–187 (2020). 32. Favre, A. et al. The role of the uplift of the Qinghai‐Tibetan Plateau for the
evolution of Tibetan biotas. Biol. Rev. 90, 236–253 (2015). 33. Su, T. et al. A Middle Eocene lowland humid subtropical “Shangri-La”
ecosystem in central Tibet. Proc. Natl Acad. Sci. USA 117, 32989–32995
(2020). Methods
Thi Among them, 19 specimens are from the Dayu section,
and 25 specimens are from the Xiede section, respectively. All specimens are
deposited at the Palaeoecology Collections of Xishuangbanna Tropical Botanical
Garden (XTBG), Chinese Academy of Sciences. They were photographed with a
Nikon D700 digital camera (Nikon, Kanagawa, Japan). A stereoscope (Zeiss Smart
Zoom 5) was used to photograph the detailed morphology of the spines at the Palaeovegetation predicted by climate modelling. To produce a more applic-
able, time dependant, simulation of the Dayu environment, we employed a fully
coupled Atmosphere-Ocean General Circulation Model (AOGCM), HadCM3BL- 7 ARTICLE Nature 439, 677–681 (2006). 42. Deng, T. et al. A mammalian fossil from the Dingqing Formation in the
Lunpola Basin, northern Tibet, and its relevance to age and paleo-altimetry. Sci. Bull. 57, 261–269 (2012). 76. Sun, J. M. et al. Palynological evidence for the latest Oligocene-early Miocene
paleoelevation estimate in the Lunpola Basin, central Tibet. Palaeogeogr. Palaeoclimatol. Palaeoecol. 399, 21–30 (2014). 43. Ma, P. F. et al. Late Oligocene-early Miocene evolution of the Lunpola Basin,
central Tibetan Plateau, evidences from successive lacustrine records. Gondwana Res. 48, 224–236 (2017). 77. DeCelles, P. G., Kapp, P., Ding, L. & Gehrels, G. E. Late Cretaceous to middle
Tertiary basin evolution in the central Tibetan Plateau: Changing
environments in response to tectonic partitioning, aridification, and regional
elevation gain. Geol. Soc. Am. Bull. 119, 654–680 (2007). 44. Hempson, G. P., Archibald, S. & Bond, W. J. A continent-wide assessment of
the form and intensity of large mammal herbivory in Africa. Science 350,
1056–1061 (2015). elevation gain. Geol. Soc. Am. Bull. 119, 654–680 (2007). 45. Spicer, R. A. The formation and interpretation of plant fossil assemblages. Adv. Bot. Res. 16, 95–191 (1989). 78. Tang, H. et al. Extinct genus Lagokarpos reveals a biogeographic connection
between Tibet and other regions in the Northern Hemisphere during the
Paleogene. J. Syst. Evol. 57, 670–677 (2019). 46. Gibson, D. J. Grasses and Grassland Ecology (Oxford Univ. Press, 2009). Paleogene. J. Syst. Evol. 57, 670–677 (2019). 79. Wang, T. X. et al. Fossil fruits of Illigera (Hernandiaceae) from the Eocene of
central Tibetan Plateau. J. Syst. Evol. 59, 1276–1286 (2021). 47. Eltringham, S. K. The Hippos: Natural History and Conservation (Princeton
Univ. Press, 1999). 48. Jiang, H. et al. Oligocene Koelreuteria (Sapindaceae) from the Lunpola Basin
in central Tibet and its implication for early diversification of the genus. J. Asian Earth Sci. 175, 99–108 (2019). 80. Del Rio, C. et al. Asclepiadospermum gen. nov., the earliest fossil record of
Asclepiadoideae (Apocynaceae) from the early Eocene of central Qinghai-
Tibetan Plateau, and its biogeographic implications. Am. J. Bot. 107, 126–138
(2020). 49. Liu, J. et al. Biotic interchange through lowlands of Tibetan Plateau suture zones
during Paleogene. Palaeogeogr. Palaeoclimatol. Palaeoecol. 524, 33–40 (2019). 81. Xu, Z. Y. The Tertiary and its petroleum potential in the Lunpola Basin, Tibet. Oil Gas. Geol. 1, 153–158 (1980). 50. Jia, L. B. et al. ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31512-z 68. Karban, R. & Myers, J. H. Induced plant responses to herbivory. Annu. Rev. Ecol. Syst. 20, 331–348 (1989). 35. Liu. X. H. Paleoelevation History and Evolution of the Cenozoic Lunpola basin,
Central Tibet. Doctoral thesis (Institute of Tibetan Plateau Research, Chinese
Academy of Sciences, 2018). y
69. Huntly, N. Herbivores and the dynamics of communities and ecosystems. Annu. Rev. Ecol. Syst. 22, 477–503 (1991). 36. Xiong, Z. Y. et al. The rise and demise of the Paleogene Central Tibetan
Valley. Sci. Adv. 8, eabj0944 (2022). y
70. Asner, G. P. et al. Large-scale impacts of herbivores on the structural diversity
of African savannas. Proc. Natl Acad. Sci. USA 106, 4947–4952 (2009). y
70. Asner, G. P. et al. Large-scale impacts of herbivores on the y
j
37. Reichgelt, T., West, C. K. & Greenwood, D. R. The relation between global
palm distribution and climate. Sci. Rep. 8, 4721 (2018). 71. Sankaran, M., Augustine, D. J. & Ratnam, J. Native ungulates of diverse body
sizes collectively regulate long‐term woody plant demography and structure of
a semi‐arid savanna. J. Ecol. 101, 1389–1399 (2013). 38. Farnsworth, A. et al. Paleoclimate model-derived thermal lapse rates: towards
increasing precision in paleoaltimetry studies. Earth Planet. Sci. Lett. 564,
116903 (2021). 72. Staver, A. C. & Bond, W. J. Is there a ‘browse trap’? Dynamics of herbivore
impacts on trees and grasses in an African savanna. J. Ecol. 102, 595–602
(2014). 39. Spicer, R. A. et al. Why do foliar physiognomic climate estimates sometimes
differ from those observed? Insights from taphonomic information loss and a
CLAMP case study from the Ganges Delta. Palaeogeogr. Palaeoclimatol. Palaeoecol. 302, 381–395 (2011). 73. Bakker, E. S. et al. Combining paleo-data and modern exclosure experiments
to assess the impact of megafauna extinctions on woody vegetation. Proc. Natl
Acad. Sci. USA 113, 847–855 (2016). 40. Walter, H. Vegetation of the Earth and Ecological Systems of the Geo-biosphere
(Springer Berlin Heidelb., 1973). 74. Spicer, R. A. et al. The topographic evolution of the Tibetan Region as revealed
by palaeontology. Palaeobio. Palaeoenv. 101, 213–243 (2021). 41. Burley, J. Encyclopedia of Forest Sciences (Acad. Press, 2004) 41. Burley, J. Encyclopedia of Forest Sciences (Acad. Press, 2004). 42
Deng T et al A mammalian fossil from the Dingqing Formation in the 75. Rowley, D. B. & Currie, B. S. Palaeo-altimetry of the late Eocene to Miocene
Lunpola basin, central Tibet. ARTICLE First fossil record of Cedrelospermum (Ulmaceae) from the
Qinghai-Tibetan Plateau: implications for morphological evolution and
biogeography. J. Syst. Evol. 57, 94–104 (2019). p
Qinghai-Tibetan Plateau: implications for morphological evolution and
biogeography. J. Syst. Evol. 57, 94–104 (2019). 82. Zhang, K. X. et al. Paleogene-Neogene stratigraphic realm and sedimentary
sequence of the Qinghai-Tibet Plateau and their response to uplift of the
plateau. Sci. China Earth Sci. 53, 1271–1294 (2010). g
g
p y
y
51. Su, T. et al. No high Tibetan Plateau until the Neogene. Sci. Adv. 5, eaav2189
(2019). 83. Wu, Y. F. & Chen, Y. Y. Fossil cyprinid fishes from the late Tertiary of north
Xizang, China. Vertebrata Palasiat. 18, 15–20 (1980). 52. Zhang, Y. L., Li, B. Y. & Zheng, D. A discussion on the boundary and area of
the Tibetan Plateau in China. Geol. Res. 21, 1–8 (2002). 84. Wu, F. X., Miao, D. S., Chang, M. M., Shi, G. L. & Wang, N. Fossil climbing
perch and associated plant megafossils indicate a warm and wet central Tibet
during the late Oligocene. Sci. Rep. 7, 878 (2017). 53. Yao, T. D. et al. From Tibetan Plateau to Third Pole and Pan-Third Pole. Bull. Chin. Acad. Sci. 32, 924–931 (2017). 54. Spicer, R. A., Farnsworth, A. & Su, T. Cenozoic topography, monsoons and
biodiversity conservation within the Tibetan Region: an evolving story. Plant
Divers. 42, 229–254 (2020). 85. Cai, C. Y., Huang, D. Y., Wu, F. X., Zhao, M. & Wang, N. Tertiary water
striders (Hemiptera, Gerromorpha, Gerridae) from the central Tibetan Plateau
and their palaeobiogeographic implications. J. Asian Earth Sci. 175, 121–127
(2017). 55. Liu, X. H., Xu, Q. & Ding, L. Differential surface uplift: Cenozoic
paleoelevation history of the Tibetan Plateau. Sci. China Earth Sci. 59,
2105–2120 (2016). 86. Low, S. L. et al. Oligocene Limnobiophyllum (Araceae) from the central
Tibetan Plateau and its evolutionary and palaeoenvironmental implications. J. Syst. Palaeontol. 18, 415–431 (2020). 56. Ding, L., Li, Z. Y. & Song, P. P. Core fragments of Tibetan Plateau from
Gondwanaland united in Northern Hemisphere. Bull. Chin. Acad. Sci. 32,
945–950 (2017). 87. Bell, A. D. & Bryan, A. Plant Form: An Illustrated Guide to Flowering Plant
Morphology (Timber Press, 2008). 57. Deng, T. & Ding, L. Paleoaltimetry reconstructions of the Tibetan Plateau:
progress and contradictions. Natl Sci. Rev. 2, 417–437 (2015). p
gy
88. Zanne, A. E. et al. References 1. Reich, P. B. et al. The evolution of plant functional variation: traits, spectra,
and strategies. Int. J. Plant Sci. 164, S143–S164 (2003). 34. Scientific Expedition Team to the Qinghai-Xizang Plateau. Vegetation of
Xizang (Tibet) (Sci. Press, 1988). 8 8 NATURE COMMUNICATIONS | (2022) 13:3787 | https://doi.org/10.1038/s41467-022-31512-z | www.nature.com/naturecommunicatio ARTICLE Three keys to the radiation of angiosperms into freezing
environments. Nature 506, 89–92 (2014). 89. Revell, L. J. phytools: an R package for phylogenetic comparative biology (and
other things). Methods Ecol. Evol. 3, 217–223 (2012). 58. Li, S. F. et al. Orographic evolution of northern Tibet shaped vegetation and
plant diversity in eastern Asia. Sci. Adv. 7, eabc7741 (2021). 59. Ding, L. et al. The Andean-type Gangdese Mountains: Paleoelevation record
from the Paleocene–Eocene Linzhou Basin. Earth Planet. Sci. Lett. 392,
250–264 (2014). 90. Paradis, E. & Schliep, K. ape 5.0: an environment for modern phylogenetics
and evolutionary analyses in R. Bioinformatics. 35, 526–528 (2019). 91. Harmon, L. J., Weir, J. T., Brock, C. D., Glor, R. E. & Challenger, W. GEIGER:
investigating evolutionary radiations. Bioinformatics. 24, 129–131 (2008). 60. Deng, T. et al. Review: implications of vertebrate fossils for paleo-elevations of
the Tibetan Plateau. Glob. Planet. Change 174, 58–69 (2019). 92. Maddison, W. P. Confounding asymmetries in evolutionary diversification
and character change. Evolution 60, 1743–1746 (2006). g
61. Westerhold, T. et al. An astronomically dated record of Earth’s climate and its
predictability over the last 66 million years. Science 369, 1383–1387 (2020). 93. Forest, C. E., Molnar, P. & Emanuel, K. A. Palaeoaltimetry from energy
conservation principles. Nature 374, 347–350 (1995). 62. Nemani, R. R. et al. Climate-driven increases in global terrestrial net primary
production from 1982 to 1999. Science 300, 1560–1563 (2003). 94. Valdes, P. J. et al. The BRIDGE HadCM3 family of climate models:
HadCM3@ Bristol v1.0. Geosci. Model Dev. 10, 3715–3743 (2017). p
63. Hopkins, W. G. Introduction to Plant Physiology (John Wiley & Sons, 1999). HadCM3@ Bristol v1.0. Geosci. Model Dev. 10, 3715–3743 (2 64. Sun, J. M., Liu, W. G., Liu, Z. H. & Fu, B. H. Effects of the uplift of the Tibetan
Plateau and retreat of Neotethys ocean on the stepwise aridification of mid-
latitude Asian interior. Bull. Chin. Acad. Sci. 32, 951–958 (2017). 95. Gough, D. O. Solar interior structure and luminosity variations. Sol. Phys. 74,
21–34 (1981). 96. Foster, G. L., Royer, D. L. & Lunt, D. J. Future climate forcing potentially
without precedent in the last 420 million years. Nat. Commun. 8, 14845
(2017). 65. Zong, G. F. Cenezoic Mammals and Environment of Hengduan Mountains
Region (China Ocean Press, 1996). g
66. Deng, T. et al. Additional information Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41467-022-31512-z. reconstruction in the Loess Plateau. Quat. Sci. Rev. 25, 945–959 (2006). Correspondence and requests for materials should be addressed to Tao Su. Correspondence and requests for materials should be addressed to Tao Su. 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
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the copyright holder. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/. Innovation Promotion Association, CAS (No. Y2021105), and the West Light Founda-
tion, CAS (No. 2020000023). Innovation Promotion Association, CAS (No. Y2021105), and the West Light Founda-
tion, CAS (No. 2020000023). p
g
The authors declare no competing interests. p y
gy
100. Lu, H. Y. et al. Phytoliths as quantitative indicators for the reconstruction of
past environmental conditions in China II: palaeoenvironmental 100. Lu, H. Y. et al. Phytoliths as quantitative indicators for the reconstruction of
past environmental conditions in China II: palaeoenvironmental
reconstruction in the Loess Plateau. Quat. Sci. Rev. 25, 945–959 (2006). Acknowledgements Peer review information Nature Communications thanks Deke Xu and the other,
anonymous, reviewer(s) for their contribution to the peer review of this work. Peer
reviewer reports are available. We are grateful to members of the Paleoecology Research Group at Xishuangbanna
Tropical Botanical Garden (XTBG) and Institute of Vertebrate Paleontology and
Paleoanthropology (IVPP) who participated in numerous fossil collection expeditions on
the Tibetan Plateau. We thank Professor Houyuan Lu and Professor Caroline A.E. Strömberg for contributions to the identification of phytoliths; the Central Laboratory of
Public Technology Service Center of XTBG for help with photography. This work is
supported by National Natural Science Foundation of China (NSFC) (Nos. 41988101 and
41922010), the Second Tibetan Plateau Scientific Expedition programme (No. 2019QZKK0705), Natural Environment Research Council of the UK (NERC) (Nos. 41661134049 and NE/P013805/1), the Strategic Priority Research Program of the Chi-
nese Academy of Sciences (CAS) (Nos. XDA20070301 and XDB26000000), Youth
Innovation Promotion Association, CAS (No. Y2021105), and the West Light Founda-
tion, CAS (No. 2020000023). Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints Competing interests 99. McInerney, F. A., Strömberg, C. A. E. & White, J. W. C. The Neogene
transition from C3 to C4 grasslands in North America stable carbon isotope
ratios of fossil phytoliths. Paleobiology 37, 23–49 (2011). ARTICLE An Oligocene giant rhino provides insights into
P
th i
l ti
C
Bi l 4 639 (2021) 66. Deng, T. et al. An Oligocene giant rhino provides insights in
Paraceratherium evolution. Commun. Biol. 4, 639 (2021). 97. Cox, P. M. Description of the “TRIFFID” Dynamic Global Vegetation Model. 1–16 (Met Office Hadley Centre, 2001). Paraceratherium evolution. Commun. Biol. 4, 639 (2021). 98. Cox, P., Huntingford, C. & Harding, R. A canopy conductance and
photosynthesis model for use in a GCM land surface scheme. J. Hydrol. 212,
79–94 (1998). 67. Young, T. P., Stanton, M. L. & Christian, C. E. Effects of natural and simulated
herbivory on spine lengths of Acacia drepanolobium in Kenya. Oikos 101,
171–179 (2003). 9 NATURE COMMUNICATIONS | (2022) 13:3787 | https://doi.org/10.1038/s41467-022-31512-z | www.nature.com/naturecommunicatio 9 Author contributions T.S and Z.-K.Z. designed research. X.-W.Z., U.G., R.A.S., K.W.T., and T.S. assembled the
data for the manuscript and led the writing process. X.-W.Z., U.G., F.-X.W., P.-R.C.,
C.D.R., J.L., J.H., X.-T.X., S.-T.Z., K.W.T., T.D., Z.-K.Z. and T.S. collected fossil data. U.G. coded the presence of spine in the phylogeny and conducted the trait ancestral
reconstruction. R.A.S., A.F., S.-F.L., and P.J.V. performed the numerical climate and
elevation model analyses. X.-W.Z. and T.S. wrote the first draft of the paper. X.-W.Z.,
U.G., R.A.S., K.W.T., and T.S. revised the manuscript. All authors discussed and com-
mented on the manuscript. T.S and Z.-K.Z. designed research. X.-W.Z., U.G., R.A.S., K.W.T., and T.S. assembled the
data for the manuscript and led the writing process. X.-W.Z., U.G., F.-X.W., P.-R.C.,
C.D.R., J.L., J.H., X.-T.X., S.-T.Z., K.W.T., T.D., Z.-K.Z. and T.S. collected fossil data. U.G. coded the presence of spine in the phylogeny and conducted the trait ancestral
reconstruction. R.A.S., A.F., S.-F.L., and P.J.V. performed the numerical climate and
elevation model analyses. X.-W.Z. and T.S. wrote the first draft of the paper. X.-W.Z.,
U.G., R.A.S., K.W.T., and T.S. revised the manuscript. All authors discussed and com-
mented on the manuscript. © The Author(s) 2022 NATURE COMMUNICATIONS | (2022) 13:3787 | https://doi.org/10.1038/s41467-022-31512-z | www.nature.com/naturecommunications 10 NATURE COMMUNICATIONS | (2022) 13:3787 | https://doi.org/10.1038/s41467-022-31512-z | www.nature.com/naturecommunicatio
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Pharmacological neuroenhancement and the ability to recover from stress – a representative cross-sectional survey among the German population
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Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37
https://doi.org/10.1186/s13011-018-0174-1 Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37
https://doi.org/10.1186/s13011-018-0174-1 Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37
https://doi.org/10.1186/s13011-018-0174-1 (2018) 13:37 Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (
https://doi.org/10.1186/s13011-018-0174-1 Open Access RESEARCH RESEARCH
Open Access
Pharmacological neuroenhancement and
the ability to recover from stress – a
representative cross-sectional survey
among the German population
Christiana Bagusat1*, Angela Kunzler1,2, Jennifer Schlecht1, Andreas G. Franke3, Andrea Chmitorz1,2†
and Klaus Lieb1,2† Christiana Bagusat1*, Angela Kunzler1,2, Jennifer Schlecht1, Andreas G. Franke3, Andrea Chmitorz1,2†
and Klaus Lieb1,2† Christiana Bagusat1*, Angela Kunzler1,2, Jennifer Schlecht1, Andreas G. Franke3, Andrea Chmitorz1,2†
and Klaus Lieb1,2† Abstract Background: Pharmacological neuroenhancement (PNE) refers to the use of psychoactive substances without doctor’s
prescription to enhance cognitive performance or to improve mood. Although some studies have reported that drugs
for PNE are also being used to cope with stressful life situations, nothing is known about the relationship of PNE and
resilience, i.e. the ability to recover from stress. This study aimed at investigating the relationship of PNE and resilience
in the first representative population sample. Methods: A cross-sectional survey in a representative sample of 1128 adults (age ≥18 yrs.) living in Germany was
conducted. The use of PNE and related attitudes, perceptions and behaviours were assessed by structured
interviews and self-report questionnaires. Stepwise logistic regression with backward elimination was conducted to
identify potential risk factors for PNE use. Results: Lifetime prevalence for the use of stimulating prescription drugs without medical indication was 4.3%, 10.2%
for stimulating illicit drugs, 20.3% for mood modulating prescription drugs, and 23.4% for cannabis. Coping with
stressful situations was more frequently reported as underlying motive for using stimulant or mood modulating
prescription drugs than stimulating illicit drugs or cannabis. The individual perceived stress increased the risk of using
stimulating prescription drugs (OR: 2.86; 95% Cl: 1.49–5.46) and the individual ability to recover from stress decreased
the risk of using any substance for PNE and especially mood modulating prescription drugs (OR: .62; 95% Cl: .47–.81). Conclusions: The non-medical use of prescription drugs for PNE appears to be more prevalent in subjects who are
less resilient to stress. Tailored resilience interventions that improve the ability to adapt to and recover from stressors
may prevent the use of prescription medication for PNE. Further research should disentangle the association between
psychological resilience and PNE as well as examine the efficacy of resilience interventions in the prevention of PNE. Keywords: Pharmacological neuroenhancement, Resilience, Stress coping, Illicit drugs, Prescription dru * Correspondence: bagusat@uni-mainz.de * Correspondence: bagusat@uni-mainz.de
†Andrea Chmitorz and Klaus Lieb contributed equally to this work. 1Department of Psychiatry and Psychotherapy, University Medical Center
Mainz, Untere Zahlbacher Str. 8, 55131 Mainz, Germany
Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37 Page 2 of 16 Page 2 of 16 (2018) 13:37 Bagusat et al. Substance Abuse Treatment, Prevention, and Policy Background Depending on the authors of scientific articles some
consider PNE to be „a bad thing “which could harm in-
dividuals and change – in a negative way - society, how-
ever, others would favour the use of PNE drugs based on
the ancient wish of mankind to enhance oneself [22, 23]. In order to overcome limitations of previous surveys
on PNE use regarding resilience factors, we performed a
quantitative study on the relationship between the ability
to recover from stress and PNE use in a representative
sample of the German population. In addition, we
assessed the association of PNE use with three well-evi-
denced resilience factors (self-efficacy, locus of control
and optimism) [37, 38]. dividuals and change – in a negative way - society, how-
ever, others would favour the use of PNE drugs based on
the ancient wish of mankind to enhance oneself [22, 23]. Research on the use of PNE as a strategy to cope with
stressful life situations has been rather neglected so far. There is some evidence that PNE is associated with the
level of perceived stress. Middendorff and colleagues
[11] conducted a survey with over 7.000 students in
Germany. Among those students who reported feeling
no or a low pressure to perform at university, only 3%
have already used prescription or illicit drugs for PNE at
least once, whereas 9% of students feeling high pressure
to perform have already used such medication or drugs
[11]. In an online survey among the Swiss population,
PNE was also positively associated with frequent stress
in the past 12 months [10]. Other studies could demon-
strate correlations between perceived stress and PNE
among specific professions [24]. Surgeons’ pressure to
perform at work and stress in private life were positively
associated with PNE or mood enhancement [16]. In
addition, work-related stress increased the willingness to
use PNE-drugs in a study among university teachers
[24]. Schröder and colleagues [25] compared users and
non-users of PNE in physicians, publicists, advertising
experts and programmers in Germany in the last 12
months. They found higher cognitive stress symptoms
among PNE-users compared to non-users. Background in subjects who are able to recover from stress to vary-
ing degrees. This ability is closely related to the con-
struct of psychological resilience, i.e., the well-observed
phenomenon that many people do not or only temporar-
ily become mentally ill despite significant adversity (e.g.,
[26–29]). Although previously considered as a stable
personality trait (e.g., a “hardy” person), resilience is
nowadays seen as modifiable outcome or dynamic
process with personality as one of many risk or protect-
ive factors for maintaining or regaining mental health
[30, 31]. Resilience is partially determined or predicted
by multiple resilience factors [30], i.e., resources which
protect a person from the potential negative effect of en-
countered stressors by modifying the individual’s re-
sponse to stress and adversities [32, 33]. These include
internal factors, such as (resilience-conducive) personal-
ity traits (e.g., optimism, hardiness), beliefs (e.g., self-effi-
cacy), as well as external factors such as social support
or socioeconomic status [34, 35]. Although there is evi-
dence that some of the internal and external resilience
factors are related to PNE use [11, 24, 36] the effect of
the individual ability to recover from stress on PNE use
is unknown so far. g
Pharmacological neuroenhancement (PNE) refers to the
use of psychoactive substances without medical indica-
tion to enhance cognitive performance or to improve
mood [1–3]. Several studies have investigated the preva-
lence of PNE in different at risk populations such as
pupils [4–6], students [4, 7–13], academics [14], chess
players [15] or physicians [16]. International studies
found divergent lifetime prevalences between 1 and 20%
[4, 7, 16–20] which are dependent on the sample being
investigated, the definition of PNE, the drugs questioned,
and the survey technique used. Most studies have inves-
tigated PNE as a mode to enhance cognitive perform-
ance by the use of stimulating prescription or illicit
drugs such as modafinil and methylphenidate or am-
phetamines, respectively. Other studies have also mea-
sured the use of mood modulating drugs such as
antidepressants by non-depressed healthy subjects to
improve mood or reduce nervousness and found life-
time prevalence rates between 5 and 15% [1, 16]. Data about the effectiveness of such substances are
very different [2, 21]. Participants and procedure We conducted a cross-sectional representative survey of
the
German
population
between
August
19th
and
September 19th 2016 regarding attitudes, perceptions
and behaviours referring to PNE. Face-to-face interviews
of approximately 30 min length were conducted in 1128
people at participant’s place (minimum age: 18 years). The individuals were selected by the “Institut für
Demoskopie Allensbach” as they met criteria of the
quota sample based on the German official statistics re-
garding central socio-demographic factors [39]. This
procedure was chosen to increase the generalizability of
the results for the German population. Participants were
informed by professional interviewers about the objec-
tives of the study, the procedure of data storage and
confirmed their voluntary participation verbally. To help
to assure confidentiality, there was no written consent. To ensure that each question is comprehensible, the
standardized questionnaire was pretested and optimised. All these studies investigated the associations between
perceived life stress and prevalence rates for PNE use. However, no study assessed the substance use for PNE The interviewers were trained uniformly to answer
further questions if necessary for example if there were Page 3 of 16 Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13 Page 3 of 16 (2018) 13:37 mood, relieve anxiety or nervousness, or to manage stress. Please tick every drug if you have already taken it for the
above-mentioned purpose without medical indication. If
you have already taken a drug, please indicate when and
how often.” The list of substances included prescription
drugs, such as methylphenidate (e.g., Ritalin®) or modafi-
nil, and illicit substances such as cocaine or amphet-
amines. All
substances
were
clustered
into
three
substance groups (stimulants, mood enhancer and can-
nabis). For a more differentiated consideration “stimu-
lants” were divided into “prescription stimulants” and
“illicit stimulants” for further analysis. If one substance
of a substance group was used at least once for PNE, the
respondent was asked to answer further questions about
the use. If more than one substance of a substance group
was used, it was asked to choose the most important
substance of that group and the respondent had to an-
swer further questions about the use only for that sub-
stance of the group. uncertainties in the understanding of terms such as “bet-
ter cognitive performance”. Hence they especially clari-
fied that the questions regarding PNE do not concern
the intake of medication by doctor’s prescription. Assessment of reasons and goals for PNE Assessment of reasons and goals for PNE
Beside the frequency of intake, participants were also
asked for the reason of their use and which goals they
pursued by the intake of the substance. The two ques-
tions were asked for one substance of each substance
group. If they used multiple substances of one group
(stimulants, mood enhancer, cannabis) they were asked
to indicate reasons and goals only for the use of the
most important one. They were asked: “For what reason
did you take this drug? Please tick for every reason stated
to what extend this applies to you.” and “What were you
trying to achieve by taking this drug? Please tick for every
reason stated to what extend this applies to you.” Users
could rate the importance of different reasons and goals
for the intake of the substance on a five-point Likert
scale (1 = I totally agree; 5 = I totally disagree) e.g., “I
wanted to improve mood” or” I wanted to be able to han-
dle stressful situations better”. The coding of items was
reversed (1 = I totally disagree; 5 = I totally agree) before
calculating mean values thus higher values mean higher
importance of the reason or goal for the substance use. As individuals might be part of different substance
groups there are no p-values to estimate statistical sig-
nificance of mean values. Questionnaires to assess the ability to recover from stress, Questionnaires to assess the ability to recover from stress,
perceived stress and related psychological resilience factors
To allow for analyses of the association between PNE
and the ability to cope with stress, different assessment
scales were used: Assessment of prevalence of PNE and reasons and goals
for intake The questionnaire to assess the prevalence of PNE as
well as goals and attitudes towards PNE by our group
was based on experiences with earlier surveys [4, 16]
and other published data. Participants and procedure The ques-
tionnaire included a section assessing socio-demographic
data such as gender, age, school education, current or last
professional position, employment status, working hours,
shift work, federal state of residence, size place of residence
and soft enhancer intake. Also there were questions regarding the perception of
the topic PNE in the media, ethical questions and
socio-psychological aspects which will be published
separately. Beside questions that were directly asked by the inter-
viewer, the questionnaire also included a section that
had to be completed written and returned hidden in an
envelope to the interviewer to increase the reliability of
the prevalence rates of PNE use. As these questions re-
ferred to the individual consumption of psychoactive
substances
to
increase
individual
performance,
this
procedure guaranteed confidentiality for the respondent
regarding these sensitive questions. The study was ap-
proved by the local Ethics Committee (Landesärztekam-
mer Rheinland-Pfalz, No 837.209.14, 9448F), and there
was no remuneration of the participating subjects. Assessment of prevalence of PNE To estimate the prevalence of PNE, we asked two main
questions: The first question referred to enhancement by
the intake of freely available substances that can be
bought in supermarkets or pharmacies without any pre-
scription such as energy drinks, Ginkgo biloba or caf-
feine tablets. The interviewer asked: “There are various
substances mentioned on this list. Are there any that you
have taken or are currently taking to improve your men-
tal performance, improve your mood, relieve anxiety or
nervousness, or manage stress? You only have to tell me
the corresponding numbers from the list.” The second
question referred to the use of prescription and/or illicit
drugs. It was assessed within the self-completed an-
onymous paper-and pencil-section. Out of 1128 respon-
dents, 86 had never heard of the phenomenon of PNE. Thus they were assigned to the group of “non-users” in
the further analysis. Hence 1042 had been asked for
their use of prescription/illicit substances for PNE. Perceived Stress Scale (PSS-4) [42] Analyses were performed with SPSS for Windows, Version
17.0. To guarantee representativeness to the highest
possible standard, data of respondents included in the
final sample was weighted for the area and federal
states of Germany, size of the town, gender, school
education, age and profession. N values of weighted
data were rounded. Differences between users or sub-
groups of users and non-users in sociodemographic
variables (gender, age, education, current or last pro-
fessional position, employment status, working hours,
shift work, federal state of residence, size place of
residence and soft enhancer intake) were analysed
using chi2-test, Fisher’s exact test, t-test and Welch
test. Variables referring to the importance of reasons
and goals were recoded for analysis (1 = I totally dis-
agree; 5 = I totally agree). Means are reported with
standard deviations (SD). The PSS-4 consists of four items measuring the individ-
ual evaluation of stressful situations in the previous 12
months (item 1 “How often have you felt that you were
unable to control the important things in your life?”,
item 2. “How often have you felt confident about your ability
to handle your personal problems?”, item 3 “How often
have you felt that things were going your way?”, item 4
“How often have you felt difficulties were piling up so
high that you could not overcome them?”). The items
are rated on a five-point Likert scale (1 = never; 5 = very
often). Item 2 and 3 are reverse coded and were recoded
for the analysis. For each subject, sum scores across all
items are calculated (range: 0–16). Higher values indi-
cate more perceived stress. We used the German version
of the scale [43]. Brief Resilience Scale (BRS) [40] Higher values indicate
higher internal (items 1 and 2) or external (items 3
and 4) control beliefs. Optimism-Pessimism-2 Scale (SOP-2) [46]
The
questionnaire
consists
of
two
items
assessing
self-rated optimism (“How optimistic are you in gen-
eral?”) and pessimism (“How pessimistic are you in gen-
eral?”). The questionnaire uses a seven-point Likert scale
(optimism: 1 = not at all optimistic; 7 = very optimistic;
pessimism: 1 = not at all pessimistic; 7 = very pessimistic). To calculate the mean of the two items, reverse scoring
of the item pessimism is used (range: 1–7). Brief Resilience Scale (BRS) [40] Here, the interviewer asked: “Here are some drugs that
can be used to improve cognitive performance, improve The scale consists of six items assessing self-ratings of
the individual ability to recover from stress despite Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37 Page 4 of 16 Bagusat et al. Substance Abuse Treatment, Prevention, and Policy Page 4 of 16 (2018) 13:37 significant adversity (item 1” I tend to bounce back
quickly after hard times”, item 2” I have a hard time
making it through stressful events”, item 3” It does not
take me long to recover from a stressful event”, item 4”
It is hard for me to snap back when something bad hap-
pens”, item 5” I usually come through difficult times
with little trouble” and item 6” I tend to take a long time
to get over set-backs in my life”). The items are rated on
a five-point Likert scale (1 = strongly disagree; 5 = strongly
agree). Item 1, 3, and 5 are positively phrased; items 2, 4,
and 6 are negatively phrased. The coding of the nega-
tively phrased items is reversed in order to calculate the
mean (range: 1–5) of the six items [40]. Higher values
indicate a higher ability to recover from stress. We used
the German version of the instrument, which was re-
cently validated in a population of n = 2.609 German
participants [41]. The psychometric data of the BRS rat-
ings of the sample investigated here were part of this
validation study. “Whether at work or in my private life: What I do is
mainly determined by others”, “Fate often gets in the way
of my plans”). The questions are rated on a five-point
Likert scale (1 = does not apply at all; 5 = applies
completely). Mean scores for internal or external con-
trol are calculated (range: 1–5). Higher values indicate
higher internal (items 1 and 2) or external (items 3
and 4) control beliefs. “Whether at work or in my private life: What I do is
mainly determined by others”, “Fate often gets in the way
of my plans”). The questions are rated on a five-point
Likert scale (1 = does not apply at all; 5 = applies
completely). Mean scores for internal or external con-
trol are calculated (range: 1–5). (2018) 13:37 (2018) 13:37 Bagusat et al. Substance Abuse Treatment, Prevention, and Policy In the multivariate model, all predictors (BRS, PSS-4,
ASKU, IE internal, IE external, SOP-2) were excluded
stepwise. In order to control for potential confounders,
sociodemographic
variables
(gender,
age,
education,
current or last professional position, employment status,
working hours, shift work, federal state of residence, size
place of residence, soft enhancer intake) with a statisti-
cally significant mean difference between users or sub-
groups of users and non-users (p < .1) were included at
once (method: enter) from the first step on in the model. Continuous variables (age, BRS, PSS-4, IE external, IE
internal, SOP-2 and ASKU) were z-transformed. The
significance level was p < 0.05. illicit drugs M = 2.43, SD = 1.52; cannabis: M = 2.04,
SD = 1.43) (see Fig. 1). On the other hand, users of illicit
drugs agreed to a higher extent that their goal for the in-
take was to improve mood (stimulating illicit drugs M =
4.26, SD = 1.16; cannabis: M = 4.12, SD = 1.28) compared
to users of prescription drugs (stimulating prescription
drugs: M = 3.32, SD = 1.44; mood modulating prescription
drugs: M = 3.23, SD = 1.66). Stress, the ability to recover from stress and resilience
factors in users and non-users of substances for PNE
The ability to recover from stress and the level of per-
ceived stress differed between users (n = 435) and
non-users (n = 686) of any prescription or illicit drug
(see Additional file 1): Users had a lower ability to re-
cover from stress than non-users as measured by the
Brief Resilience Scale (BRS) (user: M = 3.19, SD = .96;
non-user: M = 3.46, SD = .93; p < 0.001). This difference
was also shown in the subgroups ‘stimulating prescrip-
tion drugs’ and ‘mood modulating drugs’ but not in the
subgroups ‘stimulating illicit drugs’ or ‘cannabis’ (see
Additional file 1). Users also reported more perceived
stress than non-users as measured by the Perceived
Stress Scale (PSS-4) (user: M = 7.46, SD = 2.87; non-user:
M = 6.70, SD = 2.79, p < .01). The same results were
found for all subgroups of PNE users (i.e., stimulating
prescription
drugs,
stimulating
illicit
drugs,
mood
modulating drugs and cannabis) (see Additional file 1). Furthermore, compared to non-users, users showed
lower values in the three resilience factors. (2018) 13:37 As indicated
by the mean scores in the Optimism-Pessimism-2 Scale
(SOP-2), users were less optimistic than non-users (user:
M = 4.83,
SD = 1.21;
non-user:
M = 5.08,
SD = 1.19,
p < .01) overall as well as in all subgroups of PNE users
(see Additional file 1). Prevalence rates for the use of substances for PNE Most participants were familiar with the phenomenon of
taking drugs or other substances for PNE (n = 1042;
92.4%). Table 2 summarizes the prevalence rates of the
use of prescription and illicit drugs for PNE in the repre-
sentative sample. As one person is assigned to a substance-group via
lifetime use of relevant substances, it is possible that
individuals are part of several clustered substance-
groups (Table 2). Overall, lifetime use (“at least once”) of any prescrip-
tion or illicit drug for cognitive or mood enhancement
was 38.8%, with a last year, last month, and last week
prevalence of 19.1%, 10.1%, and 8.5%, respectively (Table 2). Regarding different substance categories, the highest life-
time prevalence was found for cannabis (23.4%) followed
by mood modulating prescription drugs (20.3%) and stimu-
lating illicit drugs (10.2%). Stimulating prescription drugs
had the lowest lifetime prevalence (4.3%) compared to the
other substance groups (see Table 1). However, last year,
last month and last week prevalence was higher for mood
modulating prescription drugs (10.6% / 5.9% / 5.6%) than
for cannabis (8.6% / 3.7% / 2.8%), stimulating illicit drugs
(3.8% / 1.3% / 0.6%) and stimulating prescription drugs
(2.2% / 0.8% / 0.3%). In addition, users showed lower self-efficacy beliefs
than non-users (user: M = 3.89, SD = .70; non-user: M =
4.02, SD = .72, p < .01), as assessed by the Short Scale for
Measuring General Self-efficacy Beliefs (ASKU). This re-
sult was found in all subgroups of PNE users (see
Additional file 1). Furthermore, compared to non-users,
users had lower internal locus of control (user: M = 4.11,
SD = .74; non-user: M = 4.23, SD = .69, p < .05) and
higher external locus of control (user: M = 2.59, SD = .83;
non-user: M = 2.38, SD = .82, p < .01), as measured by the
Short Scale for the Assessment of Locus of Control (IE). For internal locus of control, the same difference was
only identified for the subgroups ‘mood modulating
drugs’
and
‘cannabis’
but
not
for
the
subgroups
‘stimulating prescription drugs’ and ‘stimulating illicit
drugs’ (see Additional file 1). External locus of control
was higher in users compared to non-users in all sub-
groups (Additional file 1). Sample characteristics The total sample consists of n = 1128 subjects. Table 1
gives
sociodemographic
information
for
users
and
non-users of PNE. Short Scale for Measuring General Self-efficacy Beliefs
(ASKU) [44] In order to assess the associations between PNE use
and the ability to recover from stress, perceived stress
and resilience factors (self-efficacy, control beliefs and
optimism), stepwise logistic regression with backward
elimination was conducted to determine predictors of
PNE consumption using the most parsimonious model. Prior to multivariate analyses, means of users (and re-
spective subgroups) with nonusers were compared using
t-tests for continuous variables to assess the associations
between each of the potential predictors (BRS, PSS-4,
ASKU, IE internal, IE external, SOP-2) with PNE use. To
test for multicollinearity, associations between the pre-
dictor variables (Pearson correlations) were examined
and
the
variance
inflation
index
(VIF)
calculated. According to the literature, the correlations should
not exceed .80, the VIF should not exceed 10 [47]. The questionnaire consists of three positively worded
items assessing self-rated confidence in the individual
ability to achieve intended results (“I can rely on my own
abilities in difficult situations”, “I am able to solve most
problems on my own”, “I can usually solve even challen-
ging and complex tasks well”) rated on a five-point Likert
scale (1 = does not apply at all; 5 = applies completely). Mean scores are used for analysis (range: 1–5). Higher
values indicate higher self-efficacy. Short Scale for the Assessment of Locus of Control (IE) [45]
The four-item scale assesses internal and external con-
trol beliefs (internal control beliefs: “I’m my own boss”,
“If I work hard, I will succeed”; external control beliefs: Page 5 of 16 Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37 Reasons and goals for PNE Coping with stressful situations was a relevant motive
for the intake of substances for PNE, but this varied be-
tween substance groups. Users of prescription drugs
agreed to a higher extent that stress coping was a reason
for their use (stimulating prescription drugs: M = 3.57,
SD = 1.39; mood modulating prescription drugs: M =
3.16, SD = 1.65) than users of illicit drugs (stimulating Bagusat et al. Reasons and goals for PNE Substance Abuse Treatment, Prevention, and Policy (2018) 13:37 Page 6 of 16 Table 1 Sociodemographic characteristics for users and non-users in the representative survey of the German population
Any medication or drug (n = 435)
Non-User (n = 686)
p-value
Weighteda
Unweighted
Weighteda
Unweighted
% (n)
95% CI
% (n)
% (n)
95% CI
% (n)
min
max
min
max
Age
47.7 (17.7)
46.0
49.3
48.0 (17.6)
53.4 (18.1)
52.0
54.8
52.9 (17.9)
0.01
Gender
0.06
Male
51.7 (225)
47.0
56.4
49.9 (217)
46.0 (316)
42.3
49.7
45.9 (315)
Female
48.3 (210)
43.6
53.0
50.1 (218)
54.0 (371)
50.3
57.7
54.1 (372)
Education
0.82
No formal degree
1.4 (6)
0.3
2.5
1.2 (5)
1.6 (11)
0.7
2.6
1.3 (9)
Secondary modern schoolb
31.3 (133)
26.9
35.7
24.4 (104)
33.2 (227)
29.7
36.8
26.1 (178)
Middle schoolc
30.4 (129)
25.9
34.7
31.5 (134)
30.9 (211)
27.4
34.4
31.2 (213)
University-entrance diplomad
17.7 (75)
14.0
21.3
23.7 (101)
17.7 (121)
14.9
20.6
22.9 (156)
University degree
19.3 (82)
15.5
23.1
19.2 (82)
16.5 (113)
13.8
19.3
18.5 (126)
Current or last professional position
0.23
No employment yet
5.3 (23)
3.2
7.4
5.8 (25)
4.5 (31)
3.0
6.1
5.0 (34)
Skilled worker
14.4 (62)
11.1
17.7
14.4 (62)
14.2 (97)
11.6
16.8
13.5 (92)
Executive employee
10.7 (46)
7.7
13.6
10.9 (47)
14.2 (97)
11.6
16.8
14.5 (99)
Non-executive employee
43.3 (187)
38.6
48.0
42.1 (182)
44.0 (300)
40.3
47.7
43.4 (296)
Civil servants
5.6 (24)
3.4
7.7
6.7 (29)
6.5 (44)
4.6
8.3
7.6 (52)
Self-employed
6.0 (26)
3.8
8.3
6.5 (28)
3.4 (23)
2.0
4.7
3.7 (25)
Other
14.8 (64)
11.5
18.2
13.7 (59)
13.2 (90)
10.7
15.7
12.3 (84)
Shift work
0.14
Yes
15.4 (44)
11.2
19.6
15.5 (44)
20.0 (75)
15.9
24.0
19.7 (74)
No
84.6 (241)
80.4
88.8
84.5 (240)
80.0 (301)
76.0
84.1
80.3 (302)
Weekly working hours
0.73
Currently not working
34.0 (147)
29.6
38.5
34.0 (147)
44.8 (307)
41.1
48.5
44.8 (307)
< 20 h
6.7 (19)
3.8
9.6
6.3 (18)
4.3 (16)
2.2
6.4
4.3 (16)
20–29
8.8 (25)
5.45
12.1
8.5 (24)
9.9 (37)
6.9
13.0
9.4 (35)
30–40
50.5 (144)
44.2
56.3
50.4 (143)
50.4 (188)
45.3
55.5
49.9 (186)
41–50
24.9 (71)
19.9
29.9
25.4 (72)
25.7 (96)
21.3
30.2
26.5 (99)
> 50
9.1 (26)
5.8
12.5
9.5 (27)
9.7 (36)
6.7
12.7
9.9 (37)
Size place of residence (inhabitants)
0.01
< 2.000
7.8 (34)
5.3
10.3
6.9 (30)
8.3 (57)
6.2
10.4
6.6 (45)
2.000–20.000
29.7 (129)
25.4
34.0
29.2 (127)
37.3 (256)
33.7
40.9
34.5 (237)
20.000–100.000
25.8 (112)
21.6
29.9
26.9 (117)
28.7 (197)
25.3
32.1
31.7 (218)
> 100.000
36.8 (160)
32.3
41.3
37.0 (161)
36.8 (176)
32.3
41.3
27.2 (187)
Soft enhancer intake
0.01
yes
86.3 (372)
83.1
89.6
86.3 (372)
49.5 (334)
45.7
53.3
50.4 (341)
no
13.7 (59)
6.6
20.8
13.7 (59)
50.5 (341)
46.7
54.3
49.6 (336)
N = 1128. Reasons and goals for PNE Data are given in % with numbers of subjects in parentheses. For age, means with SD in parentheses are given. aweighted according to the distribution
of the general population in Germany as reported by the German Office of National Statistics; bequivalent to German “Hauptschule” degree after 9 years of formal
education; cequivalent to German “Realschule” degree after 10 years of formal education; dequivalent to German general or subject-specific. “Hochschulreife” or
“Fachhochschulreife” degree (entrance qualifications for university or university of applied sciences) after 11. Twelve or 13 years of formal education; p-values:
statistically significant differences between the respective group of users and non-users (α = .05); weighted n is rounded; weighted % refers to valid answers Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37 Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37
Pa Page 7 of 16 Table 2 Use of prescription and illicit drugs to enhance cognitive performance or mood without medical indication
Substance group / Single substancesa
nb
Lifetime %c (n)
Last year %c (n)
Last month %c (n)
Last week %c (n)
Stimulating prescription drugs
1115
4.3 (48)
2.2 (25)
0.8 (9)
0.3 (4)
Prescription drug containing amphetamines
1112
1.7 (19)
0.8 (9)
0.3 (4)
–
Methylphenidate
1110
2.2 (25)
1.1 (12)
0.3 (3)
0.1 (1)
Anti-dementia drug
1110
1.0 (11)
0.6 (6)
0.2 (2)
0.2 (2)
Modafinil
1.107
0.4 (4)
0.1 (1)
0.1 (1)
0.1 (1)
Stimulating illicit drugs
1118
10.2 (114)
3.8 (42)
1.3 (14)
0.6 (6)
Cocaine
1114
6.1 (68)
1.9 (21)
0.3 (4)
0.1 (1)
Amphetamines
1115
6.9 (77)
2.5 (28)
0.8 (9)
0.3 (4)
Meth-Amphetamines
1111
2.0 (22)
0.6 (6)
0.1 (1)
0.1 (1)
Mood modulating prescription drugs
1110
20.3 (225)
10.6 (118)
5.9 (66)
5.6 (62)
Anti-depressant
1096
8.5 (93)
4.0 (44)
1.6 (18)
1.5 (16)
Beta blocker
1080
8.5 (92)
5.2 (56)
4.1 (45)
4.0 (43)
Benzodiazepines
1088
8.9 (98)
3.5 (38)
0.9 (10)
0.7 (8)
Cannabis
1109
23.4 (260)
8.6 (94)
3.7 (40)
2.8 (30)
Any medication or drug
1121
38.8 (435)
19.1 (214)
10.1 (113)
8.5 (95)
N = 1128
aAs multiple selections were possible and some individuals used several substances, values could not be added up per substance group; bN refers to valid values,
i.e. Stress, the ability to recover from stress and resilience the resilience factor optimism was the main factor
explaining stimulating illicit drug use, but not the ability
to recover from stress, perceived stress level or the other
resilience factors. Here, each additional unit on the
SOP-2 (i.e. the increasing level of optimism of the sub-
ject) decreased the likelihood for the use of stimulating
illicit drugs compared to non-use by 37% (OR: .63; 95%
CI: .47–.86). The final model explained 45% of the vari-
ance (R2: .45) (4). Prior to multivariate logistic regression analyses, we ex-
amined the correlations between predictor variables
(BRS, PSS-4, ASKU, SOP-2, IE) to assess for potential
multicollinearity (Additional file 2). We found medium
to high correlations between the predictor variables
(range: −.39 to .62). However, the correlations were
below the recommended cut-off (.80) indicating that
these variables all assess different constructs and multi-
collinearity can be excluded. The VIF is > 10 which is
below the recommended cut-off providing additional
evidence against multicollinearity. Table 5 shows the first and the last step of the adjusted
multivariate logistic regression model for users of canna-
bis compared to non-users. Here again, only the degree
of optimism as measured by the SOP-2 remained as pre-
dictor in the final model (step 6) meaning that the resili-
ence factor optimism, but not the ability to recover from
stress, the perceived stress level or the other resilience
factors were main factors explaining the use of cannabis. Overall, each additional unit on the SOP-2 (i.e. the in-
creasing level of optimism of the subject) decreases the
likelihood of cannabis use by 26% (OR: .74; 95% CI:
.59–.92). The final model explained 33% of the variance
(R2: .33) (Table 5). Table 5 shows the first and the last step of the adjusted
multivariate logistic regression model for users of canna-
bis compared to non-users. Here again, only the degree
of optimism as measured by the SOP-2 remained as pre-
dictor in the final model (step 6) meaning that the resili-
ence factor optimism, but not the ability to recover from
stress, the perceived stress level or the other resilience
factors were main factors explaining the use of cannabis. Table 3 shows the first and the last step of the adjusted
multivariate logistic regression model for mood modu-
lating prescription drugs. In that subgroup, only the BRS
remained as predictor in the final model (step 6). Reasons and goals for PNE all observations without missing values in the respective question; cWeighted according to the distribution of the general population in Germany as reported
by the German office of national statistics, n refers to the absolute frequency and % refers to the relative frequency of participants that have taken the respective
substance ever in their life, in the last year, last month or last week aAs multiple selections were possible and some individuals used several substances, values could not be added up per substance group; bN refers to valid values,
i.e. all observations without missing values in the respective question; cWeighted according to the distribution of the general population in Germany as reported
by the German office of national statistics, n refers to the absolute frequency and % refers to the relative frequency of participants that have taken the respective
substance ever in their life, in the last year, last month or last week aAs multiple selections were possible and some individuals used several substances, values could not be added up per substance group; bN refers to valid values,
i.e. all observations without missing values in the respective question; cWeighted according to the distribution of the general population in Germany as reported
by the German office of national statistics, n refers to the absolute frequency and % refers to the relative frequency of participants that have taken the respective
substance ever in their life, in the last year, last month or last week Stress, the ability to recover from stress and resilience Thus,
the ability to recover from stress, but not the other pre-
dictors was the main factor explaining the use of mood
modulation prescription drugs. Each additional unit on
the BRS (i.e. the increasing ability to recover from stress)
decreases the likelihood for the use of mood modulating
prescription drugs by 38% (OR: .62; 95% CI: .47–.81)
(see Table 3). The final model explained 36% of the vari-
ance (R2: .36) (3). Overall, each additional unit on the SOP-2 (i.e. the in-
creasing level of optimism of the subject) decreases the
likelihood of cannabis use by 26% (OR: .74; 95% CI:
.59–.92). The final model explained 33% of the variance
(R2: .33) (Table 5). Table 6 shows the first and the last step of the adjusted
multivariate logistic regression model for users of stimu-
lating prescription drugs compared to non-users. Only
the level of perceived stress as measured by the PSS-4
remained as predictor in the final model (step 6). This
means that perceived stress, but not the ability to re-
cover from stress or any of the resilience factors was the
main factor explaining stimulating prescription drug use. Table 4 shows the first and the last step of the adjusted
multivariate logistic regression model for users of stimu-
lating illicit drugs compared to non-users. Only the de-
gree of optimism as measured by the SOP-2 remained as
predictor in the final model (step 6). This means that Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37 Page 8 of 16 Fig. 1 Coping with stressful situations as a reason for the substance intake. Mean scores in the item “What was the reason for the intake? To
cope with stressful situations.” Likert scale average (1- I totally disagree to 5 – I totally agree). Stimulating prescription drugs: use of prescription
drugs containing amphetamines, methylphenidate, modafinil and/or anti-dementia drugs; Stimulating illicit drugs: use of cocaine, amphetamines
and/ or meth-amphetamines; Mood modulating prescription drugs: use of anti-depressants, beta blocker and/ or benzodiazepines); Cannabis: use
of cannabis; Any drug: use of any of the listed substances Fig. 1 Coping with stressful situations as a reason for the substance intake. Mean scores in the item “What was the reason for the intake? To
cope with stressful situations.” Likert scale average (1- I totally disagree to 5 – I totally agree). Stress, the ability to recover from stress and resilience Stimulating prescription drugs: use of prescription
drugs containing amphetamines, methylphenidate, modafinil and/or anti-dementia drugs; Stimulating illicit drugs: use of cocaine, amphetamines
and/ or meth-amphetamines; Mood modulating prescription drugs: use of anti-depressants, beta blocker and/ or benzodiazepines); Cannabis: use
of cannabis; Any drug: use of any of the listed substances Each unit on the PSS-4 (i.e. the increasing level of per-
ceived stress) increases the likelihood for the use of
stimulating prescription drugs almost three times (OR:
2.89; 95% CI: 1.49–5.46). The final model explains 45%
(R2: .45) of variance in that subgroup (Table 6). Each unit on the PSS-4 (i.e. the increasing level of per-
ceived stress) increases the likelihood for the use of
stimulating prescription drugs almost three times (OR:
2.89; 95% CI: 1.49–5.46). The final model explains 45%
(R2: .45) of variance in that subgroup (Table 6). higher prevalence rates found in this study by including
more users with different motives for the substance intake. In this study, we decided to use a broader definition of
PNE since it refers not only to the objective of
achieving higher cognitive performance, but also to
reaching a mental state which allows coping with
daily tasks. In contrast, previous studies only assessed
the intake of prescription stimulants [4, 13, 19] or
prescription medication [1]. Discussion This is the first study providing data on the use of pre-
scription and illicit drugs for PNE in a representative
sample in Germany. In addition, it contributes to previ-
ous studies in the field of PNE use by investigating the
relationship between PNE use and the ability to recover
from stress for the first time. Second, the use of psychoactive substances varies be-
tween different study populations. Previous studies were
limited to specific groups such as students or pupils,
whereas our survey delivers representative data for the
German adult population. We found a lifetime prevalence rate of any substance
use for PNE of 38.8% which was higher compared to
previous studies (1–20%) [4, 7, 16–20] . There are several
possible explanations for the higher prevalence rates in
our survey compared to other studies. First of all, we used
a broader definition of PNE which referred not only to
PNE use for enhancing cognitive performance but also to
improving mood or reducing nervousness without med-
ical indication. This broader definition may explain the Third, by using a closed envelope technique for sensi-
tive questions, we provided a high degree of confidenti-
ality for the respondents which may have resulted in a
higher and more reliable prevalence rate compared to
studies using less anonymous techniques. Indeed, previ-
ous studies providing a higher degree of confidentiality,
such as online surveys or surveys using the randomized
response technique, revealed higher prevalence rates Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37 Page 9 of 16 Table 3 Adjusted multivariate model of factors associated with use of mood modulating prescription drugs
Variables
step 1a
step 6a
Coeff. (SE)
OR (95% CI)
p
VIF
Coeff. Discussion However, it is likely that the majority of cannabis
users took the substance many years ago only for a short
period of time since the last year prevalence already de-
clined to 9%. Last week prevalence (which can be con-
sidered as an indicator for current regular use) was only
3% for cannabis. In comparison, mood modulating pre-
scription drugs seem to play a more important role in
everyday life as we found a last week prevalence of 6%. But nevertheless the use of both substance groups is
associated with “stress” in a broader context, as both
user-groups named “stress coping” as an important goal
for the substance use. Neither the ability to recover from stress nor perceived
stress were associated with the use of stimulating illicit
drugs or cannabis in this study. However, in a previous
study among German students, participants using can-
nabis for the purpose of cognitive enhancement reported
more stress than non-users as they perceived the pres-
sure to perform as more burdening [48]. Since these
analyses referred to different stress measurements and
were not controlled for other factors (as in our multi-
variate model), it is not possible to directly compare the
results. We also identified an association between the
use of stimulating illicit drugs or cannabis and a higher
level of pessimism. This is in line with previous studies
describing pessimism as a risk factor of illicit drug con-
sumption [49]. y
y
p
A major goal of this study was to assess the relation-
ship of substance use and perceived stress, the ability to
cope with stress and three resilience factors (self-efficacy,
locus of control and optimism [37, 38]). The individual
ability to recover from stress (as measured by the BRS)
was associated with a lower risk of mood modulating
drug use. Furthermore, subjects reporting high perceived
stress (as measured by the PSS-4) were more likely to
use stimulating prescription drugs such as methylphen-
idate, modafinil or amphetamines. These results provide
evidence for a slightly differential use of stimulating pre-
scription drugs and mood modulating drugs. With re-
gard to the effect sizes, the adjusted multivariate models
used explain 36% of the variance for the use of mood
modulating prescription drugs and 45% of the variance
for the use of stimulating prescription drugs. Regarding
the predictor variables we found clinical meaningful ef-
fects. Discussion (SE)
OR (95% CI)
p
BRS
−.43 (.18)
.65 (.46–.93)
.02
2.02
−.48 (.14)
.62 (.47–.81)
<.001
PSS-4
−.12 (.18)
.88 (.63–1.25)
.48
1.77
–
–
–
SOP-2
−.09 (.17)
.91 (.65–1.28)
.60
1.70
–
–
–
ASKU
.04 (.20)
1.04 (.71–1.52)
.84
1.72
–
–
–
IE-I
.09 (.18)
1.09 (.77–1.55)
.63
1.63
–
–
–
IE-E
.27 (.18)
1.31 (.92–1.86)
.14
1.60
–
–
–
Sex:
Male
Reference
1.15
Reference
Female
−.31 (.29)
.73 (.42–1.29)
.28
–
–
–
Age
.54 (.20)
1.72 (1.16–2.55)
.01
1.11
.53 (.20)
1.70 (1.15–2.50)
.01
Education:
No formal degree
Reference
1.11
Reference
Secondary modern school
−.73 (1.45)
.48 (.028–8.29)
.61
–
–
–
Middle school
−.17 (.35)
.84 (.42–.1.69)
.63
–
–
–
University-entrance diploma
−.16 (.42)
.85 (.38–1.93)
.70
–
–
–
University degree
−.17 (.45)
.84 (.35–2.02)
.70
–
–
–
Current or last professional position:
Skilled worker
Reference
1.05
Reference
Executive employee
.12 (.57)
1.13 (.37–3.44)
.83
–
–
–
Non-executive employee
.46 (.46)
1.58 (.64–3.92)
.32
–
–
–
Civil servants
−.33 (.77)
.72 (.16–.3.24)
.67
–
–
–
Self-employed
.78 (.64)
2.17 (.62–7.63)
.23
–
–
–
Other
.13 (.57)
1.14 (.37–3.46)
.82
–
–
–
Place of residence:
North Rhine-Westphalia
Reference
1.07
Reference
Hamburg
.55 (.78)
1.73 (.37–7.98)
.48
–
–
–
Lower Saxony
.09 (.50)
1.09 (.41–2.90)
.85
–
–
–
Bremen
.47 (1.77)
1.60 (.05–51.41)
.79
–
–
–
Schleswig Holstein
.27 (.71)
1.32 (.33–5.24)
.70
–
–
–
Hesse
−.24 (.56)
.78 (.26–2.35)
.78
–
–
–
Rhineland-Palatine
.30 (.68)
1.35 (.36–5.07)
.66
–
–
–
Baden-Wuerttemberg
.16 (.43)
1.17 (.50–2.75)
.71
–
–
–
Bavaria
−.13 (.43)
.88 (.38–2.03)
.77
–
–
–
Saarland
−.1.73 (1.49)
.18 (.01–3.30)
.25
–
–
–
Berlin
−.56 (.78)
.57 (.12–2.63)
.47
–
–
–
Brandenburg
1.18 (.76)
3.24 (.71–14.81)
.13
–
–
–
Mecklenburg-Western Pomerania
n. a. n. a. n. a. n. a. –
–
–
Saxony
−1.07 (.77)
.35 (.08–1.56)
.17
–
–
–
Saxony-Anhalt
−1.25 (.89)
.29 (.05–1.65)
.16
–
–
–
Thuringia
−2.69 (1.34)
.07 (.005–.94)
.05
–
–
– Table 3 Adjusted multivariate model of factors associated with use of mood modulating prescription drugs Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37
Page 10 of 16 Bagusat et al. Discussion Substance Abuse Treatment, Prevention, and Policy (2018) 13:37 Page 10 of 16 Table 3 Adjusted multivariate model of factors associated with use of mood modulating prescription drugs (Continued)
Variables
step 1a
step 6a
Coeff. (SE)
OR (95% CI)
p
VIF
Coeff. (SE)
OR (95% CI)
p
R2b
Soft enhancer intake:
No
Reference
1.06
Reference
Yes
2.29 (.35)
9.89 (5.02–19.51)
<.001
2.25 (.34)
9.49 (4.86–18.52)
.62
Logistic regression with backward elimination of factors associated with use of mood modulating prescription drugs (users n = 114; non-users = 686). First step
and final step. n.a. = no data available
aStep 1 (full model) and step 6 (reduced model) in multivariate logistic regression using backward variable selection. Predictors were z-standardized before being
included in regression analysis. Sociodemographic variables significant in the analyses of mean differences (sex, age, education, current or last professional
position, size place of residence, soft enhancer intake) were included as a block in multivariate backward logistic regression. Results are weighted according to the
distribution of the general population in Germany as reported by the German office of national statistics. bNagelkerkes R2 in step 6 of stepwise backward selection;
Coeff. standardised regression coefficient, SE standard error, OR odds ratio, CI confidence interval, p p value, VIF variance inflation factor (based on multivariate linear
regression), BRS Brief Resilience Scale, PSS-4 Perceived Stress Scale, SOP-2 Optimism-Pessimism-2 Scale, ASKU Short Scale for Measuring General Self-efficacy Beliefs, IE-I
Short Scale for the Assessment of Locus of Control, internal control beliefs; IE-E: Short Scale for the Assessment of Locus of Control, external control beliefs ariate model of factors associated with use of mood modulating prescription drugs (Continued) with respect to sensitive questions as compared to
standard, non-anonymous surveys [7, 15, 16]. the lower CI the likelihood would still be 50% higher. Our results are also in line with previous research on
the effect of risk factors on PNE use. In a previous study
on surgeons [16], we found that ORs that were lower or
in a similar range (e.g., ‘pressure to perform at work’: OR
1.29, 95% CI: 1.00 to 1.67; ‘gross income’: OR 1.34, 95%
CI: 1.09 to 1.64). At first sight, cannabis seems to play the most import-
ant role for PNE in our survey as we found the highest
lifetime prevalence (23%) compared to other substance
groups. Discussion The OR for each additional unit on the BRS was
.62 (a decrease of 38%) for mood modulating prescrip-
tion drugs with a 95% CI of .47 to .81 (53% to 19%). This
means that there is a 95% probability that the population
parameter lies within the interval, when considering the
lower CI that would result in a decrease in the likelihood
of 19%. The OR for each additional unit on the PSS-4
was 2.89 (an almost threefold increase) for stimulating
prescription drugs with a 95% CI of 1.49 to 5.46 (a 1.5
to 5.5-fold increase), which means that when considering Being pessimistic could be connected with the goal to
improve mood which was a more important goal for the
use of illicit than for prescription drugs. This could indi-
cate different patterns of substance use: prescription
drugs are rather used goal-oriented as an instrument in
stressful situations as a coping strategy whereas illicit
drugs are rather used with more general underlying
goals such as improving mood. This corresponds with
results of a study that indicates that stress coping is a
more prevalent goal for the use of prescription medica-
tion than for the use of illicit drugs. This study also
showed that improving mood measured by the goal “eu-
phoria” seems to be more important for the use of illicit
drugs than for prescription medication [50]. We did not Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37 Page 11 of 16 Table 4 Adjusted multivariate model of factors associated with use of stimulating illicit drugs
Variables
step 1a
step 6a
Coeff. (SE)
OR (95% CI)
p
VIF
Coeff. Discussion (SE)
OR (95% CI)
p
BRS
−.14 (.24)
.87 (.55–1.40)
.57
1.99
–
–
–
PSS-4
−.01 (.23)
1.00 (.64–1.55)
.99
1.79
–
–
–
SOP-2
−.34 (.21)
.71 (.48–1.07)
.10
1.72
−.46 (.16)
.63 (.47–.86)
<.01
ASKU
−.11 (.25)
.89 (.55–1.45)
.65
1.71
–
–
–
IE-I
.07 (.22)
1.07 (.69–1.66)
.77
1.61
–
–
–
IE-E
.13 (.24)
1.13 (.71–1.80)
.60
1.63
–
–
–
Sex:
Male
Reference
1.14
Reference
Female
−1.17 (.36)
.31 (.15–.63)
.001
−1.11 (.35)
.33 (.17–.66)
<.01
Age
−.90 (.26)
.41 (.24–.68)
.001
1.19
−.92 (.26)
.40 (.24–.66)
<.001
Education:
No formal degree
Reference
1.15
Reference
Secondary modern school
2.80 (1.24)
16.48 (1.45–187.61)
.02
2.65 (1.22)
14.09 (1.30–152.86)
.3
Middle school
−.12 (.43)
.89 (.38–2.07)
.78
–
–
–
University-entrance diploma
−.62 (.51)
.54 (.20–1.45)
.22
–
–
–
University degree
−.85 (.66)
.43 (.12–1.57)
.20
–
–
–
Current or last professional position:
Skilled worker
Reference
1.06
Reference
Executive employee
−.07 (.71)
.93 (.23–3.70)
.92
–
–
–
Non-executive employee
.73 (.51)
2.07 (.76–5.63)
.15
–
–
–
Civil servants
−.07 (1.08)
.93 (.22–7.74)
.95
–
–
–
Self-employed
.78 (.84)
2.17 (.42–11.2)
.35
–
–
–
Other
.39 (.61)
1.47 (.44–4.89)
.53
–
–
–
Place of residence:
North Rhine-Westphalia
Reference
1.10
Reference
Hamburg
2.20 (.95)
9.00 (1.39–58.06)
.02
2.14 (.94)
8.53 (1.37–53.35)
.02
Lower Saxony
−.11 (.749
.89 (.21–3.83)
.89
–
–
–
Bremen
3.35 (1.46)
28.36 (1.64–491.68)
.02
3.22 (1.43)
25.08 (1.53–411.61)
.02
Schleswig Holstein
−.37 (1.10)
.69 (.08–5.91)
.74
–
–
–
Hesse
1.00 (.62)
2.72 (.80–9.23)
.11
–
–
–
Rhineland-Palatine
.82 (1.00)
2.27 (.32–16.04)
.41
–
–
–
Baden-Wuerttemberg
.66 (.56)
1.93 (.64–5.79)
.24
–
–
–
Bavaria
.004 (.612)
1.00 (.30–3.33)
.99
–
–
–
Saarland
n.a. n.a. n.a. n.a. n.a. n.a. n.a. Berlin
1.66 (7.43)
5.28 (1.23–22.66)
.03
1.53 (.73)
4.62 (1.11–19.27)
.04
Brandenburg
2.04 (1.13)
7.71 (.84–70.71)
.07
–
–
–
Mecklenburg-Western Pomerania
.86 (.86)
2.36 (.44–12.83)
.32
–
–
–
Saxony
.62 (.77)
1.85 (.41–8.36)
.32
–
–
–
Saxony-Anhalt
−1.43 (1.42)
.24 (.02–3.89)
.31
–
–
–
Thuringia
.02 (1.16)
1.02 (.11–9.9)
.99
–
–
–
Soft enhancer intake:
No
Reference
1.12
Reference
Yes
3.02 (.60)
20.39 (6.35–65.52)
<.001
3.03 (.60)
20.73 (6.46–66.47)
<.001
Logistic regression with backward elimination of factors associated with use of stimulating illicit drugs (users n = 114; non-users = 686). First step
final step. n.a. Discussion = no data available
Further notes see Table 3 Table 4 Adjusted multivariate model of factors associated with use of stimulating illicit drugs Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37 Page 12 of 16 Table 5 Adjusted multivariate model of factors associated with use of cannabis
Variables
step 1a
step 6a
Coeff. (SE)
OR (95% CI)
p
VIF
Coeff. (SE)
OR (95% CI)
p
BRS
−.13 (.16)
.88 (.64–1.21)
.44
1.96
–
–
–
PSS-4
−.06 (.15)
.94 (.70–1.27)
.68
1.75
–
–
–
SOP-2
−.14 (.15)
.87 (.66–1.16)
.34
1.68
−.31 (.11)
.74 (.59–.92)
.01
ASKU
−.11 (.17)
.89 (.64–1.25)
.51
1.73
–
–
–
IE-I
−.12 (.15)
.89 (.66–1.12)
.44
1.65
–
–
–
IE-E
.14 (.15)
1.15 (.86–1.54)
.34
1.54
–
–
–
Sex:
Male
Reference
1.11
Reference
Female
−.67 (.24)
.51 (.32–.82)
.51
−.63 (.23)
.53 (.34–.84)
.01
Age
−.69 (.17)
.50 (.36–.70). Discussion <.001
1.14
−.68 (.17)
.50 (.36–.70)
<.001
Education:
No formal degree
Reference
1.16
Reference
Secondary modern school
1.68 (.96)
5.38 (.81–35.62)
.08
–
–
–
Middle school
−.02 (.32)
.99 (.53–1.82)
.96
–
–
–
University-entrance diploma
−.14 (.37)
.87 (.42–1.80)
.71
–
–
–
University degree
.60 (.39)
1.82 (.85–3.90)
.12
–
–
–
Current or last professional position:
Skilled worker
Reference
Reference
Executive employee
−.49 (.47)
.61 (.24–1.53)
.29
1.05
–
–
–
Non-executive employee
.05 (.36)
1.05 (.52–2.14)
.89
–
–
–
Civil servants
−.10 (.59)
.91 (.29–2.88)
.87
–
–
–
Self-employed
−.29 (.60)
.75 (.23–2.43)
.63
–
–
–
Other
.15 (.43)
1.16 (.50–2.70)
.73
–
–
–
Place of residence:
North Rhine-Westphalia
Reference
1.05
Reference
Hamburg
.94 (.69)
2.56 (.67–9.77)
.17
–
–
–
Lower Saxony
.01 (.45)
1.01 (.42–2.43)
.98
–
–
–
Bremen
.29 (1.15)
1.34 (.14–12.60)
.80
–
–
–
Schleswig Holstein
.01 (.71)
1.01 (.25–4.07)
.99
–
–
–
Hesse
.71 (.43)
2.04 (.87–4.74)
.10
–
–
–
Rhineland-Palatine
1.11 (.59)
3.04 (.95–9.74)
.06
–
–
–
Baden-Wuerttemberg
.66 (.39)
1.92 (.89–4.15)
.10
–
–
–
Bavaria
.34 (.38)
1.40 (.67–2.95)
.37
–
–
–
Saarland
−1.62 (1.06)
.20 (.03–1.58)
.13
–
–
–
Berlin
.62 (.55)
1.86 (.63–5.49)
.26
–
–
–
Brandenburg
1.63 (.72)
5.10 (1.24–20.92)
.03
–
–
–
Mecklenburg-Western Pomerania
.31 (.73)
1.36 (.32–5.72)
.68
–
–
–
Saxony
−.03 (.55)
1.03 (.35–3.00)
.96
–
–
–
Saxony-Anhalt
−1.87 (1.03)
.16 (.02–1.17)
.07
–
–
–
Thuringia
−.17 (.74)
.84 (.20–3.61)
.82
–
–
–
Soft enhancer intake:
No
Reference
1.09
Reference
Yes
1.79 (.26)
5.96 (3.55–10.01)
<.001
1.80 (.26)
6.05 (3.63–10.08)
<.001
Logistic regression with backward elimination of factors associated with use of cannabis (users n = 260; non-users = 686). First step and final s
n.a. = no data available
Further notes see Table 3 Table 5 Adjusted multivariate model of factors associated with use of cannabis Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37
Page 13 of 16 Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37 Page 13 of 16 Table 6 Adjusted multivariate model of factors associated with use of stimulating prescription drugs
Variables
step 1a
step 6a
Coeff. (SE)
OR (95% CI)
p
VIF
Coeff. Acknowledgements The authors want to thank Elisabeth Hildt, Oliver Quiring, Markus Schäfer,
Sabine Pohl and Hannes Boelsen for their supportive cooperation within the
common research project. We also thank Thomas Petersen from the “Institut
für Demoskopie Allensbach” for consulting and data collection and all
participating subjects for cooperation. Fourth, we did not adjust for multiple testing which
may increase the risk of type 1 error inflation. However,
our main results regarding the relationship of substance
use and perceived stress, the ability to cope with stress
and three resilience factors, the respective p-values of
the odds ratios are between p = .006 and p < .001. We
therefore infer that our main results are robust and not
affected by alpha inflation. Abbreviations ASKU: Short Scale for Measuring General Self-Efficacy Beliefs; BRS: Brief
Resilience Scale; CI: Confidence intervall; Coeff: Standardised regression
coefficient; IE-E: Short Scale for the Assessment of Locus of Control,
external control beliefs.; IE-I: Short Scale for the Assessment of Locus of
Control, internal control beliefs.; M: Mean; OR: Odds ratio; PNE: Pharmacological
neuroenhancement; PSS-4: Perceived Stress Scale; SD: Standard deviation;
SE: standard error; SOP-2: Optimism-Pessimism-2 Scale; VIF: Variance
inflation factor Funding The study was funded by the Federal Ministry of Education and Research No. 01GP1303A (BMBF). Availability of data and materials
h d
d
d
l
d d The datasets used and analysed during the current study are available from
the corresponding author on reasonable request. Fifth, data were collected cross-sectionally in this
study. As a consequence, the analyses are only explora-
tive and causal inferences about the associations found
cannot be drawn. The effects of low ability to recover
from stress and high perceived stress on PNE use and
the potential positive effects of PNE use on these vari-
ables cannot be disentangled. This limits the conclusions
in this study and underlines the importance of conduct-
ing longitudinal studies in this field. Authors’ contributions CB made substantial contributions to conception and design, acquisition of
data, analysis and interpretation of data and has been involved in drafting
the manuscript. AK made substantial contributions to analysis and interpretation
of data and has been involved in drafting the manuscript. JS made substantial
contributions to analysis and interpretation of data and has been involved in
drafting the manuscript. AGF made substantial contributions to conception and
design of data and has been involved in drafting the manuscript. AC made
substantial contributions to conception, analysis and interpretation of data and
has been involved in drafting the manuscript. KL made substantial contributions
to conception and design and has been involved in drafting the manuscript. All
authors have given final approval of the version to be published and agreed to
be accountable for all aspects of the work. Additional files Third, in this study we only used the BRS as proxy
measure for resilience as outcome by assessing the abil-
ity to recover from stress. To date, the BRS is the only
scale that was developed based on an outcome definition
of resilience [53]. Other existing resilience scales are
either based on a trait-oriented approach (e.g., Disposi-
tional Resilience Scale [DRS]) [54]or focus on measuring
the availability of resources and protective factors to
maintain or regain mental health despite significant adver-
sities (e.g., Connor-Davidson Resilience Scale [CD-RISC])
[55]. To assess resilience as defined (i.e., mental health
despite stress), the individual stressor exposure should
also be considered when measuring resilience, as sug-
gested by Kalisch and colleagues [56]. As this was not pos-
sible in the current study, future representative surveys on
resilience and PNE use are required. Additional file 1: Sample characteristics per substance group. (DOCX 70 kb)
Additional file 2: Correlations between the ability to recover from stress,
perception of stress and resilience factors. (DOCX 27 kb) Additional file 1: Sample characteristics per substance group. (DOCX 70 kb)
Additional file 2: Correlations between the ability to recover from stress,
perception of stress and resilience factors. (DOCX 27 kb) Discussion (SE)
OR (95% CI)
p
BRS
−.72 (.42)
.49 (.21–1.12)
.09
2.11
–
–
–
PSS-4
.78 (.43)
2.19 (.95–5.05)
.07
1.75
1.05 (.33)
2.86 (1.49–5.46)
<.01
SOP-2
.01 (.38)
1.00 (.48–2.13)
.98
1.76
–
–
–
ASKU
.07 (.5)
1.07 (.40–2.82)
.90
1.76
–
–
–
IE-I
.18 (.43)
1.19 (.52–2.74)
.68
1.66
–
–
–
IE-E
−.16 (.38)
.86 (.40–1.81)
.68
1.60
–
–
–
Sex:
Male
Reference
1.15
Reference
Female
−.15 (.60)
.86 (.27–2.76)
.80
–
–
–
Age
−.89 (.47)
.41 (.17–1.03)
.06
1.21
.53 (.20)
1.70 (1.15–2.50)
<.01
Education:
No formal degree
Reference
1.11
Reference
Secondary modern school
n.a. n.a. n.a. –
–
–
Middle school
−1.21 (.83)
.30 (.06–1.53)
.15
–
–
–
University-entrance diploma
.07 (.81)
1.08 (.22–5.28). .93
–
–
–
University degree
−.61 (1.09)
.54 (.06–4.61)
.57
–
–
–
Current or last professional position:
Skilled worker
Reference
Reference
Executive employee
2.16 (1.25)
8.69 (.75–100.30)
.08
1.05
–
–
–
Non-executive employee
1.00 (1.15)
2.72 (.29–25.62)
.38
–
–
–
Civil servants
1.78 (1.75)
5.92 (.19–181.33)
.31
–
–
–
Self-employed
2.74 (1.41)
15.44 (.98–242.95)
.05
–
–
–
Other
1.76 (1.42)
5.81 (.36–94.30)
.22
–
–
–
Place of residence:
North Rhine-Westphalia
Reference
1.10
Reference
Hamburg
.07 (1.55)
1.07 (0.05–22.47)
.96
–
–
–
Lower Saxony
−1.67 (1.59)
.19 (.01–4.26)
.29
–
–
–
Bremen
n.a. n.a. n.a. –
–
–
Schleswig Holstein
.17 (1.53)
1.18 (.06–23.60)
.91
–
–
–
Hesse
.10 (1.09)
1.10 (.13–9.27)
.93
–
–
–
Rhineland-Palatine
.70 (1.38)
2.01 (.13–30.15)
.61
–
–
–
Baden-Wuerttemberg
.51 (.86)
1.66 (.31–9.03)
.56
–
–
–
Bavaria
−.61 (.97)
.54 (.08–3.63)
.53
–
–
–
Saarland
n.a. n.a. n.a. –
–
–
Berlin
n.a. n.a. n.a. –
–
–
Brandenburg
1.389 (2.18
3.98 (.06–286.58)
.53
–
–
–
Mecklenburg-Western Pomerania
n.a. n.a. n.a. –
–
–
Saxony
−.09 (1.23)
.92 (.08–10.23)
.94
–
–
–
Saxony-Anhalt
n.a. n.a. n.a. –
–
–
Thuringia
n.a. n.a. n.a. −2.62 (1.32)
.07 (.01–.97)
.05
Soft enhancer intake:
No
Reference
1.09
Reference
Yes
2.63 (1.12)
13.83 (1.55–123.54)
.02
2.25 (.34)
9.49 (4.86–18.52)
<.001
Logistic regression with backward elimination) of factors associated with use of stimulating prescription drugs (users n = 48; non-users = 686). Firs
and final step. n.a. Discussion = no data available
Further notes see Table 3 Table 6 Adjusted multivariate model of factors associated with use of stimulating prescription drugs Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37 Page 14 of 16 Page 14 of 16 Page 14 of 16 (2018) 13:37 find an association of any substance use for PNE with
the degree of self-efficacy as was described in an earl-
ier study [51]. [58]. In the future, randomized controlled intervention
studies to support the individual ability to recover from
stress have to be performed in order to demonstrate that
PNE use can be reduced in at risk populations. This study has several limitations. First, we only
assessed adults over 18 years of age which does not
allow any conclusions for younger people that were
identified as an important at risk population for PNE
use [52]. In sum, our results provide evidence that PNE is
not only used to enhance cognitive performance, im-
prove mood or reduce nervousness, but also to cope
with stress. PNE as a strategy for stress management
and measures to prevent its intake should be further
investigated. Second, as we conducted the survey with a quota and
not a probability sample, our data can only be general-
ized to the population in Germany, but not to other
countries. Ethics approval and consent to participate Based on the results of this study, interventions fostering
the ability to recover from stress and reducing perceived
stress could have the potential to prevent PNE use at an
early stage. We are currently investigating the evidence
base (randomized controlled trials) of resilience trainings
available so far [57] and have discussed a methodological
framework of the suitable design of resilience trainings The study was approved by the local Ethics Committee (Landesärztekammer
Rheinland-Pfalz, No 837.209.14, 9448F). Participants were informed about the
objectives of the study, procedures of data storage, the voluntary nature of
study participation and their right to withdraw at any time. Verbal consent
was obtained to guarantee confidentiality. References The construct of resilience: a critical evaluation
and guidelines for future work. Child Dev. 2000;71(3):543–62. and guidelines for future work. Child Dev. 2000;71(3):543–62. 32. Fletcher D, Sarkar M. Psychological Resilience. Eur Psychol. 2013;18(1):12–23. 33. Rutter M. Resilience in the face of adversity. Protective factors and resistance
to psychiatric disorder. Br J Psychiatry J Ment Sci. 1985;147:598–611. 6. Wolff W, Brand R. Subjective stressors in school and their relation to
neuroenhancement: a behavioral perspective on students’ everyday life
‘doping’. Subst Abuse Treat Prev Policy. 2013;8:23. 6. Wolff W, Brand R. Subjective stressors in school and their relation to
neuroenhancement: a behavioral perspective on students’ everyday life
‘doping’. Subst Abuse Treat Prev Policy. 2013;8:23. 34. Reivich K, Shatté A. The resilience factor: seven essential skills for overcoming
life’s inevitable obstacles. New York: Broadway Books; 2002. 7. Dietz P, Striegel H, Franke AG, Lieb K, Simon P, Ulrich R. Randomized response
estimates for the 12-month prevalence of cognitive-enhancing drug use in
university students. Pharmacotherapy. 2013;33(1):44–50. 35. Southwick SM, Litz BT, Charney DS, Friedman MJ. Resilience and mental
health: challenges across the lifespan. Cambridge: Cambridge Press; 2011. 35. Southwick SM, Litz BT, Charney DS, Friedman MJ. Resilience and mental
health: challenges across the lifespan. Cambridge: Cambridge Press; 2011. 36. Franke AG, Schwarze C, Christmann M, Bonertz C, Hildt E, Lieb K. Charakteristika
von Studierenden, die pharmakologisches Neuroenhancement mit
Stimulanzien betreiben: Eine Pilotstudie [characteristics of university
students using stimulants for cognitive enhancement: a pilot study]. Psychiatr Prax. 2012;39(04):174–80. 8. Eickenhorst P, Vitzthum K, Klapp BF, Groneberg D, Mache S. Neuroenhancement
among German University students: motives, expectations, and relationship with
psychoactive lifestyle drugs. J Psychoactive Drugs. 2012;44(5):418–27. psychoactive lifestyle drugs. J Psychoactive Drugs. 2012;44(5):418–27 9. Lengvenyte A, Strumila R, Grikiniene J. Use of cognitive enhancers among
medical students in Lithuania. Nord Stud Alcohol Dr. 2016;33(2):173–87. 37. Southwick SM, Charney DS. Resilience: the science of mastering life’s greatest
challenges. Cambridge: Cambridge University Press; 2012. 10. Maier LJ, Haug S, Schaub MP. Prevalence of and motives for pharmacological
neuroenhancement in Switzerland--results from a national internet panel. Addict Abingdon Engl. 2016;111(2):280–95. 38. Stewart DE, Yuen T. A systematic review of resilience in the physically ill. Psychosomatics. 2011;52(3):199–209. 11. Middendorff E, Poskowsky J, Isserstedt W. Formen der Stresskompansation
und Leistungssteigerung bei Studierenden. HISBUS-Befragung zur Verbreitung
und zu Mustern von Hirndoping und Medikamentenmissbrauch. Hannover:
Forum Hochschule; 2012. 39. Destatis. Mikrozensus 2015 [Internet]. 2016 [cited 2017 Sep 1]. Competing interests 19. Smith ME, Farah MJ. Are prescription stimulants ‘smart pills’? The epidemiology
and cognitive neuroscience of prescription stimulant use by normal healthy
individuals. Psychol Bull. 2011;137(5):717–41. The authors declare that they have no competing interests. 20. Wilens TE, Adler LA, Adams J, Sgambati S, Rotrosen J, Sawtelle R, et al. Misuse
and diversion of stimulants prescribed for ADHD: a systematic review of the
literature. J Am Acad Child Adolesc Psychiatry. 2008;47(1):21–31. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. 21. Repantis D, Schlattmann P, Laisney O, Heuser I. Modafinil and methylphenidate
for neuroenhancement in healthy individuals: a systematic review. Pharmacol
Res. 2010;62(3):187–206. References 1. DAK Versorgungsmanagement. DAK-Gesundheitsreport 2015. Berlin:
medhochzwei-Verlag; 2015. Arbeitsschutz und Arbeitsmedizin, editor. Dortmund; 2015. 26. Bonanno GA, Westphal M, Mancini AD. Resilience to loss and potential trauma. Annu Rev Clin Psychol. 2011;7:511–35. 2. Franke AG, Bagusat C, Rust S, Engel A, Lieb K. Substances used and
prevalence rates of pharmacological cognitive enhancement among
healthy subjects. Eur Arch Psychiatry Clin Neurosci. 2014;264(Suppl 1):
S83–90. 2. Franke AG, Bagusat C, Rust S, Engel A, Lieb K. Substances used and
prevalence rates of pharmacological cognitive enhancement among
healthy subjects. Eur Arch Psychiatry Clin Neurosci. 2014;264(Suppl 1):
S83–90. 27. Feder A, Charney D, Collins K. Neurobiology of resilience. In: Resilience and
Mental Health. Cambridge: Cambridge University Press; 2011. 28. Kalisch R, Baker DG, Basten U, Boks MP, Bonanno GA, Brummelmann E, et al. The resilience framework as a strategy to combat stress-related disorders. Nat Hum Behav. 2017;1:784–90. 3. Greely H, Sahakian B, Harris J, Kessler RC, Gazzaniga M, Campbell P, et al. Towards responsible use of cognitive-enhancing drugs by the healthy. Nature. 2008;456(7223):702–5. 3. Greely H, Sahakian B, Harris J, Kessler RC, Gazzaniga M, Campbell P, et al. Towards responsible use of cognitive-enhancing drugs by the healthy. Nature. 2008;456(7223):702–5. 29. Sapienza JK, Masten AS. Understanding and promoting resilience in children
and youth. Curr Opin Psychiatry. 2011;24(4):267–73. 4. Franke AG, Bonertz C, Christmann M, Huss M, Fellgiebel A, Hildt E, et al. Non-medical use of prescription stimulants and illicit use of stimulants for
cognitive enhancement in pupils and students in Germany. Pharmacopsychiatry. 2011;44(2):60–6. 4. Franke AG, Bonertz C, Christmann M, Huss M, Fellgiebel A, Hildt E, et al. Non-medical use of prescription stimulants and illicit use of stimulants for
cognitive enhancement in pupils and students in Germany. Pharmacopsychiatry. 2011;44(2):60–6. 30. Bonanno GA, Diminich ED. Annual research review: positive adjustment to
adversity – trajectories of minimal–impact resilience and emergent resilience. J Child Psychol Psychiatry. 2013;54(4):378–401. 5. Liakoni E, Schaub MP, Maier LJ, Glauser G-V, Liechti ME. The use of
prescription drugs, recreational drugs, and ‘soft enhancers’ for cognitive
enhancement among Swiss secondary school students. PLoS One. 2015;
10(10):e0141289. 5. Liakoni E, Schaub MP, Maier LJ, Glauser G-V, Liechti ME. The use of
prescription drugs, recreational drugs, and ‘soft enhancers’ for cognitive
enhancement among Swiss secondary school students. PLoS One. 2015;
10(10):e0141289. y
y
y
31. Luthar S, Cicchetti D, Becker B. The construct of resilience: a cri 31. Luthar S, Cicchetti D, Becker B. Received: 2 July 2018 Accepted: 7 October 2018 25. Schröder H, Köhler T, Knerr P, Kühne S, Moesgen D, Klein M. Der Einfluss
psychischer Belastungen am Arbeitsplatz auf das Neuroenhancement -
empirische Untersuchungen an Erwerbstätigen. BAuA - Bundeanstalt für
Arbeitsschutz und Arbeitsmedizin, editor. Dortmund; 2015. Author details
1
f 1Department of Psychiatry and Psychotherapy, University Medical Center
Mainz, Untere Zahlbacher Str. 8, 55131 Mainz, Germany. 2German Resilience
Center (DRZ) gGmbH, Untere Zahlbacher Str. 8, 55131 Mainz, Germany. 3University of Applied Labour Studies, Bundesagentur für Arbeit,
Seckenheimer Landstr. 16, D-68163 Mannheim, Germany. 22. Hildt E, Franke AG. Cognitive Enhancement - An interdisciplinary perspective. Netherlands: Springer; 2013. 23. Douglas T. Human enhancement and supra-personal moral status. Philos
Stud. 2011;162(3):473–97. 24. Wiegel C, Sattler S, Göritz AS, Diewald M. Work-related stress and cognitive
enhancement among university teachers. Anxiety Stress Coping. 2016;29(1):
100–17. Received: 2 July 2018 Accepted: 7 October 2018 Consent for publication
Not applicable Consent for publication
Not applicable Page 15 of 16 Page 15 of 16 Page 15 of 16 Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37 Bagusat et al. Substance Abuse Treatment, Prevention, and Policy Competing interests References Available from:
https://www.destatis.de/DE/ZahlenFakten/GesellschaftStaat/Bevoelkerung/
Mikrozensus.html 40. Smith BW, Dalen J, Wiggins K, Tooley E, Christopher P, Bernard J. The brief
resilience scale: assessing the ability to bounce back. Int J Behav Med. 2008;
15(3):194–200. 12. Ram SS, Hussainy S, Henning M, Jensen M, Russell B. Prevalence of cognitive
enhancer use among New Zealand tertiary students. Drug Alcohol Rev. 2016;35(3):345–51. 41. Chmitorz A, Wenzel M, Stieglitz R-D, Kunzler A, Bagusat C, Helmreich I, et al. Population-based validation of a German version of the brief resilience scale. PLoS One. 2018;13(2):e0192761. 13. Webb JR, Valasek MA, North CS. Prevalence of stimulant use in a sample of
US medical students. Ann Clin Psychiatry. 2013;25(1):27–32. y
y
14. Maher B. Poll results: look who’s doping. Nature. 2008;452(7188):674–5. 42. Warttig SL, Forshaw MJ, South J, White AK. New, normative, English-sample
data for the short form perceived stress scale (PSS-4). J Health Psychol. 2013;18(12):1617–28. 15. Franke AG, Dietz P, Ranft K, Balló H, Simon P, Lieb K. The use of pharmacologic
cognitive enhancers in competitive chess. Epidemiol Camb Mass. 2017;28(6):
e57–8. 43. Stächele HP. Taschenatlas Stress. Linkenheim-Hochstetten: Aesopus Verlag; 2013. 16. Franke AG, Bagusat C, Dietz P, Hoffmann I, Simon P, Ulrich R, et al. Use of
illicit and prescription drugs for cognitive or mood enhancement among
surgeons. BMC Med. 2013;11:102. 44. Beierlein C, Kemper CJ, Kovaleva A, Rammstedt B. Ein Messinstrument zur
Erfassung subjektiver Kompetenzerwartungen – Allgemeine Selbstwirksamkeit
Kurzskala (ASKU) [Internet]. Köln: GESIS; 2012 [cited 2016 Apr 18]. Available
from: https://pub.uni-bielefeld.de/publication/2575629 17. Castaldi S, Gelatti U, Orizio G, Hartung U, Moreno-Londono AM, Nobile M, et
al. Use of cognitive enhancement medication among northern Italian
university students. J Addict Med. 2012;6(2):112–7. 45. Kovaleva A, Beierlein C, Kemper CJ, Rammstedt B. Eine Kurzskala zur Messung
von Kontrollüberzeugung: Die Skala Internale-Externale-Kontrollüberzeugung-4
(IE-4) [Internet]. 2012 [cited 2014 Aug 19]. Available from: http://pub. uni-bielefeld.de/publication/2575676 18. McCabe SE, Knight JR, Teter CJ, Wechsler H. Non-medical use of prescription
stimulants among US college students: prevalence and correlates from a
national survey. Addict Abingdon Engl. 2005;100(1):96–106. Page 16 of 16 Bagusat et al. Substance Abuse Treatment, Prevention, and Policy (2018) 13:37 46. Kemper CJ, Beierlein C, Kovaleva A, Rammstedt B. Eine Kurzskala zur Messung
von Optimismus-Pessimismus – Die Skala Optimismus-Pessimismus-2 (SOP2)
[Internet]. Köln: GESIS; 2012 [cited 2014 Aug 19]. Available from: http://pub.uni-
bielefeld.de/publication/2575655 47. Eid M, Gollwitzer M, Schmitt M, Regressionsanalyse M. In: Eid M, Gollwitzer M,
Schmitt M, editors. Statistik und Forschungsmethoden. References Basel: Beltz Verlag;
2010. p. 602–98. 48. Franke AG, Roser P, Lieb K, Vollmann J, Schildmann J. Cannabis for cognitive
enhancement as a new coping strategy? Results from a survey of students
at four universities in Germany. Subst Use Misuse. 2016;51(14):1856–62. 49. Wray TB, Dvorak RD, Hsia JF, Arens AM, Schweinle WE. Optimism and
pessimism as predictors of alcohol use trajectories in adolescence. J Child Adolesc Subst Abuse. 2013;22(1):58–68. 50. Brand R, Wolff W, Ziegler M. Drugs as instruments: describing and testing a
behavioral approach to the study of Neuroenhancement. Front Psychol. 2016;7:1226. 51. Maier LJ, Haug S, Schaub MP. The importance of stress, self-efficacy, and
self-medication for pharmacological neuroenhancement among employees
and students. Drug Alcohol Depend. 2015;156:221–7. 52. Wilens TE, Gignac M, Swezey A, Monuteaux MC, Biederman J. Characteristics
of adolescents and young adults with ADHD who divert or misuse their
prescribed medications. J Am Acad Child Adolesc Psychiatry. 2006;45(4):
408–14. 53. Windle G, Bennett KM, Noyes J. A methodological review of resilience
measurement scales. Health Qual Life Outcomes. 2011;9:8. 54. Bartone PT. Test-retest reliability of the dispositional resilience scale-15, a
brief hardiness scale. Psychol Rep. 2007;101(3(1)):943–4. 54. Bartone PT. Test-retest reliability of the dispositional resil brief hardiness scale. Psychol Rep. 2007;101(3(1)):943–4. 55. Connor KM, Davidson JRT. Development of a new resilience s 55. Connor KM, Davidson JRT. Development of a new resilience scale: the Connor-
Davidson resilience scale (CD-RISC). Depress Anxiety. 2003;18(2):76–82. 56. Kalisch R, Müller MB. Tüscher O. A conceptual framework for the neurobiological
study of resilience. Behav Brain Sci. 2015:1–49. 57. Helmreich I, Kunzler A, Chmitorz A, König J, Binder H, Wessa M, et al. Psychological interventions for resilience enhancement in adults (protocol). Cochrane Database Syst Rev. 2016. 58. Chmitorz A, Kunzler A, Helmreich I, Tüscher O, Kalisch R, Kubiak T, et al. Intervention studies to foster resilience – a systematic review and proposal
for a resilience framework in future intervention studies. Clin Psychol Rev. 2018 Feb 1;59:78–100.
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Genetic variability of Pantaneiro horse using RAPD-PCR markers
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Revista Brasileira de Zootecnia
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Andréa Alves do Egito1, Beatriz Helena Fuck2,3, Concepta McManus2, Samuel Rezende
Paiva2, Maria do Socorro Maués Albuquerque2, Sandra Aparecida Santos4, Urbano Gomes
Pinto de Abreu4, Joaquim Augusto da Silva5, Fabiana Tavares Pires de Souza Sereno6,
Arthur da Silva Mariante1 Andréa Alves do Egito1, Beatriz Helena Fuck2,3, Concepta McManus2, Samuel Rezende
Paiva2, Maria do Socorro Maués Albuquerque2, Sandra Aparecida Santos4, Urbano Gomes
Pinto de Abreu4, Joaquim Augusto da Silva5, Fabiana Tavares Pires de Souza Sereno6,
Arthur da Silva Mariante1 rapa Recursos Genéticos e Biotecnologia, Caixa Postal 02372, CEP: 70770-900, Brasília-DF. ersidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Caixa Postal 04508, CEP: 70910-900, Brasília-DF. Embrapa Recursos Genéticos e Biotecnologia, Caixa Postal 02372, CEP: 70770-900, Brasília-DF. 1 Embrapa Recursos Genéticos e Biotecnologia, Caixa Postal 02372, CEP: 70770-900, Brasília-DF. 1 Embrapa Recursos Genéticos e Biotecnologia, Caixa Postal 02372, CEP: 70770-900, Brasília-DF. 2 Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Caixa Postal 04508, CEP: 70910-900, Brasília-DF. 3 Ministério da Defesa, Exército Brasileiro, Academia Militar das Agulhas Negras, Seção Veterinária Rodovia Presidente Dutra Km 306 CEP
27534-970, Resende-RJ. Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Caixa Postal 04508, CEP: 70910-900, Brasília-DF. Ministério da Defesa, Exército Brasileiro, Academia Militar das Agulhas Negras, Seção Veterinária Rodovia Presidente Dutra Km 30
27534-970 Resende-RJ 4 Embrapa Pantanal, Caixa Postal 109, CEP: 79320-900, Corumbá-MS. 5 Associação Brasileira dos Criadores de Cavalo Pantaneiro, Rua Joaquim Murtinho, 1070, CEP: 78175-000, Poconé-MT. 6 Bolsista do CNPq, Pós-doutorado na Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Caixa Postas 04508,
CEP 70910 900 B
íli
DF 5 Associação Brasileira dos Criadores de Cavalo Pantaneiro, Rua Joaquim Murtinho, 1070, CEP: 78175-000, Poconé-MT. 6 Bolsista do CNPq, Pós-doutorado na Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Caixa Postas 04508,
CEP: 70910-900, Brasília-DF. ç
q
6 Bolsista do CNPq, Pós-doutorado na Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Caixa Postas 04508,
CEP: 70910-900, Brasília-DF. ABSTRACT - Blood samples were collected from Pantaneiro Horses in five regions of Mato Grosso do Sul and Mato Grosso
States. Arabian, Mangalarga Marchador and Thoroughbred were also included to estimate genetic distances and the existing
variability among and within these breeds by RAPD-PCR (Random Amplified Polymorphic DNA – Polymerase Chain
Reaction) molecular markers. From 146 primers, 13 were chosen for amplification and 44 polymorphic bands were generated. The analysis of molecular variance (AMOVA) indicated that the greatest portion of detected variability was due to differences
between individuals within populations (75.47%). Andréa Alves do Egito1, Beatriz Helena Fuck2,3, Concepta McManus2, Samuel Rezende
Paiva2, Maria do Socorro Maués Albuquerque2, Sandra Aparecida Santos4, Urbano Gomes
Pinto de Abreu4, Joaquim Augusto da Silva5, Fabiana Tavares Pires de Souza Sereno6,
Arthur da Silva Mariante1 Analysis of the genetic variability between pairs of populations presented
higher estimates for the five Pantaneiro populations with the Arabian breed, while lowest estimates were presented by pairs
formed among the Pantaneiro populations with the Mangalarga Marchador. Highest genic diversity was shown by the
Pantaneiro (0.3396), which also showed highest genetic distance with the Arabian and lowest with Mangalarga Marchador
breed. UPGMA dendrogram showed distinct differences between naturalized (Pantaneiro and Mangalarga Marchador) and
exotic (Arabian and Thoroughbred) breeds. In the dendrogram generated by UPGMA method, the similarity matrix generated
by the Jaccard coefficient showed distinction between the naturalised breeds, Pantaneiro and Mangalarga Marchador, and the
exotic breeds, Árab and English Thoroughbred. Results suggest that the Pantaneiro presents a higher genetic variability than
the other studied breeds and has a close relationship with the Mangalarga Marchador. Key Words: AMOVA, Arabian, English Thoroughbred, genetic diversity, genetic resource, Mangalarga Marchador Revista Brasileira de Zootecnia
© 2007 Sociedade Brasileira de Zootecnia
ISSN impresso: 1516-3598
ISSN on-line: 1806-9290
www.sbz.org.br Revista Brasileira de Zootecnia
© 2007 Sociedade Brasileira de Zootecnia
ISSN impresso: 1516-3598
ISSN on-line: 1806-9290
www.sbz.org.br R. Bras. Zootec., v.36, n.4, p.799-806, 2007 Correspondências devem ser enviadas para: concepta@unb.br Introduction Five populations of Pantaneiro Horses were studied,
from the following townships: Barra dos Bugres, Cuiabá
and Poconé, in Mato Grosso State and Campo Grande and
Corumbá, in Mato Grosso do Sul State (Table 1). These
townships were chosen because of the large number of
registered animals (Barra dos Bugres and Poconé) and the
presence of conservation nuclei (Campo Grande, Corumbá
and Cuiabá). The Pantaneiro horse is found in the Brazilian Pantanal
and is well adapted to temporary local flooding conditions. It is used for transporting man in the field and leading with
cattle in the region. These animals, descendents of horses
brought to Brazil by the European colonizers after the
discovery. They adapted and multiplied for over two
hundred years and natural selection helped to define the
traits found in the breed today (Sereno, 1997; Mariante
& Cavalcante, 2000). Animals from the Arabian, English Thoroughbred and
Mangalarga Marchador were included as outgroups
(Table 1), because some breeders earlier in the 20th century
used the first two breeds to improve some conformation
traits of the Pantaneiro (Balieiro, 1971; Beck, 1985; Santos,
1993). The Mangalarga Marchador was included to ratify
the relationship observed by Cothran et al. (1998). From 1900 the breed underwent many crosses with the
English-Arabian, Arabian and English Thoroughbred, to
improve conformation and increase stature (Balieiro, 1971;
Beck, 1985). These, associated with diseases such as
Tripanosomiasis and Equine Infectious Anemia were
responsible for the decrease in the population of this
breed. At present, studies developed by the Brazilian
Association of Pantaneiro Horse Breeders and
conservation programs maintain several nuclei of the
breed (Mariante & Cavalcante, 2000; Santos, 2001). Genomic DNA extraction from blood cells of 352 animals
was based on Miller et al. (1988) with slight modifications. A total of 146 decamer oligonucleotide primers (Operon
Technologies Inc., Alameda, Calif.: A, B, E, J, K, N, S and
AB) were screened using unique samples of DNA from
distinct animals and breeds. Primers were designated as
useful if they yielded well-amplified, distinguishable
polymorphic bands. A total of 13 primers (OP AB4, OP A4,
OP A8, OP A11, OP A12, OP A16, OP A19, OP E2, OP E5, OP
E19, OP N3, OP S8 and OP S18) were selected and used to
amplify DNA from all individuals (Table 1). Introduction Farm animal genetic diversity is required to meet current
production needs in various environments, to allow
sustained genetic improvement and to facilitate rapid
adaptation to changing breeding objectives (Notter, 1999). Genetic characterization is the first step in breed conservation
and may have implications for future breeding strategies
(Solis et al. 2005). Cothran et al. (1998), using biochemical
markers, observed that a specific population of Pantaneira
horses was not influenced by recent crosses with other
breeds. Nevertheless, Miserani et al. (2002) found phenotypic
variations between horses from various locations within
the Pantanal. Sereno (2002), using microsatellite markers,
found a high degree of diversity within the Pantaneiro horse
nucleus in the Nhumirim farm, of Embrapa Pantanal and
concluded that the proximity to the Pura Raça Espanhola
horse may indicate Iberian origin. RAPD-PCR reactions were carried out in a final volume
of 13 µl, with 20 mM Tris-HCl (pH 8.4); 50 mM KCl; 3.5 mM
MgCl2; 200 µM of each dNTP; 8% BSA 2.5mg/ml; 0.4 µM of
the arbitrary primer; 1.5 U Taq DNA polymerase (Phoneutria
Biotechnology & Services) and 9 ηg DNA. Reactions were
performed in a MJ Research (PTC100) thermocycler under
40 cycles of 15 second duration at 95ºC, 30 seconds at 35ºC,
1 minute at 72ºC and at the end of the cycle 7 minutes at 72ºC,
for complete extension of the amplified products. Genetic similarity within and between individuals was
estimated from RAPD data with the Jaccard Coefficient
(Sneath & Sokal, 1973), using NTSYS-pc (Numerical
Taxonomy and Multivariate Analysis System), version
2.0 (Exeter Software, Setauked, N.Y.). The genetic
similarity indices were used to generate, using UPGMA
(Sokal & Michener, 1958), a dendrogram based on the
samples used. Random Amplified Polymorphic DNA (RAPD) analysis
is a technique based on the Polymerase Chain Reaction
(PCR) amplification of discrete regions of genome with
short oligonucleotide primers of arbitrary sequence. The
method is simple and quick to perform and no prior knowledge
of the genetic make-up of the organism in question is
required (Lynch & Milligan, 1994). This technique has been
used to detect genetic diversity in equines (Bailey & Lear,
1994; Shiue et al., 1999; Apostolidis et al., 2001). Variabilidade genética do cavalo Pantaneiro utilizando marcadores RAPD-
PCR RESUMO - Amostras de sangue foram coletadas de cavalos Pantaneiros de cinco regiões dos estados de Mato Grosso do
Sul e Mato Grosso. As raças Mangalarga Marchador, Árabe e Puro-Sangue Inglês (PSI) usando marcadores moleculares RAPD-
PCR (Random Amplified Polymorphic DNA – Polymerase Chain Reaction) foram incluídas no intuito de se calcular as
distâncias genéticas e comparar a variabilidade genética entre e dentro de cada uma das raças. Dos 146 primers escrutinados,
13 foram escolhidos para amplificação com cada um dos indivíduos das oito populações, gerando um total de 44 bandas
polimórficas. Os resultados encontrados na Análise de Variância Molecular (AMOVA) indicam que grande parte da variabilidade
genética detectada se deve a diferenças entre indivíduos dentro de populações (75,47%). Na análise da estimativa dos percentuais
de variabilidade genética entre pares de populações, foram observados maiores valores para os pares formados entre as cinco
populações de cavalo Pantaneiro e a raça Árabe, enquanto os menores percentuais ocorreram entre Pantaneiro e Mangalarga
Marchador. Maior índice de diversidade gênica foi observado na raça Pantaneiro (0,3396). No dendrograma gerado pelo método
UPGMA, a partir da matriz de similaridade obtida pelo coeficiente de Jaccard, houve distinção entre as raças naturalizadas
(Pantaneiro e Mangalarga Marchador) e as exóticas (Árabe e PSI). Os resultados encontrados sugerem que o Pantaneiro
apresenta maior variabilidade genética que os de outras raças e está estreitamente relacionado ao Mangalarga Marchador. Palavras-chave: AMOVA, Árabe, diversidade genética, Mangalarga Marcador, Puro-Sangue Inglês, recurso genético Correspondências devem ser enviadas para: concepta@unb.br Egito et al. 800 © 2007 Sociedade Brasileira de Zootecnia Introduction Nei‘s genetic distances (1972 and 1978) between breeds
and populations were calculated using the POPGENE
program (Population Genetic Analysis) version 1.31 (Yeh et
al., 1999) that establishes standardized genetic distance
matrices (Nei, 1972) and matrices of genetic distances This study aimed to investigate diversity among
Pantaneiro horses from five nuclei using RAPD (Williams
et al., 1990; Welsh & McClelland, 1990) markers and
comparing them with other horse populations in Brazil. © 2007 Sociedade Brasileira de Zootecnia Genetic variability of Pantaneiro horse using RAPD-PCR markers 801 Table 1 -
Distribution of samples analysed in RAPD reactions
Population
Breed
Number of horses
Total
Males
Females
Pan BB
Pantaneira – Barra dos Bugres
14
28
42
Pan Cu
Pantaneira – Cuiabá
22
26
48
Pan Po
Pantaneira – Poconé
40
08
48
Pan CG
Pantaneira – Campo Grande
06
25
31
Pan Co
Pantaneira – Corumbá
21
27
48
A
Arabian
10
35
45
MM
Mangalarga Marchador
17
25
42
Thoroughbred
English Thoroughbred
31
17
48
Total
352 corrected for small samples (Nei, 1978). All cluster analyses
were conducted using UPGMA and the resulting clusters
were expressed as dendrograms. The analyses were carried
out in three stages: (i) all population breeds (Pantaneiro,
Arabian, Mangalarga Marchador and Thoroughbred); (ii)
only Pantaneiro populations; and (iii) pairs of populations
of all breeds. The POPGENE program als o generated genetic
diversity indices for each breed, as a function of the
means of their allelic frequencies, based on Nei (1973). Confidence values for each node of the dendrogam were
estimated from 10.000 random resamplings of the data
(bootstrap) using TFPGA (Tools for Population Genetics
Analyses, version 1.3) (Miller, 1997). breed-specific RAPD markers for Arabian/Thoroughbred
and Lavradeiro horses, respectively. The AMOVA analysis showed that the greatest part
of the total genetic variation (75.47%) was due to
differences between individuals within breeds and that
between breeds variance accounted for 24.53%. Using
only the Pantaneiro, populations accounted for 16.26% of
the total variation and with pairs of populations this
variation accounted for between 24.54% (Poconé and
Corumbá populations) and 11.10% (Poconé and Barra do
Bugres populations) (Table 2). Similar results were found
in native cattle breeds using RAPD markers (Serrano,
2001; Spritze 2003). The analysis of molecular variance (AMOVA)
(Schneider et al., 2000) was used to separate the variance
among and within populations using the ARLEQUIN
program. Introduction Although this test was not originally designed for
analysis of RAPD data, it has been used in other similar
studies (Jaggi et al., 2000; Lougheed et al., 2000; Tansley &
Brown, 2000). When all breeds were considered, greatest differences
were observed between the Pantaneiro and Arabian, while
comparisons with the Thoroughbred and Mangalarga
Marchador showed fewer differences (Table 2). According
to Cothran et al. (1998) the greater similarity between the
Pantaneiro and Mangalarga Marchador breeds, when
compared with Thoroughbred and Arabian, shows that all
the Brazilian breeds studied are closely related to each
other. Comparing Pantaneiro populations with Mangalarga
Marchador, the Mangalarga Marchador/Poconé pair showed
a high level of variability, Mangalarga Marchador/ Corumbá
and Mangalarga Marchador/Campo Grande pair comparison
showed lower variability than most comparisons within the
Pantaneiro. When the Pantaneiro was considered a single
population, paired comparisons showed: 25.91% between
breed variability for Pantaneiro/Arabian, 12.84% for
Pantaneiro/Mangalarga Marchador and 20.01% for
Pantaneiro/Thoroughbred. © 2007 Sociedade Brasileira de Zootecnia Results and Discussion In general, greater
variability between pairs of populations was accompanied
by greater distances between the same pairs, for example
Poconé/Corumbá, Campo Grande/Corumbá and Campo
Grande/Cuiabá. The Barra do Bugres/Poconé pair showed
less variability and closer distance. Exchange of germplasm
should preferentially be carried out between nuclei with
greater distances and high variability to maintain genetic
variation within the breed. The exchange of germplasm
between Barra do Bugres and Poconé, Corumbá and Cuiabá
or Cuiabá and Poconé may compromise variation levels as
there are lower estimated values between these populations
than within the breed as a whole. Townships geographically
more distant do not present highest distances and genetic
variability (P<0.05). This may be due to exchange of animals
between different studs. diversity indices than the Mangalarga Marchador, possibly
because of the intense use of a few stallions (such as the
stallion Taiamã in Poconé). This animal is held as a prime
example of the breed and is widely used in the region. This
single stallion sired 15% of the animals registered by the
ABCCP. Although remote, the possibility of crossbreeding
between the Pantaneiro and Mangalarga Marchador must
be considered to justify the relationship between them. Since Campo Grande and Cuiabá are two large urban centers
situated outside the Pantanal Matogrossense, this allowed
greater interaction with Mangalarga Marchador breeders,
a breed widely used in Brazil. Nevertheless it is important
to note that the samples studied here came from Pantaneiro
breeders, who carefully control their herds for the last 20
years or so. The equine species has a long generation
interval, about 12 years, which means this control only
exists for about 2 generations. The results for the AMOVA and genetic distances
calculated using Nei (1972) show a direct relationship
between the Pantaneiro and Mangalarga Marchador breeds,
for each population individually as well as the five
populations considered together. The population from
Corumbá showed a lower distance measure from the
Mangalarga Marchador than the other populations in the
same breed. Similar results were found by Cothran et al. (1998) using samples from Corumbá, Campo Grande and Rio
Verde who showed that three populations formed a cluster
with the Mangalarga and a larger cluster with Mangalarga
Marchador and Campolina. These findings are in agreement
with the history of the origin of the naturalized Brazilian
breeds, which have common ancestor: animals introduced
by European colonizers. The genetic distances expresses the degree of
divergence between populations. Results and Discussion Of 146 primers studied, 60 (41.1%) yielded polymorphic
bands and 47 yielded monomorphic bands (32.19%), as well
as 39 (26.71%) bands that were poorly amplified or did not
amplify. Primers were selected based on number of
polymorphic bands and amplification quality. A total of 44
polymorphic bands were considered with an average 3.38
bands/primer. Bailey & Lear (1994) studying Arabian and
Thoroughbred breeds found an average of 3.6 polymorphic
bands/primer in a RAPD assay. Martins (1996), studying
three Brazilian breeds (Lavradeiro, Crioulo and Campolina)
found 2.9 bands/primer, using 29 markers. Apostolidis
(2001) found 10.2 bands/primer and 51 polymorphic bands
in Greek horses (Thessalian, Skyros Pony, Pinia, Cretan and
Andravida). This author did not find any specific markers,
while Bayley & Lear (1994) and Martins (1996) did find The greatest index of genetic diversity was observed
when the five populations were considered as one
(Table 3), but when considered separately each Pantaneiro
population showed lower diversity index than that of the
breed. According to Cothran et al. (1998) individual and
population estimates of genetic variation of the Pantaneiro
indicate that variation levels are normal for horses and © 2007 Sociedade Brasileira de Zootecnia © 2007 Sociedade Brasileira de Zootecnia 802 Egito et al. Table 2 -
Estimates of genetic variability percentages due to differences between populations by AMOVA1
Item
Pan BB
Pan CG
Pan Co
Pan Cu
Pan Po
A
MM
Pan CG
21.81
Pan Co
22.89
23.72
Pan Cu
20.04
22.76
16.43
Pan Po
11.10
22.38
24.54
17.71
A
42.07
35.52
36.83
27.91
38.99
MM
25.78
17.43
11.44
20.41
31.94
24.23
Thoroughbred
28.82
25.64
25.39
27.15
27.44
12.62
23.17
1Abbreviations – see Table 1. Table 3 - Genic diversity for populations (*h), from allelic
frequencies using Nei (1973)/POPGENE
Population
Nº of animals
(*h)
Pantaneiro
217
0.3396
Pantaneiro – Barra dos Bugres
42
0.2365
Pantaneiro – Campo Grande
31
0.2973
Pantaneiro – Corumbá
48
0.2697
Pantaneiro – Cuiabá
48
0.2936
Pantaneiro – Poconé
48
0.2500
Arabian
45
0.2814
Mangalarga Marchador
42
0.2710
Thoroughbred
48
0.2617 Table 3 - Genic diversity for populations (*h), from allelic
frequencies using Nei (1973)/POPGENE there is no indication of inbreeding. The populations from
Campo Grande and Cuiabá had the highest values for the
genic diversity index, while the lowest was for those from the
Barra do Bugres and Poconé, which had lower indices than
the Arabian and Thoroughbred breeds. © 2007 Sociedade Brasileira de Zootecnia Results and Discussion Higher values were found
for the pairs formed between each of the five Pantaneiro
populations and the Arabian. Smaller, but still high values,
were found for distances between Pantaneiro and
Thoroughbred populations, which may mean that past
crossbreeding no longer has much influence on the
Pantaneiro breed. These results were expected as they
represent pairs of comparisons with distinct breeds but do As can be seen in Table 3, and in agreement with
Cothran et al. (1998), the Mangalarga Marchador showed a
lower genic diversity index than the Pantaneiro. The animals
from Barra dos Bugres, Poconé and Corumbá, showed lower © 2007 Sociedade Brasileira de Zootecnia Genetic variability of Pantaneiro horse using RAPD-PCR markers 803 not agree with Sereno (2002) or Cothran et al. (1998) who
found greater distances for the Pantaneiro/Thoroughbred
pair than the Pantaneiro/Arabian. In this study only the
Pantaneiro from Cuiabá had a higher distance compared with
the Thoroughbred than the Arab. The present study used
217 samples from a greater number of subpopulations,
although used the same population studied by Sereno (2002)
(Corumbá), and a greater part of those studied by Cothran et
al. (1998), Campo Grande and Corumbá. Therefore the
divergence in results may be caused by genetic variability
introduced in the subpopulations of Barra dos Bugres, Cuiabá
and Poconé (not previously studied). was also included in this cluster and was grouped with the
Corumbá Pantaneiro, which was the only node with a
confidence level higher than 70%. The dendrogram in Figure 1 is in agreement with that
found by Cothran et al. (1998) and Sereno (2002), where a
distinct separation between naturalized (Pantaneiro and
Mangalarga Marchador) and exotic breeds (Arab and
Thoroughbred) is noted. The Pantaneiros from Corumbá
were paired with the Mangalarga Marchador, in agreement
with the AMOVA and genetic distances, which indicated a
close relationship between these two populations. The Jaccard similarity coefficient (Figure 2) with all
animals analyzed individually, showed a clear distinction
between the naturalized (Pantaneiro and Mangalarga
Marchador) and exotic breeds (Arab and Thoroughbred). Ninety seven percent of the Pantaneiro horses were in a
single cluster and generally, examining sub-clusters, at
least 60% of the horses from a single region were grouped
in each individual sub-cluster. © 2007 Sociedade Brasileira de Zootecnia Abbreviations see Table 1.
All values were significant (P<0.05). Results and Discussion The exceptions were the
horses from Corumbá and Cuiabá which had 52 and 65%
respectively, of the samples in clusters of other Pantaneiro Table 4 shows standardized and corrected genetic
distances due to Nei (1972 and 1978, respectively). The
corrected distances were lower than the standardized, but
proportionally no differences between the relationships
were noted. Highest genetic distances were observed
between the five Pantaneiro populations and the Arabian,
followed by Pantaneiro and Thoroughbred. The lowest value
for the regular genetic distance (below diagonal) was between
the Corumbá/Mangalarga Marchador pair. The Barra do
Bugres/Poconé, Barra do Bugres/Corumbá and Barra do
Bugres/Cuiabá pairs also showed low values. When only the
five Pantaneiro populations were considered (above
diagonal), the greatest distances were between Corumbá/
Poconé, Corumbá/Campo Grande and Campo Grande/Cuiabá. Figure 1 - Dendrogram from UPGMA method for genetic identity
using Nei (1972)1. 1Abbreviations – see Table 1. Coefficient
0.75
0.81
0.86
0.92
0.97
Pan BB
Pan Cu
MM
Pan Co
Árabian
Thoroughbred
Pan Po
Pan CG The Thoroughbred had the lowest genic diversity
index. This is in agreement with the result found by
Bowling & Clark (1985), who studied Thoroughbred,
Arabian, Standardbred, Morgan, Quarter Horse, Paso
Fino and Peruvian Paso . They observed a lower
biochemical marker variation in Thoroughbred and
concluded that this was due to a Stud Book that has been
closed for more than 200 years, as well as intense selection
for a single trait: speed in flat races. Figure 1 - Dendrogram from UPGMA method for genetic identity
using Nei (1972)1. 1Abbreviations – see Table 1. The Pantaneiro populations grouped together
(Figure 1) in a single cluster. The Mangalarga Marchador Table 4 -
Genetic distances among eight populations of horses using Nei (1972, below diagonal and 1978 above diagonal), program
POPGENE1
PanBB
PanCG
PanCO
PanCU
PanPO
A
MM
Thoroughbred
PanBB
****
0.1047
0.0864
0.0917
0.0852
0.2338
0.1151
0.2128
PanCG
0.1101
****
0.1509
0.1484
0.1168
0.2476
0.1365
0.2192
PanCO
0.0901
0.1564
****
0.0975
0.1550
0.2238
0.0526
0.1734
PanCU
0.0957
0.1543
0.1017
****
0.1221
0.1637
0.1064
0.1813
PanPO
0.0890
0.1225
0.1590
0.1264
****
0.2274
0.1857
0.1897
A
0.2380
0.2536
0.2281
0.1683
0.2319
****
0.1975
0.1285
MM
0.1189
0.1422
0.0566
0.1108
0.1899
0.2019
****
0.1517
Thoroughbred
0.2164
0.2245
0.1772
0.1854
0.1936
0.1327
0.1555
****
1Abbreviations – see Table 1. All values were significant (P<0.05). © 2007 Sociedade Brasileira de Zootecnia Results and Discussion Table 4 -
Genetic distances among eight populations of horses using Nei (1972, b
POPGENE1 Table 4 -
Genetic distances among eight populations of horses using Nei (1972, below diagonal and 1978 a
POPGENE1 s among eight populations of horses using Nei (1972, below diagonal and 1978 above diagonal), program nces among eight populations of horses using Nei (1972, below diagonal and 1978 above diagonal), program 804 Egito et al. Figure 2 - Dendrogram generated by UPGMA method for the Jaccard coefficient for RAPD data from 352 animals and 44 polymorphic
bands. Percentages represent the percentage of the total number of animals from a specific region or breed within that region
of the dendrogram. Pantaneiro
Mangalarga
PSI/ Árabe
0.58
0.68
0.78
0.87
0.97
Coefficient
Barra dos Bugres
67%
Poconé
67%
Campo Grande
71%
Corumbá
Cuiabá
17%
Corumbá
Cuiabá
35%
Pantaneiro
3%
Mangalarga
Marchador
62%
48% Barra dos Bugres
67% Poconé
67% Pantaneiro
Mangalarga
PSI/ Árabe Figure 2 - Dendrogram generated by UPGMA method for the Jaccard coefficient for RAPD data from 352 animals and 44 polymorphic
bands. Percentages represent the percentage of the total number of animals from a specific region or breed within that region
of the dendrogram. © 2007 Sociedade Brasileira de Zootecnia © 2007 Sociedade Brasileira de Zootecnia Genetic variability of Pantaneiro horse using RAPD-PCR markers 805 and Mangalarga Marchador populations. Also, 35% of the
individuals from Cuiabá and 48% from Corumbá, were found
in the same cluster. The Mangalarga Marchador had 76% of
its individuals in the large cluster of the Pantaneiro breed. The populations of Pantaneiro, closest to Mangalarga
Marchador, independent of the analysis were those from
Corumbá, Campo Grande and Cuiabá. BAILEY, E.; LEAR, T.L. Comparison of Thoroughbred and Arabian
horses using RAPD markers. Animal Genetics, v.25, p.105-
108, 1994 (suppl 1). BALIEIRO, E.S. Subsídio ao estudo do cavalo Pantaneiro. São Paulo. Anuário Comissão Coordenadora da Criação do Cavalo
Nacional. Brasília: Ministério da Agricultura, 1971. p.59-65. BECK, S.L. Equinos: raças, manejo e equitação. 2.ed. São Paulo:
Editora dos Criadores, 1985. 479p. BOWLING, A.T.; CLARK, R.S. Blood group and protein
polymorphism gene frequencies for seven breeds of horses in
the United States. Animal Blood Groups and Biochemical The presence of individuals from different populations
in the same cluster generated similarity matrix (Jaccard
coefficient of similarity) is probably due to the interchange
of animals between different farms. Conclusions MILLER, S.A.; DYKES, D.D.; POLESKY, H.F. A simple salting out
procedure for extracting DNA from human nucleated cells. Nucleic Acids Research, v.16, p.1215, 1988. Data generated by this RAPD study are useful in
estimating distances between and within breeds and might
help futures programs of management and conservation. Of
the breeds studied here, the Pantaniero had the highest
genetic variability. Results indicate that this breed is close
to the Mangalarga Marchador, in agreement with known
breed history which indicates a common ancestor for both
and crossbreeding using Arabian and Thoroughbred
stallions. Although there was not complete isolation
between the populations examined, genetic differences
between regions were observed indicating that a
crossbreeding strategy is required to maintain genetic
diversity and avoid inbreeding within this population. Nucleic Acids Research, v.16, p.1215, 1988. MISERANI, M.G.; McMANUS, C.; SANTOS, S.A. et al. Avaliação
dos fatores que influem nas medidas lineares do cavalo
Pantaneiro. Revista Brasileira de Zootecnia, v.31, n.1,
p.335-341, 2002. NEI, M. Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Science of United
States of America, v.70, p.3321-3323, 1973. NEI, M. Estimation of average heterozygosity and genetic distance
from a small number of individuals. Genetics, v.89, p.583-
590, 1978. NEI, M. Genetic distance between populations. American
Naturalist, v.106, p.238-292, 1972. NOTTER, D.R. The importance of genetic diversity in livestock
populations of the future. Journal of Animal Science, v.77,
p.61-69, 1999. SANTOS, S.A. Avaliação e conservação do cavalo Pantaneiro no
Pantanal Mato-grossense. In: REUNIÃO ANUAL DA
SOCIEDADE BRASILEIRA DE ZOOTECNIA, 30., 1993, Rio
de Janeiro. Anais... Rio de Janeiro: Sociedade Brasileira de
Zootecnia, 1993. p.287-312. Results and Discussion This is the case for
example of the animals from Corumbá, which use two stallions
from Cuiabá. Another important factor is the number of
markers used here; maybe an increase in the number of
bands would promote a greater distinction between groups
and individuals. Genetics (presently Animal Genetics), v.16, p.93-108, 1985. COTHRAN, E.G.; SANTOS, S.A.; MAZZA, M.C.M. et al. Genetics
of the Pantaneiro horse of the Pantanal region of Brazil. Genetics and Molecular Biology, v.21, n.3, p.343–349, 1998. Genetics and Molecular Biology, v.21, n.3, p.343 349, 1998. JAGGI, C.; WIRTH, T.; BAUR, B. Genetic variability in
subpopulations of the Asp Viper (Vispera aspis) in Swiss Jura
Mountains: implications for a conservation strategy. Biological
Conservation, v.94, p.69-77, 2000. LYNCH, M.; MILLIGAN, B.G. Analysis of population genetic
structure with RAPD markers. Molecular Ecology, v.3, n.2,
p.91-99, 1994. LOUGHEED, S.C.; GIBBS, K.A; WEATHERHEAD, P. J. A
comparison of RAPD versus microsatellite NDA markers in
population studies of the Massasauga Rattlesnake. Journal of LOUGHEED, S.C.; GIBBS, K.A; WEATHERHEAD, P. J. A
comparison of RAPD versus microsatellite NDA markers in
population studies of the Massasauga Rattlesnake. Journal of
Heredity, v.91, p.458-463, 2000. The results of the AMOVA and by genetic distances
(Nei, 1972) indicate that the Arabian and Thoroughbred
breeds are more similar within themselves than with the
Pantaneiro and Mangalarga Marchador. Although well
dispersed throughout the world and closed stud books
the Thoroughbred and Arab have similar origins as Arabs
were the foundation of the Thoroughbred (Bowling &
Clark, 1985). population studies of the Massasauga Rattlesnake. Journal of
Heredity, v.91, p.458-463, 2000. MARIANTE, A.S.; CAVALCANTE, N. Animais do descobrimento:
raças domésticas da história do Brasil. Brasília: Embrapa-
Cenargen, 2000. 232p. MARTINS, V.B. Técnica de diagnóstico com marcadores RAPD
para uso e preservação de germoplasma eqüino. Brasília:
Universidade de Brasília, 1996. 44p. Dissertação (Mestrado em
Saúde) - Universidade de Brasília, 1996. MILLER, M.P. [1997]. Tools for population genetics analyses
(TFPGA): a Windows program for the analysis of allozyme and
molecular population genetic data. Disponível em: <http://
herb.bio.nau.edul~miller/tfpga.htm>. Acesso em: 10/12/04. To CNPq for scholarship for the 3rd author. SANTOS, S.A.; McMANUS, C.M.; MARIANTE, A.S. et al. A review
of Conservation and Management of the Pantaneiro horse in
the Brazilian Pantanal. A n i m a l G e n e t i c R e s o u r c e s Acknowledgements To CNPq for scholarship for the 3rd author. Literature Cited Information, v.31, p.75 86, 2001. SCHNEIDER, S., ROESSLI, D.; EXCOFFIER, L. Arlequin version
2000: A software for population genetics data analysis. Geneva:
University of Geneva, 2000. 111p. APOSTOLIDIS, A.P.; MAMURIS, Z.; KARKAVELIA, E.;
ALIFAKIOTIS, T. Comparison of Greek breeds of horses using
RAPD markers. Journal of Animal Breeding and Genetics ,
v. 118, p. 47-56, 2001. SERENO, J.R.B.; SANTOS, S.A.; ZUCCARI, C.E.S.N.; MAZZA,
M.C.M. Establishment of a breeding season and evaluation of © 2007 Sociedade Brasileira de Zootecnia 806 Egito et al. the reproductive performance of the Pantaneiro hose in Mato SPRITZE, A.L.; EGITO, A.A.; MARIANTE, A.S.; McMANUS, C.M. Caracterização genética da raça bovina Crioulo Lageano usando
marcadores moleculares RAPD. Pesquisa Agropecuária p
p
Grosso do Sul, Brazil. A n i n a l G e n e t i c R e s o u r c e s
Information, v.21, p.43-48, 1997. Grosso do Sul, Brazil. A n i n a l G e n e t i c R e s o u r c e s
Information, v.21, p.43-48, 1997. SERENO, F.T.P.S. Caracterización genética del caballo
Pantaneiro. Córdoba: Universidad de Córdoba, 2002. 126p. Tesis (Doctoral in Genetica Animal) - Universidad de Córdoba,
2002 Brasileira, v.38, n.10, p.1157-1164, 2003. TANSLEY, S.A.; BROWN, C.R. RAPD variation in the rare and
endangered (Proteaceae): implications for their conservation. SERENO, F.T.P.S. Caracterización genética del caballo
Pantaneiro. Córdoba: Universidad de Córdoba, 2002. 126p. Pantaneiro. Córdoba: Universidad de Córdoba, 2002. 126p. Tesis (Doctoral in Genetica Animal) - Universidad de Córdoba,
2002 Biological Conservation, v.95, p.39-48, 2000. WELSH, J., MCCLELLAND, M. Fingerprinting genomes using
PCR with arbitrary primers. Nucleic Acids Research, v.18,
p.7213-7218, 1990. SERRANO, G.M.S.; EGITO, A.A.; McMANUS, C.M.; MARIANTE,
A.S. Genetic diversity and population structure of Brazilian native bovine breeds based on RAPD markers. Pesquisa
Agropecuária Brasileira, v.39, p.543-549, 2004. WILLIAMS, J.G.K.; KUBELIK, A.R.; LIVAK, K.J. et al. DNA
polymorphisms amplified by arbitrary primers are useful as
i
k
N
l i
A id
R
h
18
6531 WILLIAMS, J.G.K.; KUBELIK, A.R.; LIVAK, K.J. et al. DNA
polymorphisms amplified by arbitrary primers are useful as
genetics markers. Nucleic Acids Research, v.18, p.6531-
6535, 1990. Agropecuária Brasileira, v.39, p.543 549, 2004. SHIUE, Y.L.; BICKEL, L.A.; CAETANO, A.R. et al. A synteny
map of the horse genome comprised of 240 microsatellite and
RAPD markers. Animal Genetics, v.30, p.1-9, 1999. genetics markers. Nucleic Acids Research, v.18, p.6531-
6535, 1990. YEH, F. © 2007 Sociedade Brasileira de Zootecnia Literature Cited C.; YANG, R.; BOYLE, T. POPGENE. Microsoft Window-
based freeware for population genetic analysis: version 1.3:
manual. Edmonton: University of Alberta, 1999. 29p. SNEATH, P.H.A.; SOKAL, R.R. Numerical taxonomy : the
principles and practice of numerical classification. San
Francisco: W. H. Freeman, 1973. 573p. SOKAL, R.R.; MICHENER., C.D. A statistical method for evaluating
systematic relationships. University Kansas Science
Bulletin, v.38, n.22, p.1409-1438, 1958. p
SOLIS, A.; JUGO, B.M.; MÉRIAUX, J.C. et al. Genetic diversity
within and among four south European native horse breeds base
don microsatellite DNA analysis: implications for conservation. Journal Heredity, v.96, n.6, p.670-678, 2005. Recebido: 23/06/05
Aprovado: 27/02/07
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https://openalex.org/W4390062526
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https://adc.bmj.com/content/archdischild/early/2023/12/21/archdischild-2023-326393.full.pdf
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English
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Safety, resource use and nutritional content of home-blended diets in children who are gastrostomy fed: findings from ‘YourTube’ – a prospective cohort study
|
Archives of disease in childhood
| 2,023
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cc-by
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WHAT THIS STUDY ADDS ⇒Children receiving a home-blended diet tend to
come from less deprived areas and their parents
have higher levels of education. ⇒Children receiving a home-blended diet tend to
come from less deprived areas and their parents
have higher levels of education. Correspondence to
Professor Lorna K Fraser, Cicely
Saunders Institute, King’s
College London, London, UK;
lorna.fraser@kcl.ac.uk y
Results 180 children and families completed the
baseline data collection, with 134 (74%) and 105 (58%)
providing follow-up data at 12 and 18 months. There
were fewer gastrointestinal (GI) symptoms at all time
points in the home-blended diet group, but there was
no difference in change over time within or between
the groups. The nutritional intake of those on a home-
blended diet had higher calories per kilogram and fibre,
and both home-blended and formula-fed children have
values above the dietary reference values for most
micronutrients. Safety outcomes were similar between
groups and over time. The total costs to the statutory
sector were higher among children who were formula
fed, but the costs of purchasing special equipment for
home-blended food and the total time spent on childcare
were higher for families with home-blended diet. g
⇒Children receiving a home-blended diet have
a similar safety profile to children receiving
formula feeds. Received 30 September 2023
Accepted 2 December 2023 ⇒Home-blended diets have higher fibre intake
and are associated with a lower burden of
gastrointestinal symptoms. WHAT IS ALREADY KNOWN ON THIS TOPIC WHAT IS ALREADY KNOWN ON THIS TOPIC
⇒There is an increasing number of children
who rely on gastrostomy feeds to meet their
nutritional requirements. ⇒More parents are choosing to feed their
children home-blended diets rather than the
professional organisation recommendation of
formula feeds. ⇒There is little evidence on the safety and
nutritional intake of home-blended diets in
these children compared with those receiving
formula feeds. WHAT THIS STUDY ADDS
⇒Children receiving a home-blended diet tend to
come from less deprived areas and their parents
have higher levels of education. ⇒Children receiving a home-blended diet have
a similar safety profile to children receiving
formula feeds. ⇒Home-blended diets have higher fibre intake
and are associated with a lower burden of
gastrointestinal symptoms. HOW THIS STUDY MIGHT AFFECT RESEARCH,
PRACTICE OR POLICY
⇒Home-blended diets for children who are
gastrostomy fed should be seen as a safe
alternative to formula feeding, unless there is a
clinical contraindication. ⇒There is an increasing number of children
who rely on gastrostomy feeds to meet their
nutritional requirements. ⇒There is an increasing number of children
who rely on gastrostomy feeds to meet their
nutritional requirements. q
⇒More parents are choosing to feed their
children home-blended diets rather than the
professional organisation recommendation of
formula feeds. ⇒More parents are choosing to feed their
children home-blended diets rather than the
professional organisation recommendation of
formula feeds. Design This is a cohort study. Data were collected at
months 0, 12 and 18 from parents and clinicians using
standardised measures. 1Cicely Saunders Institute, King’s
College London, London, UK
2Health Sciences, University of
York, York, UK
3University of Sunderland,
Sunderland, UK
4University of Leeds, Leeds, UK
5University of York Centre for
Health Economics, York, UK
6Somerset Partnership NHS and
Social Care Trust, Taunton, UK Setting 32 sites across England: 28 National Health
Service trusts and 4 children’s hospices. ⇒There is little evidence on the safety and
nutritional intake of home-blended diets in
these children compared with those receiving
formula feeds. ⇒There is little evidence on the safety and
nutritional intake of home-blended diets in
these children compared with those receiving
formula feeds. Patients Children aged 6 months–18 years who were
gastrostomy fed. Main outcome measure The main outcome measure
was the PedsQL Gastrointestinal Symptoms Scales score. Secondary outcomes included quality of life, sleep (child,
parent), dietary intake, anthropometry, healthcare usage,
safety outcomes and resource use. ABSTRACT
bj
i ABSTRACT
Objective To assess the risks, benefits and resource
implications of using home-blended food in children
with gastrostomy tubes compared with currently
recommended formula feeds. ABSTRACT
Objective To assess the risks, benefits and resource
implications of using home-blended food in children
with gastrostomy tubes compared with currently
recommended formula feeds. HOW THIS STUDY MIGHT AFFECT RESEARCH,
PRACTICE OR POLICY ⇒Home-blended diets for children who are
gastrostomy fed should be seen as a safe
alternative to formula feeding, unless there is a
clinical contraindication. Conclusions Children who are gastrostomy fed a
home-blended diet have similar safety profile, adequate
nutritional intake and lower burden of GI symptoms than
formula-fed children. a requirement for enteral feeding, including
neurological conditions, congenital cardiac
disease, inherited metabolic conditions, cystic
fibrosis, gastrointestinal conditions and cancer,
with neurological conditions often the most
common.1 Trial registration number ISRCTN13977361. Trial registration number ISRCTN13977361. To cite: Fraser LK,
Bedendo A, O’Neill M, et al. Arch Dis Child Epub ahead of
print: [please include Day
Month Year]. doi:10.1136/
archdischild-2023-326393
© Author(s) (or their
employer(s)) 2023. Re-use
permitted under CC BY. Published by BMJ. In the UK, where access to healthcare is free
at the point of care, commercially produced
formula is recommended for children who are
gastrostomy fed. There is, however, a growing
number of parents who are choosing to feed
their children home-blended diets in the UK,2
and this is also common in other countries. y guest. Protected by copyright. © Author(s) (or their
employer(s)) 2023. Re-use
permitted under CC BY. Published by BMJ. ►Additional supplemental
material is published online
only. To view, please visit the
journal online (http://dx.doi.
org/10.1136/archdischild-
2023-326393). Fraser LK, et al. Arch Dis Child 2023;0:1–8. doi:10.1136/archdischild-2023-326393 Received 30 September 2023
Accepted 2 December 2023 on October 23, 2024 by guest. Protected by
http://adc.bmj.com/
Arch Dis Child: first published as 10.1136/archdischild-2023-326393 on 21 December 2023. Downloaded from Original research Arch Dis Child: first published as 10.1136/archdischild-2023-326393 on 21 D Safety, resource use and nutritional content of home-
blended diets in children who are gastrostomy fed:
findings from ‘YourTube’ – a prospective cohort study Lorna K Fraser ,1,2 Andre Bedendo,2 Mark O’Neill,2 Jo Taylor ,2 Julia Hackett,2
Karen Alice Horridge,3 Janet Cade,4 Gerry Richardson,5 Han Phung,5 Alison McCarter,6
Catherine Elizabeth Hewitt 2 ►Additional supplemental
material is published online
only. To view, please visit the
journal online (http://dx.doi.
org/10.1136/archdischild-
2023-326393). 1Cicely Saunders Institute, King’s
College London, London, UK
2Health Sciences, University of
York, York, UK
3University of Sunderland,
Sunderland, UK
4University of Leeds, Leeds, UK
5University of York Centre for
Health Economics, York, UK
6Somerset Partnership NHS and
Social Care Trust, Taunton, UK
Correspondence to
Professor Lorna K Fraser, Cicely
Saunders Institute, King’s
College London, London, UK;
lorna.fraser@kcl.ac.uk
Received 30 September 2023
Accepted 2 December 2023 ►Additional supplemental
material is published online
only. To view, please visit the
journal online (http://dx.doi.
org/10.1136/archdischild-
2023-326393). Original research Original research study, the position statements of these professional organ-
isations, including the ESPGHAN2 5 and the BDA,6 moved
to being more supportive, but still call for a more robust
evidence on the safety and nutritional content of home-
blended diets. (ESPGHAN) and the British Dietetic Association (BDA),
on potential safety issues with use of a home-blended diet,
for example increased numbers of blocked gastrostomy
tubes and gastrointestinal and stoma infections, concerns
about the nutritional content of a home-blended diet, and
questions about being able to meet the macronutrient and
micronutrient requirements of children given this type of
feed. At the start of this study, these professional bodies did
not recommend use of home-blended diets to feed children
with gastrostomies.3 4 However, during the period of this This study aimed to assess the risks, benefits and resource
implications of using home-blended food in children with
gastrostomy tubes compared with currently recommended
formula feeds. This paper reports the 12-month and
18-month follow-up findings from this study. Original research Table 1 Clinical and demographic characteristics of the cohort at baseline and at 12-month and 18-month follow-up*
Baseline (n=180)
12 months (n=134)
18 months (n=105)
Home-blended
(n=104)
Formula fed
(n=76)
Home-blended
(n=79)
Formula fed
(n=55)
Home-blended
(n=57)
Formula fed
(n=48)
Age (years)
Mean (SD)
9.2 (4.4)
10.2 (4.4)
9.2 (4.4)
10.4 (4.6)
9.4 (4.4)
10.5 (4.8)
Sex, n (%)
Female
38 (37.3)
32 (42.7)
28 (36.4)
22 (40.7)
22 (40.0)
19 (39.6)
Male
64 (62.7)
43 (57.3)
49 (63.6)
32 (59.3)
33 (60.0)
29 (60.4)
Missing
2
1
2
1
2
–
Index of Multiple Deprivation, n (%)
1 (most deprived)
15 (14.4)
17 (22.4)
8 (10.1)
8 (14.5)
5 (8.8)
5 (10.4)
2
18 (17.3)
18 (23.7)
16 (20.3)
15 (27.3)
10 (17.5)
12 (25.0)
3
18 (17.3)
16 (21.1)
16 (20.3)
15 (27.3)
13 (22.8)
14 (29.2)
4
27 (26.0)
18 (23.7)
18 (22.8)
11 (20.0)
14 (24.6)
11 (22.9)
5 (least deprived)
26 (25.0)
7 (9.2)
21 (26.6)
6 (10.9)
15 (26.3)
6 (12.5)
Parental educational qualification,
n (%)
School leaving qualifications
13 (12.7)
20 (26.7)
10 (13.0)
14 (25.5)
10 (18.2)
12 (25.0)
Further education
21 (20.6)
26 (34.2)
15 (19.5)
17 (30.9)
10 (18.2)
15 (31.2)
Higher education
67 (65.7)
26 (34.7)
52 (67.5)
21 (38.2)
35 (63.6)
20 (41.7)
Other/no educational
qualifications
1 (1.0)
4 (5.3)
0 (0.0)
3 (5.5)
0 (0.0)
1 (2.1)
Missing
2
1
2
–
2
–
Child’s ethnicity, n (%)
White British
88 (88.9)
60 (81.1)
71 (89.9)
44 (80.0)
50 (87.7)
39 (81.2)
Other
11 (11.1)
14 (18.9)
8 (10.1)
11 (20.0)
7 (12.3)
9 (18.8)
Missing
24
10
–
–
–
–
Children’s diagnostic group, n (%)
Neurological
43 (41.7)
25 (32.9)
33 (42.3)
22 (40.0)
23 (41.1)
20 (41.7)
Genetic
41 (39.8)
33 (43.4)
32 (41.0)
23 (41.8)
23 (41.1)
19 (39.6)
Congenital
11 (10.7)
10 (13.2)
9 (11.5)
6 (10.9)
7 (12.5)
6 (12.5)
Other
8 (7.8)
8 (10.5)
4 (5.1)
4 (7.3)
3 (5.4)
3 (6.2)
Missing
0
Gastrostomy type, n (%)
Button (Mini or Mic-Key)
89 (86.4)
64 (84.2)
65 (83.3)
47 (85.5)
48 (85.7)
42 (87.5)
PEG
12 (11.7)
7 (9.2)
11 (14.1)
4 (7.3)
8 (14.3)
4 (8.3)
Other
2 (1.9)
5 (6.6)
2 (2.6)
4 (7.3)
0 (0.0)
2 (4.2)
Missing
1
0
Gastrostomy duration in years
Mean (SD)
5.6 (3.9)
7.2 (4.7)
5.7 (3.9)
7.6 (4.9)
6.2 (3.9)
7.7 (5.1)
Range
0.0–15.0
0.0–18.0
Missing
5
1
Fundoplication, n (%)
No
72 (69.2)
41 (54.7)
52 (65.8)
30 (55.6)
37 (64.9)
23 (48.9)
Yes
32 (30.8)
34 (45.3)
27 (34.2)
24 (44.4)
20 (35.1)
24 (51.1)
Missing
0
1
*Some missing clinical and demographic data from baseline were updated using 12-month and 18-month data if available. *Some missing clinical and demographic data from baseline were updated using 12-month and 18-month data if available.
PEG, percutaneous endoscopic gastrostomy. INTRODUCTION The number of children who rely on gastrostomy
tube feeding is rising, with the current preva-
lence estimated at 84 per 100 000 children or
more than 10 000 children in England.1 Gastros-
tomy tube feeding is used when long-term reli-
ance on enteral feeding due to unsafe swallow
or food aversion is likely to be required. There
are many underlying conditions associated with Over the last decade, there have been
concerns
raised
by
professional
bodies,
including the European Society for Paediatric
Gastroenterology Hepatology and Nutrition Fraser LK, et al. Arch Dis Child 2023;0:1–8. doi:10.1136/archdischild-2023-326393 1 Original research PEG, percutaneous endoscopic gastrostomy. 2 Original research MATERIALS AND METHODS with the COVID-19 pandemic increasing the parent-reported
data. A short video was available for professionals and parents to
measure the mid-upper arm circumference (MUAC; see https://
www.york.ac.uk/healthsciences/research/public-health/projects/
yourtube/studyresources/). This prospective cohort study was registered (ISRCTN13977361)
and was conducted according to a published protocol.7 The
COVID-19 pandemic impacted on the recruitment to this study
when all clinical research was paused within the National Health
Service (NHS) in the UK in early 2020. This resulted in a reduc-
tion in the target sample size (from 300 to 180) and a change in
data collection schedules from 0, 9 and 18 months to 0, 12 and
18 months. Detailed information on all data collection is available in the
protocol7 and baseline publication.9 Information on resource use
and costs was also collected from the parents at all time points. Children and parents were recruited through 31 NHS sites
from August 2019 until November 2021. Data were collected at
baseline and at 12 and 18 months, with the final data collection
completing on 31 May 2023. Statistical analyses All statistical analyses were undertaken using R and an alpha
of 5%. Descriptive statistics for all clinical, demographic and
outcome information used mean, SD and 95% CI for contin-
uous data, and counts and percentages for categorical data. The primary outcome was the PedsQL Gastrointestinal Symp-
toms Scales score.10 The secondary outcomes were children’s
quality of life (DISABKIDS Short Form, EuroQol-5 Dimen-
sion Visual Analogue Scale and the five-component scale of
the five-level version of EuroQol-5 Dimension (EQ-5D-5L)),11 All children aged 6 months–<19 years who received all or
part of their nutrition via a gastrostomy tube were eligible to be
included in this study. Data were collected on paper or via online
survey tools from parents, clinicians, and where appropriate the
children or the young people themselves. Nutritional intake
data were collected using published data for formula feeds and
using myfood248 for those with a home-blended diet. Anthro-
pometric data were collected from clinician or parent report, mary outcome: PedsQL Gastrointestinal Symptoms Scale score at baseline and at 12 and 18 months. GLMM, generalised linear mixed
on October 23, 2024 by guest. Protected by copyrigh
http://adc.bmj.com/
n 21 December 2023. Downloaded from Figure 1 Primary outcome: PedsQL Gastrointestinal Symptoms Scale score at baseline and at 12 and 18 months. GLMM, generalised linear mixed
model Figure 1 Primary outcome: PedsQL Gastrointestinal Symptoms Scale score at baseline and at 12 and 18 months. GLMM, generalised linear mixed
model. Fraser LK, et al. Arch Dis Child 2023;0:1–8. Statistical analyses doi:10.1136/archdischild-2023-326393 3 Original research Nutritional content at baseline and at 12-month and 18-month follow-up
Baseline (n=180)
12 months (n=134)
18 months (n=105)
Home-blended (n=104)
Formula fed (n=76)
Home-blended (n=79)
Formula fed (n=55)
Home-blended (n=57)
Formula fed (n=48)
trients
ories per kilogram
61.3 (54.1, 68.5)
44.0 (38.9, 49.1)
60.7 (51.6, 69.8)
41.0 (35.7, 46.2)
63.4 (50.9, 75.8)
40.9 (35.1, 46.7)
ilocalories
1231.2 (1107.6, 1354.8)
1114.2 (1009.1, 1219.2)
1351.1 (1186.2, 1516.0)
1154.5 (1034.7, 1274.2)
1443.8 (1239.1, 1648.5)
1172.6 (1036.4, 1308.7)
calories from protein
14.3 (13.3, 15.3)
12.8 (12.0, 13.7)
14.7 (13.4, 16.1)
13.8 (12.7, 15.0)
14.3 (13.2, 15.4)
13.7 (12.5, 14.9)
calories from carbohydrate
45.5 (43.1, 47.8)
47.9 (47.2, 48.6)
43.9 (41.4, 46.4)
47.6 (45.5, 49.7)
43.1 (40.0, 46.2)
48.1 (45.6, 50.6)
calories from fat
40.2 (37.5, 42.8)
38.0 (36.9, 39.1)
41.4 (38.5, 44.2)
37.5 (35.0, 40.0)
42.4 (38.8, 45.9)
37.2 (34.4, 40.1)
ount
hydrate (g)
139.3 (123.6, 154.9)
134.8 (121.3, 148.3)
145.2 (127.8, 162.6)
138.4 (121.9, 154.9)
151.5 (132.9, 170.1)
142.1 (122.9, 161.4)
n (g)
44.0 (39.0, 48.9)
35.9 (31.4, 40.4)
49.1 (42.7, 55.4)
39.7 (34.2, 45.3)
50.7 (43.4, 58.0)
40.2 (33.9, 46.6)
55.6 (48.9, 62.3)
46.3 (42.3, 50.4)
64.0 (52.9, 75.0)
47.8 (42.4, 53.2)
70.6 (55.8, 85.4)
48.0 (41.9, 54.1)
g)
14.1 (11.8, 16.4)
6.3 (4.9, 7.7)
14.5 (11.7, 17.4)
6.9 (4.9, 9.0)
16.5 (13.2, 19.7)
7.2 (5.0, 9.4)
er kilogram
hydrate (g/kg)
7.0 (6.0, 7.9)
5.3 (4.7, 5.9)
6.6 (5.6, 7.6)
4.8 (4.2, 5.3)
6.6 (5.5, 7.7)
4.8 (4.1, 5.4)
n (g/kg)
2.2 (1.9, 2.5)
1.3 (1.2, 1.5)
2.2 (1.9, 2.5)
1.3 (1.2, 1.5)
2.2 (1.8, 2.6)
1.3 (1.2, 1.5)
kg)
2.8 (2.4, 3.1)
1.9 (1.6, 2.1)
2.9 (2.3, 3.5)
1.8 (1.4, 2.2)
3.2 (2.3, 4.0)
1.8 (1.3, 2.3)
ories (%DRV)
76.5 (67.7, 85.3)
61.9 (55.5, 68.3)
83.5 (71.7, 95.2)
64.6 (57.0, 72.2)
88.8 (72.8, 104.8)
66.0 (57.7, 74.2)
rients
DRV)*
284.9 (235.8, 333.9)
253.4 (221.9, 284.8)
313.9 (253.6, 374.2)
265.5 (234.3, 296.7)
369.6 (237.1, 502.0)
278.1 (242.1, 314.1)
(%DRV)
151.7 (131.3, 172.0)
242.0 (216.6, 267.3)
164.0 (141.2, 186.8)
249.8 (220.7, 279.0)
157.4 (129.7, 185.1)
256.6 (223.8, 289.5)
n D (%DRV)
55.2 (44.2, 66.2)
120.4 (107.1, 133.8)
55.2 (43.6, 66.9)
124.9 (112.2, 137.7)
55.8 (40.4, 71.1)
130.2 (114.8, 145.7)
m (%DRV)
125.5 (110.1, 140.8)
144.6 (120.8, 168.5)
139.3 (120.1, 158.5)
165.2 (129.3, 201.2)
138.8 (117.1, 160.6)
171.4 (132.0, 210.9)
%DRV)
120.1 (104.5, 135.8)
136.0 (122.8, 149.1)
129.3 (113.2, 145.4)
147.5 (134.3, 160.7)
126.3 (106.8, 145.7)
152.4 (134.9, 169.9)
anese (%DRV)
136.0 (112.7, 159.2)
120.1 (98.8, 141.4)
134.6 (114.1, 155.2)
141.2 (112.0, 170.5)
157.5 (125.5, 189.4)
145.0 (113.0, 177.0)
%DRV)
124.0 (106.9, 141.2)
211.8 (176.9, 246.6)
143.0 (118.7, 167.3)
221.1 (183.9, 258.3)
134.8 (109.1, 160.5)
220.3 (180.3, 260.3)
g
19
5
16
3
13
1
at baseline: 26; at 12 months: 19; at 18 months: 15. Statistical analyses on October 23, 2024 by guest. Protected by copyright. http://adc.bmj.com/
d as 10.1136/archdischild-2023-326393 on 21 December 2023. Downloaded from Graphical summaries were used to show trends in primary
and secondary outcomes over time. When appropriate, group
comparisons used analysis of variance and Pearson’s χ2 tests. Summaries were provided overall and by the two groups of
interest using baseline allocation: those who are 100% formula
fed and those with any amount of home-blended feeds. RESULTS 180 children and families completed the baseline data collection,
with 134 (74%) and 105 (58%) providing follow-up data at 12
and 18 months, respectively (online supplemental figure 1). The clinical and demographic information at baseline and at
12 and 18 months (table 1) shows that while the two groups
were similar in terms of age, sex and underlying diagnoses, the
children who were receiving a home-blended diet tended to
come from areas of lower deprivation and have parents with
higher levels of education. Children from areas with higher
levels of deprivation were less likely to complete the study and
the parents who continued with the study had higher educa-
tional qualifications. Patient and public involvement
P
h
hild Parents whose children were gastrostomy fed (n=7) were
involved in the development and management of this study. They
prioritised outcomes, helped develop appropriate recruitment
methods, including the use of social media, and contributed to
the study materials. They also chose the study title ‘YourTube’
and are actively involved in interpreting the study findings and
dissemination. Propensity scores were used to balance the sample for demo-
graphic baseline data13 14 using the Index of Multiple Depriva-
tion score and calculated using package WeightIt V.0.13.1. The
propensity score weights were applied in a generalised linear
mixed model (GLMM) using the PedsQL total score measured
at baseline as the outcome; group, age, sex and diagnosis as
fixed effects; and recruitment site as a random effect. Assump-
tions were checked using graphical and GLMM inspection of
Akaike information criterion values. Inferential analyses were
not performed on secondary outcomes due to the large amount
of outcome data collected and concerns over multiple testing. Fraser LK, et al. Arch Dis Child 2023;0:1–8. doi:10.1136/archdischild-2023-326393 Statistical analyses Additional missing due to inconsistent data (eg, extreme values). ary reference value 4 on October 23, 2024 by guest. Protected by copyr
http://adc.bmj.com/
Arch Dis Child: first published as 10.1136/archdischild-2023-326393 on 21 December 2023. Downloaded from Original research Parenting Morale Index,12 child (parent report) and parental
sleep (Patient-Reported Outcomes Measurement Information
System, or PROMIS), Body Mass Index Standard Deviation
Score (BMISDS), MUAC, nutritional intake (total kilocalories,
kilocalories per kilogram and % of energy from macronutrients
considering the dietary reference value (DRV)), macronutrients
(protein, carbohydrate, fat and Association of Analytical Chem-
ists (AOAC) fibre) and micronutrients (vitamin B12, vitamin D,
folate, calcium, iron, manganese, zinc), and safety outcomes
(number of children reporting and the number of occurrences of
gut-intestinal infection, stoma site infection and tube blockage;
gastrostomy tube needing replacement; pneumonia; and acci-
dent and emergency (A&E) attendances in the last 12 months
and the number of children reporting any and the number of
occurrences). an NHS and personal and social services (PSS) perspective. To
calculate the total cost for each child, a micro-costing frame-
work was used. A multiple imputation model was employed
with the number of chains were considered using a two-step
approach.15 The unit cost of formula food was acquired from
the British National Formulary website,16 while the unit cost
for health and social care services was derived from Personal
Social Services Research Unit (PSSRU) 2021.15 The cost of
equipment purchased exclusively for home-blended diet and the
total time associated with childcare, categorised into time spent
on preparing and administering food, time spent on preparing
and administering medications, and time spent on caring for
gastrostomy, were also collected and provided using complete
case analysis. an NHS and personal and social services (PSS) perspective. To
calculate the total cost for each child, a micro-costing frame-
work was used. A multiple imputation model was employed
with the number of chains were considered using a two-step
approach.15 The unit cost of formula food was acquired from
the British National Formulary website,16 while the unit cost
for health and social care services was derived from Personal
Social Services Research Unit (PSSRU) 2021.15 The cost of
equipment purchased exclusively for home-blended diet and the
total time associated with childcare, categorised into time spent
on preparing and administering food, time spent on preparing
and administering medications, and time spent on caring for
gastrostomy, were also collected and provided using complete
case analysis. Primary outcome Primary outcome among children with formula-fed diet than those with home-blended
diet: £16 386 vs £12 028 per annum at baseline, £18 049 vs £14 357
per annum at 12 months, and £8345 vs £5887 per half-year at 18
months, respectively (see tables 4 and 5). The cost of formula food
mainly contributed to such differences. As a trade-off, families in
home-blended group spent an estimated £294 in the previous 12
months (at baseline), £176 in the previous 12 months (at 12 months)
and £97 in the previous 6 months (at 18 months) on kitchen equip-
ment for blending and storing blended food. They also spent an
average of 88 min (at baseline), 85 min (at 12 months) and 103 min
(at 18 months) higher per day caring for children than those in the
formula-fed group. Figure 1 shows the change over time in the primary outcome,
PedsQL Gastrointestinal Symptoms Scales score. The results from
the GLMM model (figure 1) showed that overall the formula-fed
group had more gastrointestinal symptoms than the home-blended
group; however, there was no significant effect of time, and the
change over time was not different between groups. DISCUSSION
h
l This large, prospective, national cohort study of children who
were gastrostomy fed has shown that those who were gastros-
tomy fed a home-blended diet had similar safety profile, adequate
nutritional intake and lower burden of gastrointestinal symp-
toms compared with formula-fed children. The home-blended
diet was associated with lower costs for the statutory sector, but
came with increased expenses for the families with equipment
costs and childcare time, along with a small home-made food
cost. The health-related quality of life outcomes for parents and
children were similar between the two groups. Safety outcomes The lower burden of gastrointestinal symptoms in the home-
blended diet group was maintained across the time period
of this study, and these findings are consistent with the small
number of published studies that have reported gastrointestinal
symptoms.17 The mean numbers of proximal safety outcomes were similar
between the groups and over time, that is, the number of gastrostomy
tube replacements (table 3): home-blended versus formula fed: 3.0
vs 3.4, 3.2 vs 3.2, and 1.8 vs 1.5 at 0, 12 and 18 months. The mean
number of visits to A&E and episodes of pneumonia was also similar
between the groups; gut and stoma infections were not significantly
different between the groups at baseline and at 12 months (table 3). Children who required gastrostomy feeds are often fragile and
at risk of recurrent infections, and concerns over the additional
risk of using a home-blended diet have been discussed.5 In this
study, there was no evidence of an increase in the number of
stoma site, gut infections or pneumonia in the home-blended
diet group compared with the formula-fed group. Anthropometry The BMISDS and MUAC were highly variable within the groups,
but the mean BMISDS was in the normal range in both groups and
across time (online supplemental figure 2). Original research Original research on October 23, 2024 by
http://adc.bmj.com/
Arch Dis Child: first published as 10.1136/archdischild-2023-326393 on 21 December 2023. Downloaded from among children with formula-fed diet than those with home-blended
diet: £16 386 vs £12 028 per annum at baseline, £18 049 vs £14 357
per annum at 12 months, and £8345 vs £5887 per half-year at 18
months, respectively (see tables 4 and 5). The cost of formula food
mainly contributed to such differences. As a trade-off, families in
home-blended group spent an estimated £294 in the previous 12
months (at baseline), £176 in the previous 12 months (at 12 months)
and £97 in the previous 6 months (at 18 months) on kitchen equip-
ment for blending and storing blended food. They also spent an
average of 88 min (at baseline), 85 min (at 12 months) and 103 min
(at 18 months) higher per day caring for children than those in the
formula-fed group. Nutritional content Information was available for ~86% of the children at baseline and
at 12 and 18 months (table 2). Those missing nutritional data were
all in the home-blended group. While the overall EQ-5D-5L scores in both groups were similar,
more parents reported health issues on dimensions such as pain/
discomfort and anxiety/depression at all time points (see online
supplemental table 1 and online supplemental figure 3). The data
on the other secondary outcomes are shown in online supplemental
table and figures. The patterns of nutritional intake in the two groups were similar
across time: ►
Macronutrient content: The fibre intake was higher in the
home-blended group. The kilocalorie intake per kilogram
of bodyweight was higher in the home-blended diet group,
with similar per cent of diet in both groups from fat, protein
and carbohydrate. ►
Micronutrient content: Both the home-blended and formu-
la-fed children had values above the DRV for vitamin B12,
folate, vitamin D, calcium, iron, manganese and zinc. Only
vitamin D was insufficient in the home-blended group. Health economic analyses The objective of the health economic evaluation was to assess
the costs, resource use and the associated health-related quality
of life of providing a home-blended diet compared with a
formula-fed diet only to children with gastrostomy tubes. We
compared the costs of providing formula and healthcare under Table 3 Safety outcomes at baseline and at 12-month and 18-month follow-up
Home-blended:
any follow-up
(n=104)
Formula fed: any
follow-up (n=76)
Home-blended: 12
months (n=79)
Formula fed: 12
months (n=55)
Home-blended: 18
months (n=57)
Formula fed: 18
months (n=48)
Number of gut infections during the
last 12 months
Mean (95% CI)
1.5 (0.9, 2.1)
2.7 (1.5, 3.9)
1.1 (0.8, 1.4)
2.4 (0.7, 4.2)
1.3 (−0.1, 2.8)
1.2 (0.5, 2.0)
Number of stoma site infections during
the last 12 months
Mean (95% CI)
1.3 (0.8, 1.9)
3.0 (1.8, 4.2)
1.5 (−0.1, 3.1)
2.1 (1.0, 3.2)
1.2 (0.7, 1.8)
1.9 (0.6, 3.2)
Number of tube blockages during the
last 12 months
Mean (95% CI)
2.9 (2.0, 3.8)
4.7 (−0.1, 9.5)
3.0 (1.7, 4.2)
6.7 (−6.9, 20.3)
1.8 (0.9, 2.7)
2.0 (−0.3, 4.3)
Number of replacements during the last
12 months
Mean (95% CI)
3.0 (2.6, 3.4)
3.4 (2.9, 3.8)
3.2 (2.8, 3.7)
3.2 (2.8, 3.7)
1.8 (1.5, 2.1)
1.5 (1.3, 1.7)
Number of times with pneumonia
during the last 12 months
Mean (95% CI)
1.6 (1.2, 2.1)
3.2 (−0.0, 6.4)
1.7 (1.1, 2.2)
1.1 (0.8, 1.5)
1.4 (0.8, 2.0)
1.5 (0.6, 2.4)
Number of visits to A&E
Mean (95% CI)
1.1 (0.7, 1.5)
1.4 (0.7, 2.1)
0.9 (0.5, 1.3)
0.9 (0.5, 1.2)
0.5 (0.3, 0.7)
0.6 (0.3, 0.8)
A&E, accident and emergency. ,
y g
y
py g
j Table 3 Safety outcomes at baseline and at 12-month and 18-month follow-up Fraser LK, et al. Arch Dis Child 2023;0:1–8. doi:10.1136/archdischild-2023-326393 5 on October 23, 2024 by guest. Protected by copyr
http://adc.bmj.com/
Arch Dis Child: first published as 10.1136/archdischild-2023-326393 on 21 December 2023. Downloaded from Original research Health economic outcomes The total costs to the NHS and PSS, comprising the cost of formula
food and the cost of health and social care services, were higher Table 4 Health and social care resource use at baseline and at 12-month and 18-month follow-up
Baseline*
12 months*
18 months†
Health and social
care resource use,
mean (SD)
Home-blended (n=104)
Formula fed (n=76)
Home-blended (n=79)
Formula fed (n=55)
Home-blended (n=57)
Formula fed (n=48)
General practitioner
visit
0.54 (0.50)
0.47 (0.50)
2.32 (9.05)
1.11 (1.33)
1.00 (1.54)
1.08 (1.54)
Paediatrician visit
2.66 (2.50)
2.96 (4.85)
2.46 (3.23)
2.05 (1.69)
1.18 (0.95)
1.15 (1.05)
Speech and language
therapist visit
5.40 (11.30)
2.93 (6.44)
5.05 (9.52)
5.09 (11.02)
2.70 (5.77)
3.42 (8.84)
Physiotherapist visit
11.47 (20.63)
6.13 (11.82)
10.14 (14.08)
7.04 (10.74)
5.77 (7.61)
4.23 (7.87)
Community children
nurse team visit
6.42 (9.50)
6.03 (8.75)
5.63 (10.54)
5.67 (7.87)
2.63 (6.96)
3.31 (7.82)
Dietitian visit
3.58 (3.51)
3.32 (2.94)
2.99 (2.99)
2.95 (1.87)
1.28 (1.19)
1.71 (1.56)
Hospital night stay
5.13 (10.09)
5.95 (12.38)
8.63 (31.96)
6.98 (17.41)
1.63 (3.67)
7.44 (20.00)
A&E visit
1.26 (2.17)
1.36 (3.08)
0.92 (1.72)
0.87 (1.26)
0.51 (0.87)
0.63 (0.94)
*Annual resource use. †Semiannual resource use. A&E, accident and emergency. Fraser LK, et al. Arch Dis Child 2023;0:1–8. doi:10.1136/archdischild-2023-326393 6 on October 23, 2024 by guest. Protec
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Arch Dis Child: first published as 10.1136/archdischild-2023-326393 on 21 December 2023. Downloaded from Original research
Table 5 Total cost per gastrostomy-fed child under the PSS and NHS perspective (£, 2021)
Baseline*
12 months*
18 months†
Cost component, mean (SD)
Home-blended
(n=104)
Formula fed (n=76) Home-blended (n=79) Formula fed (n=55) Home-blended (n=57)
Formula fed
(n=48)
Formula food
2315 (3679)
6485 (4770)
2489 (4528)
8016 (7291)
1070 (1414)
3429 (3496)
Health and social care services
9713 (11 688)
9901 (15 163)
12 047 (30 473)
10 033 (16 460)
3747 (4003)
4550 (9438)
Total
12 028 (13 307)
16 386 (15 704)
14 537 (31 495)
18 049 (18 132)
5887 (5447)
8345 (10 971)
*Annual cost. †Semiannual cost. NHS, National Health Service. 18-month study. There was an increase in financial costs to
families of feeding a home-blended diet, and given the evidence
that families who used a home-blended diet have higher levels
of education and live in areas of lower deprivation, future poli-
cies should address inequalities in access. Strengths and limitations Supplemental material This content has been supplied by the author(s). It
has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have
been peer-reviewed. Any opinions or recommendations discussed are solely those
of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and
responsibility arising from any reliance placed on the content. Where the content
includes any translated material, BMJ does not warrant the accuracy and reliability
of the translations (including but not limited to local regulations, clinical guidelines,
terminology, drug names and drug dosages), and is not responsible for any error
and/or omissions arising from translation and adaptation or otherwise. This has been the largest study of home-blended diets in children
with gastrostomies to date17 and the study used parent-prioritised
outcomes. Study retention was good despite the impact of the
pandemic; however, more of the home-blended group did come
from areas of lower deprivation and the parents had higher levels
of education. As this is an observational cohort study and not a
randomised controlled trial, there should be caution over the causal
implications. Consent for long-term follow-up using routine data
was obtained from the participants of this study. Hospital use and
survival are the main outcomes possible using this follow-up. Open access This is an open access article distributed in accordance with the
Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits
others to copy, redistribute, remix, transform and build upon this work for any
purpose, provided the original work is properly cited, a link to the licence is given,
and indication of whether changes were made. See: https://creativecommons.org/
licenses/by/4.0/. Patient consent for publication Not required. Ethics approval This study involves human participants and was approved by the
NHS Research Ethics Committee and Health Research Authority (REF 19/YH/0028). Participants gave informed consent to participate in the study before taking part. Ethics approval This study involves human participants and was approved by the
NHS Research Ethics Committee and Health Research Authority (REF 19/YH/0028). Participants gave informed consent to participate in the study before taking part. Provenance and peer review Not commissioned; externally peer reviewed. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement Data are available upon reasonable request. Data
may be available upon request to the PI if covered in the REC approval. Data availability statement Data are available upon reasonable request. Data
may be available upon request to the PI if covered in the REC approval. Twitter Lorna K Fraser @lornafraser10 The economic analyses have shown that while using a home-
blended diet is associated with a reduction in costs to the NHS
and PSS, there is an associated increase in costs and time on care
to families. This may in part explain why families in this study who
used a home-blended diet tended to be from areas of lower depri-
vation. There is no financial cost to the family of formula feeds
in the UK, but the distribution of costs may be different in other
healthcare systems. The additional costs and time required to use a
home-blended diet may mean that this is not a viable option for some
families. Although there is an increase in the number of commercial
companies producing prepackaged blended food, further research
is required in terms of the impact of symptoms from prepackaged
blended foods. Children with complex disabilities are already at risk
of inequalities in access to health and social care, so future services
and policies relating to enteral feeding must address the potential
financial impact of a home-blended diet. The safety profile of home-
blended diets should be useful to inform policies in schools and
hospitals where parents report varying levels of support for the use
of home-blended diets.19 Acknowledgements We would like to thank all the children and parents who
participated in this study and the healthcare professionals who recruited to this
study. We would also like to thank the members of our parent advisory board and
the clinical and academic experts on our study steering committee. Contributors LKF drafted the first version of the manuscript. LKF, CEH, JT, GR,
KAH and JC contributed to the conception of this study. LKF, CEH, GR, KAH, JC, JT
and AM contributed to the design of the study. Data collection was led by MO’N, JT,
JH and LKF. Analyses were undertaken by AB, HP and LKF. All authors reviewed the
manuscript prior to submission. LF is the guarantor. Funding This study is funded by the National Institute for Health Research Health
Technology Assessment programme (ref 17/76/06). This was a commissioned call
so the funder identified the topic area for this study but had no role in the study
conduct, data analyses or conclusions of the study. Competing interests JC is Director of Dietary Assessment, which supports
myfood24. Competing interests JC is Director of Dietary Assessment, which supports
myfood24. Health economic outcomes Home-blended diets
should be seen as a safe alternative to commercial formula,
unless there is a specific clinical contraindication. These data
show that home-blended diets can provide similar nutritional
intake to commercial formula in children who require gastros-
tomy feeding. High-quality studies are required to address any
differences in long-term outcomes for children who are fed
home-blended diets. One of the main concerns raised by professionals about the nutri-
tional adequacy of home-blended diets relates to the viscosity of
the feeds required to get through the tubes and therefore the large
volumes that may be required to maintain an adequate calorific
intake.5 18 In this study, the calorific intake was higher in the home-
blended group across the time period while maintaining adequate
anthropometric measures. Previous research has shown that gastros-
tomy-fed children may be able to tolerate higher volumes of home-
blended feeds than formula.18 The micronutrient content in this
current study was also relatively stable and above DRV for all, apart
from vitamin D, which is similar to what cross-sectional studies have
shown.5 13 Children who require specific nutritional content in their
diet, for example ketogenic diets, may require more input from
dietitians. on October 23, 2024 by guest. Protec
http://adc.bmj.com/
1 December 2023. Downloaded from Original research Jo Taylor http://orcid.org/0000-0001-5898-0900
Catherine Elizabeth Hewitt http://orcid.org/0000-0002-0415-3536 9 Fraser LK, Bedendo A, O’neill M, et al. Yourtube’ the role of different diets in
gastrostomy-fed children: baseline findings from a prospective cohort study. Dev Med
Child Neurol 2023. 10 Varni JW, Bendo CB, Denham J, et al. PedsQL gastrointestinal symptoms scales
and gastrointestinal worry scales in pediatric patients with functional and
organic gastrointestinal diseases in comparison to healthy controls. Qual Life Res
2015;24:363–78. 10 Varni JW, Bendo CB, Denham J, et al. PedsQL gastrointestinal symptoms scales
and gastrointestinal worry scales in pediatric patients with functional and
organic gastrointestinal diseases in comparison to healthy controls. Qual Life Res
2015;24:363–78. REFERENCES 1 Pardy C, Kharma N, Lau R, et al. Point prevalence of gastrostomy in a paediatric
population. J Pediatr Gastroenterol Nutr 2021;72:528–31. 11 Devlin NJ, Shah KK, Feng Y, et al. Valuing health-related quality of life: an EQ-5D-5L
value set for England. Health Econ 2018;27:7–22. 2 Breaks A, Bloch S, Smith C. Determinants in parents’ decision to use blended diets
with gastrostomy-fed children and young people: a mixed methods study. Clin Nutr
ESPEN 2022;51:288–94. 12 Benzies KM, Trute B, Worthington C, et al. Assessing psychological well-being in
mothers of children with disability: evaluation of the parenting morale index and
family impact of childhood disability scale. J Pediatr Psychol 2011;36:506–16. 3 British Dietetic Association. Practice toolkit; liquidised food via gastrostomy tube. 2017. Available: https://www.bda.uk.com/professional/practice/liquidisedtoolkit
[Accessed 1 Sep 2017]. 13 Hron B, Fishman E, Lurie M, et al. Health outcomes and quality of life indices of
children receiving blenderized feeds via enteral tube. J Pediatr 2019;211:139–45. 4 Romano C, van Wynckel M, Hulst J, et al. European society for paediatric
gastroenterology, hepatology and nutrition guidelines for the evaluation and
treatment of gastrointestinal and nutritional complications in children with
neurological impairment. J Pediatr Gastroenterol Nutr 2017;65:242–64. 14 Rosenbaum PR, Rubin DB. The central role of the propensity score in observational
studies for causal effects. Biometrika 1983;70:41–55. 15 Fraser LK, Gibson-Smith D, Jarvis S, et al. Estimating the current and future prevalence
of life-limiting conditions in children in England. Palliat Med 2021;35:1641–51. 5 Köglmeier J, Assecaira I, Banci E, et al. The use of blended diets in children with
enteral feeding tubes: a joint position paper of the ESPGHAN committees of allied
health professionals and nutrition. J Pediatr Gastroenterol Nutr 2023;76:109–17. 16 British national Formulary. British National Formulary; 2020. 17 McCormack S, Patel K, Smith C. Blended diet for enteral tube feeding in young
people: a systematic review of the benefits and complications. J Hum Nutr Diet
2023;36:1390–405. g
7 Taylor J, O’Neill M, Maddison J, et al. “’Your tube’: the role of different diets in
children who are gastrostomy Fed: protocol for a mixed methods exploratory
sequential study”. BMJ Open 2019;9:e033831. 18 Phillips G. Patient and carer experience of blended diet via gastrostomy: a qualitative
study. J Hum Nutr Diet 2019;32:391–9. 19 Maddison J, Taylor J, O’Neill M, et al. Outcomes for gastrostomy-fed children and
their parents: qualitative findings from the ’your tube’ study. Dev Med Child Neurol
2021;63:1099–106. CONCLUSIONS Children who were fed a home-blended diet maintained an
adequate nutritional intake and had no increase in safety events
when compared with children who were formula fed in this ORCID iDs
Lorna K Fraser http://orcid.org/0000-0002-1360-4191 ORCID iDs
Lorna K Fraser http://orcid.org/0000-0002-1360-4191 ORCID iDs 7 Fraser LK, et al. Arch Dis Child 2023;0:1–8. doi:10.1136/archdischild-2023-326393 REFERENCES 19 Maddison J, Taylor J, O’Neill M, et al. Outcomes for gastrostomy-fed children and
their parents: qualitative findings from the ’your tube’ study. Dev Med Child Neurol
2021;63:1099–106. 8 Carter MC, Albar SA, Morris MA, et al. Development of a UK online 24-H dietary
assessment tool: myfood24. Nutrients 2015;7:4016–32. 8 8
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Pharmacodynamic Impact of Carboxylesterase 1 Gene Variants in Patients with Congestive Heart Failure Treated with Angiotensin-Converting Enzyme Inhibitors
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PloS one
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cc-by
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RESEARCH ARTICLE OPEN ACCESS Citation: Nelveg-Kristensen KE, Bie P, Ferrero L,
Bjerre D, Bruun NE, Egfjord M, et al. (2016)
Pharmacodynamic Impact of Carboxylesterase 1
Gene Variants in Patients with Congestive Heart
Failure Treated with Angiotensin-Converting
Enzyme Inhibitors. PLoS ONE 11(9): e0163341. doi:10.1371/journal.pone.0163341 Editor: Katriina Aalto-Setala, University of Tampere,
FINLAND Received: February 3, 2016
Accepted: September 7, 2016
Published: September 23, 2016 Copyright: © 2016 Nelveg-Kristensen 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. Methods Patients with CHF, and clinical indication for ACEIs were categorized according to their
CES1 genotype. Differences in mean plasma ATII/ATI ratios between genotype groups
after ACEI dose titration, expressed as the least square mean (LSM) with 95% confidence
intervals (CIs), were assessed by analysis of variance. Data Availability Statement: Data Availability:
Only summarized data (analyses, means ect.)
can be accessed directly and are put forward in
the accompanying manuscript. However, data on
an individual level, such as the data underlying
the current study are restricted by Danish law,
i.e., the Act on Processing of Personal Data and
the rules of confidentiality in the Danish health
legislation, which is emphasized for healthcare
professionals in Denmark. Data can only be
made available for researchers who meet certain Karl Emil Nelveg-Kristensen1*, Peter Bie2, Laura Ferrero3, Ditte Bjerre3, Niels E. Bruun1,5,
Martin Egfjord4, Henrik B. Rasmussen3‡, Peter R. Hansen1, INDICES Consortium¶ a1111 1 Department of Cardiology, Gentofte University Hospital, Gentofte, Denmark, 2 Institute of Molecular
Medicine, University of Southern Denmark, Odense, Denmark, 3 Institute of Biological Psychiatry, Mental
Health Centre Sct. Hans, Copenhagen University Hospital, Roskilde, Denmark, 4 Department of
Nephrology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark, 5 Clinical Institute,
Aalborg University, Aalborg, Denmark ‡ Lead author of the INDICES Consortium
¶ Membership of the INDICES Consortium is provided in the Acknowledgments. ‡ Lead author of the INDICES Consortium
¶ Membership of the INDICES Consortium is provided in the Acknowledgments. * karl.emil.kristensen@regionh.dk ‡ Lead author of the INDICES Consortium
¶ Membership of the INDICES Consortium is provided in the Acknowledgments. * karl.emil.kristensen@regionh.dk Background Variation in the carboxylesterase 1 gene (CES1) may contribute to the efficacy of ACEIs. Accordingly, we examined the impact of CES1 variants on plasma angiotensin II (ATII)/
angiotensin I (ATI) ratio in patients with congestive heart failure (CHF) that underwent
ACEI dose titrations. Five of these variants have previously been associated with drug
response or increased CES1 expression, i.e., CES1 copy number variation, the variant of
the duplicated CES1 gene with high transcriptional activity, rs71647871, rs2244613, and
rs3815583. Additionally, nine variants, representatives of CES1Var, and three other CES1
variants were examined. Editor: Katriina Aalto-Setala, University of Tampere,
FINLAND PharmacodynamicImpact of
Carboxylesterase 1 Gene Variants in Patients
with Congestive Heart Failure Treated with
Angiotensin-Converting Enzyme Inhibitors Karl Emil Nelveg-Kristensen1*, Peter Bie2, Laura Ferrero3, Ditte Bjerre3, Niels E. Bruun1,5,
Martin Egfjord4, Henrik B. Rasmussen3‡, Peter R. Hansen1, INDICES Consortium¶ Karl Emil Nelveg-Kristensen1*, Peter Bie2, Laura Ferrero3, Ditte Bjerre3, Niels E. Bruun1,5,
Martin Egfjord4, Henrik B. Rasmussen3‡, Peter R. Hansen1, INDICES Consortium¶ Introduction Activation of the renin-angiotensin-aldosterone system (RAAS) plays a pivotal role in cardio-
vascular disease and treatment with angiotensin-converting enzyme inhibitors (ACEIs), which
inhibit the hydrolytic conversion of angiotensin I (ATI) to angiotensin II (ATII), forms an
important part of the treatment for congestive heart failure (CHF), hypertension, and ischemic
heart disease. ACEI treatment, however, is associated with substantial variability in efficacy,
which cannot solely be explained by individual differences in clinical characteristics [1–8]. Although genetic diversity may contribute to such variability there is as yet very limited evi-
dence available on this clinically important subject [9]. Most ACEIs are ester prodrugs, which are hydrolyzed to their active metabolites by hepatic
carboxylesterase 1 (CES1) [10–12]. The activity of CES1 has been associated with marked indi-
vidual variability and variants in the CES1 gene (CES1) as well as pharmacological CES1 inhibi-
tion have been shown to influence the CES1 hydrolytic capacity, and hence the activation of
ACEIs [13–18]. The structure of CES1 is complex. For example, CES1 is subjected to duplica-
tion. The duplicated version of CES1 is designated CES1A2, while CES1A1 is the original gene
copy [19]. Duplication of CES1 has been associated with the pharmacokinetics of irinotecan in
a dosage-dependent manner [20]. The haplotype of CES1A2 with the ‘active promoter’, which
is characterized by having two Sp1 transcription factor binding sites, has been associated with a
higher transcriptional level of CES1 that may lead to increased CES1 activity [18, 19]. On the
other hand, a well-established non-synonymous missense single nucleotide polymorphism
(SNP), rs71647871 (Gly143Glu), in CES1A1 has been associated with decreased CES1 activity
and reduced bioactivation of trandolapril [13]. In addition to ACEIs, CES1 is also important to
the metabolism of clopidogrel, the anticoagulant prodrug dabigatran exitelate, and the central
acting psychostimulant methylphenidate [21–23]. In this regard, rs2244613, which is located
in a CES1A1 intronic region, has been associated with decreased bioavailability of dabigatran,
the activated metabolite of dabigatran exitelate, and reduced bleeding in dabigatran etxitelate-
treated patients, and rs3815583 in the CES1A1 promoter, has been linked to appetite reduction
among ADHD patients treated with methylphenidate e [24, 25]. CES1A1 also harbors a set of
SNPs in its upstream part that are in strong LD with each other, including a SNP with a poten-
tial effect on the amount of enzyme produced, due to its localization in the Kozak sequence of
the gene. Conclusion These findings indicate that the included single variants of CES1 do not significantly influ-
ence plasma ATII/ATI ratios in CHF patients treated with ACEIs and are unlikely to be pri-
mary determinants of ACEI efficacy. Competing Interests: The authors have declared
that no competing interests exist. CES1 Gene Variants and ACEI Treatment qualified for further assessment. In the fully adjusted analysis of effects of rs2302722 on
plasma ATII/ATI ratios, the difference in mean ATII/ATI ratio between the GG genotype
and the minor allele carriers (GT and TT) was not significant, with a relative difference in
LSMs of 0.67 (95% CI 0.43–1.07; P = 0.10). Results of analyses that only included enala-
pril-treated patients remained non-significant after Bonferroni correction for multiple parallel
comparisons (difference in LSM 0.60 [95% CI 0.37–0.98], P = 0.045). criteria for access to confidential data. Requests
for these and other data may be made on an
individual basis to the corresponding author
(E-mail: karl.emil.kristensen@regionh.dk). Funding: The project INDICES (INDIvidualised
drug therapy based on pharmacogenomics: focus
on carboxylesterase 1, CES1) aims at developing
strategies for individualized treatment with
methylphenidate and angiotensin converting
enzyme inhibitors. It is supported by grant 10-
092792/DSF from the Danish Council for Strategic
Research, Programme Commission on Individuals,
Disease and Society. The funder had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. Results A total of 200 patients were recruited and 127 patients (63.5%) completed the study. The
mean duration of the CHF drug dose titration was 6.2 (SD 3.6) months. After ACEI dose
titration, there was no difference in mean plasma ATII/ATI ratios between subjects with the
investigated CES1 variants, and only one previously unexplored variation (rs2302722) 1 / 18 PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 Plasma angiotensin analyses The plasma concentrations of ATI and ATII were determined by radioimmunoassay as
describedpreviously [31, 32]. Specific anti-ATI and anti-ATII antibodies, i.e., Ab-3-20008939
and Ab-5–030682 raised in rabbits were used in final dilutions of at least 1:100.000. There was
<0.1% cross reactivity between these antibodies for ATI and ATII, but Ab-5–030682 cross-
reacts with shorter bioactive angiotensins [31]. In brief, plasma samples were acidified by 4%
acetic acid, extracted by use of C-18 Sep-Pak cartridges (Waters, Hedehusene, Denmark) and
dried overnight. After elution, samples and antibodies were incubated for 24 hours, and known
amounts of 125Iodine (I)-labeled ATI and ATII were then added for another 24 hours of incu-
bation. Sediments of free antigen were obtained by adding a charcoal-plasma suspension fol-
lowed by centrifugation. Finally, the radioactivity of the supernatant representing 125I bound
antibodies was measured, the ATI and ATII concentrations were determined, and plasma ATI/
ATII ratio was calculated. Subjects In the period 2012–2014 patients 18 years of age with CHF of any cause and a left ventricular
ejection fraction 45% who were referred to the CHF outpatient clinic at Gentofte University
Hospital, Copenhagen, Denmark, for initiation or dose titration of ACEIs and other CHF
drugs were recruited for the study. Patients were routinely followed by outpatient consultations
every second to fourth week until completion of CHF drug dose titration. Main exclusion crite-
ria were treatment with captopril or lisinopril that are not metabolized by CES1 [10], treatment
with angiotensin II type I receptor blockers, pregnancy, malignant disease, CHF requiring hos-
pitalization, and baseline serum creatinine 150 mmol/l. At baseline blood samples (10 ml)
were collected in EDTA-containing tubes for CES1 genotyping. Patients were followed until
they had been titrated to maximal tolerable doses of ACEIs, beta-blockers, and aldosterone
antagonists, for at least two weeks or a maximum of 2 months [30]. On the day of study termi-
nation, blood samples (10 ml) were collected for determination of plasma ATI and ATII levels,
and the plasma ATII/ATI ratio was calculated. Blood samples were collected during day time
(9:00 AM–2:00 PM) in pre-chilled tubes containing EDTA and aprotinine, immediately centri-
fuged, and plasma stored at -20°C until analysis. The patients rested for 15–20 minutes in a sit-
ting position prior to blood sample collection and the time of the last preceding ACEI drug
ingestion was carefully recorded. Patients who stopped ACEI treatment during the follow up
period were excluded and the causes for ACEI discontinuation, e.g., adverse events or non-
compliance, were registered. Introduction To our knowledge, there are no reports available on the relationship between CES1
variants and pharmacodynamic effects of ACEIs and it is notable that the plasma ATII/ATI PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 2 / 18 CES1 Gene Variants and ACEI Treatment ratio is closely correlated to circulating levels of active ACEI metabolites [26–29]. In this study
we therefore examined the influence of the above-mentioned genetic variations in CES1 on
the plasma ATII/ATI ratio in ACEI-treated patients with CHF including nine of the SNPs in
the upstream part of CES1A1, which form the so-called CES1Var. In addition, three selected
CES1 variants that were not suspected to have a functional impact themselves were included as
potential markers of causal genetic variants. Genetic analyses Genomic DNA was extracted from the EDTA-stabilized blood samples using the Maxwell1stru-
ment (Promega Corporation, Madison, WI, USA). Subsequently, we determined the total num-
ber of copies of CES1A1 and CES1A2 using a commercially available assay based on duplex real- 3 / 18 PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 CES1 Gene Variants and ACEI Treatment time PCR (Thermo Fisher Scientific,Waltham, MA USA). This assay targeted intron 11 (Taq-
Man1 copy number assay Hs00139541_cn) in CES1A1 and CES1A2. Since this region is identi-
cal in CES1A1 and CES1A2 the assay determined the number of copies of both of these gene
versions. Deletion of CES1A1 has not been reported. Hence, all individuals in our study were
assumed to harbor two CES1A1 copies. With this assumption, a copy number of three or four as
determined by the above assay signifiedpresence of one or two CES1A2 copies. For samples lacking CES1A2 two overlapping long range PCRs were carried out allowing
for the amplification of all CES1A1 exons. The first of these long PCRs amplified a 12.5 kb frag-
ment of the gene containing its promoter and exon 1–5. The second long PCR amplified a 19.2
kb CES1A1 fragment containing exon 6–14. For samples with three or four copies, i.e. samples
harboring CES1A1 as well as CES1A2, the 12.5 kb fragment of CES1A1 as well as the corre-
sponding CES1A2 fragment with the approximately same size was amplified. The long PCR for
amplification of the CES1A1 fragment containing exon 6–14 is unable to distinguish CES1A1
from CES1A2 and was therefore not applicable for analysis of samples containing both of these
gene versions. The sequences of the forward and reverse primers for amplification of the 12.5
kb fragment of CES1A1 were 5’-ACTATGGGGGGACGGAGTTCA-3’ and 5’-CCAGTCCT
GAATTCAGGTATTGTAAT CA-3’. The 12.5 kb fragment of CES1A2 was amplified using the
same reverse primer and a forward primer with the sequence 5’- CAGGAGCTATTGAGA
GATGGAATCAT-3’. For amplification of the 19.2 kb fragment of CES1A1 we used a forward
and reverse primer with the sequences 5’-CTGATTACAATACCT GAATTCAGGAC-3’ and
5’-GTATTTCTGCTCATTATGGT CACG-3’, respectively. The amplified fragments were sub-
jected to Sanger sequencing in order to determine the genotypes of the following CES1A SNPs:
rs3815583, rs12149373, rs12149371, rs12149322, rs12149370, rs12149368, rs111604615,
rs566557773, rs201577108, rs12149366, rs56278207, rs71647871, rs2302722, rs2244614, and
rs2244613. We also identified the CES1A2 variant with increased transcriptional activity, i.e.,
the haplotype carrying the ‘active promoter’ [18]. Genetic analyses Our procedure was validated using a variety
of different approaches. Notably, we showed that the CES1A2 specific primers did not support
amplification of samples that did not harbor this gene variant as determined by the real time
PCR describedabove, and traces of CES1P1 (the CES1-related pseudogene) or other undesired
sequences were not detected in the Sanger chromatograms (not shown). The single nucleotide variations rs12149368, rs12149373, rs12149371, rs12149322, rs12149370,
rs111604615, rs566557773, rs201577108, and rs12149366, which all are located in the 5’ UTR or
exon 1 of CES1A1,constitute a major haploblock. The variation rs12149368 resides at a position
immediately upstream to the initiation codon of CES1A1,thereby altering the Kozak sequence in
this gene. The variations rs111604615, rs201577108 and rs12149366 all cause an amino acid
change. Consequently, we hypothesized that the abovementioned major haploblock was associ-
ated with altered CES1 activity [33]. The single nucleotide variation rs56278207, which is located
in intron 1 of CES1A1,is in LD with the variations in the major haploblock and servedas an addi-
tional marker of this block. Similarly, rs2244614 of CES1A1,which is in linkage disequlibrium
with rs2244613, was included in the analysis. Finally, intronic rs2302722 was selected as another
marker since its genotype proportion was compatible with those expectedunder Hardy-Weinberg
equilibrium (HWE) and the minor allele frequency (MAF) of this single nucleotide variation was
found to exceed 0.05. Alignment of sequences and detection of genetic variants were done using Lasergene
(DNASTAR, Madison, WI, USA). For mapping we applied Geneious v7.1.5 (Biomatters, Auck-
land, New Zealand). CES1A1 sequences were aligned to Hg19 (GRh37.p13) and CES1A2
sequences were aligned with AB119998.1. The CES1A2 variant with the “active promoter” was
identified based upon previously published sequence information [18]. Measures of pairwise
linkage disequilibrium (LD) expressed as r2 were calculated and visualized using Haploview 4 / 18 PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 CES1 Gene Variants and ACEI Treatment v4.2 [34]. The LD blocks were identified using the confidence interval method implemented
in Haploview. The Chi-square test was used to examine whether genotype proportions corre-
sponded to those expected under Hardy-Weinberg equilibrium. v4.2 [34]. The LD blocks were identified using the confidence interval method implemented
in Haploview. The Chi-square test was used to examine whether genotype proportions corre-
sponded to those expected under Hardy-Weinberg equilibrium. Ethics This study was approved by the Danish National Committee on Health Research Ethics (Pro-
tocol no.: H-4-2012-094) and the Danish data protection agency (I-suite no.: 01825 and identi-
fication no.: GEH-2012-032), respectively. All participants gave their written informed consent
before inclusion. Statistics One-way analyses of variance (ANOVA) were applied to model the differences between geno-
type groups in means for the plasma ATII/ATI ratio. Results from the ANOVA models values
were given as least square means (LSMs). To fit the assumption of a normally distributed out-
come variable, values of the plasma ATII/ATI ratio were transformed into a base 10 logarith-
mic scale before statistical assessment. Initially, all genetic variants were assessed in univariate
analyses. Only variants that produced a univariate significancelevel of P0.30 were further
investigated in multivariate ANOVA models adjusted for age, sex, height, blood pressure, treat-
ment with spironolactone or eplerenone, treatment with beta-blockers and, ultimately, also
adjusted for the timespan from last ACEI tablet ingestion until blood sample collection at the
day of study termination. The single nucleotide variants under examination were bi-allelic and
thus giving rise to three genotypes each. Hence, these variants were initially included as three-
level variables. By contrast, the CES1A2 haplotype with the ‘active promoter’ was exclusively
examined as a binary (active vs. inactive) variable [18]. In the adjusted models, the genetic vari-
ants with three genotypes were condensed into dichotomous variables, i.e., carriers or non-car-
riers of the minor allele, to increase the statistical power of the study. Sensitivity analyses were
done in a sub-group of enalapril-treated patients. The absence of interaction between variables
and the assumption of exposure group equality of the standard deviation were fulfilledif not
otherwisestated. Due to multiple testing, we used a Bonferroni-correctedsignificance level of
P0.01 (standard P-value of 0.05 divided by the number of independent analyses) for evalua-
tion of the association between CES1 variants and the outcome measure. SNPs located in a hap-
loblock are not independent and for such SNPs we therefore only corrected once. Variants
with low minor allele frequencies (MAFs) were excluded from the association analyses. We
used a Bonferroni-corrected significance level of P0.003 for the assessment of P values in the
Hardy Weinberg equilibrium (HWE) tests since these included all 15 examined CES1 SNP
genotypes and the copy number variation. Analyses and data management were performed in
SAS version 9.3 (SAS Institute Inc. Cary, North Carolina). Study population and CES1 genetics A total of 200 patients with CHF were recruited for the study. Hereof, 50 (25%) patients were
excluded because they discontinued their ACEI treatment before the final CHF drug dose
titration was achieved, 9 (4.5%) failed to show up for scheduled appointments, 8 (4.0%) were
excluded due to severe comorbidity (liver cirrhosis [n=1] and cancer [n=4]) or potential ACEI
adverse effects (kidney failure [n=1], hyperkalemia [n=1], and symptomatic hypotension
[n=1]), and 6 (3.0%) patients died, leaving 127 (63.5%) subjects available for analysis. Of these
127 patients, 99 (78%) were treated with enalapril, 24 (19%) with ramipril, and 4 (3%) with 5 / 18 PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 CES1 Gene Variants and ACEI Treatment Table 1. Baseline characteristics of the analyzed study population (n = 127). Female (n, [%])
30 (23.0)
Age (mean [SD] years)
68.50 (11.7)
Systolic BP (mmHg, mean [SD])
125.31 (17.9)
Diastolic BP (mmHg, mean [SD])
73.12 (11.3)
Height (cm, mean [SD])
176.48 (9.0)
BMI (kg/m2, mean [SD])
26.29 (4.6)
NYHA class (mean [SD])
1.46 (0.6)
EF (mean [SD])
0.38 (0.11)
PCI (n, %)
38 (29.9)
CABG (n, %)
21 (16.5)
DM type 1 (n, %)
2 (1.5)
DM type 2 (n, %)
20 (15.7)
Atrial fibrillation (n, %)
54 (42.5)
IHD (n, %)
66 (52.0)
COPD (n, %)
20 (15.7)
History of moking (n, %)
75 (59.1)
Enalapril (n, %)
99 (78.0)
Ramipril (n, %)
24 (18.9)
Trandolapril (n, %)
4 (3.1)
Beta-blockers (n, %)
110 (86.6)
Aldosterone inhibitors (n, %)
43 (38.9)
BMI: body mass index; BP: blood pressure; CABG: coronary artery bypass graft surgery; COPD: chronic
obstructive pulmonary disease; DM: diabetes mellitus;
EF: Left ventricular ejection fraction; IHD: ischemic heart disease; NYHA: New York Heart Association; PCI:
percutaneous coronary intervention; SD: standard deviation. doi:10.1371/journal.pone.0163341.t001 Table 1. Baseline characteristics of the analyzed study population (n = 127). doi:10.1371/journal.pone.0163341.t001 trandolapril, respectively. The mean duration of the CHF drug dose titration period was 6.2
(SD 3.6) months. Other baseline characteristics of subjects in the total and analyzed study pop-
ulation are shown in Table 1. As shown in Fig 1, there was a time-dependent and inverse rela-
tionship between plasma ATI levels and the plasma ATII/ATI ratio (P for interaction = 0.026). The distribution of CES1A2 with 2 (n = 89), 3 (n = 34), and 4 (n = 4) CES1 copies, respectively,
were in HWE (P = 0.73). Study population and CES1 genetics Allele frequencies of the CES1A1 variants are shown in Table 2. Plasma levels of ATI and ATII, and the plasma ATII/ATI ratio, respectively, in subjects with
each of the CES1 variants are shown in Table 3. The single nucleotide variants at rs12149373,
rs12149322, rs111604615, rs566557773, rs201577108, rs12149366 were not in HWE
(P<0.003) and the frequencies of the minor allele at rs71647871 and the “active promoter” of
CES1A2 were low (0.01 [n=2] and 0.03 [n=2], respectively) (Table 2). Accordingly, these three
variants were not considered for further statistical analysis. Similarly, data on the CES1A2
‘active promoter’ was not included in Table 2 as this haplotype was rare in our study popula-
tion. The reference alleles of rs56278207, rs2244613 and rs2244614 were not the major alleles
[35]. The majority of the investigated SNPs within CES1A1 were highly correlated as evidenced
by high R2 values in the pairwisecomparisons as shown in Fig 2. Data were missing for 41 indi-
viduals in the analyses of rs2302722, rs2244614, and rs2244613, respectively, which reflected
that the long PCR for amplification of exon 6–14 of CES1A1 is unable to discriminate CES1A1
from CES1A2 and therefore was not applicable for analysis of CES1A1 in individuals carrying
CES1A2 (Table 2). 6 / 18 PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 CES1 Gene Variants and ACEI Treatment Fig 1. Plasma angiotensin I (ATI) concentration and plasma ATII/ATI ratio after angiotensin-
converting enzyme inhibitor (ACEI) ingestion in patients with congestive heart failure after
completion of dose titration. Values are plotted against the time in hours from ACEI (enalapril, ramipril, or
trandolapril) ingestion until blood sample collection. Δ: Plasma ATII/ATI ratio, □: Plasma ATI concentrations. doi:10.1371/journal.pone.0163341.g001 Fig 1. Plasma angiotensin I (ATI) concentration and plasma ATII/ATI ratio after angiotensin-
converting enzyme inhibitor (ACEI) ingestion in patients with congestive heart failure after
completion of dose titration. Values are plotted against the time in hours from ACEI (enalapril, ram
trandolapril) ingestion until blood sample collection. Δ: Plasma ATII/ATI ratio, □: Plasma ATI concen
doi:10.1371/journal.pone.0163341.g001 doi:10.1371/journal.pone.0163341.g001 SNP: single nucleotide polymorphism; MAF: minor allele frequency; HWE: Hardy-Weinberg equilibrium. AF: minor allele frequency; HWE: Hardy-Weinberg equilibrium. SNP: single nucleotide polymorphism; MAF: minor allele frequency; HWE: Hardy-Weinberg equilibrium.
d i 10 1371/j
l
0163341 t002 Influence of CES1 gene variations on the plasma ATII/ATI ratio There were no statistical differences between the means of plasma ATII/ATI ratios from sub-
jects with any of the investigated CES1 variations when these were included either as three- or
two-level variables, respectively. Only rs2302722 (P = 0.27) complied with the pre-specified
limit (P <0.30) for further assessment in multivariate ANOVA models (Table 3). Mean plasma
ATII/ATI ratios among subjects with each of the three genotypes at rs2302722 and the loga-
rithmic-transformed values of the plasma ATII/ATI ratios used for the statistical analyses are
provided in Fig 3. As only two subjects were homozygous for the minor allele (T) at rs2302722,
the GT and TT genotypes were analyzed as a single group. In the fully adjusted analysis of the
effect of the rs2302722 genotype, the mean plasma ATII/ATI ratios for the GG genotype (LSM Table 2. CES1A1 variants: allele and genotype frequencies for the analyzed study population (n = 127). SNP
Position
Reference allele
Minor allele
Major allele
MAF
Missing (n)
Genotype (n/n/n)
P-value (HWE)
rs3815583
Promoter
T
G
T
0.20
7
8/32/80
0.068
rs12149373
Promoter
A
G
A
0.23
7
15/24/81
0.000
rs12149371
Promoter
A
G
A
0.19
7
9/28/83
0.007
rs12149322
Promoter
G
C
G
0.21
7
11/28/81
0.001
rs12149370
Promoter
A
G
A
0.18
7
5/34/83
0.556
rs12149368
Promoter
C
G
C
0.14
7
6/22/92
0.007
rs111604615
Exon
G
C
G
0.20
7
11/27/82
0.001
Rs566557773
Exon
C
T
C
0.20
7
11/27/82
0.001
rs201577108
Exon
T
C
T
0.20
7
11/27/82
0.001
rs12149366
Exon
T
G
T
0.20
7
10/27/83
0.002
rs56278207
Intron
-
-
T
0.33
9
18/42/58
0.034
rs71647871
Exon
G
A
G
0.01
4
0/2/121
0.927
rs2302722
Intron
G
T
G
0.10
41
2/13/71
0.160
rs2244614
Intron
C
C
T
0.31
41
8/37/41
0.933
rs2244613
Intron
C
C
A
0.04
41
0/7/79
0.694 . CES1A1 variants: allele and genotype frequencies for the analyzed study population (n = 127). Table 2. CES1A1 variants: allele and genotype frequencies for the analyzed st PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 7 / 18 CES1 Gene Variants and ACEI Treatment Table 3. Plasma angiotensin I (ATI) and ATII concentrations, and plasma ATII/ATI ratio. Gene variation
Genotype
AT I (pg/ml; mean[SD])
AT II (pg/ml; mean[SD])
AT II/I-ratio (mean[SD])
P- Valuea
P-Valueb
CES1A2 promoter
-
-
Weak
234.7 (31.2)
8.3 (8.6)
0.072 (0.134)
Strongc
59.1 (1.4)
4.2 (2)
0.071 (0.035)
-
-
-
Copy number
0.47
0.22
2
257.1 (258.3)
8.5 (8.3)
0.056 (0.049)
3
242.8 (354.6)
8.3 (6.9)
0.081 (0.133)
4
296.3 (383.7)
8.1 (2.4)
0.065 (0.044)
rs3815583 (T>G)
0.81
0.62
TT
233.0 (244.8)
8.3 (7.6)
0.067 (0.093)
TG
266.2 (353.5)
8.5 (9.2)
0.057 (0.044)
GG
290.6 (386.9)
7.7 (3.2)
0.066 (0.058)
rs71647871 (G>A)
-
-
GG
253.6 (289.2)
8.5 (7.9)
0.063 (0.08)
GA
171.4 (169.1)
8.5 (5.2)
0.068 (0.036)
AA
-
-
-
rs2244613 (C>A)
0.42
-
AA
267.4 (66.5)
8.6 (8.5)
0.056 (0.051)
CA
119.9 (112.3)
6.6 (7.3)
0.054 (0.015)
CC
-
-
-
rs2244614 (C>T)
0.64
0.39
TT
210.3 (188.4)
8.1 (7.7)
0.056 (0.055)
CT
261.9 (300.6)
8.6 (9.6)
0.056(0.047)
CC
258.3 (236.4)
9.3 (6.1)
0.06 (0.031)
rs12149373 (A>G)
0.09
0.23
AA
225.6 (242.2)
8 (7.5)
0.066 (0.091)
AG
329.7 (386.2)
9.5 (10.3)
0.044 (0.031)
GG
219.6 (311.8)
7.9 (3.8)
0.085 (0.07)
rs12149371 (A>G)
0.75
0.58
AA
224.8 (242.8)
8 (7.4)
0.066 (0.09)
AG
284.1 (365.4)
9.3 (9.7)
0.06 (0.054)
GG
225.6 (242.2)
7.7 (3.9)
0.059 (0.059)
rs12149322 (G>C)
0.49
0.63
GG
211.6 (227.3)
8 (7.5)
0.066 (0.091)
GC
317.1 (399.0)
9.5 (9.7)
0.052 (0.039)
CC
225.6 (242.2)
7.5 (3.6)
0.079 (0.077)
rs12149370 (A>G)
0.69
0.65
AA
225.6 (295.3)
8 (7.5)
0.066 (0.091)
AG
304.2 (377.9)
9.2 (8.9)
0.058 (0.051)
GG
172.5 (169.8)
6.9 (2.9)
0.073 (0.07)
rs12149368 (C>G)
0.80
0.88
CC
247.5 (295.3)
8.2 (7.5)
0.063 (0.086)
CG
214.4 (196.2)
9 (9.8)
0.07 (0.058)
GG
332.9 (421.3)
8 (3.7)
0.063 (0.068)
rs111604615 (G>C)
0.77
0.65
GG
233.4 (250.8)
8.1 (7.5)
0.065 (0.091)
GC
273.1 (356.8)
9.3 (9.8)
0.054 (0.038)
(Continued) Table 3. Plasma angiotensin I (ATI) and ATII concentrations, and plasma ATII/ATI ratio. PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 8 / 18 SD: standard deviation. doi:10.1371/journal.pone.0163341.t003 PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 SD: standard deviation. aGenetic variants included as three leveled variables. CES1 Gene Variants and ACEI Treatment Table 3. (Continued)
Gene variation
Genotype
AT I (pg/ml; mean[SD])
AT II (pg/ml; mean[SD])
AT II/I-ratio (mean[SD])
P- Valuea
P-Valueb
CC
269.9 (353.9)
7.8 (3.9)
0.079 (0.078)
rs566557773 (C>T)
0.90
0.65
CC
233.4 (250.8)
8.1 (7.5)
0.065 (0.091)
CT
259.7 (356.4)
9.4 (9.8)
0.063 (0.053)
TT
310.3 (351.6)
7.5 (3.7)
0.058 (0.055)
rs201577108 (T>C)
0.90
0.86
TT
233.4 (250.8)
8.1 (7.5)
0.065 (0.091)
TC
256.7 (356.4)
9.4 (9.8)
0.063 (0.053)
CC
310.3 (351.6)
7.5 (3.7)
0.058 (0.055)
rs12149366 (T>G)
0.67
0.93
TT
237.7 (252.3)
8.1 (7.4)
0.065 (0.09)
TG
236.5 (352.2)
9.2 (9.9)
0.065 (0.052)
GG
336.7 (358.9)
7.9 (3.7)
0.055 (0.057)
rs56278207 (ins[T])
0.78
0.62
TT
247.7 (311)
8.8 (7.9)
0.073 (0.105)
-T
244.2 (271.1)
8.6 (9)
0.06 (0.051)
—
210.3 (239.3)
6.1 (3.3)
0.05 (0.029)
rs2302722 (C>T)
0.27
0.10
GG
279.1 (274.2)
8.7 (8.9)
0.055 (0.052)
GT
122.5 (130.9)
5.5 (3)
0.065 (0.038)
TT
278.5 (125.9)
16.5 (9.2)
0.058 (0.007) SD: standard deviation. Fig 2. Linkage disequilibrium (LD) relationships between single nucleotide polymorphisms (SNPs)
of CES1A1. The top of the figure shows SNPs and the bottom of the figure shows LD relationships as well as
LD blocks of highly coupled variants. The strength of LD was determined by R2 statistics. doi:10.1371/journal.pone.0163341.g002 Fig 2. Linkage disequilibrium (LD) relationships between single nucleotide polymorphisms (SNPs)
of CES1A1. The top of the figure shows SNPs and the bottom of the figure shows LD relationships as well as
LD blocks of highly coupled variants. The strength of LD was determined by R2 statistics. Fig 2. Linkage disequilibrium (LD) relationships between single nucleotide polymorphisms (SNPs)
of CES1A1. The top of the figure shows SNPs and the bottom of the figure shows LD relationships as well as
LD blocks of highly coupled variants. The strength of LD was determined by R2 statistics. doi:10.1371/journal.pone.0163341.g002 doi:10.1371/journal.pone.0163341.g002 9 / 18 PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 CES1 Gene Variants and ACEI Treatment CES1 Gene Variants and ACEI Treatment Fig 3. Distribution of the plasma angiotensin II (ATII)/ATI ratios (A) and logarithmic-transformed ATII/
ATI ratios (B) according to CES1A1 rs2302722 genotypes. doi:10.1371/journal.pone.0163341.g003 Fig 3. Distribution of the plasma angiotensin II (ATII)/ATI ratios (A) and logarithmic-transformed ATII/
ATI ratios (B) according to CES1A1 rs2302722 genotypes. doi:10.1371/journal.pone.0163341.g003 doi:10.1371/journal.pone.0163341.g003 0.041 [95% confidence interval (CI) 0.031–0.055]) and the minor allele carriers, i.e., GT and
TT (LSM 0.059 [95% CI 0.037–0.096]) did not significantly differ from each other, with a rela-
tive difference in LSMs of 0.67 (95% CI 0.43–1.07; P = 0.10). In analyses restricted to only
include enalapril-treated patients that constituted the vast majority of our study population
(7 8%), the relative difference between LSMs for the GG (0.039 [95% CI 0.028–0.054]) and GT
(0.064 [95% CI 0.036–0.112]) remained non-significant after correction for multiple compari-
sons (0.60 [95% CI 0.37–0.98], P = 0.045). There were no enalapril-treated individuals who
were homozygous for the minor allele (TT) of rs2302722. Hence, this genotype was not repre-
sented in the latter analysis. Numerous variants
within the entire gene encoding CES1 have been reported of which some have been shown to
reduce or increase CES1 transcription and CES1 activity [18, 19, 36]. However, as yet only very
few CES1 variants have been associated with altered pharmacokinetics of ACEIs and clinical
outcomes among ACEI-treated patients. The non-conservative single nucleotide substitution
variation at codon 143 in exon 4 of CES1A1 that results in a change of the nucleotide G to an
A (rs71647871) was associated with marked reduction of CES1 in vitro activity and complete
inhibition of the hydrolytic conversion of trandolapril to the active metabolite trandolaprilat The HWE testing of several of the SNPs produced low P values which could reflect genotyp-
ing inaccuracies or non-random sampling of our study population. However, since the applied
genotyping procedure has been extensively validated in our laboratory without giving rise to
suspected co-amplification of undesired DNA fragments, e.g., fragments of CES1P1 or mixed
reads in the Sanger sequencing chromatograms (data not shown), it is highly unlikely that geno-
typing inaccuracies were primary determinants of these low P values. Instead, we focused our
attention on the potential pathophysiological functions of CES1. Besides having a role in the
hydrolytic conversion of various drugs, CES1 is involved in several endogenous physiological
processes, e.g., hydrolytic conversion of cholesteryl esters and triacylglycerol,[39–43]fatty acyl
coenzyme A hydrolysis,[44] and fatty acid ethyl ester synthase activity,[44, 45] and may there-
fore be implicated in development of cardiovascular disease, e.g. by contributing to dyslipidemia
associated with increased risk of ischaemic heart disease.[46] Accordingly, the low P values
observedupon HWE testing of several of the SNPs could reflect that patients with certain CES1
genotypes and CHF disease subtypes were preferentially recruited in the study. Also, the scien-
tific value of analyses of SNPs that are in strong LD may be limited as these to some extent rep-
resent redundant analyses. However, due to the observedminor differences in MAFs between
the SNPs in the LD block, which are all solitary markers of the haplotype carryingCES1VAR,
we found it reasonable to report the results of all the included genetic variations. In the present
study, although the subanalysis of enalapril-treated patients produced a P value of 0.045, the
results of the fully adjusted models of rs2302722 did not reach sufficient levels of significance,
particularly not after Bonferroni correction for multiple parallel comparisons. CES1 Gene Variants and ACEI Treatment results remained non-significant based on the Bonferroni-correctedsignificance level for mul-
tiple comparisons. To our knowledge, we are the first to investigate the impact of CES1 varia-
tion on a pharmacodynamic outcome parameter of ACEI treatment and although more studies
are clearly warranted, the results suggest that the investigated variants in CES1 are unlikely to
be major determinants of ACEI efficacy. j
y
Due to the pivotal role of CES1 in the hydrolytic activation of most ACEIs and an increased
focus on individually tailored cardiovascular treatment, pharmacogenetic research on the
importance of CES1 variation for the response to ACEI treatment, as well as studies of CES1-
mediated drug-druginteractions, has lately received increasing interest. Numerous variants
within the entire gene encoding CES1 have been reported of which some have been shown to
reduce or increase CES1 transcription and CES1 activity [18, 19, 36]. However, as yet only very
few CES1 variants have been associated with altered pharmacokinetics of ACEIs and clinical
outcomes among ACEI-treated patients. The non-conservative single nucleotide substitution
variation at codon 143 in exon 4 of CES1A1 that results in a change of the nucleotide G to an
A (rs71647871) was associated with marked reduction of CES1 in vitro activity and complete
inhibition of the hydrolytic conversion of trandolapril to the active metabolite trandolaprilat
[13]. This variation has also been associated with reduced in vitro hydrolysis of other ACEI
prodrugs, i.e., enalapril, ramipril, perindopril, moexipril, and fosinopril [36, 37]. To our knowl-
edge, only the SNP -816 A>C at CES1A2 has been associated with a clinical outcome measure
in patients treated with ACEIs, i.e., a reduction of the antihypertensive effect of imidapril in a
relatively small (n = 105) study of Japanese patients with hypertension [38]. However, a subse-
quent study of Japanese cancer patients revealed the SNP -816 A>C to reside in CES1P1 and
being absent or rare in CES1A2, thus questioning the exact nature of the association with the
response to imidapril [20]. Due to the pivotal role of CES1 in the hydrolytic activation of most ACEIs and an increased
focus on individually tailored cardiovascular treatment, pharmacogenetic research on the
importance of CES1 variation for the response to ACEI treatment, as well as studies of CES1-
mediated drug-druginteractions, has lately received increasing interest. Discussion In this study of patients with CHF that underwent dose titration with ACEIs that are activated
by CES1, we investigated the impact of a total of 17 selected CES1 variations on the plasma
ATII/ATI ratio, a proximal pharmacodynamic marker of ACEI activity. Genetic analysis con-
firmed a high level of LD between several of the investigated CES1 variations. Furthermore, we
found no significant association between the examined genotypes and the plasma ATII/ATI
ratio, when data were assessed in univariate unbalanced ANOVA models. In the subsequent
multivariate analyses of the effect of rs2302722 (the only CES1 variant that qualified for final
analyses) and in models that exclusively included enalapril-treated patients, respectively, PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 10 / 18 Accordingly, the negative findings of the present study do not
allow for conclusions on effects of CES1 variations on specific effects of ACEIs. Although the therapeutic actions of ACEIs are considered to represent a drug class effect,
the molecular structures of these drugs are distinct, which may affect their individual pharma-
cokinetic and pharmacodynamic properties [53, 54]. Also, the tissue penetration has been
shown to vary between ACEIs as a consequence of their respective lipophilicity [55]. A previ-
ous in vitro study also suggested that the efficacyof CES1-mediated hydrolysis of enalapril was
inferior to that of ramipril and trandolapril, and that these drugs exhibited different types of
CES1 enzyme kinetics [14]. Likewise, another recent study of healthy volunteers showed a 20%
reduction of enalaprilat (the activated form of enalapril) concentration in subjects homozygous
for the minor allele at rs71647871, whereas no observableeffect of this SNP was found on the
pharmacokinetics of quinapril [37]. Clopidogrel is also a substrate for CES1, and after ingestion
more than 90% of this prodrug is hydrolyzed to an inactive metabolite by hepatic CES1, thus
escaping cytochrome P450-mediated activation [23]. Importantly, in vitro studies have shown
that enalpril and trandolapril inhibited the CES1-mediated hydrolysis of clopidogrel to the
deesterifiedand inactive metabolite, which was translated into an increased risk of clinically
significant bleeding in patients with acute myocardial infarction co-treated with clopidogrel
and ACEIs [17]. However, these results have subsequently been challenged and the sum of cur-
rent evidence would appear to indicate that although CES1 variation may account for some
variability of CES1 enzymatic activity between individuals, the frequency and the effect size of
the variants of CES1 are likely to be small. Hence, these variants may have limited clinical rele-
vance [56]. Nonetheless,
rs2302722 represented the most promising pharmacogenetic variant of our current enquiry. In the current study, we did not find any association between CES1 variants and the plasma
ATII/ATI ratio when all ACEIs were included in the model, and the results did not change
when genetic variants were condensed into dichotomous variables or in multivariate analyses PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 11 / 18 CES1 Gene Variants and ACEI Treatment where only enalapril-treated patients were included, respectively. These results are in line with a
recently published in vitro study that found no association between CES1 copy number variation
and CES1 activity, and where the nonsynonymous CES1 variants G19V, S83L and A270S had
no influence on ACEI activation by CES1 [36]. Conversely, our results may be contrary to previ-
ously published results on a significant effect of rs2244613 on dabigatran activation and bleed-
ing, and the association between rs3815583 and appetite reduction in patients treated with
methylphenidate, respectively [24, 25]. However, the potential impact of these two genetic varia-
tions on CES1 expression and CES1 hydrolytic activity towards ACEIs or other substrates is not
yet known, and such observedeffects might reflect LD with causal genetic variants related to the
efficacyand safety of these drugs. Also, although the correlation between plasma ACE activity
and the systemic (plasma) ATII/ATI ratio is well documented, systemic activation of the renin-
angiotensin system does not necessarily reflect tissue specific ACE activity, which is also depen-
dent on the disease etiologyand other pathogenic mechanisms [28, 29, 47]. In this regard, a pre-
vious study has found that myocardial, but not systemic, pulmonary or renal ACE activities
were increased in a rodent model of CHF, and human cardiac ACE gene expression has been
shown to be increased among patients with CHF compared to persons with normal hearts [48,
49]. Intriguingly, there are also studies to suggest that tissue specific conversion of ATI to ATII
increases over time despite treatment with ACEIs, and that intracellular ATII synthesis as well
as the mediation of the more prolonged genomic effects of ATII, e.g., translation of growth fac-
tors and immunomodulatory cytokines, is independent of ACE activation and ATII receptor
type 1 binding, respectively [50, 51]. Furthermore, an aldosterone escape has been reported
in several studies with rising systemic levels of aldosterone during prolonged treatment with
ACEIs in patients with CHF [52]. Conclusion The present study of patients with CHF that underwent ACEI dose titration did not support an
association between a range of CES1 variants and ACEI pharmacodynamics measured by the
plasma ATII/ATI ratio. These findings indicate that the investigated variants in CES1 are
unlikely to be primary determinants of ACEI efficacy. Strengths and limitations The present study was restricted by a relatively small sample size and hence had limited statisti-
cal power. Consequently, we were unable to do analyses stratified for individual ACEIs and 12 / 18 PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 CES1 Gene Variants and ACEI Treatment ACEI doses, respectively. However, we found no significant differences in maintenance doses
of enalapril (the most frequently used ACEI) between the investigated CES1 variants (not
shown). In addition, low P values were obtained by HWE testing of several SNPs which com-
plicated the interpretation of some of the findings. In contrast to previous studies on plasma
ATI and ATII levels in ACEI-treated patients, where subjects rested in a supine position before
blood samples were collected, we applied a sitting position that may have influenced AT levels
[57]. However, the plasma ATII/ATI ratio and the relationship between this ratio and circulat-
ing levels of ATI during ACEI treatment observedin the present study were comparable to
previous findings [29, 58]. Also, environmental factors may have affected the impact of CES1
variants and ACEI treatment on the plasma ATII/ATI ratio, including dietary habits, and
results might have been influenced by CES1-dependent interactions with endogenous CES1
substrates and other CES1-metaboliseddrugs often used for patients with CHF, e.g., simva-
statin and carvedilol[59–62]. Furthermore, patients with CHF frequently have altered drug
pharmacokinetics owing to, e.g., intestinal congestion, reduced organ perfusion, and impaired
renal and hepatic drug clearance, which also may have affected the results [63]. Also, as the
current study did not examine all known genetic variants of CES1, we cannot exclude the possi-
bility that undetermined CES1 variants may have contributed to the variability in ACEI pro-
drug activation and subsequent pharmacodynamics. PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 Acknowledgments The project INDICES (INDIvidualised drug therapy based on pharmacogenomics: focus on
carboxylesterase 1, CES1) aims at developing strategies for individualizedtreatment with meth-
ylphenidate and angiotensin converting enzyme inhibitors. It is supported by grant 10-092792/
DSF from the Danish Council for Strategic Research, Programme Commission on Individuals,
Disease and Society. List of all partners in the INDICES Consortium: List of all partners in the INDICES Consortium: 1. Henrik Berg Rasmussen, Lead Author of the INDICES Consortium (Email: 1. Henrik Berg Rasmussen, Lead Author of the INDICES Consortium (Email:
henrik.berg.rasmussen@regionh.dk), Institute of Biological Psychiatry, Mental Health
Centre Sct. Hans, Copenhagen University Hospital, Roskilde, Denmark. henrik.berg.rasmussen@regionh.dk), Institute of Biological Psychiatry, Mental Health
Centre Sct. Hans, Copenhagen University Hospital, Roskilde, Denmark. 2. Ditte Bjerre, Institute of Biological Psychiatry, Mental Health Centre Sct. Hans, Copenhagen
University Hospital, Roskilde, Denmark. 2. Ditte Bjerre, Institute of Biological Psychiatry, Mental Health Centre Sct. Hans, Copenhagen
University Hospital, Roskilde, Denmark. 3. Majbritt Busk Madsen, Institute of Biological Psychiatry, Mental Health Centre Sct. Hans,
Copenhagen University Hospital, Roskilde, Denmark. 4. Laura Ferrero, Institute of Biological Psychiatry, Mental Health Centre Sct. Hans, Copenha-
gen University Hospital, Roskilde, Denmark. 5. Kristian Linnet, Section of Forensic Chemistry, Department of Forensic Medicine, Faculty
of Health Sciences, University of Copenhagen,Denmark. 5. Kristian Linnet, Section of Forensic Chemistry, Department of Forensic Medicine, Faculty
of Health Sciences, University of Copenhagen,Denmark. 6. Ragnar Thomsen, Section of Forensic Chemistry, Department of Forensic Medicine, Faculty
of Health Sciences, University of Copenhagen,Denmark. 13 / 18 PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 CES1 Gene Variants and ACEI Treatment 7. Gesche Jürgens, Department of Clinical Pharmacology, Bispebjerg University Hospital,
Copenhagen, Denmark. 8. Kim Dalhoff, Department of Clinical Pharmacology, Bispebjerg University Hospital,
Copenhagen, Denmark. 9. Claus Stage, Department of Clinical Pharmacology, Bispebjerg University Hospital, Copen-
hagen, Denmark. 10. Hreinn Stefansson, CNS Division, deCODE Genetics, Reykjavik, Iceland. 11. Thomas Hankemeier, The Leiden/Amsterdam Center for Drug Research LACDR, Leiden
University, Gorlaeus laboratories, Leiden, The Netherlands. 12. Rima Kaddurah-Daouk, Department of Psychiatry and Behavioral Sciences, Duke Univer-
sity, Durham, NC, USA. 13. Søren Brunak, Center for Biological Sequence Analysis, Technical University of Denmark,
Kgs. Lyngby, Denmark. 14. Olivier Taboureau, Center for Biological Sequence Analysis, Technical University of Den-
mark, Kgs. Lyngby, Denmark. 15. Grace Shema Nzabonimpa, Center for Biological Sequence Analysis, Technical University
of Denmark, Kgs. Lyngby, Denmark. 16. PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 Acknowledgments Tine Houmann, Centre for Child and Adolescent Mental Health, Mental Health Services
in the Capital Region of Denmark. 17. Pia Jeppesen, Centre for Child and Adolescent Mental Health, Mental Health Servicesin
the Capital Region of Denmark. 18. Kristine Kaalund-Jørgensen, Centre for Child and Adolescent Mental Health, Mental
Health Servicesin the Capital Region of Denmark. 19. Peter Riis Hansen, Department of Cardiology, Copenhagen University Hospital, Hellerup,
Denmark. 20. Karl Emil Nelveg-Kristensen, Department of Cardiology, Copenhagen University Hospital,
Hellerup, Denmark. 21. Anne Katrine Pagsberg, Centre for Child and Adolescent Mental Health, Mental Health
Servicesin the Capital Region of Denmark. 22. Kerstin Plessen Centre for Child and Adolescent Mental Health, Mental Health Servicesin
the Capital Region of Denmark. 23. Poul-Erik Hansen, Department of Science, Systems and Models, Roskilde University, Ros-
kilde, Denmark. 24. Thomas Werge, Institute of Biological Psychiatry, Mental Health Centre Sct. Hans, Copen-
hagen University Hospital, Roskilde, Denmark. 25. Jørgen Dyrborg, Centre for Child and Adolescent Mental Health, Mental Health Services
in the Capital Region of Denmark. 26. Maj-Britt Lauritzen, Centre for Child and Adolescent Mental Health, Mental Health Ser-
vices in the Capital Region of Denmark. 14 / 18 PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 CES1 Gene Variants and ACEI Treatment Author Contributions Author Contributions
Conceptualization: KENK PB NEB HBR PRH. Data curation: KENK LF DB HBR PRH. Formal analysis: KENK PB LF DB HBR PRH. Funding acquisition: HBR PRH. Investigation: KENK PB LF DB HBR PRH. Methodology:KENK PB LF DB HBR PRH. Project administration: KENK HBR PRH. Resources: KENK PB NEB HBR PRH. Software: PB HBR. Supervision:PB NEB ME HBR PRH. Validation: PB HBR. Visualization: KENK LF HBR PRH Author Contributions
Conceptualization: KENK PB NEB HBR PRH. Data curation: KENK LF DB HBR PRH. Formal analysis: KENK PB LF DB HBR PRH. Funding acquisition: HBR PRH. Investigation: KENK PB LF DB HBR PRH. Methodology:KENK PB LF DB HBR PRH. Project administration: KENK HBR PRH. Resources: KENK PB NEB HBR PRH. Software: PB HBR. Supervision:PB NEB ME HBR PRH. Validation: PB HBR. Visualization: KENK LF HBR PRH. Writing – original draft: KENK HBR PRH. References Zhu HJ, Appel DI, Johnson JA, Chavin KD,Markowitz JS. (2009) Role of carboxylesterase 1 and
impact of natural genetic variants on the hydrolysis of trandolapril. Biochem Pharmacol 77: 1266–72. doi: 10.1016/j.bcp.2008.12.017 PMID: 19185566 14. Thomsen R, Rasmussen HB, Linnet K,Consortium I. (2014) In vitro drug metabolism by human carbox-
ylesterase 1: focus on angiotensin-converting enzyme inhibitors. Drug Metab Dispos 42: 126–33. doi:
10.1124/dmd.113.053512 PMID: 24141856 15. Takai S, Matsuda A, Usami Y, Adachi T, Sugiyama T, Katagiri Y, et al. (1997) Hydrolytic profile for
ester- or amide-linkage by carboxylesterases pI 5.3 and 4.5 from human liver. Biol Pharm Bull 20:
869–73. PMID: 9300133 16. Sato Y, Miyashita A, Iwatsubo T,Usui T. (2012) Simultaneous absolute protein quantification of carbox-
ylesterases 1 and 2 in human liver tissue fractions using liquid chromatography-tandem mass spec-
trometry. Drug Metab Dispos 40: 1389–96. doi: 10.1124/dmd.112.045054 PMID: 22504157 17. Kristensen KE, Zhu HJ, Wang X, Gislason GH, Torp-Pedersen C, Rasmussen HB, et al. (2014) Clopi-
dogrel bioactivation and risk of bleeding in patients cotreated with angiotensin-converting enzyme
inhibitors after myocardial infarction: a proof-of-concept study. Clin Pharmacol Ther 96: 713–22. doi:
10.1038/clpt.2014.183 PMID: 25222620 18. Yoshimura M, Kimura T, Ishii M, Ishii K, Matsuura T, Geshi E, et al. (2008) Functional polymorphisms
in carboxylesterase1A2 (CES1A2) gene involves specific protein 1 (Sp1) binding sites. Biochem Bio-
phys Res Commun 369: 939–42. doi: 10.1016/j.bbrc.2008.02.120 PMID: 18328811 19. Fukami T, Nakajima M, Maruichi T, Takahashi S, Takamiya M, Aoki Y, et al. (2008) Structure and char-
acterization of human carboxylesterase 1A1, 1A2, and 1A3 genes. Pharmacogenet Genomics 18:
911–20. PMID: 18794728 20. Sai K, Saito Y, Tatewaki N, Hosokawa M, Kaniwa N, Nishimaki-Mogami T, et al. (2010) Association of
carboxylesterase 1A genotypes with irinotecan pharmacokinetics in Japanese cancer patients. Br J
Clin Pharmacol 70: 222–33. doi: 10.1111/j.1365-2125.2010.03695.x PMID: 20653675 21. Laizure SC, Parker RB, Herring VL,Hu ZY. (2014) Identification of carboxylesterase-dependent dabi-
gatran etexilate hydrolysis. Drug Metab Dispos 42: 201–6. doi: 10.1124/dmd.113.054353 PMID:
24212379 22. Zhu HJ, Appel DI, Peterson YK, Wang Z,Markowitz JS. (2010) Identification of selected therapeutic
agents as inhibitors of carboxylesterase 1: potential sources of metabolic drug interactions. Toxicology
270: 59–65. doi: 10.1016/j.tox.2010.01.009 PMID: 20097249 23. Zhu HJ, Wang X, Gawronski B, Brinda B, Angiolillo D,Markowitz J. (2013) Carboxylesterase 1 as a
determinant of clopidogrel metabolism and activation. J Pharmacol Exp Ther 344: 665–72. doi: 10. 1124/jpet.112.201640 PMID: 23275066 24. References 1. Kober L, Torp-Pedersen C, Carlsen JE, Bagger H, Eliasen P, Lyngborg K, et al. (1995) A clinical trial of
the angiotensin-converting-enzyme inhibitor trandolapril in patients with left ventricular dysfunction
after myocardial infarction. Trandolapril Cardiac Evaluation (TRACE) Study Group. N Engl J Med 333:
1670–6. PMID: 7477219 2. Webb AJ, Fischer U, Mehta Z,Rothwell PM. (2010) Effects of antihypertensive-drug class on interindi-
vidual variation in blood pressure and risk of stroke: a systematic review and meta-analysis. Lancet
375: 906–15. doi: 10.1016/S0140-6736(10)60235-8 PMID: 20226989 3. Elliott WJ. (1996) Higher incidence of discontinuation of angiotensin converting enzyme inhibitors due
to cough in black subjects. Clin Pharmacol Ther 60: 582–8. PMID: 8941032 4. Schelleman H, Stricker BH, De Boer A, Kroon AA, Verschuren MW, Van Duijn CM, et al. (2004) Drug-
gene interactions between genetic polymorphisms and antihypertensive therapy. Drugs 64: 1801–16. PMID: 15301563 5. Materson BJ. (2007) Variability in response to antihypertensive drugs. Am J Med 120: S10–20. 6. Svanstrom H, Pasternak B, Melbye M,Hviid A. (2015) Use of different types of angiotensin converting
enzyme inhibitors and mortality in systolic heart failure. Int J Cardiol 182: 90–6. doi: 10.1016/j.ijcard. 2014.12.092 PMID: 25576729 7. van Vark LC, Bertrand M, Akkerhuis KM, Brugts JJ, Fox K, Mourad JJ, et al. (2012) Angiotensin-con-
verting enzyme inhibitors reduce mortality in hypertension: a meta-analysis of randomized clinical trials
of renin-angiotensin-aldosterone system inhibitors involving 158,998 patients. Eur Heart J 33: 2088–
97. doi: 10.1093/eurheartj/ehs075 PMID: 22511654 8. Savarese G, Costanzo P, Cleland JG, Vassallo E, Ruggiero D, Rosano G, et al. (2013) A meta-analy-
sis reporting effects of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers in
patients without heart failure. J Am Coll Cardiol 61: 131–42. PMID: 23219304 9. Talameh JA,Lanfear DE. (2012) Pharmacogenetics in chronic heart failure: new developments and
current challenges. Curr Heart Fail Rep 9: 23–32. doi: 10.1007/s11897-011-0076-2 PMID: 22135185 10. DrugBank. Available: http://www.drugbank.ca/. 11. Rasmussen HB, Bjerre D, Linnet K, Jurgens G, Dalhoff K, Stefansson H, et al. (2015) Individualization
of treatments with drugs metabolized by CES1: combining genetics and metabolomics. Pharmacoge-
nomics 16: 649–65. doi: 10.2217/pgs.15.7 PMID: 25896426 15 / 18 PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 CES1 Gene Variants and ACEI Treatment 12. Merali Z, Ross S,Pare G. (2014) The pharmacogenetics of carboxylesterases: CES1 and CES2
genetic variants and their clinical effect. Drug Metabol Drug Interact 29: 143–51. doi: 10.1515/dmdi-
2014-0009 PMID: 24988246 13. PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 References PMID: 8865076 33. Yamada S, Richardson K, Tang M, Halaschek-Wiener J, Cook VJ, Fitzgerald JM, et al. (2010) Genetic
variation in carboxylesterase genes and susceptibility to isoniazid-induced hepatotoxicity. Pharmaco-
genomics J 10: 524–36. doi: 10.1038/tpj.2010.5 PMID: 20195289 34. Barrett JC, Fry B, Maller J,Daly MJ. (2005) Haploview: analysis and visualization of LD and haplotype
maps. Bioinformatics 21: 263–5. PMID: 15297300 35. dbSNP Short Genetic Variations (NCBI). Available: http://www.ncbi.nlm.nih.gov/SNP. 35. dbSNP Short Genetic Variations (NCBI). Available: h 36. Wang X, Wang G, Shi J, Aa J, Comas R, Liang Y, et al. (2015) CES1 genetic variation affects the acti-
vation of angiotensin-converting enzyme inhibitors. Pharmacogenomics J. 37. Tarkiainen EK, Tornio A, Holmberg MT, Launiainen T, Neuvonen PJ, Backman JT, et al. (2015) Effect
of carboxylesterase 1 c.428G > A single nucleotide variation on the pharmacokinetics of quinapril and
enalapril. Br J Clin Pharmacol. 38. Geshi E, Kimura T, Yoshimura M, Suzuki H, Koba S, Sakai T, et al. (2005) A single nucleotide polymor-
phism in the carboxylesterase gene is associated with the responsiveness to imidapril medication and
the promoter activity. Hypertens Res 28: 719–25. PMID: 16419644 39. Ghosh S. (2000) Cholesteryl ester hydrolase in human monocyte/macrophage: cloning, sequencing,
and expression of full-length cDNA. Physiol Genomics 2: 1–8. PMID: 11015575 40. Blais DR, Lyn RK, Joyce MA, Rouleau Y, Steenbergen R, Barsby N, et al. (2010) Activity-based protein
profiling identifies a host enzyme, carboxylesterase 1, which is differentially active during hepatitis C
virus replication. J Biol Chem 285: 25602–12. doi: 10.1074/jbc.M110.135483 PMID: 20530478 41. Crow JA, Middleton BL, Borazjani A, Hatfield MJ, Potter PM,Ross MK. (2008) Inhibition of carboxyles-
terase 1 is associated with cholesteryl ester retention in human THP-1 monocyte/macrophages. Bio-
chim Biophys Acta 1781: 643–54. doi: 10.1016/j.bbalip.2008.07.005 PMID: 18762277 42. Okazaki H, Igarashi M, Nishi M, Tajima M, Sekiya M, Okazaki S, et al. (2006) Identification of a novel
member of the carboxylesterase family that hydrolyzes triacylglycerol: a potential role in adipocyte
lipolysis. Diabetes 55: 2091–7. PMID: 16804080 43. Dolinsky VW, Gilham D, Alam M, Vance DE,Lehner R. (2004) Triacylglycerol hydrolase: role in intra-
cellular lipid metabolism. Cell Mol Life Sci 61: 1633–51. PMID: 15224187 44. Bencharit S, Edwards CC, Morton CL, Howard-Williams EL, Kuhn P, Potter PM, et al. (2006) Multisite
promiscuity in the processing of endogenous substrates by human carboxylesterase 1. J Mol Biol 363:
201–14. PMID: 16962139 45. Bora PS, Guruge BL, Miller DD, Chaitman BR,Ruyle MS. References Pare G, Eriksson N, Lehr T, Connolly S, Eikelboom J, Ezekowitz MD, et al. (2013) Genetic determi-
nants of dabigatran plasma levels and their relation to bleeding. Circulation 127: 1404–12. doi: 10. 1161/CIRCULATIONAHA.112.001233 PMID: 23467860 25. Bruxel EM, Salatino-Oliveira A, Genro JP, Zeni CP, Polanczyk GV, Chazan R, et al. (2013) Association
of a carboxylesterase 1 polymorphism with appetite reduction in children and adolescents with atten-
tion-deficit/hyperactivity disorder treated with methylphenidate. Pharmacogenomics J 13: 476–80. doi: 10.1038/tpj.2012.25 PMID: 22688218 26. Juillerat L, Nussberger J, Menard J, Mooser V, Christen Y, Waeber B, et al. (1990) Determinants of
angiotensin II generation during converting enzyme inhibition. Hypertension 16: 564–72. PMID:
2172161 27. Biollaz J, Schelling JL, Jacot Des Combes B, Brunner DB, Desponds G, Brunner HR, et al. (1982)
Enalapril maleate and a lysine analogue (MK-521) in normal volunteers; relationship between plasma
drug levels and the renin angiotensin system. Br J Clin Pharmacol 14: 363–8. PMID: 6289859 28. Delacretaz E, Nussberger J, Puchler K, Wood AJ, Robinson PR, Waeber B, et al. (1994) Value of dif-
ferent clinical and biochemical correlates to assess angiotensin converting enzyme inhibition. J Cardio-
vasc Pharmacol 24: 479–85. PMID: 7528305 29. Nussberger J, Brunner D, Keller I,Brunner HR. (1992) Measurement of converting enzyme activity by
antibody-trapping of generated angiotensin II. Comparison with two other methods. Am J Hypertens
5: 393–8. PMID: 1326287 30. McMurray JJ, Adamopoulos S, Anker SD, Auricchio A, Bohm M, Dickstein K, et al. (2012) ESC guide-
lines for the diagnosis and treatment of acute and chronic heart failure 2012: The Task Force for
the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of 16 / 18 PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 CES1 Gene Variants and ACEI Treatment Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC. Eur J
Heart Fail 14: 803–69. doi: 10.1093/eurjhf/hfs105 PMID: 22828712 Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC. Eur J
Heart Fail 14: 803–69. doi: 10.1093/eurjhf/hfs105 PMID: 22828712 31. Plovsing RR, Wamberg C, Sandgaard NC, Simonsen JA, Holstein-Rathlou NH, Hoilund-Carlsen PF,
et al. (2003) Effects of truncated angiotensins in humans after double blockade of the renin system. Am J Physiol Regul Integr Comp Physiol 285: R981–91. PMID: 12869368 32. Sandgaard NC,Bie P. (1996) Natriuretic effect of non-pressor doses of endothelin-1 in conscious
dogs. J Physiol 494 (Pt 3): 809–18. PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 References (1996) Purification and characterization of
human heart fatty acid ethyl ester synthase/carboxylesterase. J Mol Cell Cardiol 28: 2027–32. PMID:
8899561 46. Law MR, Wald NJ,Rudnicka AR. (2003) Quantifying effect of statins on low density lipoprotein choles-
terol, ischaemic heart disease, and stroke: systematic review and meta-analysis. BMJ 326: 1423. PMID: 12829554 47. Hoshida S, Kato J, Nishino M, Egami Y, Takeda T, Kawabata M, et al. (2001) Increased angiotensin-
converting enzyme activity in coronary artery specimens from patients with acute coronary syndrome. Circulation 103: 630–3. PMID: 11156871 48. Studer R, Reinecke H, Muller B, Holtz J, Just H,Drexler H. (1994) Increased angiotensin-I converting
enzyme gene expression in the failing human heart. Quantification by competitive RNA polymerase
chain reaction. J Clin Invest 94: 301–10. PMID: 8040271 49. Hirsch AT, Talsness CE, Schunkert H, Paul M,Dzau VJ. (1991) Tissue-specific activation of cardiac
angiotensin converting enzyme in experimental heart failure. Circ Res 69: 475–82. PMID: 1650297 50. Zhuo JL,Li XC. (2011) New insights and perspectives on intrarenal renin-angiotensin system: focus on
intracrine/intracellular angiotensin II. Peptides 32: 1551–65. doi: 10.1016/j.peptides.2011.05.012
PMID: 21699940 51. Sharman DC, Morris AD,Struthers AD. (2007) Gradual reactivation of vascular angiotensin I to angio-
tensin II conversion during chronic ACE inhibitor therapy in patients with diabetes mellitus. Diabetolo-
gia 50: 2061–6. PMID: 17676311 17 / 18 PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 CES1 Gene Variants and ACEI Treatment 52. Struthers AD. (1996) Aldosterone escape during angiotensin-converting enzyme inhibitor therapy in
chronic heart failure. J Card Fail 2: 47–54. PMID: 8798105 53. Furberg CD,Pitt B. (2001) Are all angiotensin-converting enzyme inhibitors interchangeable? J Am
Coll Cardiol 37: 1456–60. PMID: 11300461 54. Feldman RD, Hussain Y, Kuyper LM, McAlister FA, Padwal RS,Tobe SW. (2014) Intraclass Differ-
ences Among Antihypertensive Drugs. Annu Rev Pharmacol Toxicol. 55. Shah AD,Arora RR. (2005) Tissue angiotensin-converting enzyme inhibitors: are they more effective
than serum angiotensin-converting enzyme inhibitors? Clin Cardiol 28: 551–5. PMID: 16405197 56. Cressman AM, Macdonald EM, Fernandes KA, Gomes T, Paterson JM, Mamdani MM, et al. (2015) A
population-based study of the drug interaction between clopidogrel and angiotensin converting
enzyme inhibitors. Br J Clin Pharmacol. 57. Williams GH, Cain JP, Dluhy RG,Underwood RH. (1972) Studies of the control of plasma aldosterone
concentration in normal man. I. Response to posture, acute and chronic volume depletion, and sodium
loading. J Clin Invest 51: 1731–42. PMID: 4338121 58. Waeber B, Nussberger J, Perret L, Santoni JP,Brunner HR. PLOS ONE | DOI:10.1371/journal.pone.0163341
September 23, 2016 References (1989) Experience with perindopril in nor-
mal volunteers. Arch Mal Coeur Vaiss 82 Spec No 1: 35–41. PMID: 2549899 59. Laizure SC, Herring V, Hu Z, Witbrodt K,Parker RB. (2013) The role of human carboxylesterases in
drug metabolism: have we overlooked their importance? Pharmacotherapy 33: 210–22. doi: 10.1002/
phar.1194 PMID: 23386599 60. Takahashi S, Katoh M, Saitoh T, Nakajima M,Yokoi T. (2009) Different inhibitory effects in rat and
human carboxylesterases. Drug Metab Dispos 37: 956–61. doi: 10.1124/dmd.108.024331 PMID:
19225040 61. Wang X, Zhu HJ,Markowitz JS. (2015) Carboxylesterase 1-mediated drug-drug interactions between
clopidogrel and simvastatin. Biol Pharm Bull 38: 292–7. doi: 10.1248/bpb.b14-00679 PMID: 25747989 62. Singh BN,Malhotra BK. (2004) Effects of food on the clinical pharmacokinetics of anticancer agents:
underlying mechanisms and implications for oral chemotherapy. Clin Pharmacokinet 43: 1127–56. PMID: 15568891 63. Ogawa R, Stachnik JM,Echizen H. (2014) Clinical pharmacokinetics of drugs in patients with heart fail-
ure: an update (part 2, drugs administered orally). Clin Pharmacokinet 53: 1083–114. doi: 10.1007/
s40262-014-0189-3 PMID: 25248847 63. Ogawa R, Stachnik JM,Echizen H. (2014) Clinical pharmacokinetics of drugs in patients with heart fail-
ure: an update (part 2, drugs administered orally). Clin Pharmacokinet 53: 1083–114. doi: 10.1007/
s40262-014-0189-3 PMID: 25248847 18 / 18
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Crystal Structure of Cryptosporidium parvum Pyruvate Kinase
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Abstract Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This
tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding
of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase
structures. Pyruvate kinase of Cryptosporidium parvum is exceptional among known enzymes of protozoan origin in that it
exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate
kinase from C. parvum has been solved by molecular replacement techniques and refined to 2.5 A˚ resolution. In the active
site a glycerol molecule is located near the c-phosphate site of ATP, and the protein structure displays a partially closed
active site. However, unlike other structures where the active site is closed, the a6’ helix in C. parvum pyruvate kinase
unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a
phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the C. parvum pyruvate kinase
structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is
formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues
303–320) of the second monomer at the interface of these monomers. Both cysteine residues are unique to C. parvum, and
the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains
unknown at this time. Citation: Cook WJ, Senkovich O, Aleem K, Chattopadhyay D (2012) Crystal Structure of Cryptosporidium parvum Pyruvate Kinase. PLoS ONE 7(10): e46875. doi:10.1371/journal.pone.0046875 Editor: Inari Kursula, Helmholtz Centre for Infection Research, Germany Received February 20, 2012; Accepted September 10, 2012; Published October 9, 2012 Received February 20, 2012; Accepted September 10, 2012; Published October 9, 2012 Copyright: 2012 Cook et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2012 Cook et al. This is an open-access article distributed under the terms of the Creative Commons
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: This work was in part supported by a research grant to DC from American Foundation for AIDS Research (amFAR) no. 106493-25-RGGN. No additional
external funding was received for this study. 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: debasish@uab.edu Early studies suggested that C. parvum depends heavily on
glycolysis for energy [11,12]. All of the enzymes in the glycolytic
pathway were found in the cytoplasmic fraction of the oocysts
[12]. Subsequently, genome sequencing revealed genes encoding
all glycolytic enzymes, but genes encoding the Krebs cycle
enzymes and the components of the mitochondrial complexes I
to IV were missing. The lack of functional mitochondria further
underscores the reliance of the organism on glycolysis [13]. However, biochemical and structural studies of glycolytic enzymes
of C. parvum are quite limited. In contrast, the glycolytic pathway
has been studied in considerable detail in trypanosomatid parasites
[10,14], and two enzymes in particular, glyceraldehyde 3-
phosphate dehydrogenase and pyruvate kinase (PyK), have been
targeted for designing antitrypanosomal drugs [15,16]. More
recently, PyK has been shown to be an effective target for
antibacterial agents against methicillin resistant Staphylococcus aureus
[17,18]. Crystal Structure of Cryptosporidium parvum Pyruvate
Kinase William J. Cook1, Olga Senkovich1, Khadijah Aleem3, Debasish Chattopadhyay2,4* 1 Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America, 2 Center for Biophysical Sciences and Engineering,
University of Alabama at Birmingham, Birmingham, Alabama, United States of America, 3 Ronald E. McNair Scholar Program, University of Alabama at Birmingham,
Birmingham, Alabama, United States of America, 4 Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America PLOS ONE | www.plosone.org General Description and Quality of CpPyK Structure General Description and Quality of CpPyK Structure
The asymmetric unit in the crystal structure contains two
monomers (A & B) of CpPyK related by non-crystallographic two-
fold symmetry (Fig. 1). The complete tetramer is formed with their
symmetry partners related by the crystallographic 2-fold axis along
the c axis (Fig. 2A). The root mean square deviation (r.m.s.d.)
between monomers A and B is 0.38 A˚ for all 485 Ca atoms. The
final model includes residues 23–32, 42–507 and 518–526 for each
chain. The arrangement of the monomers in the tetramer is
similar to that seen in other PyKs. The two monomers in the
asymmetric unit form the major interface and bury approximately
2000 A˚ 2 of surface area. Each CpPyK molecule consists of four domains: N (residues
23–32), A (42–112 and 212–389), B (113–211) and C (390–526). The A-domain constitutes the central part of the molecule and
forms a parallel (a/b)8 barrel (Fig. 2B). The B-domain contains
nine b strands that form an antiparallel b-barrel. The active site
is located at the interface of the A and B domains, and residues
from both domains participate in substrate binding. The C-
domain is composed of five b strands surrounded by five a-
helices. The allosteric site for binding the effector molecule is
located in the C-domain. The A domains of the two monomers
A and B form the major interface (also referred to as large
interface or A-A interface). The C-domains of monomers
related by the crystallographic 2-fold symmetry form a smaller
interface called C-C interface (Fig. 2A). The N domain is mostly
disordered in PyK structures; the only portion visible in this
structure is a short a helix referred to as N-helix (residues 23–
32; Fig. 2B). Although the electron density for the polypeptide
chain connecting the N-helix to the A-domain (residues 33–41)
was missing, the helix for each monomer could be unambig-
uously assigned to the respective monomer (Figs. 1 and 2B). Furthermore, the N-helix in this structure is in a similar position
as in 3MA8, in which the linker peptide was modeled. This
short helix is involved in a unique interaction in CpPyK not
seen in any other PyK (discussed below). Three-dimensional structures of PyK from mammalian, bacte-
rial and parasitic organisms have been reported [20,24,27,28,29]. Among the parasitic proteins, L. Introduction The protozoan parasite Cryptosporidium parvum, one of the
causative agents of human cryptosporidiosis, belongs to the
Coccidia subgroup in the phylum apicomplexa and causes
waterborne diseases worldwide [1,2]. Cryptosporidium oocysts
can withstand common water treatment methods, including
chlorination, and major outbreaks of cryptosporidiosis caused by
contamination of drinking water have been reported [3,4]. Although in healthy adults Cryptosporidium infection results in
self limited diarrhea, an outbreak in 1993 in Milwaukee, WI
affected an estimated 400,000 people [5]. Moreover, as an
opportunistic infection it can be fatal in immunocompromised
individuals, such as persons infected with HIV, especially those
without access to highly active antiretroviral therapy [6]. Despite the global spread of the parasite, therapeutic options for
effective treatment of C. parvum infection are limited [7]. Very few
potential drug targets are known, because metabolic pathways and
regulatory molecules that are key to the survival of the parasite are
largely uncharacterized [8]. Therefore, expanding our knowledge
of major biochemical pathways that are known to be important in
protozoa, such as glycolysis, is of considerable interest [9,10]. PyK catalyzes the last step of glycolysis, in which the phosphoryl
group of phosphoenolpyruvate (PEP) is transferred to ADP to form
pyruvate and ATP, and serves as a major regulator of glycolysis
[9]. There are some notable differences among PyKs from various
species. In mammals four PyK isozymes are expressed, but
Trypanosomes encode only one, [19,20,21]. In at least two 1 PLOS ONE | www.plosone.org October 2012 | Volume 7 | Issue 10 | e46875 October 2012 | Volume 7 | Issue 10 | e46875 Structure of Pyruvate Kinase members
of
apicomplexa,
Toxoplasma
gondii
and
Plasmodium
falciparum, a second PyK localized in the apicoplast has been
discovered in addition to the cytoplasmic form [22,23]. However,
only the cytoplasmic form has been identified in C. parvum (http://
cryptodb.org/cryptodb/). was in the same range as reported for T. gondii PyK (Figs. S2A and
S2B). It should be noted that the conditions used in our assay may
not represent the optimal conditions for CpPyK activity. Enzyme
activity remained unchanged in the presence of 1 mM tris(2-
carboxyethyl)phosphine (TCEP), a strong reducing agent that is
relatively resistant to oxidation, and up to 5 mM dithiothreitol
(DTT) (Fig. S2B). A hallmark of PyK is the allosteric regulation of its activity by
various phosphorylated sugars [24]. PyKs from various organ-
isms use different effector molecules for regulation. Results and Discussion CpPyK used for crystallographic study contained amino acid
residues 2–526 and 14 additional amino terminal residues derived
from the expression vector, which includes a T7-tag. The
calculated molecular weight for a monomer of this CpPyK
construct is 57.67 kDa. In a previous study we reported that the
oligomeric state of CpPyK could not be confirmed based on the
results of size exclusion chromatography [32]. However, the
majority of PyKs are tetrameric, and CpPyK also displays a
tetrameric assembly in the crystal structure. Examination of a
partially purified protein preparation on an analytical gel filtration
column showed that the elution volume of the major peak fraction
containing CpPyK was similar to that of purified Catalase (Mr
232 kDa). Chromatograms showing the elution profiles are
presented in Figs. S1A and S1B. SDS-PAGE analysis of the
fractions (Fig. S1C) indicates that CpPyK eluted at a volume (9.5–
12 ml) expected for a tetramer. Thus CpPyK exists mainly as a
tetrameric protein but may remain in equilibrium with other
oligomeric states. Electron density was generally excellent in the A and C
domains, except for a long loop in the C-domain (residues 508–
517) that was completely disordered. This loop is involved in
binding of effector molecules (usually fructose biphosphates) and is
typically not ordered unless the effector molecule is bound. The
density was much weaker in domain B, especially for residues 118–
150, 167–179, and 186–200, and the high B-factors reflect this. For monomer A the average B-factor for domains N, A and C is
52.0 A˚ 2, while the average B-factor for domain B is 92.5 A˚ 2. The
corresponding average B-factors for monomer B are 52.5 and
109.2 A˚ 2, respectively. A summary of the data collection and refinement statistics is
presented in Table 1. The overall quality of the structure of
CpPyK is excellent (see Table 1 and Materials and Methods). Only Gly123 in each monomer exhibits phi, psi angles in non-
allowed regions of the Ramachandran plot. Electron density for
these two residues was extremely weak. The final model also
contains two sulfate ions, SULF1 and SULF2 (corresponding to
SO4 527 and SO4 528 in the deposited coordinates), and two
glycerol molecules, GOL1 and GOL2 (GOL530 and GOL 531 Although detailed kinetic analysis was not performed, we
confirmed that the purified protein was enzymatically active in the
pH range 5.5–7.5. General Description and Quality of CpPyK Structure mexicana pyruvate kinase (LmPyK)
is the most thoroughly studied (13 out of 18 entries in the protein
data bank), and structural consequences of binding substrates and
allosteric regulators have been established from a series of crystal
structures [20,28,30]. Crystal structures of PyK from three other
parasites, T. gondii, Trypanosoma cruzi and P. falciparum (PDBID:
3KHD; unpublished) have also been reported [25,31]. These
studies demonstrated that the structures of PyKs are influenced by
the presence or absence of ligands at specific sites in the protein
and by the crystallization conditions [25,30]. Recently an
unpublished crystal structure of CpPyK was deposited in the
protein data bank (PDBID: 3MA8). We have independently
determined the structure using crystals grown under conditions
significantly different from those reported for 3MA8. Here we
describe the CpPyK structure and compare it with structures of
PyK from other organisms and the one reported in 3MA8. Introduction For example,
mammalian enzymes are strongly regulated by fructose 1, 6-
bisphosphate (F-1,6 BP), but trypanosomal PyK remains rela-
tively unaffected by F-1,6 BP and is activated by submicromolar
concentrations of fructose 2, 6-bisphosphate (F-2,6 BP) [20]. Cytoplasmic
PyK
of
T. gondii
is
activated
by
glucose
6-
phosphate and F-1,6 BP [11,25]. Notably, C. parvum PyK
(CpPyK) is exceptional, as it showed no allosteric property
[11]. Phosphosugars, including glucose 1-phosphate, glucose 6-
phosphate, fructose 6-phosphate, ribose 5-phosphate, F-1,6 BP
and F-2,6BP, had no effect on the enzyme activity [11]. The
only other known PyK that lacks allosteric activity is the
mammalian muscle isozyme M1 [26]. CpPyK Crystal Structure Contains a Disulfide Linked N-
helix CpPyK Crystal Structure Contains a Disulfide Linked N-
helix in the deposited coordinates), associated with each chain. In
addition there are six acetate ions and 149 water molecules
(Table 2). A new feature of the CpPyK structure is that the two monomers
in the asymmetric unit are linked by two right-handed disulfide
bonds between Cys26 of one monomer and Cys312 of the other
(Fig. 1). Analysis of the crystal structure using the SSBOND server
(http://hazeslab.med.ualberta.ca/forms/ssbond.html)
[35]
also
predicted only this pair of disulfide bonds. These two cysteine
residues (out of a total of 21) are unique to Cryptosporidium
(Fig. 3). Since no disulfide bond has been described in other PyK
structures, and Denton et al. [11] reported that pyruvate kinase
activity in C. parvum extract was enhanced by reducing agent, we
maintained reducing conditions throughout the purification steps
(see Materials and Methods). In fact, addition of reducing agents
during purification was found to be necessary to prevent protein
aggregation. Interestingly, although the disulfide bond appears to
be exposed in the crystal structure, it was not reduced. However,
any functional role of the N-domain has been ruled out, since
removal of this domain had no effect on the enzymatic activity of
human PyK [36]. Moreover, the structural significance of this
domain is likely to be minimal, since in some crystal structures of
full length PyKs a large portion of the N-domain remains
disordered [25]. It should be noted that Cys312 is located in the
long helix (residues 303–320) that is involved in interactions with
the other monomer in the asymmetric unit across the large
interface between adjacent A domains (Fig. 1) and is connected to
the a6’-helix (residues 293–302), which changes conformation
upon substrate binding. Results of primary sequence alignment using the CLUSTALW
server (http://www.ebi.ac.uk/) [33,34] show that identity between
PyKs from various species varies roughly in the range 40–66%
based on the evolutionary relationship between the organisms. Thus, pair-wise sequence identity between the pyruvate kinase of
C. parvum and those of human, E. coli, L. mexicana, P. falciparum and
T. gondii are 39, 42, 41, 51 and 54%, respectively, while the P. falciparum and T. gondii sequences are 66% identical. The overall
architecture of CpPyK is similar to the structures of PyKs from
other organisms. However, the orientation of the B-domain with
respect to the A and C domains varies rather widely in various
PyK structures. Results and Discussion Initial reaction velocity was measured using
conditions described for similar enzymes (see Materials and
Methods), and the Vmax of approximately 0.04 mM NADH/min PLOS ONE | www.plosone.org October 2012 | Volume 7 | Issue 10 | e46875 2 Structure of Pyruvate Kinase Figure 1. CpPyK asymmetric unit. Monomers A and B comprise the asymmetric unit and are related by a noncrystallographic 2-fold axis
perpendicular to the plane of the paper. The domains in each monomer are colored as follows: N - cyan (residues 23–32), A - wheat (residues 42–112
and 212–389), B - magenta (residues 113–211), and C - light green (residues 390–526). The sulfate ions are shown as stick models; the sulfate ion
bound at the effector site in each monomer is labeled SULF1. The sulfur atoms of cysteine residues 26 and 312 in each monomer are shown as orange
balls; the disulfide is indicated by CC. The unwound helix a6’ is shown in red in both monomers. The A domains from these monomers form the
major protein-protein interface in the tetramer. doi:10.1371/journal.pone.0046875.g001 Figure 1. CpPyK asymmetric unit. Monomers A and B comprise the asymmetric unit and are related by a noncrystallographic 2-fold axis
perpendicular to the plane of the paper. The domains in each monomer are colored as follows: N - cyan (residues 23–32), A - wheat (residues 42–112
and 212–389), B - magenta (residues 113–211), and C - light green (residues 390–526). The sulfate ions are shown as stick models; the sulfate ion
bound at the effector site in each monomer is labeled SULF1. The sulfur atoms of cysteine residues 26 and 312 in each monomer are shown as orange
balls; the disulfide is indicated by CC. The unwound helix a6’ is shown in red in both monomers. The A domains from these monomers form the
major protein-protein interface in the tetramer. doi:10.1371/journal.pone.0046875.g001 CpPyK Crystal Structure Contains a Disulfide Linked N-
helix Therefore, pair-wise alignment of the entire
molecule by superposition is influenced by the relative orientation
of the B domains. For example, the r.m.s.d. between the A
monomers of CpPyK and P. falciparum PyK is 0.86 A˚ , while the
r.m.s.d. for the combined A and C domains only is 0.67 A˚ . On the
other hand, the r.m.s.d. values between the A monomers of
CpPyK and those of the truncated (PDBID: 3GG8) and full length
versions (PDBID:3EOE) of T. gondii PyKs are 0.65 and 0.83 A˚ . When the B domain is removed from these structures the r.m.s.d. values
lie
around
0.6–0.7 A˚ . The
r.m.s.d. values
for
the
superposition of the A and C domains of CpPyK with LmPyK
(PDBID: 1PKL) and human PyK (PDBID: 3GQY) are 0.76 A˚ and
0.85 A˚ , respectively. October 2012 | Volume 7 | Issue 10 | e46875 PLOS ONE | www.plosone.org 3 Structure of Pyruvate Kinase Figure 2. CpPyk tetramer and monomer. (A) The tetramer is
generated by a crystallographic 2-fold axis. Domains of monomer A are
colored the same as in Fig. 1. Symmetry related monomers are shown in
green and orange. A minor interface is formed by the C-domains of the
symmetry partners. (B) Monomer A. The domains are colored as follows:
N
li ht
i k A
B
t
C
li ht
Th t
lf t CpPyK Active Site is Partially Closed The sulfur atoms in the
disulfide bond between cysteine residues 26 and 312 are shown as
orange balls. Glycerol and acetate ions are shown as stick models. The
unwound helix a6’ is shown in red. The location of the missing effector
loop is indicated. The loop representing the Cryptosporidium-specific
insertion in the primary sequence is also labeled. Apart from the orientation of the B-domain, the major
conformational difference between these structures is in the
residue range 293–302 in CpPyK (a6’ shown in red in Fig. 2B);
in both LmPyK structures the corresponding region is a-helical,
but in CpPyK the helix is completely unwound in the A monomer
and contains only a short helical stretch in the B monomer
(Fig. 4B). Comparison with the structure of human PyK showed
that it is very similar to the CpPyK structure. There is no
significant difference in the active site. There is an extended loop
in the CpPyK structure due to a characteristic 6 residue insertion
(residues 259–264; Fig. 2B, Fig. 3 and Fig. 4A) found only in
Cryptosporidium sequences. This loop lies at the exterior of the
molecule. Unwinding of a6’ Helix in the Active Site of CpPyK g
p y
Based on the crystal structures of PyKs with and without various
ligands, a model for the structural rearrangement at the active site
has been proposed [28,30]. According to this model, the transition
from the inactive to active state involves a rigid body rotation of
the A- and C-domains of 6u around a pivot point at the base of the
ab-barrel of domain A. Upon binding of substrates in the active
site, the side chain of a conserved arginine residue (Arg310 in
LmPyK, corresponding to Arg342 in CpPyK) moves into the
vicinity of the active site of the adjacent subunit at the large
interface, where it forms two stabilizing hydrogen bonds with
backbone carbonyls of arginine and glycine residues (correspond-
ing to Arg294 and Gly295 in CpPyK) located in the a6’ helix. This
interaction across the A-A interface appears to be functionally
important, since residues in the a6’ helix (294RGDLGME300 in
CpPyK) are highly conserved in all PyKs, and mutation of Arg310
in LmPyK results in the loss of enzymatic activity [36]. In the
present structure, even though there is no substrate, analog or
sulfate ion bound at the active site, Arg342 assumes a conforma- Figure 2. CpPyk tetramer and monomer. (A) The tetramer is
generated by a crystallographic 2-fold axis. Domains of monomer A are
colored the same as in Fig. 1. Symmetry related monomers are shown in
green and orange. A minor interface is formed by the C-domains of the
symmetry partners. (B) Monomer A. The domains are colored as follows:
N - light pink, A - orange, B - magenta, C - light green. The two sulfate
ions are labeled SULF1 and SULF2. The N-helix of the B monomer (cyan)
is included in order to show the disulfide bond. The sulfur atoms in the
disulfide bond between cysteine residues 26 and 312 are shown as
orange balls. Glycerol and acetate ions are shown as stick models. The
unwound helix a6’ is shown in red. The location of the missing effector
loop is indicated. The loop representing the Cryptosporidium-specific
insertion in the primary sequence is also labeled. doi:10.1371/journal.pone.0046875.g002 Figure 2. CpPyk tetramer and monomer. (A) The tetramer is
generated by a crystallographic 2-fold axis. Domains of monomer A are
colored the same as in Fig. 1. Symmetry related monomers are shown in
green and orange. CpPyK Active Site is Partially Closed Specific structural changes are observed in PyKs in response to
binding of substrate and effector molecules. Moreover, structural
changes resulting from differences in crystallization conditions
have also been reported. For example, differences in the structures
of full length and truncated versions of T. gondii PyK have been
attributed to different crystallization conditions [25]. From this
consideration CpPyK crystals are quite comparable to the LmPyK
crystal grown from ammonium sulfate in acidic buffer (pH 4.0–
4.6) at 4uC [28]. Although these latter crystals were grown in the
presence of F-1,6BP, only sulfate ions were located in the effector
binding site as well as at the sites for binding PEP and ATP. Crystals of the apo form of LmPyK (without any added substrate,
effector or analog) were also grown at a low pH (4.8) in the
presence of ammonium sulfate. Fig. 4A shows superposition of
these two LmPyK structures with the CpPyK structure. The only
significant deviations in the A and C-domains of these three
structures are in the two regions where there is a 6-residue
insertion in the sequence. Notably, the orientation of the B-
domain relative to the A-domain in the CpPyK structure is more
similar to the LmPyK structure that has sulfate ions bound in the
active site (PDBID: 3E0V), while in the apo-LmPyK structure the
orientation is markedly different. Therefore, the active site of
CpPyK appears to mimic the partially closed conformation
observed in the LmPyK sulfate-bound form (PDBID: 3E0V). This LmPyK structure has two sulfate ions in the active site
occupying the positions for the b and c-phosphate groups of ATP,
but the CpPyK structure contains no sulfate ion at these positions. Instead, there is a glycerol molecule (GOL1) located in the active
site of CpPyK at nearly the same position occupied by the ATP c-
phosphate. Figure 2. CpPyk tetramer and monomer. (A) The tetramer is
generated by a crystallographic 2-fold axis. Domains of monomer A are
colored the same as in Fig. 1. Symmetry related monomers are shown in
green and orange. A minor interface is formed by the C-domains of the
symmetry partners. (B) Monomer A. The domains are colored as follows:
N - light pink, A - orange, B - magenta, C - light green. The two sulfate
ions are labeled SULF1 and SULF2. The N-helix of the B monomer (cyan)
is included in order to show the disulfide bond. Unwinding of a6’ Helix in the Active Site of CpPyK A minor interface is formed by the C-domains of the
symmetry partners. (B) Monomer A. The domains are colored as follows:
N - light pink, A - orange, B - magenta, C - light green. The two sulfate
ions are labeled SULF1 and SULF2. The N-helix of the B monomer (cyan)
is included in order to show the disulfide bond. The sulfur atoms in the
disulfide bond between cysteine residues 26 and 312 are shown as
orange balls. Glycerol and acetate ions are shown as stick models. The
unwound helix a6’ is shown in red. The location of the missing effector
loop is indicated. The loop representing the Cryptosporidium-specific
insertion in the primary sequence is also labeled. doi:10.1371/journal.pone.0046875.g002 PLOS ONE | www.plosone.org October 2012 | Volume 7 | Issue 10 | e46875 4 Structure of Pyruvate Kinase Table 1. Data-collection and refinement statistics. Table 1. Data-collection and refinement statistics. Crystal data
Space Group
P21212
Unit cell parameters (A˚)
a = 129.9, b = 136.9, c = 77.2
Vm (A˚3 Da21)
3.04
Solvent content (%)
59.6
Data collection
Resolution range (A˚)
50.0–2.50 (2.54–2.50)a
No. of reflections
202,725 (9168)
No. of unique reflections
47,438 (2177)
Multiplicity
4.3 (4.3)
Completeness (%)
97.8 (98.9)
Rmerge (%)
7.1 (51.4)
Mean I/s(I)
12.1 (1.0)
Refinement statistics
Resolution range (A˚)
49.71–2.50 (2.565–2.50)
Reflections (working set)
45,005 (3101)
Reflections (test set)
2344 (148)
R value (working set)
0.213 (0.273)
Free R value
0.249 (0.333)
No. of protein atoms
7292
No. of sulphate ions
5
No. of water molecules
149
Estimated coordinate error based on R value (A˚)
0.39
Estimated coordinate error based on free R value (A˚)
0.26
R.m.s. deviations from ideal values
Bond lengths (A˚)
0.005
Bond angles (u)
0.92
Mean B value (A˚2)
61.7
Structure quality
Ramachandran most favored (%)
96.87
Ramachandran allowed (%)
2.92
Ramachandran outliers (%)
0.21
Rotomer outliers (%)
0.24
aValues in parentheses are for the outermost resolution shell. d i 10 1371/j
l
0046875 t001 aValues in parentheses are for the outermost resolution shell. doi:10.1371/journal.pone.0046875.t001 group of Ser362. In the B monomer only the Glu272 side chain is
involved in hydrogen bonding with the glycerol, and additional
hydrogen bonds are formed with water molecules and an acetate
ion. In the activator-bound human PyK (PDBID: 3GQY) an L-
tartaric acid is found in the same position. Unwinding of a6’ Helix in the Active Site of CpPyK tion somewhat similar to that observed in substrate-bound PyK
structures with its side chain pointed toward the active site of the
other monomer (Fig. 5). A similar conformation is observed when
sulfate ions are bound in the active site of LmPyK [28]. On the
other hand, while unwinding of the a6’ helix was observed when
the sulfate ions were removed from the crystals of LmPyk, in
CpPyK the a6’ helix remains unwound in both monomers, and
the carbonyl of Gly295 is far away from the Arg342 side chain. Instead, the Arg342 side chain forms a long hydrogen bond with
the carbonyl of Arg294. Arg294 is also hydrogen bonded to the
Thr328 carbonyl oxygen through its side chain. Unwinding of the
a6’ helix is therefore consistent with the absence of sulfate ion from
the active site [28]. Nevertheless, the CpPyK active site remained
in a partially closed conformation. The GOL1 molecule at the
ATP binding site in the CpPyK crystal structure, therefore, has a
similar effect as the sulfate ion in the active site. In the A monomer
the glycerol (GOL1) forms hydrogen bonds with the side chain
nitrogen of Asn76, the side chain of Glu272 and the hydroxyl tion somewhat similar to that observed in substrate-bound PyK
structures with its side chain pointed toward the active site of the
other monomer (Fig. 5). A similar conformation is observed when
sulfate ions are bound in the active site of LmPyK [28]. On the
other hand, while unwinding of the a6’ helix was observed when
the sulfate ions were removed from the crystals of LmPyk, in
CpPyK the a6’ helix remains unwound in both monomers, and
the carbonyl of Gly295 is far away from the Arg342 side chain. Instead, the Arg342 side chain forms a long hydrogen bond with
the carbonyl of Arg294. Arg294 is also hydrogen bonded to the
Thr328 carbonyl oxygen through its side chain. Unwinding of the
a6’ helix is therefore consistent with the absence of sulfate ion from
the active site [28]. Nevertheless, the CpPyK active site remained
in a partially closed conformation. The GOL1 molecule at the
ATP binding site in the CpPyK crystal structure, therefore, has a
similar effect as the sulfate ion in the active site. In the A monomer
the glycerol (GOL1) forms hydrogen bonds with the side chain
nitrogen of Asn76, the side chain of Glu272 and the hydroxyl Sulfate Binding to CpPyK Each monomer in the asymmetric unit of CpPyK binds two
sulfate ions at equivalent positions, one in the C-domain and the
other at the interface of the A and C domains (Fig. 2B). The sulfate
ion in the C-domain (SULF1) occupies a position corresponding to
the 6-phosphate of the effector molecule in different PyKs
(location E in Fig 4A). This sulfate ion forms hydrogen bonds
with hydroxyl groups from Thr432, Thr434 and Thr437, as well
as one hydrogen bond with the N atom of Thr437 (Fig. 6A). The
corresponding residues in other PyKs are always serine or
threonine, with one exception in E. coli, where one of the
threonine residues (Thr434 in CpPyK) is replaced by glycine. In October 2012 | Volume 7 | Issue 10 | e46875 PLOS ONE | www.plosone.org 5 Structure of Pyruvate Kinase the r.m.s.d. for the dimers is 0.95 A˚ . As expected, the individual
monomers in these two structures have very similar overall
structure (r. m. s. d. 0.65 A˚ for A monomers) except for the
orientation of the N-helices and the B-domains (Fig. 7). Only a
short stretch of N-helix was observed in our structure, and there is
continuous electron density connecting the helix in each monomer
to the side chain of Cys312 of the other (Figure S3). In 3MA8 the
helix is joined to the A-domain, and no disulfide bond was
modeled. However, the major difference between these structures
is the orientation of the B-domain of each monomer with respect
to the corresponding A-domain; thus, the r.m.s.d. between the A
monomers of these structures (after removal of the B-domains) is
reduced to 0.57 A˚ . The only sulfate ion modeled in the 3MA8
structure superimposes with SULF1 in the effector site of our
structure (Fig. 7). Interestingly, the a6’ helices in both monomers
of 3MA8 are intact, although the active site is unoccupied. Therefore, at least some of the differences between these two
structures may have resulted from the difference in crystallization
conditions or the presence of a glycerol molecule in the active site
of our structure or a combination of both. Table 2. Contacts for acetate ions. Expression and Purification Expression, purification and crystallization of CpPyK have been
described previously [32]. Briefly, the coding sequence for CpPyK
[13] was cloned into the BamHI/HindIII restriction sites in pET21a
vector (Novagen). The reported primary sequence (Q5CSM7) for
CpPyK
in
the
protein
database
(http://www.uniprot.org/
uniprot/) contains 532 amino acids. However, based on alignment
with PyK sequences from various organisms using CLUSTALW
[33,34] we assigned the methionine residue at position 7 as the first
residue of CpPyK. The recombinant CpPyK used in this study
contained
residues
2–526,
a
vector-derived
T7
tag
(MASMTGGQQMG) and three additional residues (RSG) at
the N-terminus. Recombinant CpPyK was expressed in E. coli BL21(DE3) cells
in LB medium containing 50 mg/ml ampicillin and 0.2% glucose;
induction was initiated with 0.4 mM isopropyl thio-b-galactopyr-
anoside when the optical density of the culture reached 0.7–0.8,
and the culture was grown overnight at 22uC. For purification, the
frozen cell pellet was suspended in lysis buffer (50 mM Tris HCl,
0.1 M sodium chloride, 10 mM DTT, 0.1 mM phenylmethyl
sulfonylfluoride, 1 mM benzamidine hydrochloride and 0.1 mg/
ml lysozyme) and incubated at 4uC for 1 hr. The suspension was
treated
with
DNAase
I
(1 mg/ml
final)
and
subjected
to
centrifugation at 20,000 rpm for 30 min. Solid ammonium sulfate
was added slowly to the clear supernatant with stirring; the pellet
resulting
from
25–40%
ammonium
sulfate
saturation
was
suspended in 50 mM Tris HCl, 2.5 mM b-mercaptoethanol
(BME), and 1 mM benzamidine hydrochloride (pH 8.2) and
dialyzed overnight at 4uC against the same buffer. Recombinant
CpPyK was further purified from the dialysate by anion exchange
chromatography on a DEAE Sephacel column and eluted using a
linear gradient of sodium chloride (0–0.5 M). Fractions containing
CpPyK were pooled, concentrated and subjected to size exclusion
chromatography on a preparative Superdex200 column equili- The second sulfate ion (SULF2) occupies a small pocket at the
interface of the A and C domains and is hydrogen-bonded to two
NZ atoms (Lys464 and Lys470), the hydroxyl oxygen of Thr471
(all in domain C), and the peptide N atom of Thr103, which
belongs to domain A (Fig. 6B). Three of these residues are unique
to Cryptosporidium, and Thr471 is found only in another
apicomplexan parasite, T. gondii. Sulfate Binding to CpPyK Residue (Atom name)
Distance (A˚ )
Acetate 531 (A)
O
Gol529 (O3)
3.10
O
Asn76 (ND2)
2.90
Acetate 532 (A)
O
His79 (ND1)
3.12
O
Arg198 (NH1)
2.32
Acetate 533 (A)
O
Arg93 (NH1)
3.09
OXT
Arg93 (NH1)
3.16
Acetate 531 (B)
O
Gol529 (O3)
2.64
O
Asn76 (ND2)
2.93
Acetate 532 (B)
O
His79 (ND1)
3.32
O
Arg198 (NH1)
2.67
Acetate 533 (B)
O
Lys404 (NZ)
3.21
OXT
Water 682
3.03
doi:10.1371/journal.pone.0046875.t002 Residue (Atom name)
Distance (A˚ ) Further biochemical studies are needed to evaluate the
regulatory mechanisms of Cryptosporidium PyK. The crystal
structure reported here does not reveal any obvious difference in
the active site of CpPyK as compared to the human enzyme. Additional biochemical and structural analyses of the parasitic
protein will be necessary to identify mechanistic and structural
features that may be targets for drug development. doi:10.1371/journal.pone.0046875.t002 Materials and Methods spite of the presence of the sulfate ion, the loop between the two C-
terminal b strands in the molecule (residues 508–517) is disordered
as seen in other PyK structures without any effector molecule. As
discussed earlier, CpPyK is not activated by fructose biphosphates
or a number of other phosphosugars [11]. However, the effector
site is capable of binding a sulfate ion. CpPyK may, therefore, use
a different effector molecule or a different regulatory mechanism
[11]. This hypothesis is further supported by the observation that
in LmPyK four pairs of stabilizing salt bridge interactions between
Asp482-Arg493 and Lys484-Glu498 are formed in the tetramer
when F-2,6BP is bound in the effector site [30]. In CpPyK the
aspartate residue in the first pair is replaced by valine (Val509),
and the glutamate residue in the second pair is replaced by
Pro526. Therefore, neither salt bridge would be possible in
CpPyK. spite of the presence of the sulfate ion, the loop between the two C-
terminal b strands in the molecule (residues 508–517) is disordered
as seen in other PyK structures without any effector molecule. As
discussed earlier, CpPyK is not activated by fructose biphosphates
or a number of other phosphosugars [11]. However, the effector
site is capable of binding a sulfate ion. CpPyK may, therefore, use
a different effector molecule or a different regulatory mechanism
[11]. This hypothesis is further supported by the observation that
in LmPyK four pairs of stabilizing salt bridge interactions between
Asp482-Arg493 and Lys484-Glu498 are formed in the tetramer
when F-2,6BP is bound in the effector site [30]. In CpPyK the
aspartate residue in the first pair is replaced by valine (Val509),
and the glutamate residue in the second pair is replaced by
Pro526. Therefore, neither salt bridge would be possible in
CpPyK. Crystallization and Data Collection Conditions for growing large single crystals were identified in a
limited screening effort using only 196 conditions at 4uC and
22uC. Plate-shaped crystals exceeding 1 mm in the longest
dimension were grown by the hanging drop vapor diffusion
technique at 4uC using 0.4–0.8 M ammonium sulfate and 0.1 M
sodium acetate buffer; the protein concentration was 7 mg/ml. Typically, these crystals grew at highly acidic pH ranging from 3.8
to 4.2 and reached maximum size in a week. The crystal used for
structure analysis was grown at pH 4.0 with 0.65 M ammonium
sulfate. Crystals were also grown by pre-incubating CpPyK with a
non-hydrolyzable ATP analog (adenyl-5’-yl imidodiphosphate,
3 mM), 5 mM magnesium chloride and 5 mM pyruvic acid. Using a coupled enzyme assay we confirmed that recombinant
CpPyK used for crystallization was enzymatically active [37]. In
this assay P. falciparum lactate dehydrogenase (PfLDH) was used to
couple the oxidation of pyruvic acid generated in the reaction
catalyzed by CpPyK. CpPyK activity was measured in the pH
range 5.5–7.5. Measurement of CpPyK activity at lower pH was
technically difficult, because activity of PfLDH significantly
dropped at lower pH. A typical assay mixture contained
2.5 mM phosphoenol pyruvate, 1 mM ADP, 5 mM magnesium
chloride, 10 mM KCl, 0.2 mM NADH, 1 mg CpPyK and 0.18 mg
PfLDH in 50 mM HEPES buffer, pH 7.0. The rate of decrease in
absorbance at 340 nm was followed for 1 min at 22uC in a UV X-ray diffraction data were collected at SBC 19BM beam line at
the Advanced Photon Source synchrotron facility. For data
collection, the crystal was soaked in a cryoprotecting solution
containing 25% glycerol (v/v) in the reservoir solution for Figure 4. Comparison of CpPyK with LmPyK. (A) Superposition of A monomers of CpPyK (red), L. mexicana PyK without sulfate ion in the active
site (1PKL, green), and L. mexicana PyK with sulfate ion in the active site (3E0V, blue). Sulfate ions bound at the effector binding site (labeled E) in all
three structures are shown as stick models: CpPyK (yellow and red), 1PKL (green) and 3E0V (blue). Sulfate ions in the active site area (labeled A) in
3E0V are also shown in blue. One additional sulfate in the CpPyK structure at the interface of the C and A domains is shown in magenta. Comparison with 3MA8 As expected, the structure of CpPyK described here is very
similar to the structure deposited in the PDB (3MA8) by the
structural genomics consortium. The two protein samples differ in
the sequence of the tag attached at the N-terminus. Crystallization
conditions reported for 3MA8 were also significantly different
from the conditions in which we crystallized CpPyK. While
crystals for 3MA8 were obtained at 20Cu using polyethylene glycol
3350 at pH 7.5, crystals used in our study were grown at 4uC using
ammonium sulfate as precipitant at a much lower pH (4.0). As in
our structure there are two molecules in the asymmetric unit of
3MA8. The packing of the dimers is similar in both structures, and October 2012 | Volume 7 | Issue 10 | e46875 PLOS ONE | www.plosone.org 6 Structure of Pyruvate Kinase Structure of Pyruvat PLOS ONE | www.plosone.org
7
October 2012 | Volume 7 | Issue 10 | e4687 October 2012 | Volume 7 | Issue 10 | e46875 October 2012 | Volume 7 | Issue 10 | e46875 7 Structure of Pyruvate Kinase Figure 3. Primary sequences of pyruvate kinases from various organisms were aligned using the CLUSTALW program [33,34]. The
labeling of secondary structural elements corresponds to the CpPyK structure. The two black triangles indicate the cysteine residues involved in the
disulfide bond. The stars mark the characteristic 6-residue insertion preceding the b5 strand in domain A of CpPyK. doi:10.1371/journal.pone.0046875.g003 spectrophotometer (Beckman Coulter DU640) (Fig. S1A). Enzyme
activity was also measured in the presence of 1, 2 and 5 mM DTT
and in the presence of 1 mM TCEP (Fig. S1B). brated with 50 mM Tris HCl, 0.1 M sodium chloride, and
2.5 mM BME, pH 8.2. Fractions containing purified CpPyK
eluted in a peak. Previously, we reported that the oligomeric state
of CpPyK could not be ascertained from the results of size
exclusion chromatography [32]. We subjected a partially purified
preparation of CpPyK and catalase (Mr 232 KDa) to size
exclusion chromatography separately on an analytical sizing
column, Superdex 200 10/30 (GE life sciences). As shown in
supplementary Fig. S1, the elution volumes were comparable. spectrophotometer (Beckman Coulter DU640) (Fig. S1A). Enzyme
activity was also measured in the presence of 1, 2 and 5 mM DTT
and in the presence of 1 mM TCEP (Fig. S1B). Crystallization and Data Collection The glycerol
molecule in the active site of CpPyK is shown as a ball and stick model. The two areas of the protein structures affected by the insertions in L. mexicana and C. parvum sequences are labeled. (B) Unwinding of helix a6’ in both monomers of CpPyK (red and yellow) as compared to 3E0V (cyan)
and 1PKL (green). Residues 293 and 302 for CpPyK are labeled. doi:10.1371/journal.pone.0046875.g004 Figure 4. Comparison of CpPyK with LmPyK. (A) Superposition of A monomers of CpPyK (red), L. mexicana PyK without sulfate ion in the active
site (1PKL, green), and L. mexicana PyK with sulfate ion in the active site (3E0V, blue). Sulfate ions bound at the effector binding site (labeled E) in all
three structures are shown as stick models: CpPyK (yellow and red), 1PKL (green) and 3E0V (blue). Sulfate ions in the active site area (labeled A) in
3E0V are also shown in blue. One additional sulfate in the CpPyK structure at the interface of the C and A domains is shown in magenta. The glycerol
molecule in the active site of CpPyK is shown as a ball and stick model. The two areas of the protein structures affected by the insertions in L. mexicana and C. parvum sequences are labeled. (B) Unwinding of helix a6’ in both monomers of CpPyK (red and yellow) as compared to 3E0V (cyan)
and 1PKL (green). Residues 293 and 302 for CpPyK are labeled. doi:10.1371/journal.pone.0046875.g004 October 2012 | Volume 7 | Issue 10 | e46875 PLOS ONE | www.plosone.org 8 Structure of Pyruvate Kinase Figure 5. Cartoon drawing of monomers A (light pink) and B
(light cyan) showing the orientation of the Arg342 side chain
from monomer. A. Unwinding of the a6’ helix of monomer B results
in the movement of the main chain carbonyl group of Gly295 too far
away for interaction with Arg342 of monomer A. Arg294, Thr328 and
Gln329 of the B monomer are also shown. doi:10.1371/journal.pone.0046875.g005 approximately 5 min at 4uC and then placed in a nitrogen stream
maintained at 100K. A total of 360 images was collected on a
2106210 mm2 CCD detector (MAR Research) at a crystal-to-
detector distance of 200 mm, with 20 s exposure for each 0.5u
oscillation frame. Intensity data were processed using the program
package HKL2000 [38]. A crystal grown in the presence of ATP
analog and pyruvic acid was also used for data collection. Crystallization and Data Collection This
crystal diffracted to ,2.8 A˚ resolution and was isomorphous with
the apo-form. However, the crystal suffered severe radiation
damage, and a data set could not be collected. Structure Determination and Refinement The structure of CpPyK was solved using the molecular
replacement routines in CNS [39], using search models based on
the L. mexicana PyK structure (1PKL) [20]. For the cross rotation
search, three models were tested: the entire monomer (residues 1–
479), a model containing domains A and C (residues 1–73 and
175–479), and domain B (residues 74–174). The entire monomer
and the model containing domains A and C gave solutions with
strong single peaks in the cross rotation, but no solution was
obtained with domain B. The solution using only domains A and
C was clearly better; the highest RF-function value was 0.0691,
while the next highest value was only 0.0432 (corresponding values
for the entire monomer were 0.0653 and 0.524, respectively). Translation functions were calculated with both solutions using
data from 15 to 4 A˚ . The highest translation function T value for
the model containing domains A and C was 0.322, while the T
value for the entire monomer was only 0.209. Therefore, the
model containing domains A and C was subjected to rigid body
and then simulated annealing refinement, and 2Fo–Fc maps were
calculated to identify the location of domain B. It was clear that
domain B had moved as a rigid body, and the density was of
sufficient quality to allow placement of the entire domain into the Figure 5. Cartoon drawing of monomers A (light pink) and B
(light cyan) showing the orientation of the Arg342 side chain
from monomer. A. Unwinding of the a6’ helix of monomer B results
in the movement of the main chain carbonyl group of Gly295 too far
away for interaction with Arg342 of monomer A. Arg294, Thr328 and
Gln329 of the B monomer are also shown. doi:10.1371/journal.pone.0046875.g005 Figure 6. Binding of sulfate ions in CpPyK. Potential hydrogen bond donors and acceptors are indicated by dotted lines with distances in A˚. (A)
SULF1 and nearby residues in the allosteric site. (B) SULF2 and nearby residues. doi:10.1371/journal.pone.0046875.g006 Figure 6. Binding of sulfate ions in CpPyK. Potential hydrogen bond donors and acceptors are indicated by dotted lines with distances in A˚. (A)
SULF1 and nearby residues in the allosteric site. (B) SULF2 and nearby residues. doi:10.1371/journal.pone.0046875.g006 October 2012 | Volume 7 | Issue 10 | e46875 PLOS ONE | www.plosone.org 9 Structure of Pyruvate Kinase Figure 7. Cartoon drawing showing superposition of C. parvum
pyruvate kinase structures. Acknowledgments C. parvum cDNA was obtained from the NIH AIDS Reagent Program. X-
ray diffraction data were collected at Argonne National Laboratory,
Structural Biology Center at the Advanced Photon Source. Argonne is
operated by University of Chicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research
under contract DE-AC02-06CH11357. Supporting Information Figure S1
Characterization of CpPyK. (A) Chromatogram
showing elution of catalase (Mr 232 kDa) from Superdex 200 10/
30 column. The flow rate was 0.5 ml/min, and fractions of 2 ml
were collected. (B) Chromatogram showing elution of CpPyK
from the same column at the same flow rate and fraction size. (C)
SDS PAGE electrophoresis pattern of fractions collected in S1B. Twenty microliters of fractions 1–12 were boiled with an equal
volume of 2X SDS sample denaturing buffer, and 10 ml mixtures
were subjected to electrophoresis on 12% polyacrylamide gel
containing 1% SDS. Lanes are labeled with the corresponding
fraction number. The lane labeled M shows the standards with
respective molecular weights shown in kDa. The gel was stained
with Coomassie Blue. (TIF) Figure S1
Characterization of CpPyK. (A) Chromatogram
showing elution of catalase (Mr 232 kDa) from Superdex 200 10/
30 column. The flow rate was 0.5 ml/min, and fractions of 2 ml
were collected. (B) Chromatogram showing elution of CpPyK
from the same column at the same flow rate and fraction size. (C)
SDS PAGE electrophoresis pattern of fractions collected in S1B. Twenty microliters of fractions 1–12 were boiled with an equal
volume of 2X SDS sample denaturing buffer, and 10 ml mixtures
were subjected to electrophoresis on 12% polyacrylamide gel
containing 1% SDS. Lanes are labeled with the corresponding
fraction number. The lane labeled M shows the standards with
respective molecular weights shown in kDa. The gel was stained
with Coomassie Blue. (TIF) Figure S2
Analysis of enzymatic activity of CpPyK. (A)
CpPyK activity was measured by monitoring absorbance at
340 nm for 1 min at 22uC. Reactions were performed in 1 ml of
50 mM HEPES buffer, pH 7.0. (B) The effect of reducing agents
on CpPyK activity was determined by incubating the enzyme in
the presence of reducing agents (1, 2 and 5 mM DTT or 1 mM
TCEP). Reaction velocities calculated as the rate of oxidation of
NADH are shown along the Y-axis. (TIF) Figure 7. Cartoon drawing showing superposition of C. parvum
pyruvate kinase structures. Our structure is orange, except for the B
domain, which is magenta; 3MA8 is light green. Sulfate ions are shown
as stick models (our structure - yellow and red; 3MA8 - green). The a6’
helix in our structure is highlighted in red. doi:10.1371/journal.pone.0046875.g007 Figure S3
Electron density maps showing the disulfide
bonds in CpPyK. Supporting Information All residues (23–32) in the N-helix of each
monomer were truncated to alanine except residue 26, which was
truncated to glycine, and the model was refined using Refmac5
[43]. Refined coordinates were used for calculating 2Fo–Fc
(magenta) and Fo-Fc (yellow) electron density maps, which were
displayed using Coot [40]. (A, B) Residues 26 and 29 of one
monomer and residue 312 of the other monomer are labeled. The
2Fo–Fc map is contoured at 1s, and the Fo-Fc map is contoured at
4.5s level. Large residual electron density peaks are observed in
locations occupied by the sulfur atoms of Cys26 and Met29 in our
model. partial model. Multiple cycles of refinement and rebuilding
allowed replacement of non-identical residues and rebuilding of
several regions where there were deletions or insertions compared
to the L. mexicana structure. The graphics program Coot was used
for model-building [40]. Residues 1–22, 33–41 and 508–517 could
not be modeled due to very weak electron density in these areas. Refinement of the structure was performed by simulated
annealing using CNS with the stereochemical parameter files
defined by Engh and Huber [41]. No sigma cutoff was applied to
the data. Five percent of the data were randomly selected and
removed prior to refinement for analysis of the free R factor. The
two subunits in the asymmetric unit were restrained by the non-
crystallographic symmetry throughout the simulated annealing
refinement. As the refinement progressed, water molecules were
added by using the water-picking routine in CNS, which searched
the peaks in the 2Fo–Fc map using a 3 sigma cutoff for density and
checked distance criteria for reasonable hydrogen-bond donors
and acceptors. All water molecules were subsequently verified by
inspection of the maps. In the final stage of refinement we
removed the noncrystallographic symmetry restraints and used the
translation/libration/screw (TLS) [42] and restrained refinement
option in REFMAC5 [43]. TLS parameters were generated using
the TLS Motion Determination (TLSMD) server (http://skuld. bmsc.washington.edu/˜tlsmd/) [44,45]. Validation of the final Structure Determination and Refinement Our structure is orange, except for the B
domain, which is magenta; 3MA8 is light green. Sulfate ions are shown
as stick models (our structure - yellow and red; 3MA8 - green). The a6’
helix in our structure is highlighted in red. doi:10.1371/journal.pone.0046875.g007 model with MolProbity [46] produced a Clash Score of 8.87 (98th
percentile for 271 structures in the resolution range 2.5060.25 A˚ )
and an overall score of 1.66 (99th percentile for 6960 structures in
the same resolution range). Atomic coordinates and structure
factors for CpPyK have been deposited in the Protein Data Bank
(PDBID:4DRS). Fig. 3 was prepared using ESPript [47]; all other
figures were prepared using Pymol [48]. References 31. Morgan HP, McNae IW, Nowicki MW, Zhong W, Michels PA, et al. (2011) The
trypanocidal drug suramin and other trypan blue mimetics are inhibitors of
pyruvate kinases and bind to the adenosine site. J Biol Chem 286: 31232–31240. 10. Verlinde CL, Hannaert V, Blonski C, Willson M, Pe´rie´ JJ, et al. (2001)
Glycolysis as a target for the design of new anti-trypanosome drugs. Drug Resist
Update 4:1–14. 32. Senkovich O, Speed H, Grigorian A, Bradley K, Ramarao CS, et al. (2005)
Crystallization of three key glycolytic enzymes of the opportunistic pathogen
Cryptosporidium parvum. Biochim Biophys Acta 1750: 166–172. 11. Denton H, Brown SM, Roberts CW, Alexander J, McDonald V, et al. (1996)
Comparison of the phosphofructokinase and pyruvate kinase activities of
Cryptosporidium parvum, Eimeria tenella and Toxoplasma gondii. Mol Biochem Parasitol
76: 23–29. 33. Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, et al. (2010) A new
bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res 38
Suppl: W695–699. 12. Entrala E, Mascaro´ C (1997) Glycolytic enzyme activities in Cryptosporidium
parvum oocysts. FEMS Microbiol Lett 151: 51–57. 34. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, et al. (2007)
ClustalW and ClustalX version 2.0. Bioinformatics 23: 2947–2948. 13. Abrahamsen MS, Templeton TJ, Enomoto S, Abrahante JE, Zhu G, et al. (2004) Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 304: 441–445. 35. Hazes B, Dijkstra BW. (1988) Model building of disulfide bonds in proteins with
known three-dimensional structure. Protein Eng 2: 119–125. known three-dimensional structure. Protein Eng 2: 119–125 14. Barrett MP, Mottram JC, Coombs GH (1999) Recent advances in identifying
and validating drug targets in trypanosomes and leishmanias. Trends Microbiol
7: 82–88. 36. Valentini G, Chiarelli LR, Fortin R, Dolzan M, Galizzi A, et al. (2002) Structure
and function of human erythrocyte pyruvate kinase. Molecular basis of
nonspherocytic hemolytic anemia. J Biol Chem 277: 23807–23814. 15. Aronov AM, Suresh S, Buckner FS,Van Voorhis WC,Verlinde CL, et al. (1999)
Structure-based design of submicromolar, biologically active inhibitors of
trypanosomatid glyceraldehyde-3-phosphate dehydrogenase. Proc Natl Acad
Sci USA 96: 4273–4278. p
y
y
J
37. Taber RL, Campbell A, Spencer S. (1998) A simple experiment demonstrating
the allosteric regulation of yeast pyruvate kinase. Biochem Educ 26: 73–76. 38. Otwinoski Z, Minor W (1997) Processing of X-ray diffraction data collected in
oscillation mode. Methods Enzymol 276: 307–326. 16. References 24. Valentini G, Chiarelli L, Fortin R, Speranza ML, Galizzi A, et al. (2000) The
allosteric regulation of pyruvate kinase. J Biol Chem 275: 18145–18152. 1. Tzipori S, Ward H (2002) Cryptosporidiosis: biology, pathogenesis and disease. Microbes Infect 4: 1047–1058. g
py
J
25. Bakszt R, Wernimont A, Allali-Hassani A, Mok MW, Hills T, et al. (2010) The
crystal structure of Toxoplasma gondii pyruvate kinase 1. PLoS One 5: e12736. 2. Tzipori S, Griffiths JK (1998) Natural history and biology of Cryptosporidium
parvum. Adv Parasitol 40: 5–36. 26. Ikeda Y, Tanaka T, Noguchi T (1997) Conversion of non-allosteric pyruvate
kinase isozyme into an allosteric enzyme by a single amino acid substitution. J Biol Chem 272: 20495–20501. 3. Dillingham RA, Lima AA, Guerrant RL (2002) Cryptosporidiosis: epidemiology
and impact. Microbes Infect 4: 1059–1066. 4. Baldursson S, Karanis P (2010) Waterborne transmission of protozoan parasites:
review of worldwide outbreaks - an update 2004–2010. Water Res. 45: 6603–
6614. 27. Mattevi A, Valentini G, Rizzi M, Speranza ML, Bolognesi M, et al. (1995)
Crystal structure of Escherichia coli pyruvate kinase type I: molecular basis of the
allosteric transition. Structure 3: 729–741. 5. Corso P, Kramer MH, Blair KA, Addiss DG, Davis JP, et al. (2003) Cost of
illness in the 1993 waterborne Cryptosporidium outbreak, Milwaukee, Wisconsin. Emerg Infect Dis 9: 426–431. 28. Tulloch LB, Morgan HP, Hannaert V, Michels PAM, Fothergill-Gilmore LA,
et al. (2008) Sulphate removal induces a major conformational change in
Leishmania mexicana pyruvate kinase in the crystalline state. J Mol Biol 383: 615–
626. 6. Nannini EC, Okhuyesen PC (2002) HIV1 and the gut in the era of highly active
antiretroviral therapy. Curr Gastroenterol Rep 4: 392–398. 29. Larsen TM, Benning MM, Rayment I, Reed GH (1998) Structure of the
bis(Mg2+)-ATP-oxalate complex of the rabbit muscle pyruvate kinase at 2.1 A˚
resolution: ATP binding over a barrel. Biochemistry 37: 6247–6255. 7. Collinet-Adler S, Ward HD (2010) Cryptosporidiosis: environmental, therapeu-
tic and preventive challenges. Eur J Clin Microbiol Infect Dis 29: 927–935. 8. Coombs GH (1999) Biochemical peculiarities and drug targets in Cryptosporidium
parvum: lessons from other coccidian parasites. Parasitol Today, 15: 333–338. 30. Morgan HP, McNae IW, Nowicki MW, Hannaert V, Michels PAM, et al. (2010) Allosteric mechanism of pyruvate kinase from Leishmania mexicana uses a
rock and lock model. J Biol Chem 285: 12892–12898. 9. Fothergill-Gilmore LA, Michels PAM (1993) Evolution of glycolysis. Prog
Biophys Mol Biol 59: 105–235. References Nowicki MW, Tulloch LB, Worrall L, McNae IW, Hannaert V, et al. (2008)
Design, synthesis and trypanocidal activity of lead compounds based on
inhibitors of parasite glycolysis. Bioorg Med Chem 16: 5050–5061. 39. Bru¨nger AT, Adams PD, Clore GM, Delano WL, Gros P, et al. (1998)
Crystallography & NMR system: A new software suite for macromolecular
structure determination. Acta Crystallogr D Biol Crystallogr 54: 905–921. 17. Zoraghi R, Worrall L, See RH, Strangman W, Popplewell WL, et al. (2011)
Methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase as a target for
bis-indole alkaloids with antibacterial activities. J Biol Chem 286: 44716–44725. y
g
y
g
40. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development
of Coot. Acta Crystallogr D Biol Crystallogr 66: 486–501. 40. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Feature of Coot. Acta Crystallogr D Biol Crystallogr 66: 486–501. 41. Engh RA, Huber R (1991) Accurate bond and angle parameters for X-ray
protein structure refinement. Acta Crystallogr A 47: 392–400. 18. Axerio-Cilies P, See RH, Zoraghi R, Worral L, Lian T, et al. (2012)
Cheminformatics-driven discovery of selective, nanomolar inhibitors for
staphylococcal pyruvate kinase. ACS Chem Biol 7: 350–359. 42. Schomaker V, Trueblood KN (1968) On the rigid-body motion of molecules in
crystals. Acta Crystallogr B 24: 63–76. 19. Hall ER, Cottam GL (1978) Isozymes of pyruvate kinase in vertebrates: their
physical, chemical, kinetic and immunological properties. Int J Biochem 9: 785–
793. 43. Murshudov GN, Skuba´k P, Lebedev AA, Pannu NS, Steiner RA, et al. (2011)
REFMAC5 for the refinement of macromolecular crystal structures. Acta
Crystallogr D Biol Crystallogr 67: 355–367. 20. Rigden DJ, Phillips SE, Michels PA, Fothergill-Gilmore LA (1999) The structure
of pyruvate kinase from Leishmania mexicana reveals details of the allosteric
transition and unusual effector specificity. J Mol Biol 291: 615–635. 44. Painter J, Merritt EA (2006a) Optimal description of a protein structure in terms
of multiple groups undergoing TLS motion. Acta Crystallogr D Biol Crystallogr
62: 439–450. 21. Lakhdar-Ghazal F, Blonski C, Willson M, Michels P, Perie J (2002) Glycolysis
and proteases as targets for the design of new anti-trypanosome drugs. Curr Top
Med Chem 2: 439–456. 45. Painter J, Merritt EA (2006b) TLSMD web server for the generation of multi-
group TLS models. J Appl Crystallogr 39: 109–111. 46. Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral GJ, et al. Author Contributions Conceived and designed the experiments: DC. Performed the experiments:
DC OS KA. Analyzed the data: DC WJC OS. Wrote the paper: DC WJC
OS. October 2012 | Volume 7 | Issue 10 | e46875 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 10 Structure of Pyruvate Kinase References (2007)
MolProbity: all-atom contacts and structure validation for proteins and nucleic
acids. Nucleic Acids Res 35: W375–W383. 22. Saito T, Nishi M, Lim MI, Wu B, Maeda T, et al. (2008) A novel GDP-
dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the
apicoplast and the mitochondrion. J Biol Chem 28: 14041–14052. 47. Gouet P, Courcelle E, Stuart DI, Metoz F (1999) ESPript: multiple sequence
alignments in PostScript. Bioinformatics 15: 305–308. 23. Maeda T, Saito T, Harb OS, Roos DS, Takeo S, et al. (2009) Pyruvate kinase
type-II isozyme in Plasmodium falciparum localizes to the apicoplast. Parasitol Int
58: 101–105. 48. Delano WL (2002) The PyMOL Molecular Graphics System. http://www. pymol.org. October 2012 | Volume 7 | Issue 10 | e46875 PLOS ONE | www.plosone.org 11
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Multi-dimensional Classification via Selective Feature Augmentation
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Multi-dimensional Classification via Selective Feature
Augmentation Bin-Bin Jia 1,2,3 Min-Ling Zhang 1,3
1 School of Computer Science and Engineering, Southeast University, Nanjing 210096, China
2 College of Electrical and Information Engineering, Lanzhou University of Technology, Lanzhou 730050, China
3 Key Laboratory of Computer Network and Information Integration, Southeast University, Ministry of Education, China Abstract: In multi-dimensional classification (MDC), the semantics of objects are characterized by multiple class spaces from differ-
ent dimensions. Most MDC approaches try to explicitly model the dependencies among class spaces in output space. In contrast, the re-
cently proposed feature augmentation strategy, which aims at manipulating feature space, has also been shown to be an effective solu-
tion for MDC. However, existing feature augmentation approaches only focus on designing holistic augmented features to be appended
with the original features, while better generalization performance could be achieved by exploiting multiple kinds of augmented features. In this paper, we propose the selective feature augmentation strategy that focuses on synergizing multiple kinds of augmented features. Specifically, by assuming that only part of the augmented features is pertinent and useful for each dimension′s model induction, we de-
rive a classification model which can fully utilize the original features while conduct feature selection for the augmented features. To val-
idate the effectiveness of the proposed strategy, we generate three kinds of simple augmented features based on standard kNN, weighted
kNN, and maximum margin techniques, respectively. Comparative studies show that the proposed strategy achieves superior perform-
ance against both state-of-the-art MDC approaches and its degenerated versions with either kind of augmented features. ine learning, multi-dimensional classification, feature augmentation, feature selection, class dependencies. Keywords: Machine learning, multi-dimensional classification, feature augmentation, feature selection, class depen Citation: B. B. Jia, M. L. Zhang. Multi-dimensional classification via selective feature augmentation. Machine Intelligence Research,
vol.19, no.1, pp.38–51, 2022. http://doi.org/10.1007/s11633-022-1316-5 source allocation[11], etc. Fig. 1 shows an illustrative ex-
ample of MDC on vehicle classification. Research Article
Manuscript received April 17, 2021; accepted September 8, 2021
Recommended by Associate Editor Hui Yu
Colored figures are available in the online version at https://link.
springer.com/journal/11633
© The Author(s) 2022 19(1), February 2022, 38-51
DOI: 10.1007/s11633-022-1316-5 19(1), February 2022, 38-51
DOI: 10.1007/s11633-022-1316-5 Machine Intelligence Research
www.mi-research.net 2 Related work In order to validate the effect-
iveness of SFAM, three simple kinds of augmented fea-
tures are generated by making use of standard kNN,
weighted kNN, and maximum margin techniques, respect-
ively. After that, for each dimension, SFAM derives a
classification model which can take full advantage of the
original features via
regularization and conduct fea-
ture selection for the augmented features via
regulariz-
ation (i.e., selective feature augmentation). Experimental ℓ2
ℓ1
set and usually suffers high computational complexity
due to a possibly large number of classes. In fact, one of
the key challenges for MDC studies is how to model de-
pendencies among class spaces in appropriate ways. Ex-
isting works mainly focus on modeling class dependencies
in output space, such as capturing pairwise class depend-
encies[12−14], specifying chaining order over class spaces[15, 16],
learning a directed acyclic graph (DAG) structure for
class spaces[17−19], and partitioning class spaces into
groups[2], etc. Recently, feature augmentation strategy,
which aims at manipulating feature space, has been
shown as an effective solution for MDC. This strategy en-
riches the original feature space with a set of new fea-
tures that are generated by making use of some well-es-
tablished techniques, e.g., kNN[20] or deep learning[21]. Ex-
isting works only focus on how to design more informat-
ive augmented features, while it might be beneficial to ex-
ploit multiple kinds of augmented features generated by
making use of different techniques. In this paper, we pro-
pose the selective feature augmentation strategy, which
makes the first attempt to synergize multiple kinds of
augmented features. The strategy is abbreviated as
SFAM, i.e., selective feature augmentation for multi-di-
mensional classification, in the following parts of this pa-
per for brevity. Specifically, SFAM assumes only part of
augmented features are pertinent and useful for each di-
mension′s model induction. In order to validate the effect-
iveness of SFAM, three simple kinds of augmented fea-
tures are generated by making use of standard kNN,
weighted kNN, and maximum margin techniques, respect-
ively. After that, for each dimension, SFAM derives a
classification model which can take full advantage of the
original features via
regularization and conduct fea-
ture selection for the augmented features via
regulariz-
ation (i.e., selective feature augmentation). Experimental O(q2)
The MDC problem can be solved via an independent
decomposition strategy, where a total of q multi-class
classifiers are learned independently, one per dimension. 1 Introduction Traditional supervised learning tasks usually charac-
terize the semantics of objects with one output variable,
i.e., single-output learning, among which multi-class clas-
sification is one of the most important learning frame-
works. However, in some real-world applications, it is bet-
ter to use multiple output variables to characterize the
rich semantics of objects, which results in the problem of
multi-output learning[1]. Here, when the type of each out-
put variable is restricted to discrete-valued, the multi-di-
mensional classification (MDC) framework is obtained[2, 3]. Under the MDC setting, each object is represented by a
single instance while associated with multiple class vari-
ables, each corresponding to a specific class space charac-
terizing the object′s semantics along one specific dimen-
sion. Specifically, the MDC problem widely exists in
many
application
scenarios,
such
as
bioinforma-
tics[4, 5], text classification[6, 7], computer vision[8−10], re- X ∈Rd
Y = C1 × C2 × · · · × Cq
Y
Cj = {cj
1, cj
2, · · · , cj
Kj} (1 ≤j ≤q)
Kj
D = {(xi, yi) | 1 ≤i ≤m}
xi = [xi1, xi2, · · · , xid]T ∈X
yi = [yi1, yi2, · · · , yiq]T ∈Y
xi
yij ∈Cj
f : X 7→Y
D
f(x∗) ∈Y
x∗
Formally speaking, let
be the d-dimensional
feature space and
be the output
space. Here,
corresponds to the Cartesian product of q
class spaces
which con-
sists of
possible classes respectively. Given a set of
MDC training examples
, where
is a d-dimensional feature
vector and
is the q-dimension-
al class vector associated with
with each element
, the MDC task aims to learn a predictive model
from
which can return a proper class vec-
tor
for unseen instance
. It is obvious that the MDC problem can be solved di-
mension by dimension, i.e., training a multi-class classifi-
er for each class space. However, this independent decom-
position strategy does not consider potential dependen-
cies among class spaces which might impact the generaliz-
ation performance of the resulting model. The MDC
problem can also be solved by a single multi-class classifi-
er, where each distinct class combination is regarded as a
new class. However, this powerset-like strategy cannot
consider class combinations not appearing in the training 39 B. B. Jia and M. L. Zhang / Multi-dimensional Classification via Selective Feature Augmentation (a)
(b)
(c)
(d)
Fig. 1 Introduction 1 An
illustrative
example
of
multi-dimensional
classification: Vehicle classification. For a vehicle, it can be
classified from the type dimension (with possible classes car,
SUV, bus, truck, etc.), from the brand dimension (with possible
classes Audi, Benz, YUTONG, JAC, etc.), and from the color
dimension (with possible classes black, white, red, blue, etc.). Here, (a) is a red Audi car, (b) is a black Benz SUV, (c) is a red
YUTONG bus, and (d) is a white JAC truck. results demonstrate that SFAM achieves superior per-
formance against both state-of-the-art MDC approaches
and its degenerated versions with either kind of augmen-
ted features. The rest of this paper is organized as follows. Firstly,
related works on multi-dimensional classification are
briefly discussed. Secondly, technical details of SFAM are
introduced. Thirdly, experimental results of comparative
studies are reported. Finally, we conclude this paper. (b) (a) (c) 2 Related work (c) (d) The most related learning framework to multi-dimen-
sional classification is the widely studied multi-label clas-
sification (MLC)[22−24], which can be regarded as a special
case of MDC when the type of class variable in each di-
mension is restricted to binary-valued. However, MDC
usually assumes heterogeneous class spaces, which are
used to characterize the rich semantics of objects from
different dimensions, while MLC usually assumes homo-
geneous class space in which multiple concepts are relev-
ant to the polysemous objects. Fig. 1 An
illustrative
example
of
multi-dimensional
classification: Vehicle classification. For a vehicle, it can be
classified from the type dimension (with possible classes car,
SUV, bus, truck, etc.), from the brand dimension (with possible
classes Audi, Benz, YUTONG, JAC, etc.), and from the color
dimension (with possible classes black, white, red, blue, etc.). Here, (a) is a red Audi car, (b) is a black Benz SUV, (c) is a red
YUTONG bus, and (d) is a white JAC truck. ℓ2
ℓ1
set and usually suffers high computational complexity
due to a possibly large number of classes. In fact, one of
the key challenges for MDC studies is how to model de-
pendencies among class spaces in appropriate ways. Ex-
isting works mainly focus on modeling class dependencies
in output space, such as capturing pairwise class depend-
encies[12−14], specifying chaining order over class spaces[15, 16],
learning a directed acyclic graph (DAG) structure for
class spaces[17−19], and partitioning class spaces into
groups[2], etc. Recently, feature augmentation strategy,
which aims at manipulating feature space, has been
shown as an effective solution for MDC. This strategy en-
riches the original feature space with a set of new fea-
tures that are generated by making use of some well-es-
tablished techniques, e.g., kNN[20] or deep learning[21]. Ex-
isting works only focus on how to design more informat-
ive augmented features, while it might be beneficial to ex-
ploit multiple kinds of augmented features generated by
making use of different techniques. In this paper, we pro-
pose the selective feature augmentation strategy, which
makes the first attempt to synergize multiple kinds of
augmented features. The strategy is abbreviated as
SFAM, i.e., selective feature augmentation for multi-di-
mensional classification, in the following parts of this pa-
per for brevity. Specifically, SFAM assumes only part of
augmented features are pertinent and useful for each di-
mension′s model induction. 2 Related work However, this intuitive strategy ignores possible depend-
encies among class spaces, and the induced model would
be suboptimal. An improved strategy is learning the q
multi-class classifiers in a chaining order, where predic-
tions of preceding classifiers are used as extra features by
the subsequent ones[15, 16]. However, the chaining order
would largely affect the generalization performance while
determining an optimal one is NP-hard. The MDC prob-
lem can also be solved via a powerset transformation
strategy where a single multi-class classifier is learned by
regarding all distinct class combinations in the training
set as new classes. However, this intuitive strategy can-
not consider class combinations not appearing in the
training set and usually suffers high computational com-
plexity due to a large number of new classes. An im-
proved strategy is partitioning the class spaces into
groups according to conditional dependencies[2]. However,
the combinatorial nature still exists, which leads to that
the deficiencies cannot be fully addressed. A family of
MDC models called multi-dimensional Bayesian network
classifier[25] aims at learning different kinds of DAG struc-
tures over class spaces to explicitly model the class de-
pendencies. However, determining DAG structures is
computationally demanding, and only nominal features
can be tackled generally. The class dependencies can also
be modeled in a two-level strategy[12−14], where pairwise
dependencies are captured in the first level, and then
high-order dependencies are captured in the second-level
based on the predictions from the first level. However,
capturing pairwise dependencies needs
complexity,
which is very time-consuming. Machine Intelligence Research 19(1), February 2022 40 The afore-mentioned strategies mainly focus on dir-
ectly modeling class dependencies in the output space,
while the KRAM approach[20] attempts to manipulate the
feature space of MDC examples via feature augmenta-
tion by making use of kNN techniques. Helpful discrimin-
ative information is expected to be brought into feature
space which would facilitate the subsequent MDC model
induction. Based on deep learning techniques, the LEFA
approach[21] further generates better-augmented features
which can depict the inter-class dependencies and the in-
tra-class exclusiveness simultaneously. However, these ap-
proaches simply treat the original and augmented fea-
tures equally, which might be less reasonable due to vari-
ous characteristics of different features. 2 Related work Moreover, it is
usually easier to design multiple kinds of simple augmen-
ted features than a terrific one, and it might be benefi-
cial to consider synergizing the discriminative informa-
tion residing in different kinds of augmented features. Fig. 2 shows an intuitive comparison between existing fea-
ture augmentation techniques and the proposed one in
this paper. Existing works usually employ general MDC
algorithms to accomplish the training phase, while the
proposed one designs a novel training algorithm that can
accomplish the selective feature augmentation phase. xi
exi
zi
xi
exi
zi = [xi; exi]
xi
exi
feature augmentation strategy. In other words, the tech-
nical details in this section correspond to the part of the
training algorithm in Fig. 2(b). For any instance
, let
be the concatenation of all the corresponding augmented
features generated by the N augmentation models, and
corresponds to the concatenation of
and
, i.e.,
, in the following, we derive a regularized
classification model which fully utilizes original features
and employs feature selection mechanism over aug-
mented features
. ℓ1
ℓ2
(w∗
ja, b∗
ja)
For simplicity, we employ the one-vs-rest decomposi-
tion strategy for each dimension where a classification
model with both
and
regularization is derived to
solve the decomposed binary classification problems. Spe-
cifically, for the a-th decomposed binary classification
problem in the j-th dimension, we determine the optimal
model
as follows: (w∗
ja, b∗
ja) = arg min
wja,bja
L(wja, bja) + λR(wja)
(1) (1) λ
L(wja, bja)
where
is a trade-off parameter. The first term
denotes the empirical loss function. In this
paper, we employ the cross-entropy loss, which is defined
as follows: 3 Technical details of SFAM Here,
is the concatenation of
and
. Specifically, existing feature augmentation techniques focus on
designing a better augmentation model to generate more
informative augmented features, while this paper focuses on
designing a novel training algorithm that aims at synergizing
multiple kinds of augmented features. zi
xi
exi
Fig. 2 An intuitive comparison of the training phase between
existing feature augmentation techniques and the proposed one
in this paper. Here,
is the concatenation of
and
. Specifically, existing feature augmentation techniques focus on
designing a better augmentation model to generate more
informative augmented features, while this paper focuses on
designing a novel training algorithm that aims at synergizing
multiple kinds of augmented features. To optimize problem (1), we solve one of the three 3 Technical details of SFAM L(wja, bja) = −
m
∑
i=1
[
lja
i
× ln hwja,bja(zi)+
(1 −lja
i ) × ln(1 −hwja,bja(zi))
]
. This section presents how we implement the selective Augmentation
model
Augmented-features
Training algorithm
Predictive
model
Augmentation
model 1
Augmentation
model N
……
Augmented-features
Training algorithm
Predictive
model
(a) Existing feature augmentation
(b) Selective feature augmentation
(xi, yi)
(xi, yi)
(zi, yi)
(zi, yi)
xi
~
xi
~ Augmentation
model
Augmented-features
Training algorithm
Predictive
model
Augmentation
model 1
Augmentation
model N
……
Augmented-features
Training algorithm
Predictive
model
(a) Existing feature augmentation
(b) Selective feature augmentation
(xi, yi)
(xi, yi)
(zi, yi)
(zi, yi)
xi
~
xi
~
zi
xi
exi
Fig. 2 An intuitive comparison of the training phase between
existing feature augmentation techniques and the proposed one
in this paper. Here,
is the concatenation of
and
. Specifically, existing feature augmentation techniques focus on
designing a better augmentation model to generate more
informative augmented features, while this paper focuses on
designing a novel training algorithm that aims at synergizing
multiple kinds of augmented features. Augmentation
model
Augmented-features
Training algorithm
Predictive
model
(a) Existing feature augmentation
(xi, yi)
(zi, yi)
xi
~ lja
i
= 1yij=cj
a
1π
π
hwja,bja(zi)
Here,
, the predicate
returns 1 if holds
and 0 otherwise, and
is the logistic function
which is defined as follows: Predictive
model hwja,bja(zi) =
1
1 + e−(⟨wja,zi⟩+bja) Augmentation
model 1
Augmentation
model N
……
Augmented-features
Training algorithm
Predictive
model
(b) Selective feature augmentation
(xi, yi)
(zi, yi)
xi
~ ⟨·, ·⟩
R(wja)
where
returns the inner product of two vectors. The
second term
denotes the regularization term,
which is defined as follows: R(wja) = ∥θja∥2
2 + ∥eθja∥1 θja
eθja
wja
wja =
[θja; eθja]
ℓ2
ℓ1
where
and
denote the first d elements and the
last remaining elements of
, respectively, i.e.,
. It is worth noting that the
regularization
corresponds to the original features while the
regularization corresponds to the augmented features. By
doing this, we employ a feature selection mechanism over
the augmented features to synergize multiple kinds of
augmented features in a better way while the original
features are still fully utilized. In other words, the
selective feature augmentation strategy is implemented
here. (b) Selective feature augmentation zi
xi
exi
Fig. 2 An intuitive comparison of the training phase between
existing feature augmentation techniques and the proposed one
in this paper. B. B. Jia and M. L. Zhang / Multi-dimensional Classification via Selective Feature Augmentation B. B. Jia and M. L. Zhang / Multi-dimensional Classification via Selective Feature Augmentation B. B. Jia and M. L. Zhang / Multi-dimensional Classification via Selective Feature Augmentation 41 {θja}, {eθja}
{bja}
sets of parameters
and
alternately,
while the remaining parameters are fixed. Lf = 1
4
m
∑
i=1
∥exi∥2
2
(4) (4) {θja}
{eθja}
{bja}
{eθja}
{bja}
1) Optimizing w.r.t. when
and
are fixed: When
and
are fixed, the optimiza-
tion problem (1) can be equivalently reformulated as fol-
lows: ∇
where
denotes the differential operator. ∇
where
denotes the differential operator. e ∇L2(eθja)
Proof. For
, it can be calculated as ∇L2(eθja) = ∂L2(eθja)
∂eθja
= −
m
∑
i=1
(lja
i
−he
θja(zi))exi. min
θja
L1(θja) + λ ∥θja∥2
2
(2) min
θja
L1(θja) + λ ∥θja∥2
2 (2) eθja
eθ′
ja
Given any
and
, we have where where ∇L2(eθ′
ja) −∇L2(eθja)
2 =
m
∑
i=1
(
he
θ′
ja(zi) −he
θja(zi)
)
exi
2
≤
m
∑
i=1
he
θ′
ja(zi) −he
θja(zi)
2 ∥exi∥2 ≤
m
∑
i=1
1
4
⟨eθ′
ja, exi⟩−⟨eθja, exi⟩
2 ∥exi∥2 ≤
m
∑
i=1
1
4
⟨eθ′
ja −eθja, exi⟩
2 ∥exi∥2 ≤
1
4
m
∑
i=1
eθ′
ja −eθja
2 ∥exi∥2
2 . L1(θja) = −
m
∑
i=1
[
lja
i
× ln hθja(zi)+
(1 −lja
i ) × ln(1 −hθja(zi))
] and hθja(zi) =
1
1 + e−(⟨θja,xi⟩+Ci
1) . Ci
1 = ⟨eθja, exi⟩+ bja
θja
Ω1(θja)
Here,
is a constant which is not
dependent on the variables
. In this paper, we use
gradient descent to solve the optimization problem (2). Specifically, let
be the objective function, the
gradient is given as follows: ≤
1
4
Here, the second “ ” is due to the truth that the
Lipschitz constant of logistic function equals . So then,
it is easy to know: ∂Ω1(θja)
∂θja
= −
m
∑
i=1
(lja
i
−hθja(zi))xi + 2λθja. {eθja}
{θja}
{bja}
{θja}
{bja}
2) Optimizing w.r.t. when
and
are fixed: When
and
are fixed, the optimiza-
tion problem (1) can be equivalently reformulated as fol-
lows: ∇L2(eθ′
ja) −∇L2(eθja)
2
eθ′
ja −eθja
2
≤1
4
m
∑
i=1
∥exi∥2
2 □ which completes the proof. which completes the proof. min
e
θja
L2(eθja) + λ∥eθja∥1
(3) (3) eθ(t)
ja
eθja
∆eθja = eθja −eθ(t)
ja
According to Theorem 1, given any initial value
of
, let
, the following inequation al-
ways holds: where where L2(eθja) = −
m
∑
i=1
[
lja
i
× ln he
θja(zi)+
(1 −lja
i ) × ln(1 −he
θja(zi))
]
he
θja(zi) =
1
1 + e−(⟨e
θja,exi⟩+Ci
2) . L2(eθja) = −
m
∑
i=1
[
lja
i
× ln he
θja(zi)+
(1 −lja
i ) × ln(1 −he
θja(zi))
] ∇L2(eθja) −∇L2(eθ(t)
ja )
2 ≤Lf
∆eθja
2 . L2(eθja)
eθ(t)
ja
Then, the quadratic approximation of
around
can be given as follows: and bL2(eθja) ≃L2(eθ(t)
ja ) + ⟨∇L2(eθ(t)
ja ), ∆eθja⟩+ Lf
2
∆eθja
2
2 =
Lf
2
eθja −u(t)
2
2 + CLf he
θja(zi) =
1
1 + e−(⟨e
θja,exi⟩+Ci
2) . Ci
2 = ⟨θja, xi⟩+ bja
{eθja}
Here,
is a constant which is not
dependent on the variables
. In this paper, we use
the accelerated proximal gradient method[26] to solve it. Ci
2 = ⟨θja, xi⟩+ bja
{eθja}
Here,
is a constant which is not
dependent on the variables
. In this paper, we use
the accelerated proximal gradient method[26] to solve it. CLf = −1
2Lf
∇L2(eθ(t)
ja )
2
2 + L2(eθ(t)
ja )
eθja
where
is a constant
which is not dependent on variables
, and CLf = −1
2Lf
∇L2(eθ(t)
ja )
2
2 + L2(eθ(t)
ja )
eθja
where
is a constant
which is not dependent on variables
, and L2(eθja)
∇L2(eθja)
Theorem 1. For the derivable function
,
is Lipschitz continuous, and the Lipschitz con-
stant is u(t) = eθ(t)
ja −1
Lf ∇L2(eθ(t)
ja ). (5) (5) 42 Machine Intelligence Research 19(1), February 2022 Algorithm 1. The proposed SFAM approach. D
{(xi, yi) | 1 ≤i ≤m}
Input:
: MDC training set
λ
: trade-off parameter in (1)
An
1 ≤n ≤N
: augmentation model n (
)
x∗
: unseen instance
y∗
x∗
Output:
: predicted class vector for Algorithm 1. The proposed SFAM approach L2(eθja) ≤bL2(eθja)
According to the descent lemma[27], the approxima-
tion is an upper bound of the original function, i.e.,
always holds. Therefore, we can min-
imize the original function by iteratively minimizing the
approximation. Plugging the above approximation into
the optimization problem (3), we can obtain the follow-
ing iterative equation: Algorithm 1. The proposed SFAM approach. D
{(xi, yi) | 1 ≤i ≤m}
Input:
: MDC training set Algorithm 1. The proposed SFAM approach. D
{(xi, yi) | 1 ≤i ≤m}
Input:
: MDC training set g
p
p
pp
D
{(xi, yi) | 1 ≤i ≤m}
Input:
: MDC training set λ: trade-off parameter in (1) λ: trade-off parameter in (1) ( )
An
1 ≤n ≤N
: augmentation model n (
) An
1 ≤n ≤N
: augmentation model n (
) x∗: unseen instance x∗
: unseen instance
y∗
x∗
Output:
: predicted class vector for x∗
: unseen instance
y∗
x∗
Output:
: predicted class vector for eD = ∅
1)
; for i = 1 to m do
2)
for n = 1 to N do
3) for i = 1 to m do
2) eθ(t+1)
ja
=arg min
e
θja
Lf
2
eθja −u(t)
2
2 + λ
eθja
1 =
soft
(
u(t), λ
Lf
)
. (6) for n = 1 to N do
3) ∆xi
n
xi
An
4) Generate the n-th augmented features
for
the i-th training example
via
; ∆xi
n
xi
An
4) Generate the n-th augmented features
for
the i-th training example
via
; (6) end for
5)
eD = eD ∪(zi, yi)
zi = [xi; exi]
exi
∆xi
1 , · · · , ∆xi
N
6)
where
,
is the
concatenation of
; end for
5)
eD = eD ∪(zi, yi)
zi = [xi; exi]
exi
∆xi
1 , · · · , ∆xi
N
6)
where
,
is the
concatenation of
; soft(·, ·)
Here,
is the (element-wise) soft-thresholding
function which is defined as follows: end for
7)
for j = 1 to q do
8)
for a = 1 to Kj do
9)
wja = [θja; eθja] = 0, bja = 0
10) Initialize
;
Repeat
11)
θja
12) Update
by solving the optimization
problem (2) via gradient descent;
eθ(0)
ja = eθ(1)
ja = eθja r0 = r1 = 1 t = 1
13) Initialize
,
,
;
Repeat
14)
v(t)
ja
15) Obtain
according to (7);
u(t) = v(t)
ja −1
Lf ∇L2(v(t)
ja )
16) Compute
;
eθ(t+1)
ja
17) Obtain
according to (6);
rt+1 = 1 +
√
1 + 4r2
t
2
18) Compute
;
t = t + 1
19)
;
Until
20)
Convergence
eθja
eθ(t)
ja
21) Update
with
bja
22) Update
by solving the optimization
problem (8) via gradient descent; end for
7)
for j = 1 to q do
8)
for a = 1 to Kj do
9) for j = 1 to q do
8)
for a = 1 to Kj do
9) soft(x, µ) =
x −µ,
if x > µ
x + µ,
if x < −µ
0,
otherwise. p
)
θja
12) Update
by solving the optimization
problem (2) via gradient descent; θja
12) Update
by solving the optimization
problem (2) via gradient descent; O(t−2)
O(t−1)
eθ(t)
ja
v(t)
ja
In [28], it is shown that the convergence rate of the it-
erative equation in (6) can be improved to
from
if we replace
in (5) with the following
: p
( )
g
;
eθ(0)
ja = eθ(1)
ja = eθja r0 = r1 = 1 t = 1
13) Initialize
,
,
;
Repeat
14) p
v(t)
ja
Obtain
according to (7); v(t)
ja
Obtain
according to (7);
u(t) = v(t)
ja −1
Lf ∇L2(v(t)
ja )
Compute
;
eθ(t+1)
ja
Obtain
according to (6);
rt+1 = 1 +
√
1 + 4r2
t
2
Compute
;
t
t + 1; v(t)
ja
Obtain
according to (7);
u(t) = v(t)
ja −1
Lf ∇L2(v(t)
ja )
Compute
;
eθ(t+1)
ja
Obtain
according to (6);
rt+1 = 1 +
√
1 + 4r2
t
2
Compute
; v(t)
ja = eθ(t)
ja + rt−1 −1
rt
(eθ(t)
ja −eθ(t−1)
ja
)
(7)
= r1 = 1
rt =
1 +
√
1 + 4r2
t−1
2
t > 1
and
when
. (7) {bja}
{θja}
{eθja}
{θja}
{eθja}
3) Optimizing w.r.t. when
and
are fixed: When
and
are fixed, the optimiz-
ation problem (1) can be equivalently reformulated as fol-
lows: min
bja
L3(bja)
(8) Until
23)
Convergence
end for
24)
end for
25)
x′
∗s
ex∗= [∆x∗
1 ;· · · ; ∆x∗
N ]
26) Obtain
augmented features
for j = 1 to q do
27)
y∗j
28) Determine the class
according to (9);
end for
29)
y∗= [y∗1, y∗2, · · · , y∗q]T
30) Return
. A
h
b
h
l
i
i
i i (8) where where L3(bja) = −
m
∑
i=1
[
lja
i
× ln hbja(zi)+
(1 −lja
i ) × ln(1 −hbja(zi))
]
hbja(zi) =
1
1 + e−(bja+Ci
3) . L3(bja) = −
m
∑
i=1
[
lja
i
× ln hbja(zi)+
(1 −lja
i ) × ln(1 −hbja(zi))
] y∗j
28) Determine the class
according to (9); end for
29)
y∗= [y∗1, y∗2, · · · , y∗q]T
30) Return
. y∗= [y∗1, y∗2, · · · , y∗q]T
30) Return
. 4 Experiments HSS(f) = 1
p
p
∑
i=1
1
q × r(i)
EMS(f) = 1
p
p
∑
i=1
1r(i)=q
SEMS(f) = 1
p
p
∑
i=1
1r(i)≥q−1. This section conducts comparative studies, and the
obtained experimental results clearly validate the superi-
ority and effectiveness of SFAM. Firstly, Section 4.1 in-
troduces the experimental setup, including the employed
benchmark data sets, the evaluation metrics, and the
compared approaches. Then, Section 4.2 reports the de-
tailed experimental results with statistical tests. Finally,
Section 4.3 further investigates SFAM′s algorithmic
design and parameter sensitivity. For the three metrics, it is easy to know that the lar-
ger the values, the better the performance. Ten-fold
cross-validation is conducted over each benchmark data
set, where both the mean metric value as well as stand-
ard deviation are recorded for comparative studies. 2) Evaluation metrics S = {(xi, yi) | 1 ≤i ≤p}
f : X 7→Y
ˆyi = f(xi) = [ˆyi1, ˆyi2,
· · · , ˆyiq]T
xi
yi = [yi1, yi2, · · · , yiq]T
r(i) = ∑q
j=1 1yij=ˆyij
In this paper, a total of three widely-used evaluation
metrics are employed for performance evaluation, includ-
ing Hamming score (HS), exact match (EM), and sub-ex-
act match (SEM)[2, 13, 14, 20, 29]. Specifically, given the test
set
, for the MDC model
to be evaluated, let
be the predicted class vector for
while the
ground-truth one is
, then the
number of correctly predicted dimensions corresponds to
. The detailed definitions of the met-
rics are given as follows: As shown in Algorithm 1, it is worth noting that SFAM
should be regarded as a general framework and can be
coupled with any kind of augmented-features, while this
paper only aims at investigating the feasibility of syner-
gizing different kinds of augmented features. A
h
b
h
l y∗= [y∗1, y∗2, · · · , y∗q]T
30) Return
. wja
bja
x∗
ex∗
z∗= [x∗; ex∗]
As the above three alternating optimizing steps con-
verge, we can obtain the optimal values of
and
. For unseen instance
, let
be its augmented features,
then its class label in the j-th dimension can be determ-
ined based on the augmented instance
as
follows: and hbja(zi) =
1
1 + e−(bja+Ci
3) . Ci
3 = ⟨wja, zi⟩
{bja}
L3(bja)
Here,
is a constant which is not dependent
on variable
. In this paper, we use gradient descent
to solve it. Specifically, the gradient of the objective
function (i.e.,
) is given as follows: y∗j = cj
ˆa, where ˆa = arg max
1≤a≤Kj
⟨wja, z∗⟩+ bja. (9) (9) D
eD
The complete procedure of the proposed SFAM ap-
proach is summarized in Algorithm 1. Firstly, SFAM
transforms the original MDC training set
into
by
augmenting each instance′s feature space (Steps 1)−7)). ∂L3(bja)
∂bja
= −
m
∑
i=1
(lja
i
−hbja(zi)). 43 B. B. Jia and M. L. Zhang / Multi-dimensional Classification via Selective Feature Augmentation B. Jia and M. L. Zhang / Multi-dimensional Classification ℓ2
ℓ1
ℓ2
ℓ1
After that, the predictive model is induced via a classific-
ation model with both
and
regularization (Steps
8)−25)), where the
regularization and the bias term are
updated via gradient descent, and the
regularization is
updated via an accelerated proximal gradient method. Fi-
nally, the class vector of unseen instance is predicted
based on the augmented features as well (Steps 26)−30)). As shown in Algorithm 1, it is worth noting that SFAM
should be regarded as a general framework and can be
coupled with any kind of augmented-features, while this
paper only aims at investigating the feasibility of syner-
gizing different kinds of augmented features. ℓ2
ℓ1
ℓ2
ℓ1
After that, the predictive model is induced via a classific-
ation model with both
and
regularization (Steps
8)−25)), where the
regularization and the bias term are
updated via gradient descent, and the
regularization is
updated via an accelerated proximal gradient method. Fi-
nally, the class vector of unseen instance is predicted
based on the augmented features as well (Steps 26)−30)). 1) Benchmark data sets In this paper, a total of six state-of-the-art MDC ap-
proaches are employed as compared approaches, includ-
ing BR, CP, ECC[16], ESC[2], gMML[29], and SEEM[13]: In this paper, a total of ten benchmark data sets are
collected for comparative studies. Table 1 summarizes the
detailed characteristics of all benchmark data sets, includ-
ing number of examples (#Exam.), number of dimen-
sions (#Dim.), number of class labels per dimension
(#Labels/Dim.), and number of features (#Features). Here, for the “#Labels/Dim.” column, if all dimensions
contain the same number of class labels, then only this
number is recorded. Otherwise, the number of class la-
bels per dimension is recorded in turn. For the “#Fea-
tures” column, n and x denote numeric and nominal fea-
tures, respectively. i) BR solves the MDC problem via training a number
of multi-class classifiers independently, one per dimen-
sion. BR serves as the baseline when all possible class de-
pendencies are ignored. ii) CP transforms the MDC problem into a single
multi-class classification problem by treating the whole
output space as a compound one. Each distinct class com-
bination in the training set is regarded as a new class. CP
serves as the baseline when all possible class dependen-
cies in the training set are considered, but overfitting
might occur because CP cannot return class combina-
tions not appearing in the training set. Table 1 Characteristics of benchmark data sets
Data Set
#Exam. #Dim. #Labels/Dim. #Features
Flare1
323
3
3,4,2
10x
Enb
768
2
2,4
6n
WQplants
1 060
7
4
16n
WQanimals
1 060
7
4
16n
WaterQuality
1 060
14
4
16n
BeLaE
1 930
5
5
1n, 44x
Voice
3 136
2
4,2
19n
TIC2000
9 822
3
6,4,2
83x
Adult
18 419
4
7,7,5,2
5n, 5x
Default
28 779
4
2,7,4,2
14n, 6x iii) ECC solves the MDC problem via training a chain
of multi-class classifiers, one per dimension, where predic-
tions of preceding classifiers on the chain are used as ex-
tra features by the subsequent ones. iv) ESC preprocesses the MDC problem via partition-
ing the class variables into super-class, where each super-
class is used as a compound class variable. iv) ESC preprocesses the MDC problem via partition-
ing the class variables into super-class, where each super-
class is used as a compound class variable. 1) Benchmark data sets v) gMML works by learning a regression model for
each class label as well as a Mahalanobis metric which
can shorten the distance between the regression outputs
and ground-truth label vector. vi) SEEM models the class dependency via a two-level
strategy, where the pairwise and high-order class depend- Machine Intelligence Research 19(1), February 2022 Machine Intelligence Research 19(1), February 2022 44 ∆x
kNN2 = ∆x
kNN1 +
[
ζx
1 , ζx
2 , · · · , ζx
q
]T . (11) encies are modeled in the first and second levels, respect-
ively. (11) For BR, CP, ECC, ESC, and SEEM, the multi-class
base learner is implemented via LIBLINEAR[30] with the
parameter setting “L2-regularized logistic regression
(primal)” for a fair comparison. Following [2], for en-
semble approaches ECC and ESC, a total of 10 base
models are trained over 67% examples randomly selected
from the training set, and the predictive results are com-
bined via majority voting. For gMML and SEEM, the re-
commended parameters are used according to respective
literature. 1 ≤j ≤q
For the maximum margin-augmented features, SFAM
employs the real-valued predictions, which are returned
by the multi-class support vector machine to generate the
maximum
margin-augmented
features. Specifically,
SFAM
solves
the
following
maximum
margin
formulation[31] for the j-th dimension (
): min
Pj,ξj
1
2
Kj
∑
a=1
∥pja∥2
2 + γ ×
m
∑
i=1
ξji
s.t. ⟨pjai, xi⟩−⟨pja, xi⟩≥ea
ji −ξji,
1 ≤a ≤Kj, 1 ≤i ≤m x
Nk(x) = {ir | 1 ≤r ≤k}
k
x
D
vx
ja = [vx
ja(1), vx
ja(2), · · · , vx
ja(k)]T∈{0, 1}k
In order to validate the effectiveness of the selective
feature augmentation strategy for the proposed SFAM
approach, a total of three simple kinds of augmented fea-
tures are generated. Two of them are generated using
standard and weighted kNN techniques, respectively, and
the remaining one is generated using maximum margin
techniques. Specifically, the two kinds of kNN-based aug-
mented features are generated by KRAM[20]. 1) Benchmark data sets In the future, it is interesting to further
investigate synergizing multiple kinds of augmented
features which are generated by making use of more
advanced techniques such as deep learning[21]. δx
ja = ⟨Ik, vx
ja⟩
(1 ≤a ≤Kj) Ik
q
∆x
kNN1
x
where
is a column vector of all ones with length k. By
concatenating all the
counting statistics vectors, the
first kNN-augmented feature vector
based on
standard kNN techniques for can be obtained: ∆x
kNN1 =
[
δx
1 , δx
2 , · · · , δx
q
]T . (10) (10) 1) Benchmark data sets To be more
specific, for each instance , let
be the set of indices for the nearest neighbors of iden-
tified in the training set
, we can define an indicating
vector
which is
defined as follows: zi = [xi; exi] , Pj = [pj1, pj2, · · · , pjKj] ∈Rd×Kj
ξj = [ξj1, ξj2, · · · , ξjm]T
∈Rm×1
yij = cj
ai
ea
ji = 1
yij ̸= cj
a
ai ̸= a
γ
P = [P1,
P2, · · · , P q]
∆x
SVM
x
where
is
the
weight
matrix
to
be
determined,
and
is the slack variable
vector. Suppose
, then
if
(i.e.,
) and 0 otherwise. Furthermore,
is a trade-off
parameter. Let
, the maximum
margin-augmented feature vector
for each instance
can be defined as follows: ∆x
SVM = P Tx. (12) (12) D
Then, the MDC training set
can be transformed in-
to: vx
ja(r) = 1yirj=cj
a
(1 ≤r ≤k, ir ∈Nk(x)). to: 1 ≤a ≤Kj, 1 ≤j ≤q yir = [yir1, · · · , yirq]T
xir
x
vx
ja
δx
j = [δx
j1, δx
j2, · · · , δx
jKj]T
Here,
. corres-
ponds to the class vector of the neighboring MDC
example
for
. Based on
, the following discrete
version of statistics
can be
defined w.r.t. the j-th class space: eD = {(zi, yi) | 1 ≤i ≤m} zi = [xi; exi]
exi =
[
∆xi
kNN1; ∆xi
kNN2; ∆xi
SVM
]
xi
where
, and
denotes the augmented features of
. Here, we reiterate
that we make use of standard kNN, weighted kNN, and
maximum margin techniques to generate the augmented
feature only for the purpose of simplicity. The
experiments in this paper mainly aims at validating the
effectiveness of the selective feature augmentation
strategy. In the future, it is interesting to further
investigate synergizing multiple kinds of augmented
features which are generated by making use of more
advanced techniques such as deep learning[21]. zi = [xi; exi]
exi =
[
∆xi
kNN1; ∆xi
kNN2; ∆xi
SVM
]
xi
where
, and
denotes the augmented features of
. Here, we reiterate
that we make use of standard kNN, weighted kNN, and
maximum margin techniques to generate the augmented
feature only for the purpose of simplicity. The
experiments in this paper mainly aims at validating the
effectiveness of the selective feature augmentation
strategy. 1) Among all the 30 cases (10 data sets×3 evaluation 4.2 Experimental results β = [1, 1/
√
2, · · · , 1/
√
k]T
ζx
j = [ζx
j1, ζx
j2, · · · , ζx
jKj]T
Moreover, let
, a bias vector
is defined as follows: β = [1, 1/
√
2, · · · , 1/
√
k]T
ζx
j = [ζx
j1, ζx
j2, · · · , ζx
jKj]T
Moreover, let
, a bias vector
is defined as follows: ζx
j = [ζx
j1, ζx
j2, · · · , ζx
jKj]T is defined as follows: Table 2 reports the detailed experimental results with
the performance rank shown in the parentheses. Moreover, the Wilcoxon signed-ranks test[32] (at 0.05 sig-
nificance level) serves as the statistical tool to show
whether SFAM achieves better performance against the
compared approaches over the whole benchmark data
sets. The corresponding test results are summarized in
Table 3. ζx
ja = ⟨β, vx
ja⟩−min(vx
ja)
max(vx
ja) −min(vx
ja)(ζmax −ζmin) + ζmin. ζmax
ζmin
max(vx
ja) = ∑δx
ja
r=1 β(r)
min(vx
ja) = ∑k
r=r0 β(r)
r0 = k −δx
ja + 1
β(r)
β
∆x
kNN2
x
Here,
and
are two hyper-parameters and set as
0.5 and 0 respectively, and
,
where
and
denotes the r-th element of the weight vector
. Then,
the second kNN-augmented feature vector
based
on weighted kNN techniques for can be obtained: According to the reported experimental results, we
can make the following observations: 1) Among all the 30 cases (10 data sets×3 evaluation 1) Among all the 30 cases (10 data sets×3 evaluation B. B. Jia and M. L. Zhang / Multi-dimensional Classification via Selective Feature Augmentation 45 metrics), SFAM ranks first in 24 cases, second in 1 case,
third in 1 case, fourth in 1 case, fifth in 3 cases, and nev-
er ranks last. 2) BR solves the MDC problem by dealing with each
dimension independently, where potential class dependen-
cies are fully ignored. Although SFAM also induces classi-
fication models for each dimension independently, the
class dependencies can be considered by the augmented
features[20]. It is shown that SFAM achieves superior per-
formance against BR in terms of each metric, which re-
veals that considering class dependencies is important for
learning MDC models. 3) Both ECC and gMML explicitly consider the class
dependencies, where a chaining order over class spaces or
Table 2 Experimental results (mean±std. deviation) of each MDC approach. class dependencies can be considered by the augmented
features[20]. It is shown that SFAM achieves superior per-
formance against BR in terms of each metric, which re-
veals that considering class dependencies is important for
learning MDC models. 3) Both ECC and gMML explicitly consider the class
dependencies, where a chaining order over class spaces or 2) BR solves the MDC problem by dealing with each
dimension independently, where potential class dependen-
cies are fully ignored. Although SFAM also induces classi-
fication models for each dimension independently, the 4.2 Experimental results In addition, the performance rank on
each data set is also shown in the parentheses. Data set
Hamming score
SFAM
BR
CP
ECC
ESC
gMML
SEEM
Flare1
0.925±0.035(2)
0.925±0.034(2)
0.923±0.033(7)
0.926±0.034(1)
0.925±0.035(2)
0.925±0.034(2)
0.925±0.032(2)
Enb
0.865±0.036(1)
0.774±0.023(3)
0.764±0.031(5)
0.773±0.034(4)
0.754±0.029(6)
0.742±0.027(7)
0.777±0.031(2)
WQplants
0.666±0.016(1)
0.658±0.014(3)
0.649±0.016(7)
0.654±0.016(5)
0.653±0.016(6)
0.655±0.015(4)
0.661±0.023(2)
WQanimals
0.641±0.012(1)
0.631±0.013(3)
0.628±0.013(7)
0.629±0.013(6)
0.631±0.014(3)
0.630±0.015(5)
0.635±0.015(2)
WQ
0.653±0.012(1)
0.644±0.011(3)
0.625±0.011(7)
0.642±0.012(5)
0.642±0.014(5)
0.643±0.013(4)
0.646±0.014(2)
BeLaE
0.441±0.013(1)
0.427±0.017(2)
0.383±0.023(7)
0.424±0.021(3)
0.420±0.022(4)
0.417±0.020(5)
0.416±0.020(6)
Voice
0.947±0.009(1)
0.900±0.012(3)
0.898±0.011(4)
0.896±0.012(6)
0.897±0.011(5)
0.842±0.009(7)
0.910±0.011(2)
TIC2000
0.946±0.003(1)
0.915±0.006(3)
0.905±0.006(6)
0.915±0.006(3)
0.915±0.006(3)
0.895±0.007(7)
0.917±0.005(2)
Adult
0.724±0.005(1)
0.721±0.004(2)
0.709±0.004(6)
0.720±0.003(4)
0.710±0.005(5)
0.705±0.004(7)
0.721±0.004(2)
Default
0.671±0.003(3)
0.669±0.003(5)
0.669±0.004(5)
0.670±0.003(4)
0.672±0.004(1)
0.666±0.004(7)
0.672±0.003(1)
Data set
Exact match
SFAM
BR
CP
ECC
ESC
gMML
SEEM
Flare1
0.824±0.073(1)
0.821±0.075(4)
0.817±0.068(7)
0.824±0.073(1)
0.824±0.073(1)
0.821±0.075(4)
0.818±0.075(6)
Enb
0.729±0.071(1)
0.548±0.045(3)
0.529±0.063(5)
0.546±0.069(4)
0.508±0.057(6)
0.483±0.053(7)
0.554±0.063(2)
WQplants
0.100±0.037(1)
0.092±0.033(5)
0.093±0.031(3)
0.092±0.034(5)
0.093±0.036(3)
0.092±0.035(5)
0.096±0.034(2)
WQanimals
0.058±0.013(5)
0.058±0.017(5)
0.065±0.018(1)
0.059±0.017(4)
0.064±0.019(2)
0.062±0.023(3)
0.049±0.022(7)
WQ
0.009±0.006(1)
0.005±0.008(4)
0.000±0.000(7)
0.005±0.008(4)
0.005±0.008(4)
0.006±0.008(3)
0.009±0.006(1)
BeLaE
0.028±0.011(1)
0.021±0.008(7)
0.026±0.014(3)
0.023±0.010(5)
0.027±0.009(2)
0.022±0.009(6)
0.026±0.011(3)
Voice
0.897±0.018(1)
0.809±0.023(3)
0.807±0.021(4)
0.802±0.022(6)
0.803±0.019(5)
0.699±0.017(7)
0.831±0.020(2)
TIC2000
0.846±0.009(1)
0.762±0.017(3)
0.739±0.017(6)
0.762±0.017(3)
0.762±0.016(3)
0.706±0.018(7)
0.770±0.015(2)
Adult
0.284±0.010(5)
0.275±0.008(6)
0.317±0.010(1)
0.287±0.007(4)
0.312±0.011(2)
0.230±0.009(7)
0.289±0.010(3)
Default
0.185±0.006(4)
0.181±0.007(6)
0.194±0.008(1)
0.185±0.006(4)
0.187±0.007(3)
0.177±0.007(7)
0.190±0.009(2)
Data set
Sub-exact match
SFAM
BR
CP
ECC
ESC
gMML
SEEM
Flare1
0.954±0.039(5)
0.957±0.039(2)
0.954±0.039(5)
0.957±0.039(2)
0.954±0.039(5)
0.957±0.039(2)
0.960±0.033(1)
Enb
1.000±0.000(1)
1.000±0.000(1)
1.000±0.000(1)
1.000±0.000(1)
1.000±0.000(1)
1.000±0.000(1)
1.000±0.000(1)
WQplants
0.292±0.035(1)
0.286±0.044(3)
0.285±0.052(5)
0.285±0.053(5)
0.282±0.049(7)
0.286±0.053(3)
0.287±0.042(2)
WQanimals
0.246±0.034(1)
0.229±0.030(5)
0.232±0.032(3)
0.226±0.026(7)
0.231±0.029(4)
0.227±0.033(6)
0.241±0.029(2)
WQ
0.061±0.022(1)
0.047±0.019(6)
0.034±0.017(7)
0.048±0.022(4)
0.048±0.019(4)
0.049±0.024(3)
0.050±0.025(2)
BeLaE
0.137±0.024(1)
0.134±0.025(2)
0.117±0.019(7)
0.130±0.025(3)
0.128±0.024(5)
0.130±0.020(3)
0.125±0.022(6)
Voice
0.998±0.003(1)
0.991±0.006(2)
0.989±0.006(5)
0.989±0.008(5)
0.991±0.007(2)
0.985±0.011(7)
0.990±0.006(4)
TIC2000
0.993±0.002(1)
0.983±0.003(4)
0.978±0.002(6)
0.984±0.003(2)
0.984±0.003(2)
0.978±0.003(6)
0.982±0.003(5)
Adult
0.690±0.007(1)
0.685±0.009(2)
0.637±0.007(7)
0.679±0.008(4)
0.644±0.007(6)
0.669±0.008(5)
0.680±0.006(3)
Default
0.604±0.007(1)
0.601±0.006(4)
0.594±0.008(6)
0.600±0.007(5)
0.604±0.008(1)
0.593±0.008(7)
0.604±0.007(1) able 2 Experimental results (mean±std. deviation) of each MDC approach. In addition, the performance rank on
each data set is also shown in the parentheses. 2) BR solves the MDC problem by dealing with each
dimension independently, where potential class dependen-
cies are fully ignored. 4.2 Experimental results Although SFAM also induces classi-
fication models for each dimension independently, the 3) Both ECC and gMML explicitly consider the class
dependencies, where a chaining order over class spaces or Machine Intelligence Research 19(1), February 2022 46 achieves comparable performance against it in terms of
all metrics, as shown in Table 4, among the 19 cases
where the performance of SFAM is different from DeV2,
there are a total of 14 cases where the performance of
SFAM is better than DeV2. These results clearly valid-
ate that SFAM can identify the pertinent and useful fea-
tures from the three kinds of augmented features. Be-
sides, it is shown that both DeV1 and DeV2 achieve sim-
ilar results compared to SFAM. A possible reason is that
the two kinds of kNN-augmented features contain more
useful discriminative information than the maximum
margin-augmented features. Nonetheless, SFAM is able to
utilize all the available information in hand to achieve
better generalization performance. Table 3 Wilcoxon signed-ranks test for SFAM against each
compared approach where the p-values at 0.05 significance level
are also shown in the brackets. SFAM against
Evaluation metric
HS
EM
SEM
BR
win[3.91e-03]
win[3.91e-03]
win[7.81e-03]
CP
win[1.95e-03]
tie[2.75e-01]
win[7.81e-03]
ECC
win[3.91e-03]
win[3.91e-02]
win[7.81e-03]
ESC
win[7.81e-03]
tie[2.62e-01]
win[1.56e-02]
gMML
win[3.91e-03]
win[9.77e-03]
win[7.81e-03]
SEEM
win[9.77e-03]
tie[7.42e-02]
win[3.91e-02] a Mahalanobis metric is employed to accomplish this
task. It is shown that SFAM also achieves superior per-
formance against ECC and gMML in terms of each met-
ric, which validates the superiority of SFAM′s selective
feature augmentation strategy. ℓ1
eΘ = [eθ11, · · · , eθ1K1, · · · , eθq1, · · · , eθqKq]T
ℓ1
Furthermore, Fig. 3 shows the weight matrix (abso-
lute value) of the learned model w.r.t. the
regulariza-
tion. Specifically, following the notations in Section 3,
Fig. 3 shows the absolute value of the weight matrix
for the data set
Voice, TIC2000, Adult, and Default. For each figure,
each row corresponds to the binary classification model of
one class label. The first third of the columns correspond
to the discrete version of kNN-augmented features in
(10), the middle third of the columns to the continuous
version of kNN-augmented features in (11), and the last
third of the columns to the maximum margin-augmented
features in (12). It is shown that, for each third of all
columns, the diagonal element usually takes the largest
value in its corresponding row. 4.2 Experimental results Note that each element in
all the three kinds of augmented features (i.e., each
column in Fig. 3) corresponds to one class label, and each
binary classification model (i.e., each row in Fig. 3) also
corresponds to one class label. In other words, the largest
value corresponds to the augmented feature w.r.t. its own
class label. It is also shown that each binary classifica-
tion model is only related to a part of the augmented fea-
tures, where the model weights w.r.t. the two kinds of
kNN-augmented features are usually larger than the mod-
el weights w.r.t. the maximum margin-augmented fea-
tures. This observation further supports the afore-men-
tioned conjecture that the two kinds of kNN-augmented
features contain more useful discriminative information
than the maximum margin-augmented features. Besides,
we can also observe that the model weights w.r.t. the
regularization for some binary classification models are al-
most all zero, which means that not all binary classifica-
tion models rely on augmented features. 4) CP solves the MDC problem by dealing with all di-
mensions jointly via powerset transformation, which can
be viewed as optimizing exact match. ESC and SEEM
can be regarded as two improved versions of CP, where
class spaces are grouped into super-classes according to
conditional dependencies, or each pair of class spaces are
considered in the first level learning. It is shown that
SFAM still achieves comparable performance against CP,
ESC, and SEEM in terms of exact match and superior
performance against CP, ESC, and SEEM in terms of
Hamming score and sub-exact match. 1) Effectiveness of algorithmic design In this paper, SFAM generates three kinds of augmen-
ted features according to (10)−(12), respectively. To fur-
ther investigate the effectiveness of SFAM′s algorithmic
design, we also compare SFAM with its three degener-
ated versions which generate either kind of augmented
features. The one with the discrete version of kNN-aug-
mented features in (10) is denoted as DeV1, which is also
known as the KRAMd approach[20], the one with the con-
tinuous version of kNN-augmented features in (11) is de-
noted as DeV2, which is also known as the KRAMc ap-
proach[20], and another one with maximum margin-aug-
mented features in (12) is denoted as DeV3. It is worth
noting that the baseline BR actually serves as another de-
generated version without any kind of augmented fea-
tures, whose experimental results have been reported and
analyzed in Section 4.2. 2) Parameter sensitivity analysis λ
λ
λ
The regularized classification model (1) has one trade-
off parameter, . In this section, we investigate how the
performance of SFAM changes with different values of . Fig. 4 illustrates SFAM′s performance fluctuation when
ranges in {0.01, 0.1, 1, 10, 100} over data sets Flare1,
WQplants, WQanimals, and BeLaE. It is shown that the
performance of SFAM degenerates with either a small or λ
λ
λ
The regularized classification model (1) has one trade-
off parameter, . In this section, we investigate how the
performance of SFAM changes with different values of . Fig. 4 illustrates SFAM′s performance fluctuation when
ranges in {0.01, 0.1, 1, 10, 100} over data sets Flare1,
WQplants, WQanimals, and BeLaE. It is shown that the
performance of SFAM degenerates with either a small or Detailed experimental results are shown in Table 4. Table 5 summarizes the test results of the Wilcoxon
signed-ranks test (at 0.05 significance level). It is shown
that SFAM achieves superior performance against DeV1
in terms of Hamming score and exact match, and DeV3
in terms of all metrics. For DeV2, although SFAM λ
λ = 1
λ
large value of generally, and
is usually a better
choice. Therefore, we set to 1 in all the previous com-
parative studies. 5 Conclusions
Feature augmentation has been shown as an effective
strategy for solving the MDC problem. Existing works
only focus on generating better augmented features, while
it might be beneficial to exploit multiple kinds of aug-
mented features generated using different techniques. This paper makes a first attempt to synergize the dis-
criminative information residing in multiple kinds of aug-
Table 4 Experimental results (mean±std. deviation) of SFAM and its three degenerated versions. In addition, the performance rank on
each data set is also shown in the parentheses. Data set
Hamming score
SFAM
DeV1
DeV2
DeV3
Flare1
0.925±0.035(1)
0.924±0.036(4)
0.925±0.035(1)
0.925±0.035(1)
Enb
0.865±0.036(1)
0.852±0.036(2)
0.848±0.043(3)
0.807±0.029(4)
WQpla. 0.666±0.016(1)
0.664±0.016(2)
0.664±0.016(2)
0.659±0.015(4)
WQani. 0.641±0.012(1)
0.641±0.015(1)
0.641±0.015(1)
0.632±0.013(4)
WQ
0.653±0.012(1)
0.650±0.011(3)
0.652±0.010(2)
0.645±0.012(4)
BeLaE
0.441±0.013(1)
0.436±0.008(2)
0.434±0.011(4)
0.435±0.019(3)
Voice
0.947±0.009(1)
0.945±0.010(3)
0.946±0.010(2)
0.913±0.010(4)
TIC2000
0.946±0.003(2)
0.946±0.003(2)
0.947±0.003(1)
0.914±0.006(4)
Adult
0.724±0.005(1)
0.724±0.004(1)
0.724±0.005(1)
0.721±0.004(4)
Default
0.671±0.003(1)
0.671±0.003(1)
0.671±0.003(1)
0.669±0.003(4)
Data set
Exact match
SFAM
DeV1
DeV2
DeV3
Flare1
0.824±0.073(1)
0.821±0.079(4)
0.824±0.073(1)
0.824±0.073(1)
Enb
0.729±0.071(1)
0.705±0.072(2)
0.696±0.086(3)
0.613±0.058(4)
WQpla. 0.100±0.037(1)
0.097±0.038(2)
0.097±0.037(2)
0.095±0.034(4)
WQani. 0.058±0.013(3)
0.058±0.011(3)
0.061±0.016(1)
0.059±0.018(2)
WQ
0.009±0.006(1)
0.008±0.005(2)
0.008±0.005(2)
0.005±0.008(4)
BeLaE
0.028±0.011(1)
0.025±0.007(2)
0.024±0.007(3)
0.024±0.008(3)
Voice
0.897±0.018(1)
0.893±0.020(3)
0.894±0.021(2)
0.834±0.018(4)
TIC2000
0.846±0.009(3)
0.847±0.008(2)
0.849±0.008(1)
0.760±0.018(4)
Adult
0.284±0.010(1)
0.284±0.009(1)
0.284±0.010(1)
0.274±0.008(4)
Default
0.185±0.006(2)
0.185±0.006(2)
0.186±0.006(1)
0.181±0.006(4)
Data set
Sub-exact match
SFAM
DeV1
DeV2
DeV3
Flare1
0.954±0.039(1)
0.954±0.039(1)
0.954±0.039(1)
0.954±0.039(1)
Enb
1.000±0.000(1)
1.000±0.000(1)
1.000±0.000(1)
1.000±0.000(1)
WQpla. 0.292±0.035(3)
0.294±0.037(1)
0.294±0.036(1)
0.289±0.042(4)
WQani. 0.246±0.034(1)
0.244±0.031(2)
0.243±0.035(3)
0.231±0.032(4)
WQ
0.061±0.022(1)
0.059±0.019(2)
0.058±0.020(3)
0.048±0.020(4)
BeLaE
0.137±0.024(1)
0.133±0.021(4)
0.134±0.022(2)
0.134±0.023(2)
Voice
0.998±0.003(1)
0.998±0.003(1)
0.998±0.003(1)
0.993±0.006(4)
TIC2000
0.993±0.002(1)
0.992±0.002(3)
0.993±0.002(1)
0.983±0.003(4)
Adult
0.690±0.007(1)
0.690±0.007(1)
0.689±0.007(3)
0.685±0.008(4)
Default
0.604±0.007(1)
0.604±0.007(1)
0.604±0.006(1)
0.601±0.007(4)
B. B. Jia and M. L. Zhang / Multi-dimensional Classification via Selective Feature Augmentation
47 B. B. Jia and M. L. Zhang / Multi-dimensional Classification via Selective Feature Augmentation 47 λ
λ = 1
λ
large value of generally, and
is usually a better
choice. Therefore, we set to 1 in all the previous com-
parative studies. strategy for solving the MDC problem. Existing works
only focus on generating better augmented features, while
it might be beneficial to exploit multiple kinds of aug-
mented features generated using different techniques. This paper makes a first attempt to synergize the dis-
criminative information residing in multiple kinds of aug- 5 Conclusions 3 The weight matrix (absolute value) of the learned model w.r.t. the
regularization Data set: Adult
10
20
30
40
(c) Adult
50
60
2
4
6
8
10
12
14
16
18
20
0.6
0.5
0.4
0.3
0.2
0.1
0 5
10
15
20
25
30
35
40
45
Data set: Default
(d) Default
2
4
6
8
10
12
0.30
0.25
0.20
0.15
0.10
0.05
0 ℓ1
Fig. 3 The weight matrix (absolute value) of the learned model w.r.t. the
regularization catenation operation. Besides, this paper only generates
three simple kinds of augmented features to validate the
proposed selective feature augmentation strategy, it is
also worth investigating generating more kinds of aug-
mented features. mented features. Accordingly, a novel strategy named se-
lective feature augmentation is proposed, which assumes
that only part of the augmented features is pertinent and
useful for each dimension′s model induction. Comparat-
ive studies clearly validate the effectiveness of the pro-
posed strategy. 5 Conclusions Feature augmentation has been shown as an effective Machine Intelligence Research 19(1), February 2022 48 Table 5 Wilcoxon signed-ranks test for SFAM against its three variants where the p-values at 0.05 significance level are also shown in
the brackets. Table 5 Wilcoxon signed-ranks test for SFAM against its three variants where the p-values at 0.05 significance level are also shown in
the brackets. SFAM against
Evaluation metric
HS
EM
SEM
DeV1
win[3.13e-02]
win[4.69e-02]
tie[3.13e-01]
DeV2
tie [1.25e-01]
tie [3.52e-01]
tie [1.88e-01]
DeV3
win[3.91e-03]
win[7.81e-03]
win[7.81e-03]
0.5
1.0
1.5
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Fig. Acknowledgements Current feature augmentation works simply concaten-
ate the original and augmented features, though the pro-
posed SFAM has treated them differently via different
regularization terms. In fact, the original and augmented
features (even different kinds of augmented features) can
be regarded as features from different views[33]. In the fu-
ture, other ensemble strategies borrowing from multi-view
learning can also be used instead of merely using the con- This work was supported by National Science Founda-
tion of China (No. 62176055) and China University S&T
Innovation Plan Guided by the Ministry of Education. The authors wish to thank the associate editor and an-
onymous reviewers for their insightful comments and sug-
gestions. The authors thank the Big Data Center of B. B. Jia and M. L. Zhang / Multi-dimensional Classification via Selective Feature Augmentation 49 0.925
0.920
0.915
0.910
0.84
0.82
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0.950
0.945
0.940
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(a) Flare1
(b) WQplants
Data set: WQplants
Data set: Flare1
EM
HS
SEM
EM
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SEM
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(c) WQanimals
(d) BeLaE
Data set: WQanimals
Data set: BeLaE
EM
HS
SEM
EM
HS
SEM
Fig. 4 Performance SFAM changes as λ ranges in {0.01, 0.1, 1, 10, 100}
. Jia and M. L. Zhang / Multi-dimensional Classification via Selective Feature Augmentation
4 0.925
0.920
0.915
0.910
0.84
0.82
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0.01
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Data set: Flare1
EM
HS 0.67
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0.090
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Data set: WQplants
EM
HS Data set: Flare1 Data set: WQplants 0.30
0.29
0.28
0.01
0.1
1
10
100
The trade off parameter λ
SEM (a) Flare1 (b) WQplants Data set: BeLaE EM EM Southeast University for providing the facility support on
the numerical calculations in this paper. material. If material is not included in the article's Creat-
ive Commons licence and your intended use is not per-
mitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the
copyright holder. Open Access This article is licensed under a Creative Commons At-
tribution 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. To view a copy of this licence, visit http://creative-
commons.org/licenses/by/4.0/ D. Xu, Y. X. Shi, I. W. Tsang, Y. S. Ong, C. Gong, X. B.
Shen. Survey on multi-output learning. IEEE Transac-
tions on Neural Networks and Learning Systems, vol. 31,
no. 7, pp. 2409–2429, 2020. DOI: 10.1109/TNNLS.2019.294
5133.
[1] References D. Xu, Y. X. Shi, I. W. Tsang, Y. S. Ong, C. Gong, X. B. Shen. Survey on multi-output learning. IEEE Transac-
tions on Neural Networks and Learning Systems, vol. 31,
no. 7, pp. 2409–2429, 2020. DOI: 10.1109/TNNLS.2019.294
5133. [1] The images or other third party material in this art-
icle are included in the article's Creative Commons li-
cence, unless indicated otherwise in a credit line to the 50 Machine Intelligence Research 19(1), February 2022 J. Read, C. Bielza, P. Larrañaga. Multi-dimensional classi-
fication with super-classes. IEEE Transactions on Know-
ledge and Data Engineering, vol. 26, no. 7, pp. 1720–1733,
2014. DOI: 10.1109/TKDE.2013.167. [2] via stacked dependency exploitation. Science China In-
formation Sciences, vol. 63, no. 12, Article number 222102,
2020. DOI: 10.1007/s11432-019-2905-3. [14] B. B. Jia, M. L. Zhang. MD-KNN: An instance-based ap-
proach for multi-dimensional classification. In Proceed-
ings of the 25th International Conference on Pattern Re-
cognition, IEEE, Milan, Italy, pp. 126−133, 2021. DOI:
10.1109/ICPR48806.2021.9412974. [14] B. B. Jia, M. L. Zhang. Maximum margin multi-dimen-
sional classification. IEEE Transactions on Neural Net-
works and Learning Systems, published online. DOI:
10.1109/TNNLS.2021.3084373. [3] J. D. Rodríguez, A. Pérez, D. Arteta, D. Tejedor, J. A. Lozano. Using multidimensional Bayesian network classifi-
ers to assist the treatment of multiple sclerosis. IEEE
Transactions on Systems, Man, and Cybernetics − Part C,
vol. 42, no. 6, pp. 1705–1715, 2012. DOI: 10.1109/TSMCC. 2012.2217326. [4] J. H. Zaragoza, L. E. Sucar, E. F. Morales, C. Bielza, P. Larranaga. Bayesian chain classifiers for multidimensional
classification. In Proceedings of the 22nd International
Joint Conference on Artificial Intelligence, Barcelona,
Spain, pp. 2192−2197, 2011. DOI: 10.5591/978-1-57735-
516-8/IJCAI11-365. [15] J. Read, L. Martino, D. Luengo. Efficient Monte Carlo
methods for multi-dimensional learning with classifier
chains. Pattern Recognition, vol. 47, no. 3, pp. 1535–1546,
2014. DOI: 10.1016/j.patcog.2013.10.006. [16] H. Borchani, C. Bielza, C. Toro, P. Larrañaga. Predicting
human immunodeficiency virus inhibitors using multi-di-
mensional Bayesian network classifiers. Artificial Intelli-
gence in Medicine, vol. 57, no. 3, pp. 219–229, 2013. DOI:
10.1016/j.artmed.2012.12.005. [5] C. Bielza, G. Li, P. Larrañaga. Multi-dimensional classific-
ation with Bayesian networks. International Journal of
Approximate Reasoning, vol. 52, no. 6, pp. 705–727, 2011. DOI: 10.1016/j.ijar.2011.01.007. [17] H. Shatkay, F. X. Pan, A. Rzhetsky, W. J. Wilbur. Multi-
dimensional classification of biomedical text: Toward
automated, practical provision of high-utility text to di-
verse users. Bioinformatics, vol. 24, no. 18, pp. 2086–2093,
2008. DOI: 10.1093/bioinformatics/btn381. [6] J. H. References Bolt, L. C. Van Der Gaag. Balanced sensitivity func-
tions for tuning multi-dimensional Bayesian network clas-
sifiers. International Journal of Approximate Reasoning,
vol. 80, pp. 361–376, 2017. DOI: 10.1016/j.ijar.2016.07.011. [18] F. Serafino, G. Pio, M. Ceci, D. Malerba. Hierarchical mul-
tidimensional classification of web documents with multi-
webclass. In Proceedings of the 18th International Confer-
ence on Discovery Science, Springer, Banff, Canada,
pp. 236–250, 2015. DOI: 10.1007/978-3-319-24282-8_20. [7] M. Benjumeda, C. Bielza, P. Larrañaga. Tractability of
most probable explanations in multidimensional Bayesian
network classifiers. International Journal of Approximate
Reasoning, vol. 93, pp. 74–87, 2018. DOI: 10.1016/j.ijar. 2017.10.024. [19] Z. Lian, Y. Li, J. H. Tao, J. Huang, M. Y. Niu. Expression
analysis based on face regions in real-world conditions. In-
ternational Journal of Automation and Computing,
vol. 17, no. 1, pp. 96–107, 2020. DOI: 10.1007/s11633-019-
1176-9. [8] B. B. Jia, M. L. Zhang. Multi-dimensional classification
via kNN feature augmentation. Pattern Recognition,
vol. 106, Article number 107423, 2020. DOI: 10.1016/j.pat-
cog.2020.107423. [20] Z. W. He, L. Zhang, F. Y. Liu. DiscoStyle: Multi-level lo-
gistic ranking for personalized image style preference infer-
ence. International Journal of Automation and Comput-
ing, vol. 17, no. 5, pp. 637–651, 2020. DOI: 10.1007/s11633-
020-1244-1. [9] H. B. Wang, C. Chen, W. W. Liu, K. Chen, T. L. Hu, G. Chen. Incorporating label embedding and feature aug-
mentation for multi-dimensional classification. In Proceed-
ings of the 34th AAAI Conference on Artificial Intelli-
gence, AAAI, New York, USA, pp. 6178−6185, 2020. DOI:
10.1609/AAAI.V34I04.6083. [21] Y. Zhang, X. Y. Shi, S. Y. Mi, X. Yang. Image captioning
with transformer and knowledge graph. Pattern Recogni-
tion Letters, vol. 143, pp. 43–49, 2021. DOI: 10.1016/j. patrec.2020.12.020. [10] M. L. Zhang, Z. H. Zhou. A review on multi-label learning
algorithms. IEEE Transactions on Knowledge and Data
Engineering, vol. 26, no. 8, pp. 1819–1837, 2014. DOI:
10.1109/TKDE.2013.39. [22] A. H. Al Muktadir, T. Miyazawa, P. Martinez-Julia, H. Harai, V. P. Kafle. Multi-target classification based auto-
matic virtual resource allocation scheme. IEICE Transac-
tions on Information and Systems, vol. E102-D, no. 5,
pp. 898–909, 2019. DOI: 10.1587/transinf.2018NTP0016. [11] E. Gibaja, S. Ventura. A tutorial on multilabel learning. ACM Computing Surveys, vol. 47, no. 3, Article number
52, 2015. DOI: 10.1145/2716262. [23] J. Arias, J. A. Gamez, T. D. Nielsen, J. M. Puerta. A scal-
able pairwise class interaction framework for multidimen-
sional classification. International Journal of Approxim-
ate Reasoning, vol. 68, pp. 194–210, 2016. References DOI: 10.1016/
j.ijar.2015.07.007. [12] M. L. Zhang, Y. K. Li, X. Y. Liu, X. Geng. Binary relev-
ance for multi-label learning: An overview. Frontiers of
Computer Science, vol. 12, no. 2, pp. 191–202, 2018. DOI:
10.1007/s11704-017-7031-7. [24] S. Gil-Begue, C. Bielza, P. Larrañaga. Multi-dimensional
Bayesian network classifiers: A survey. Artificial Intelli-
[25] B. B. Jia, M. L. Zhang. Multi-dimensional classification
[13] 51 gence Review, vol. 54, no. 1, pp. 519–559, 2021. DOI:
10.1007/s10462-020-09858-x. B. B. Jia and M. L. Zhang / Multi-dimensional Classification via Selective Feature B. B. Jia and M. L. Zhang / Multi-dimensional Classification via Selective Feature Augmentation Bin-Bin Jia received the B. Sc. degree in
electronic information science and techno-
logy from North China Electric Power Uni-
versity, China in 2010, and the M. Sc. de-
gree in information and communication
engineering
from
Beihang
University,
China in 2013. He joined College of Elec-
trical and Information Engineering, Lan-
zhou University of Technology, China in
2013. Currently, he is a Ph. D. degree candidate in School of
Computer Science and Engineering, Southeast University,
China. gence Review, vol. 54, no. 1, pp. 519–559, 2021. DOI:
10.1007/s10462-020-09858-x. J. Huang, G. R. Li, Q. M. Huang, X. D. Wu. Learning la-
bel-specific features and class-dependent labels for multi-
label classification. IEEE Transactions on Knowledge and
Data Engineering, vol. 28, no. 12, pp. 3309–3323, 2016. DOI: 10.1109/TKDE.2016.2608339. [26] H. H. Bauschke, J. Bolte, M. Teboulle. A descent lemma
beyond Lipschitz gradient continuity: First-order meth-
ods revisited and applications. Mathematics of Operations
Research, vol. 42, no. 2, pp. 330–348, 2017. DOI: 10.1287/
moor.2016.0817. [27] His research interests include machine learning and data min-
ing, especially in multi-dimensional classification. E-mail: jiabb@seu.edu.cn
ORCID iD: 0000-0003-3302-9398 A. Beck, M. Teboulle. A fast iterative shrinkage-threshold-
ing algorithm for linear inverse problems. SIAM Journal
on Imaging Sciences, vol. 2, no. 1, pp. 183–202, 2009. DOI:
10.1137/080716542. [28] Min-Ling Zhang received the B. Sc.,
M. Sc., and Ph. D. degrees in computer sci-
ence from Nanjing University, China in
2001, 2004 and 2007, respectively. Cur-
rently, he is a professor at School of Com-
puter Science and Engineering, Southeast
University, China. In recent years, Dr. Zhang has served as the General Co-Chairs
of ACML′18, Program Co-Chairs of PAK-
DD′19, CCF-ICAI′19, ACML′17, CCFAI′17, PRICAI′16, Senior
PC member or Area Chair of AAAI 2017−2020, IJCAI
2017−2022, KDD 2021, ICDM 2015−2021, etc. E-mail: zhangml@seu.edu.cn (Corresponding author)
ORCID iD: 0000-0003-1880-5918 References He is also on the
editorial board of IEEE Transactions on Pattern Analysis and
Machine Intelligence, ACM Transactions on Intelligent Systems
and Technology, Neural Networks, Science China Information
Sciences, Frontiers of Computer Science, etc. Dr. Zhang is the
Steering Committee Member of ACML and PAKDD, Vice Chair
of CAAI Machine Learning Society, standing committee mem-
ber of the CCF Artificial Intelligence & Pattern Recognition So-
ciety. He is a Distinguished Member of CCF, CAAI, and Senior
Member of ACM, IEEE. Min-Ling Zhang received the B. Sc.,
M. Sc., and Ph. D. degrees in computer sci-
ence from Nanjing University, China in
2001, 2004 and 2007, respectively. Cur-
rently, he is a professor at School of Com-
puter Science and Engineering, Southeast
University, China. In recent years, Dr. Zhang has served as the General Co-Chairs
of ACML′18, Program Co-Chairs of PAK-
DD′19, CCF-ICAI′19, ACML′17, CCFAI′17, PRICAI′16, Senior
PC member or Area Chair of AAAI 2017−2020, IJCAI
2017−2022, KDD 2021, ICDM 2015−2021, etc. He is also on the
editorial board of IEEE Transactions on Pattern Analysis and
Machine Intelligence, ACM Transactions on Intelligent Systems
and Technology, Neural Networks, Science China Information
Sciences, Frontiers of Computer Science, etc. Dr. Zhang is the
Steering Committee Member of ACML and PAKDD, Vice Chair
of CAAI Machine Learning Society, standing committee mem-
ber of the CCF Artificial Intelligence & Pattern Recognition So-
ciety. He is a Distinguished Member of CCF, CAAI, and Senior
Member of ACM, IEEE. Z. C. Ma, S. C. Chen. Multi-dimensional classification via
a metric approach. Neurocomputing, vol. 275, pp. 1121–
1131, 2018. DOI: 10.1016/j.neucom.2017.09.057. [29] R. E. Fan, K. W. Chang, C. J. Hsieh, X. R. Wang, C. J. Lin. LIBLINEAR: A library for large linear classification. Journal of Machine Learning Research, vol. 9, pp. 1871–
1874, 2008. [30] K. Crammer, Y. Singer. On the algorithmic implementa-
tion of multiclass kernel-based vector machines. Journal of
Machine Learning Research, vol. 2, pp. 265–292, 2001. [31] J. Demšar. Statistical comparisons of classifiers over mul-
tiple data sets. Journal of Machine Learning Research,
vol. 7, pp. 1–30, 2006. [32] J. Zhao, X. J. Xie, X. Xu, S. L. Sun. Multi-view learning
overview: Recent progress and new challenges. Informa-
tion Fusion, vol. 38, pp. 43–54, 2017. DOI: 10.1016/
j.inffus.2017.02.007. [33] His research interests include machine learning and data min-
ing. E-mail: zhangml@seu.edu.cn (Corresponding author)
ORCID iD: 0000-0003-1880-5918
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https://cardiothoracicsurgery.biomedcentral.com/track/pdf/10.1186/s13019-022-01799-6
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English
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Ringed fluorodeoxyglucose uptake predicted poor prognosis after resection of pulmonary pleomorphic carcinoma
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Journal of cardiothoracic surgery
| 2,022
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cc-by
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© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom-
mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Pulmonary pleomorphic carcinoma (PPC) is a relatively rare and poorly differentiated non-small cell
carcinoma. This study aimed to investigate the clinicopathological features including programmed cell death ligand 1
(PD-L1) expression status in patients with PPC who underwent curative resection. Methods: We retrospectively studied 29 consecutive patients who had undergone anatomical lung resections for
PPC. Perioperative and pathological variables, including radiological findings, were investigated to define prognostic
factors. Methods: We retrospectively studied 29 consecutive patients who had undergone anatomical lung resections for
PPC. Perioperative and pathological variables, including radiological findings, were investigated to define prognostic
factors. Results: Overall survival (OS) rates were 71.8% at 1 year and 60.0% at 5 years. Disease-free survival (DFS) rates were
54.8% at 1 year and 43.6% at 5 years. Univariate analysis revealed that ringed fluorodeoxyglucose (FDG) uptake on
positron emission tomography/computed tomography (PET/CT) (p = 0.003), a cavity in the tumor on CT (p = 0.004),
and tumor size (> 40 mm) (p = 0.014) were poor prognostic factors for OS. Regarding DFS, ringed FDG uptake
(p = 0.002), a cavity on CT (p < 0.001), tumor size (p = 0.007), and pleural invasion (p = 0.014) were poor prognostic fac-
tors. PD-L1 expression was not a prognostic factor. Conclusion: This study showed for the first time that ringed FDG uptake on PET/CT is a poor prognostic factor of
PPC. PD-L1 expression status was not related to the prognosis. istration The study was approved by the Kobe City Medical Center General Hospital’s ethics board (N
ust 20, 2020. : Lung cancer, Pleomorphic carcinoma, Prognostic factor, PET-CT, Programmed cell death ligand 1 Keywords: Lung cancer, Pleomorphic carcinoma, Prognostic factor, PET-CT, Programmed cell dea contains at least 10% spindle and/or giant cells or a car-
cinoma consisting only of spindle and giant cells [2]. Compared to other histological types of non-small-cell
lung cancers (NSCLCs), PPC is associated with a more
aggressive clinical course and worse prognosis despite
complete resection and adjuvant chemotherapy [3, 4]. Given the current situation, we need to seek novel treat-
ment approaches, such as the administration of immune
checkpoint inhibitors (ICIs). However, due to its rarity,
there is no consensus about the clinicopathological char-
acteristics of PCP and clinical implications of a novel Shishido et al. Journal of Cardiothoracic Surgery (2022) 17:47
https://doi.org/10.1186/s13019-022-01799-6 Ringed fluorodeoxyglucose uptake
predicted poor prognosis after resection
of pulmonary pleomorphic carcinoma Ringed fluorodeoxyglucose uptake
predicted poor prognosis after resection
of pulmonary pleomorphic carcinoma Background Pulmonary pleomorphic carcinoma (PPC) is a rare
epithelial malignant tumor that accounts for approxi-
mately 0.4% of all lung cancers [1]. The 2015 World
Health Organization (WHO) classification defines PPC
as a poorly differentiated non-small cell carcinoma that *Correspondence: akihiro@kuhp.kyoto-u.ac.jp
1 Department of General Thoracic Surgery, Kobe City Medical Center
General Hospital, 2‑2‑1, Minatojimaminamimachi, Chuo‑ku, Kobe‑city,
Hyogo 6500047, Japan
Full list of author information is available at the end of the article *Correspondence: akihiro@kuhp.kyoto-u.ac.jp
1 Department of General Thoracic Surgery, Kobe City Medical Center
General Hospital, 2‑2‑1, Minatojimaminamimachi, Chuo‑ku, Kobe‑city,
Hyogo 6500047, Japan
Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom-
mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 8 Shishido et al. Journal of Cardiothoracic Surgery (2022) 17:47 biomarker, programmed cell death ligand 1 (PD-L1)
expression. Medical Center General Hospital’s ethics board (No. 20112) on August 20, 2020. Written informed consent for
this study was obtained from patients who were alive at
data collection. Medical Center General Hospital’s ethics board (No. 20112) on August 20, 2020. Written informed consent for
this study was obtained from patients who were alive at
data collection. Medical Center General Hospital’s ethics board (No. 20112) on August 20, 2020. Written informed consent for
this study was obtained from patients who were alive at
data collection. In this study, we retrospectively investigated patients
with PPC who were surgically treated in our hospital and
tried to identify the prognostic factors and the clinico-
pathological features associated with the disease. Programmed cell death ligand 1 (PD-L1) expression
was assessed on 4-μm-thick sections of formalin-fixed
paraffin-embedded tissue blocks, using anti-PD-L1 anti-
body (clone E1L3N, 1:200, Cell Signaling Technology,
Danvers, MA). We evaluated the PD-L1 tumor propor-
tion score (TPS), and the PD-L1 status was considered
positive when TPS ≥ 5% [5]. Methods We retrospectively reviewed the data of 29 PPCs (1.5%)
from a total of 1903 primary lung cancer patients sur-
gically treated at Kobe City Medical Center General
Hospital between January 2006 and April 2020. Clini-
cal information, including age, sex, smoking habits, and
clinical stage at presentation, was obtained from the
medical records. All patients underwent chest computed
tomography (CT) before the surgery, and we evaluated
tumor size, lymph nodes, the presence of the cavity in the
tumor, and the location of the tumor. A peripheral tumor
was defined as being located in the outer third of the lung
field. Positron emission tomography/CT (PET/CT) and
brain magnetic resonance imaging (MRI) were also con-
ducted to evaluate lymph nodes and distant metastases. We also addressed the ringed fluorodeoxyglucose (FDG)
uptake on PET/CT, which is defined as donut-like FDG
accumulation exclusively at the rim of a tumor regard-
less of whether or not a cavity is formed in the tumor
(Fig. 1a). All pathological materials were reviewed by
expert pathologists at our hospital. The diagnosis of PCP
was obtained by light microscopy findings and immu-
nohistochemical examinations according to the 2015
WHO classification [2]. Pathological lymph node sta-
tus, lymphovascular invasion, and pleural invasion were
also examined. The study was approved by the Kobe City Overall survival (OS) and disease-free survival (DFS)
were assessed using the Kaplan–Meier method, and uni-
variate analyses were performed using the log-rank test. The correlations between PD-L1 expression and clinico-
pathological characteristics were evaluated using Fisher’s
exact test for categorical variables and Student’s t-test
for numerical variables. Data were analyzed statistically
using EZR (Saitama Medical Center, Jichi Medical Uni-
versity, Saitama, Japan), which is a graphical user inter-
face for R (The R Foundation for Statistical Computing,
Vienna, Austria) [6]. All P values were two-sided, and
differences were considered statistically significant at
P < 0.05. Results All the clinicopathological data of the 29 patients
included in this study are shown in Table 1. There
were 20 male (69.0%) and 9 female (31.0%) patients. The median age was 70 years (range: 44–88 years). Twenty-two patients (75.9%) had a smoking history,
and 3 (10.3%) were diagnosed with PPC before sur-
gery. Regarding the CT findings, the tumor was located Fig. 1 A 72-year-old male patient with pT3N0M0 pulmonary pleomorphic carcinoma. a Fused PET/CT image obtained before surgery shows
ringed FDG uptake in the tumor (b) Axial non-contrast CT image on the same day shows peripheral solid mass without cavity formation. c The
gross specimen shows a mainly solid tumor with a crescent-like cavity. d Microscopic examination of areas in the red squares shows necrosis on the
upper left side (arrow) and spindle cell on the lower right side (triangle) (Hematoxylin and eosin staining, × 100) Fig. 1 A 72-year-old male patient with pT3N0M0 pulmonary pleomorphic carcinoma. a Fused PET/CT image obtained before surgery shows
ringed FDG uptake in the tumor (b) Axial non-contrast CT image on the same day shows peripheral solid mass without cavity formation. c The
gross specimen shows a mainly solid tumor with a crescent-like cavity. d Microscopic examination of areas in the red squares shows necrosis on the
upper left side (arrow) and spindle cell on the lower right side (triangle) (Hematoxylin and eosin staining, × 100) Page 3 of 8 Shishido et al. Journal of Cardiothoracic Surgery (2022) 17:47 Table 1 Patients preoperative, pathological, and postoperative data
BI Brinkman index, CT computed tomography, FDG fluorodeoxyglucose, Pl plerual invasion, Ly lymphative invasion, V vascular invasion, TPS tumor proportion score, DFS disease-free survival, OS overall survival, m.o. Results months, m male, f female
Case
Age
Sex
B.I
Cavity on CT
Ringed FDG uptake
Tumor size
(mm)
p-TNM
p-Stage
Pl
Ly
V
TPS (%)
DFS (m.o.)
OS (m.o.)
Outcome
1
56
m
900
No
No
34
T2N0M0
I
0
0
0
30
91
91
Alive
2
70
f
1000
No
Yes
42
T2N0M0
II
0
0
0
0
54
54
Death
3
69
f
0
No
No
32
T2N1M0
II
3
0
1
0
52
52
Alive
4
62
m
1180
No
No
20
T3N0M0
II
3
0
0
70
104
104
Alive
5
67
m
1000
No
No
25
T3N0M0
II
0
0
0
90
48
48
Alive
6
54
m
700
No
Not available
38
T2N1M0
II
2
0
1
40
70
70
Alive
7
55
m
600
No
Yes
70
T3N0M0
II
1
0
1
30
48
48
Alive
8
70
m
225
No
No
42
T2N0M0
II
0
0
0
2–3
46
46
Alive
9
72
f
0
Yes
Yes
43
T2N0M0
II
1
0
1
0
50
50
Alive
10
71
m
0
No
No
22
T1N0M0
I
0
0
0
10
49
49
Alive
11
71
f
0
No
No
15
T1N0M0
I
0
0
0
0
76
76
Alive
12
69
f
0
No
No
26
T1N0M0
I
0
0
0
0
99
99
Alive
13
78
m
1600
No
No
25
T1N2M0
III
0
0
0
90
34
34
Alive
14
62
m
0
No
No
22
T2N2M0
III
1
0
1
0
35
94
Alive
15
70
f
1600
No
No
15
T1N0M0
I
0
0
1
80
77
77
Alive
16
80
m
800
Yes
Yes
92
T4N0M0
III
0
0
0
90
3
69
Death
17
65
m
940
No
No
60
T3N0M0
II
2
0
1
50
5
41
Alive
18
88
m
1000
Yes
Yes
45
T2N0M0
II
2
0
0
70
3
3
Death
19
75
m
1100
No
No
38
T3N1M0
III
3
0
1
70
3
3
Death
20
44
m
1000
Yes
Yes
90
T4N0M1
IV
1
1
1
90
1
6
Death
21
74
m
500
No
Yes
85
T4N0M0
III
1
0
0
2–3
1
3
Death
22
49
m
600
No
Yes
24
T1N0M0
I
0
1
1
0
6
7
Death
23
80
f
0
No
No
40
T2N0M0
I
1
0
0
5
12
38
Death
24
59
f
800
Yes
Not available
55
T3N0M0
II
3
0
0
80
0
3
Death
25
78
m
2000
Yes
Yes
35
T2N2M0
III
0
0
1
80
1
1
Death
26
72
m
1560
No
Yes
41
T3N0M0
II
3
0
1
90
3
11
Alive
27
73
m
1000
Yes
Yes
42
T2N0M0
II
2
0
1
60
4
4
Death
28
72
f
880
No
No
25
T3N1M0
III
0
1
1
100
6
6
Alive
29
76
m
2320
Yes
Yes
25
T2N0M0
I
2
1
1
10
3
5
Alive Table 1 Patients’ preoperative, pathological, and postoperative data
BI Brinkman index, CT computed tomography, FDG fluorodeoxyglucose, Pl plerual invasion, Ly lymphative invasion, V vascular invasion, TPS tumor proportion score, DFS disease-free survival, OS overall survival, m.o. Results months, m male, f female
Case
Age
Sex
B.I
Cavity on CT
Ringed FDG uptake
Tumor size
(mm)
p-TNM
p-Stage
Pl
Ly
V
TPS (%)
DFS (m.o.)
OS (m.o.)
Outcome
1
56
m
900
No
No
34
T2N0M0
I
0
0
0
30
91
91
Alive
2
70
f
1000
No
Yes
42
T2N0M0
II
0
0
0
0
54
54
Death
3
69
f
0
No
No
32
T2N1M0
II
3
0
1
0
52
52
Alive
4
62
m
1180
No
No
20
T3N0M0
II
3
0
0
70
104
104
Alive
5
67
m
1000
No
No
25
T3N0M0
II
0
0
0
90
48
48
Alive
6
54
m
700
No
Not available
38
T2N1M0
II
2
0
1
40
70
70
Alive
7
55
m
600
No
Yes
70
T3N0M0
II
1
0
1
30
48
48
Alive
8
70
m
225
No
No
42
T2N0M0
II
0
0
0
2–3
46
46
Alive
9
72
f
0
Yes
Yes
43
T2N0M0
II
1
0
1
0
50
50
Alive
10
71
m
0
No
No
22
T1N0M0
I
0
0
0
10
49
49
Alive
11
71
f
0
No
No
15
T1N0M0
I
0
0
0
0
76
76
Alive
12
69
f
0
No
No
26
T1N0M0
I
0
0
0
0
99
99
Alive
13
78
m
1600
No
No
25
T1N2M0
III
0
0
0
90
34
34
Alive
14
62
m
0
No
No
22
T2N2M0
III
1
0
1
0
35
94
Alive
15
70
f
1600
No
No
15
T1N0M0
I
0
0
1
80
77
77
Alive
16
80
m
800
Yes
Yes
92
T4N0M0
III
0
0
0
90
3
69
Death
17
65
m
940
No
No
60
T3N0M0
II
2
0
1
50
5
41
Alive
18
88
m
1000
Yes
Yes
45
T2N0M0
II
2
0
0
70
3
3
Death
19
75
m
1100
No
No
38
T3N1M0
III
3
0
1
70
3
3
Death
20
44
m
1000
Yes
Yes
90
T4N0M1
IV
1
1
1
90
1
6
Death
21
74
m
500
No
Yes
85
T4N0M0
III
1
0
0
2–3
1
3
Death
22
49
m
600
No
Yes
24
T1N0M0
I
0
1
1
0
6
7
Death
23
80
f
0
No
No
40
T2N0M0
I
1
0
0
5
12
38
Death
24
59
f
800
Yes
Not available
55
T3N0M0
II
3
0
0
80
0
3
Death
25
78
m
2000
Yes
Yes
35
T2N2M0
III
0
0
1
80
1
1
Death
26
72
m
1560
No
Yes
41
T3N0M0
II
3
0
1
90
3
11
Alive
27
73
m
1000
Yes
Yes
42
T2N0M0
II
2
0
1
60
4
4
Death
28
72
f
880
No
No
25
T3N1M0
III
0
1
1
100
6
6
Alive
29
76
m
2320
Yes
Yes
25
T2N0M0
I
2
1
1
10
3
5
Alive Shishido et al. Results Journal of Cardiothoracic Surgery (2022) 17:47 Page 4 of 8 Fig. 2 Kaplan–Meier curves of overall survival (a) and disease-free survival (b) for patients with surgically resected pleomorphic carcinoma, and
Kaplan–Meier curves of overall survival (c) and disease-free survival (d) according to the ringed FDG uptake on PET/CT Fig. 2 Kaplan–Meier curves of overall survival (a) and disease-free surviva Fig. 2 Kaplan–Meier curves of overall survival (a) and disease-free survival (b) for patients with surgically resected pleom
Kaplan–Meier curves of overall survival (c) and disease-free survival (d) according to the ringed FDG uptake on PET/CT edition of the Union for International Cancer Control
[UICC]) [7]. The median tumor size was 38 mm (range:
15–92 mm). Nodal status was classified as pN0 in 22
patients (75.9%), pN1 in 4 patients (13.8%), and pN2 in
3 patients (10.3%). Lymphatic invasion was observed in
4 patients (13.8%) and vascular invasion in 15 patients
(51.7%). Pleural invasion was observed in 16 patients
(55.1%). PD-L1 expression (TPS ≥ 5%) was observed in
20 patients (69.0%). Platinum-based adjuvant chemo-
therapy was administered to 8 patients (27.6%) who
were at pStages II and III. in the peripheral field of the lung in 19 cases (65.5%). The cavity in the tumor was found in 8 patients (27.6%). FDG uptake was observed in tumors of all 27 patients
who preoperatively underwent PET/CT, and among
them, ringed uptake was found in 12 patients (44.4%). In 5 cases, CT showed no cavity, but fused PET/CT
showed ringed FDG uptake, with a representative case
shown in Fig. 1. No apparent cavity was macroscopi-
cally noted, but necrosis in the central site and tumor
cells in the peripheral site were observed in corre-
spondence with the PET/CT image (Fig. 1). The surgi-
cal procedures included 25 lobectomies, 1 bilobectomy,
and 3 pneumonectomies. The pathological stage was
I in 8 patients, II in 13, III in 7, and IV in 1 who had
been surgically treated for a single brain metastasis (8th The mean follow-up time was 4.3 years (range: 0.1–
8.7 years). One patient died of acute exacerbation of
underlying interstitial pneumonia 18 days after surgery,
9 patients of PPC recurrence, and 1 of a second lung Shishido et al. Results Journal of Cardiothoracic Surgery (2022) 17:47 Page 5 of 8 Table 2 Univariate analysis of prognostic factors associated with
disease-free survival and overall survival
CT chest computed tomography, FDG fluorodeoxyglucose, PET/CT positron
emission tomography/CT, PD-L1 programmed cell death ligand 1
Characteristic, risk factor
Disease-free
survival
Overall survival
p value
p value
Age, > 70 y
0.118
0.128
Sex, male
0.193
0.742
Smoking, former or current
0.137
0.140
Cavity on CT
< 0.001
0.004
Ringed FDG uptake on PET/CT
0.002
0.003
Tumor size, > 40 mm
0.007
0.014
pN classification, N > 0
0.445
0.374
Lymphatic invasion
0.104
0.222
Vascular invasion
0.434
0.652
Pleural invasion
0.014
0.157
PD-L1 expression, TPS ≥ 5%
0.438
0.661 Table 2 Univariate analysis of prognostic factors associated with
disease-free survival and overall survival cases, such as T3 tumors extending beyond parietal
pleura, which were frequently observed in previous stud-
ies [4, 9, 11]. Tumor relapse tends to occur within one
year of surgery [3, 4, 11, 12]. Similarly, 11 of 13 relapses
were observed within 6 months of surgery, and most
cases were detected as distant metastases. This finding
might imply that identifying such an aggressive subgroup
in this entity would lead to clarify appropriate candi-
dates for neoadjuvant or adjuvant therapy in patients
with curative resection of PPC. The strategy on adjuvant
chemotherapy in the current study does not seem con-
sistent, and no patients received pre- or postoperative
immune checkpoint inhibitors (ICIs). These facts made it
difficult to discuss implications for neoadjuvant or adju-
vant therapy in PPC. Several factors have been reported to be determi-
nants of poor outcomes in patients with PPC, such as
lymph node metastasis, tumor size, and pleural invasion
[3, 4, 8–10, 13]. However, most studies were retrospec-
tive analyses with small sample sizes with the prognostic
factors remaining unclear. In particular, the association
between imaging findings and prognostic factors of PPC
has been reported in only a few articles to date. The CT
features of PPC are almost the same as those of NSCLCs,
but the possibility of PPC might be suggested when a sub-
pleural necrotic tumor is detected with peritumoral areas
of ground-glass opacification and regional invasion to
the chest wall or mediastinum [14]. A central low-atten-
uation area or cavity on CT was reported to have a poor
prognosis and represents tumor necrosis on histopatho-
logical specimens [15]. Results Pathological massive coagula-
tion necrosis indicates aggressive tumor growth, thereby
contributing to poor prognosis [8]. On the contrary, air
bronchogram on CT was suggested to be a favorable
prognostic factor, which might reflect intact intratu-
moral bronchi without tumor invasion [16]. In addition
to the cavity in the tumor, tumor size, and pleural inva-
sion, our study showed for the first time that ringed FDG
uptake on PET/CT was a significant prognostic factor of
PPC. In 5 cases, the tumor had no cavity macroscopically
or on CT, but revealed ringed FDG uptake on PET/CT
(Fig. 1a–c). The ringed uptake supposedly indicates cen-
tral necrosis, which is an aggressive feature that was later
confirmed histologically (Fig. 1d). PET/CT might poten-
tially detect central necrosis prior to cavity formation on
CT and has important clinical implications for the prog-
nosis of PPC. cancer. During the follow-up period, PPC recurrence
was observed in 13 patients (44.8%), mediastinal lymph
node metastasis in 1 patient and distant recurrence in
12, including malignant pleural effusion in 2 patients. The OS rates after pulmonary resection were 71.8% at
1 year and 60.0% at 5 years (Fig. 2a). The DFS rates after
pulmonary resection were 54.8% at 1 year and 43.6%
at 5 years (Fig. 2b), indicating that most relapses were
observed within one year of surgery. The results of the
univariate analysis for long-term prognosis are sum-
marized in Table 2. We excluded 1 patient who died
of acute exacerbation of interstitial pneumonia shortly
after surgery. Ringed FDG uptake (p = 0.003; Fig. 2c),
a cavity on CT (p = 0.004), and tumor size (> 40 mm)
(p = 0.014) were significant poor prognostic factors
for OS. Regarding DFS, ringed FDG uptake (p = 0.002;
Fig. 2d), a cavity on CT (p < 0.001), tumor size
(> 40 mm) (p = 0.007), and pleural invasion (p = 0.014)
were significant poor prognostic factors. The distribu-
tion of clinicopathological characteristics according
to PD-L1 expression is shown in Table 3, which shows
that PD-L1 expression was significantly correlated only
with smoking history (p = 0.016). cancer. During the follow-up period, PPC recurrence
was observed in 13 patients (44.8%), mediastinal lymph
node metastasis in 1 patient and distant recurrence in
12, including malignant pleural effusion in 2 patients. The OS rates after pulmonary resection were 71.8% at
1 year and 60.0% at 5 years (Fig. 2a). Results The DFS rates after
pulmonary resection were 54.8% at 1 year and 43.6%
at 5 years (Fig. 2b), indicating that most relapses were
observed within one year of surgery. The results of the
univariate analysis for long-term prognosis are sum-
marized in Table 2. We excluded 1 patient who died
of acute exacerbation of interstitial pneumonia shortly
after surgery. Ringed FDG uptake (p = 0.003; Fig. 2c),
a cavity on CT (p = 0.004), and tumor size (> 40 mm)
(p = 0.014) were significant poor prognostic factors
for OS. Regarding DFS, ringed FDG uptake (p = 0.002;
Fig. 2d), a cavity on CT (p < 0.001), tumor size
(> 40 mm) (p = 0.007), and pleural invasion (p = 0.014)
were significant poor prognostic factors. The distribu-
tion of clinicopathological characteristics according
to PD-L1 expression is shown in Table 3, which shows
that PD-L1 expression was significantly correlated only
with smoking history (p = 0.016). Discussion In this study, we retrospectively investigated the clinico-
pathological characteristics of patients with PPC who
were surgically treated in our hospital. PPC is generally
associated with a poor prognosis with a 5-year OS rate
of 20–48% after surgical resection [3, 4, 8–10]. In the
present study, we observed a favorable long-term sur-
vival rate of 60% compared with previous studies. This is
presumably attributed to the absence of locally advanced PD-L1 on antigen-presenting cells or tumor cells, when
interacting with PD-1 on T cells, negatively regulates T
cell activation or immune response against tumor cells
[17, 18]. Accordingly, PD-L1 expression in tumors is
thought to affect tumor behavior and prognosis. PD-L1
is expressed more frequently in PPCs than in NSCLCs. Shishido et al. Discussion Journal of Cardiothoracic Surgery (2022) 17:47 Page 6 of 8 I
i
t PD L1
d i
75%
f
PPC
ti
t
A
t
l
i
f
11 000 NSCLC
Table 3 Distribution of clinicopathological characteristics according to programmed cell death ligand 1 expression
PD-L1 programmed cell death ligand 1, CT chest computed tomography, FDG fluorodeoxyglucose, PET/CT positron emission tomography/CT
Characteristics
All patients (%)
PD-L1 ≥ 5% (%)
PD-L1 < 5% (%)
p value
Age (years)
Median [range]
70 [44–88]
72 [44–88]
70 [49–72]
0.561
Sex
Male
20 (69.0)
16 (80)
4 (44.4)
0.088
Female
9 (31.0)
4 (20)
5 (55.6)
Smoking status
Never
7 (24.1)
2 (10)
5 (55.6)
0.016
Former/current
22 (75.9)
18 (90)
4 (44.4)
Cavity on CT
Absent
21 (72.4)
13 (65)
8 (89)
0.371
Present
8 (27.6)
7 (35)
1 (11)
Ringed FDG uptake on PET/CT
Absent
15 (55.6)
10 (55.6)
5 (55.6)
1
Present
12 (44.4)
8 (44.4)
4 (44.4)
Tumor size (mm)
Median [range]
38 [15–92]
38 [15–92]
32 [15–85]
0.561
pT classification
T1
6 (20.7)
3 (15.8)
3 (30)
0.072
T2
12 (41.4)
6 (31.6)
6 (60)
T3
8 (27.6)
8 (42.1)
0 (0)
T4
3 (10.3)
2 (10.5)
1 (10)
pN classification
N0
22 (75.9)
15 (75)
7 (77.8)
1
N1
4 (13.8)
3 (15)
1 (11.1)
N2
3 (10.3)
2 (10)
1 (11.1)
Pathological stage
I
8 (27.6)
5 (25)
3 (33.3)
1
II
13 (44.8)
9 (45)
4 (44.4)
III
7 (24.1)
5 (25)
2 (22.2)
IV
1 (3.5)
1 (5)
0 (0)
Lymphatic invasion
Absent
25 (86.2)
17 (85)
8 (89.9)
1
Present
4 (13.8)
3 (15)
1 (11.1)
Vascular invasion
Absent
14 (49.3)
9 (45)
5 (55.6)
0.7
Present
15 (51.7)
11 (55)
4 (44.4)
Pleural invasion
Absent
13 (44.8)
8 (40)
5 (55.6)
0.688
Present
16 (55.2)
12 (60)
4 (44.4) In a previous report, PD-L1 was expressed in 75% of
PPC cases [5] but was expressed in approximately 20%
of NSCLC [19] or adenocarcinoma cases [20]. Simi-
larly, in the present study, 69% of PPC cases expressed
PD-L1. These studies, including the current one, used
clone E1L3N and 5% as the cut-off of positivity. Younger
patients [21] and parietal pleural invasion [12] were
reportedly associated with higher PD-L1 expression in PPC patients. Availability of data and materials y
The datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request. Acknowledgements 9. Chen F, Sonobe M, Sato T, et al. Clinicopathological characteristics of sur-
gically resected pulmonary pleomorphic carcinoma. Eur J Cardiothorac
Surg. 2012;41(5):1037–42. 9. Chen F, Sonobe M, Sato T, et al. Clinicopathological characteristics of sur-
gically resected pulmonary pleomorphic carcinoma. Eur J Cardiothorac
Surg. 2012;41(5):1037–42. We greatly appreciate the contributions to this study by Chisato Miyakoshi,
who provided valuable advice on statistical analysis. We would also like to
thank Yuri Shimada, Yusuke Shima, Atsushi Matsunashi, Megumu Osaki,
Ryosuke Hirabayashi, Daichi Fujimoto, Kazuma Nagata, Atsushi Nakagawa, and
Ryo Tachikawa, who provided medical treatment to the patients. 10. Ji C, Zhong C, Fang W, Zhao H. Surgical treatment for pulmonary pleo-
morphic carcinoma: a retrospective study of 60 patients. Thorac Cancer. 2014;5(3):250–4. 10. Ji C, Zhong C, Fang W, Zhao H. Surgical treatment for pulmonary pleo-
morphic carcinoma: a retrospective study of 60 patients. Thorac Cancer. 2014;5(3):250–4. 11. Okuda K, Oda R, Suzuki A, et al. Clinicopathological factors influenced the
prognosis of surgically resected pulmonary pleomorphic carcinoma. J
Thorac Dis. 2017;9(5):1295–302. References 1. Yendamuri S, Caty L, Pine M, et al. Outcomes of sarcomatoid carcinoma of
the lung: a surveillance, epidemiology, and end results database analysis. Surgery. 2012;152(3):397–402. Author details 1 Department of General Thoracic Surgery, Kobe City Medical Center General
Hospital, 2‑2‑1, Minatojimaminamimachi, Chuo‑ku, Kobe‑city, Hyogo 6500047,
Japan. 2 Department of Diagnostic Pathology, Kobe City Medical Center Gen-
eral Hospital, Kobe, Hyogo, Japan. 3 Department of Respiratory Medicine, Kobe
City Medical Center General Hospital, Kobe, Hyogo, Japan. The current study has several limitations. First, our
analysis was based on a very small number of patients
from one institution because of the rarity of PPCs; hence,
our findings are inconclusive. Second, the retrospective
nature of this study may lead to the potential risk of sev-
eral biases. Therefore, our results should be interpreted
with consideration of these limitations, and multicenter
prospective studies are needed to validate our findings. Received: 25 December 2021 Accepted: 16 March 2022 Received: 25 December 2021 Accepted: 16 March 2022 Consent for publication Written informed consent for this study was obtained from patients who were
alive at data collection. Abbreviations
PPC P l PPC: Pulmonary pleomorphic carcinoma; WHO: World Health Organization;
NSCLC: Non-small-cell lung cancer; ICI: Immune checkpoint inhibitor; PD-L1:
Programmed cell death ligand 1; CT: Computed tomography; PET: Positron
emission tomography; MRI: Magnetic resonance imaging; FDG: Fluorodeoxy-
glucose; PD-L1: Programmed cell death ligand 1; TPS: Tumor proportion score;
OS: Overall survival; DFS: Disease-free survival. 6. Kanda Y. Investigation of the freely available easy-to-use software ‘EZR’ for
medical statistics. Bone Marrow Transplant. 2013;48(3):452–8. 7. Brierley J, Gospodarowicz MK, Wittekind C. TNM classification of malig-
nant tumours. 8th ed. Chichester: Wiley; 2017. p. 106–12. 8. Mochizuki T, Ishii G, Nagai K, et al. Pleomorphic carcinoma of the
lung: clinicopathologic characteristics of 70 cases. Am J Surg Pathol. 2008;32(11):1727–35. 8. Mochizuki T, Ishii G, Nagai K, et al. Pleomorphic carcinoma of the
lung: clinicopathologic characteristics of 70 cases. Am J Surg Pathol. 2008;32(11):1727–35. Conclusions
h b
f 2. Travis WD, Brambilla E, Nicholson AG, et al. The 2015 world health
organization classification of lung tumors: impact of genetic, clinical
and radiologic advances since the 2004 classification. J Thorac Oncol. 2015;10(9):1243–60. To the best of our knowledge, the present study showed
for the first time that ringed FDG uptake on PET/CT
was a significant prognostic factor of PPC, in addition to
a cavity on CT and tumor size, which affected both DFS
and OS. Pleural invasion affected DFS but not OS. PD-L1
expression did not have any impact on DFS or OS. 3. Raveglia F, Mezzetti M, Panigalli T, et al. Personal experience in surgical
management of pulmonary pleomorphic carcinoma. Ann Thorac Surg. 2004;78(5):1742–7. 4. Yuki T, Sakuma T, Ohbayashi C, et al. Pleomorphic carcinoma of the lung:
a surgical outcome. J Thorac Cardiovasc Surg. 2007;134(2):399–404. 5. Yvorel V, Patoir A, Casteillo F, et al. PD-L1 expression in pleomorphic,
spindle cell and giant cell carcinoma of the lung is related to TTF-1,
p40 expression and might indicate a worse prognosis. PLoS ONE. 2017;12(7):e0180346. Ethics approval and consent to participate The study was approved by the Kobe City Medical Center General Hospital’s
ethics board (No. 20112) on August 20, 2020. Discussion A meta-analysis of over 11,000 NSCLC
patients revealed that higher expression of PD-L1 was
associated with the male sex, smoking history, tumor
size, and lymph node metastases but not age [22]. The
current study showed that PD-L1 expression was asso-
ciated with smoking history but not sex, age, or tumor
size. Therefore, meta-analyses or multicenter prospective
studies are necessary to define the correlation between Shishido et al. Journal of Cardiothoracic Surgery (2022) 17:47 Page 7 of 8 PD-L1 expression and clinicopathological features. The association between PD-L1 and prognosis remains
inconclusive, although several retrospective studies
have focused on PPC. This is partly due to varieties of
the clone of antibodies as well as cut-off values, both of
which probably have a substantial influence [21, 22]. In
addition, factors regarding whether or not surgical resec-
tion is indicated and whether or not ICI was available or
is indicated would affect the analyses. The present study
only included surgical cases with or without adjuvant
chemotherapy other than ICI and did not demonstrate
any prognostic impact for PD-L1 expression even though
other cut-off values were applied (data not shown). More
recently, next generation sequencing has revealed intra-
tumoral heterogeneity in PD-L1 expression between
epithelial and sarcomatoid components, in contrast to
similarity in driver mutations [23], suggesting necessity
of careful assessment of PD-L1 in each component to
evaluate or predict the efficacy of ICI on PPC for future
studies. manuscript. YS, AA, SH, YS, KT, HH, and YT reviewed the draft. All authors have
read and approved the final manuscript. manuscript. YS, AA, SH, YS, KT, HH, and YT reviewed the draft. All authors have
read and approved the final manuscript. Funding
No funding has been received for this work. Authors’ contributions Comprehensive molecular profiling
of pulmonary pleomorphic carcinoma. NPJ Precis Oncol. 2021;5(1):57. 23. Nagano M, Kohsaka S, Hayashi T, et al. Comprehensive molecular profiling
of pulmonary pleomorphic carcinoma. NPJ Precis Oncol. 2021;5(1):57. Authors’ contributions YS was a major contributor in writing the manuscript. AA supervised the
process of writing the manuscript. SH reviewed histopathology and provided
pathological images. HH and YT supervised the process of drafting the Page 8 of 8 Shishido et al. Journal of Cardiothoracic Surgery (2022) 17:47 Shishido et al. Journal of Cardiothoracic Surgery (2022) 17:47 12. Naito M, Tamiya A, Takeda M, et al. A high PD-L1 expression in pulmonary
pleomorphic carcinoma correlates with parietal-pleural invasion and
might predict a poor prognosis. Intern Med. 2019;58(7):921–7. 13. Fishback NF, Travis WD, Moran CA, Guinee DG Jr, McCarthy WF, Koss MN. Pleomorphic (spindle/giant cell) carcinoma of the lung. A clinicopatho-
logic correlation of 78 cases. Cancer. 1994;73(12):2936–45. 14. Kim TS, Han J, Lee KS, et al. CT findings of surgically resected pleo-
morphic carcinoma of the lung in 30 patients. Am J Roentgenol. 2005;185(1):120–5. 15. Fujisaki A, Aoki T, Kasai T, et al. Pleomorphic carcinoma of the lung:
relationship between CT findings and prognosis. Am J Roentgenol. 2016;207(2):289–94. 16. Onoda H, Kimura T, Tao H, Okabe K, Matsumoto T, Ikeda E. Air broncho-
gram in pleomorphic carcinoma of the lung is associated with favorable
prognosis. Thorac Cancer. 2018;9(6):718–25. 17. Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoin-
hibitory receptor by a novel B7 family member leads to negative regula-
tion of lymphocyte activation. J Exp Med. 2000;192(7):1027–34. 18. Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement
of PD-L1 on tumor cells in the escape from host immune system and
tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A. 2002;99(19):12293–7. 19. Schmidt LH, Kümmel A, Görlich D, et al. PD-1 and PD-L1 expression in
NSCLC indicate a favorable prognosis in defined subgroups. PLoS ONE. 2015;10(8):e0136023. 20. Inamura K, Yokouchi Y, Sakakibara R, et al. Relationship of tumor PD-L1
expression with EGFR wild-type status and poor prognosis in lung adeno-
carcinoma. Jpn J Clin Oncol. 2016;46(10):935–41. 21. Imanishi N, Hirai A, Yoneda K, et al. Programmed death-ligand 1 (PD-
L1) expression in pleomorphic carcinoma of the lung. J Surg Oncol. 2018;117(7):1563–9. 22. Li H, Xu Y, Wan B, et al. The clinicopathological and prognostic signifi-
cance of PD-L1 expression assessed by immunohistochemistry in lung
cancer: a meta-analysis of 50 studies with 11,383 patients. Transl Lung
Cancer Res. 2019;8(4):429–49. 23. Nagano M, Kohsaka S, Hayashi T, et al. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub-
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https://openalex.org/W39282610
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https://zenodo.org/records/1667401/files/article.pdf
|
German
| null |
Ueber eigentümliche Doppelkörnchen (Parasiten?) in Trachomzellen
|
Deutsche medizinische Wochenschrift/Deutsche Medizinische Wochenschrift
| 1,907
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public-domain
| 1,086
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DEIJTSOHE MIEDIZINISCHhl WOCIIIENSCflRIFP DEIJTSOHE MIEDIZINISCHhl WOCIIIENSCflRIFP No. 23 No. 23 914 Dieses Dokument wurde zum persönlichen Gebrauch heruntergeladen. Vervielfältigung nur mit Zustimmung des Verlages. Ueber eigentümliche Doppelkörnchen (Pa'
rasiten?) in Trachomzellen. Ueber eigentümliche Doppelkörnchen (Pa'
rasiten?) in Trachomzellen. )
Von Prof. Dr. lt. Greeff. Ich teilte wohl sicher das gleiche Schicksal mit manchem
Forscher, als ich mich seit Jahren bemühte, zum Teil mit
Unterstützung namhafter Bakteriologen, den Erreger einer der
verbreitetsten Volkskrankheiten, des Trachoms sive Granulose,
zu finden. Die Untersuchungen waren auf einem toten Punkt ange-
kommen, als die glänzenden Entdeckungen von Schaudinn
und Hoffmann die Hoffnung erweckten, mit diesen neuen
Untersuchungsmethoden auch auf unserem Gebiete weiter zu
kommen. Die Untersuchungen, die ich mit meinem Assistenten
Dr. Clausen, als fleißigem Mitarbeiter, anstellte, im frischen
Zustand oder nach der Giemsaschen Methode oder nach der
von Levaditi
spirillenartige Gebilde zu entdecken, blieben
jedoch ebenfalls ohne positiven Erfolg. Wir haben jedoch schließlich einen andersartigen, neuen
Befund erhoben, den wir in Kürze mitteilen wollen. In den
Trachomfollikein finden sich vielfach zerstreut ganz eigen-
tümliche, große Zellen mit vielfachen Zelleinschlüssen. Th. Leber hat sich schon eingehend mit ihnen beschäftigt (cf. 25. Bericht der ophthalmologischen Gesellschaft in Heidelberg
1896). Sie sehen fast wie große Protozoen aus, sind auch da-
für gehalten worden, bis Leber, der zuerst dieser Ansicht
nicht abhold war, davon abkam und sie wegen der eigentüm-
lichen, großen Protoplasrnaeinschlüsse Körperchenzellen nannte. DEUTSCHE MEDIZINISCHE WOCHIENSOHIRIFT 915 6 Juni. 6 Juni. thelien der Conjunctiva gefunden haben, während wir sie als
Zelleinschlüsse überall in den Follikein finden konnten. Das
Vorkommen allein in den Epithelien wäre noch nicht sehr
charakteristisch, da das Trachom keine epitheli1e Erkrankung ist. k
hi
d
f Auf diese Zellen war seit langer Zeit unser Augenmerk ge-
richtet. Prof. Frosch vom Institut für Infektionskrankheiten,
dessen tätiger Mitarbeiterschaft in Rat und Tat wir uns in
letzter Zeit zu erfreuen hatten, sprach die Vermutung aus,
daß diese großen Zellen wohl nicht die Erreger des Trachoms
seien, daß es jedoch nicht unmöglich wäre, daß sie Beziehungen
zu den Erregern des Trachoms hätten. Es kam uns hier nur darauf an, in dieser vorläufigen
Mitteilung unsere Befunde bekannt zu geben, ohne für heute
Schlüsse daraus zu ziehen, da wir noch mitten in weiteren
Untersuchungen begriffen sind und noch manche Forderungen
erfüllt werden müssen, ehe wir die Gebilde sicher als die Para-
siten des Trachoms ansprechen dürfen. In der Tat ist es uns schon vor längerer Zeit gelungen, in dem
Protoplasma dieser Zellen zuerst nach Färbungen mit Giemsa
einen eigentümlichen Befund zu erheben. 1) I-lalberstädter und y. Prowazek, Ueber Zelleinschlüsse parasitIrer Natur
beim Trachom.
.rbeitn
dem Kaiserlichen Gesundheitsatnte f907, Bd. 26, H. I. Ueber eigentümliche Doppelkörnchen (Pa'
rasiten?) in Trachomzellen. In dem schwach
hellblau gefärbten Protoplasma finden sich dunkelblau oder
bald mehr rötlich gefärbte, bald rundliche oder eckige, mehr
oder weniger scharf begrenzte Einschlüsse, die einzeln liegen
oder sich zusammenfügen (Fig. 1 b). Es sind die bekannten Fig. 1. Fig. 1. Phagocytenartige, sogenannte Körperchenzelle aus einem Trachom-
follikel im hängenden Tropfen. Zeiss: Oel-Immers. I
Oc. 8. Kern mit zwei Kernkörperchen,
Zelleinschlüsse (Plasmin ?),
Doppelkörnchen. Fig. II. Doppelkörnchen, im Schnitt sich von außen an rote Blutkörperchen
anschmiegend. Fig. III. Doppelkörnchen frei im hängenden Tropfen, sich lebhaft bewegend
d
i i h
i
d l
d Fig. 1. Fig. 1. Fig. 1. Fig. 1. Fig. 1. Phagocytenartige, sogenannte Körperchenzelle aus einem Trachom-
follikel im hängenden Tropfen. Zeiss: Oel-Immers. I
Oc. 8. Kern mit zwei Kernkörperchen, Fig. 1. Phagocytenartige, sogenannte Körperchenzelle aus einem Trachom-
follikel im hängenden Tropfen. Zeiss: Oel-Immers. I
Oc. 8. Kern mit zwei Kernkörperchen p
Zelleinschlüsse (Plasmin ?), Fig. II. Doppelkörnchen, im Schnitt sich von außen an rote Blutkörperchen
anschmiegend. Fig. II. Doppelkörnchen, im Schnitt sich von außen an rote Blutkörperchen
anschmiegend. Fig. III. Doppelkörnchen frei im hängenden Tropfen, sich lebhaft bewegend
und gern zu zwei sich aneinanderlegend. Fig. III. Doppelkörnchen frei im hängenden Tropfen, sich lebhaft bewegend
und gern zu zwei sich aneinanderlegend. Gebilde. Mit stärksten Vergrößerungen sahen wir ferner da-
neben kleinste, sehr dunkle Pänktchen, die die große Neigung
haben, sich wie Doppelcoccen zu zwei aneinander zu legen
(Fig. le). Sie sind ganz erheblich kleiner als Coccen, nur
etwa 1/5 so groß und verhalten sich auch sonst ganz anders
als Coceen. Entweder sind sie rar oder massenhaft im Proto-
plasma eingeschlossen. Am besten treten sie hervor, wenn
man die mikroskopischen Bilder mit dem Zetnowschen Filter
photographiert und vergrößert (Dr. Thorner). Sowohl im
Schnitt als im hängenden Tropfen, dem man z. B. etwas
Gentianaviolett zusetzt, werden sie sichtbar. Später haben
wir sie auch im ungefärbten Zustand in lebhafter, mole-
kularer Bewegung in dem Follikelinhalt gefunden. Seltener,
aber zweifellos kommen die Gebilde auch außerhalb dieser
Zellen im Gewebe vor, so sind sie in Fig. 2 zu sehen, wie
sie sich im Trachomgewebe von außen an einige rote Blut-
körperchen anlegen. i
f
d
i d
i B li i
L b
i
d Gebilde. Mit stärksten Vergrößerungen sahen wir ferner da-
neben kleinste, sehr dunkle Pänktchen, die die große Neigung
haben, sich wie Doppelcoccen zu zwei aneinander zu legen
(Fig. le). Ueber eigentümliche Doppelkörnchen (Pa'
rasiten?) in Trachomzellen. Sie sind ganz erheblich kleiner als Coccen, nur
etwa 1/5 so groß und verhalten sich auch sonst ganz anders
als Coceen. Entweder sind sie rar oder massenhaft im Proto-
plasma eingeschlossen. Am besten treten sie hervor, wenn
man die mikroskopischen Bilder mit dem Zetnowschen Filter
photographiert und vergrößert (Dr. Thorner). Sowohl im
Schnitt als im hängenden Tropfen, dem man z. B. etwas
Gentianaviolett zusetzt, werden sie sichtbar. Später haben
wir sie auch im ungefärbten Zustand in lebhafter, mole-
kularer Bewegung in dem Follikelinhalt gefunden. Seltener,
aber zweifellos kommen die Gebilde auch außerhalb dieser
Zellen im Gewebe vor, so sind sie in Fig. 2 zu sehen, wie
sie sich im Trachomgewebe von außen an einige rote Blut-
körperchen anlegen. p
g
Die Befunde sind hier in Berlin im Laboratorium der
Augenklinik in der Charité, in dem Laboratorium des Instituts
für Infektionskrankheiten zu Berlin und während eines vier-
wöchigen Aufenthaltes in Posen in dem dortigen, unter Leitung
des Prof. Wernicke stehenden hygienischen Institut gemacht
worden. Noch mitten in den Untersuchungen darüber begriffen,
hätten wir den Zeitpunkt der Publikation vielleicht noch nicht
für gekommen erachtet, wenn nicht soeben eine Publikation
von S. Halberstädter und G. y. Prowazek erschienen
wäre1), die bei den zahlreichen Trachomkranken in Java Be-
funde erhoben haben, die den unsrigen zum mindesten sehr
ähnlich sind. Diese Autoren sprechen die Doppelkörnchen als
Parasiten an. Sie stellen sie nahe den Initialkörperchen bei
Vaccine und Scharlach. Ein wesentlicher Unterschied ist nur
der, daß diese Autoren die Initialkörperchen nur in den Epi-
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https://openalex.org/W3189255942
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https://www.researchsquare.com/article/rs-84298/v1.pdf?c=1602024329000
|
English
| null |
Electrolyte and renal disorders in patients with newly diagnosed glioblastoma
|
Future oncology
| 2,021
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cc-by
| 5,712
|
Electrolyte and renal disorders in patients with newly
diagnosed glioblastoma
Daniela Pierscianek
(
daniela.pierscianek@uk-essen.de
)
University Hospital Essen Department of Neurosurgery: Universitatsklinikum Essen Klinik fur
Neurochirurgie und Wirbelsaulenchirurgie
https://orcid.org/0000-0003-0980-7444
Marvin Darkwah Oppong
University Hospital Essen Department of Neurosurgery: Universitatsklinikum Essen Klinik fur
Neurochirurgie und Wirbelsaulenchirurgie
Yahya Ahmadipour
University Hospital Essen Department of Neurosurgery: Universitatsklinikum Essen Klinik fur
Neurochirurgie und Wirbelsaulenchirurgie
Laurèl Rauschenbach
University Hospital Essen Department of Neurosurgery and Spinal Surgery: Universitatsklinikum Essen
Klinik fur Neurochirurgie und Wirbelsaulenchirurgie
Anna Michel
University Hospital Essen Department of Neurosurgery and Spinal Surgery: Universitatsklinikum Essen
Klinik fur Neurochirurgie und Wirbelsaulenchirurgie
Sied Kebir
University Hospital Essen, Department of Neurology
Philipp Dammann
University Hospital Essen Department of Neurosurgery and Spinal Surgery: Universitatsklinikum Essen
Klinik fur Neurochirurgie und Wirbelsaulenchirurgie
Karsten H. Wrede
University Hospital Essen Department of Neurosurgery and Spinal Surgery: Universitatsklinikum Essen
Klinik fur Neurochirurgie und Wirbelsaulenchirurgie
Martin Glas
University Hospital Essen, Department of Neurology
Jörg Hense
University Hospital Essen, Department of Medical Oncology
Christoph Pöttgen
University Hospital Essen, Department of Radiotherapy
Ulrich Sure
University Hospital Essen Department of Neurosurgery: Universitatsklinikum Essen Klinik fur
Neurochirurgie und Wirbelsaulenchirurgie
Ramazan Jabbarli Electrolyte and renal disorders in patients with newly
diagnosed glioblastoma
Daniela Pierscianek
(
daniela.pierscianek@uk-essen.de
)
University Hospital Essen Department of Neurosurgery: Universitatsklinikum Essen Klinik fur
Neurochirurgie und Wirbelsaulenchirurgie
https://orcid.org/0000-0003-0980-7444
Marvin Darkwah Oppong
University Hospital Essen Department of Neurosurgery: Universitatsklinikum Essen Klinik fur
Neurochirurgie und Wirbelsaulenchirurgie
Yahya Ahmadipour
University Hospital Essen Department of Neurosurgery: Universitatsklinikum Essen Klinik fur
Neurochirurgie und Wirbelsaulenchirurgie
Laurèl Rauschenbach
University Hospital Essen Department of Neurosurgery and Spinal Surgery: Universitatsklinikum Essen
Klinik fur Neurochirurgie und Wirbelsaulenchirurgie
Anna Michel
University Hospital Essen Department of Neurosurgery and Spinal Surgery: Universitatsklinikum Essen
Klinik fur Neurochirurgie und Wirbelsaulenchirurgie
Sied Kebir
University Hospital Essen, Department of Neurology
Philipp Dammann
University Hospital Essen Department of Neurosurgery and Spinal Surgery: Universitatsklinikum Essen
Klinik fur Neurochirurgie und Wirbelsaulenchirurgie
Karsten H. Wrede
University Hospital Essen Department of Neurosurgery and Spinal Surgery: Universitatsklinikum Essen
Klinik fur Neurochirurgie und Wirbelsaulenchirurgie
Martin Glas
University Hospital Essen, Department of Neurology
Jörg Hense
University Hospital Essen, Department of Medical Oncology
Christoph Pöttgen
University Hospital Essen, Department of Radiotherapy
Ulrich Sure
University Hospital Essen Department of Neurosurgery: Universitatsklinikum Essen Klinik fur
Neurochirurgie und Wirbelsaulenchirurgie
Ramazan Jabbarli Daniela Pierscianek
(
daniela.pierscianek@uk-essen.de
) University Hospital Essen Department of Neurosurgery and Spinal Surgery: Universitatsklinikum Essen
Klinik fur Neurochirurgie und Wirbelsaulenchirurgie Conclusions Preoperative electrolyte and renal disorders are common in GBM patients. Of them, only hypochloremia
showed a strong association with GBM prognosis, independently of age, sex, extent of resection, clinical
performance status, postoperative therapy, and molecular status. Further studies are needed to evaluate the
impact of hypochloremia on GBM survival. Research Keywords: glioblastoma, hypochloremia, kidney function, electrolytes, routine laboratory parameters
Posted Date: October 6th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-84298/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read
Full License License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read
Full License Version of Record: A version of this preprint was published at Future Oncology on October 21st, 2021. See
the published version at https://doi.org/10.2217/fon-2020-1312. Page 2/17 Results Electrolyte and renal disorders at admission were present in 275 (30.6%) and 544 (60.4%) of 900 GBM
patients respectively and were more common in patients with higher age, previous comorbidities and poor
initial clinical performance status. In univariate analysis and Kaplan-Meier survival plots, presence of
hyponatremia, hypochloremia, hypocalcemia, hyperuricemia and low glomerular filtration rate were
associated with poorer survival. Multivariate analysis revealed hypochloremia as an independent
prognostic factor for overall (p=0.004) and 1-year (p=0.021) survival. Background Electrolyte disturbances and altered renal function have been linked to the prognosis of critically ill patients
and recently also of cancer patients. Little is known about the prevalence and prognostic impact of
electrolyte and renal disorders in patients with glioblastoma (GBM), the most frequent malignant primary
brain tumor. This study aimed to assess electrolyte and renal disorders in GBM patients and evaluate their
effect on patients’ outcome. Methods Patients treated for newly diagnosed GBM between 2005 and 2018 were included. Electrolytes and renal
function parameters were assessed preoperatively. Medical records of patients were retrospectively
reviewed for demographic and clinical parameters, as well as patients’ survival. 2.1. Study population In this retrospective longitudinal cohort study all patients (aged ≥ 18 years) with newly diagnosed and
histologically confirmed GBM, that were treated between July 2005 and December 2018 in the University
Hospital Essen were included. After surgery (microsurgical tumor resection or tumor biopsy), patients were
transferred to adjuvant therapies (chemotherapy, radiation), or in accordance with patients’ willingness, to
best supportive care. The study was approved by the Institutional Ethics Committee. 2.2. Data management The primary objective of the study was to evaluate the association betwee
and kidney function parameters with initial demographic/clinical characte
patients. As outcome endpoints, overall survival (OS) and 1-year survival (
Preoperative serum blood samples were routinely collected 1–2 days prior
sodium, chloride, potassium, and calcium were investigated. Kidney functi
creatinine, urea, and glomerular filtration rate (GFR) GFR was estimated ac
Diet in Renal Disease (MDRD) equation. Default laboratory cutoff values w
serum values. Medical records were reviewed for patients’ age at diagnosis
(Karnofsky Performance Scale [KPS]), treatment modalities (microsurgical
radiation and chemotherapy), MGMT and IDH1 status. The primary objective of the study was to evaluate the association between routinely analyzed electrolytes
and kidney function parameters with initial demographic/clinical characteristics and survival of GBM
patients. As outcome endpoints, overall survival (OS) and 1-year survival (1-YS) were assessed. Preoperative serum blood samples were routinely collected 1–2 days prior to surgery. For electrolytes,
sodium, chloride, potassium, and calcium were investigated. Kidney function was assessed using
creatinine, urea, and glomerular filtration rate (GFR) GFR was estimated according to the Modification of
Diet in Renal Disease (MDRD) equation. Default laboratory cutoff values were used to define aberrations in
serum values. Medical records were reviewed for patients’ age at diagnosis, sex, preoperative clinical status
(Karnofsky Performance Scale [KPS]), treatment modalities (microsurgical tumor resection, tumor biopsy,
radiation and chemotherapy), MGMT and IDH1 status. 1. Introduction Glioblastoma (GBM) is the most frequent primary malignant brain tumor in adults. Prognosis remains poor
with a median survival of 17–25 months despite multimodal therapies (1). Still, survival varies significantly
among individuals and estimation of patients’ prognosis evolves into a highly relevant aspect in the
treatment of patients with GBM. Therapies comprise tumor resection or stereotactic biopsy followed by
combined radio-/chemotherapy or solely one of both adjuvant modalities, according to the patients’ clinical
status, age, etc. Several prognostic factors have been identified in GBM, like patients’ age, clinical
performance status, methylation of O6-methylguanin-DNA-methyltransferase gene promotor (MGMT), or
mutation of the isocitrate-dehydrogenase gene 1(IDH1)(2–5). These parameters allow an early estimation
of patients’ prognosis after GBM diagnosis. However, the identification of other relevant survival markers, Page 3/17 particularly of those assessable prior to surgery, is of eminent clinical importance and might be helpful
during initial treatment planning. Electrolyte disorders and altered renal function have long been linked to patients’ prognosis in various
diseases. Aberrations in serum electrolyte levels were shown to be associated with a higher mortality in
intensive care units in critically ill adult patients (6, 7). Several recent studies additionally suggest that
electrolyte disorders might be associated with a worse prognosis in a variety of malignancies (8), e.g. in
colorectal cancer (9), lung cancer (10) and lymphoma (11, 12). To date, data on prevalence and eventual
clinical impact of preoperative disturbances in serum electrolytes and renal parameters in GBM patients is
scarce. This study aimed to assess routinely investigated electrolytes and renal parameters in GBM patients at
admission and to evaluate the impact of electrolyte and renal disorders on patients’ survival. 2.3. Statistical analysis Statistical analysis was performed using SPSS (version 26, SPSS Inc., IBM, Chicago, IL, USA) and PRISM
(version 5.0, GraphPad Software Inc., San Diego, CA, USA). First, all assessed variables were investigated in
univariate analyses. Continuous, non-normally distributed data were presented as median with the
corresponding interquartile range (IR, 25. – 75. percentile) whereas categorical data were reported as
absolute numbers and their percentages. For categorical data, Fisher’s exact or chi-square test was used, as Page 4/17 Page 4/17 applicable. The Mann-Whitney-U test was performed for continuous variables. Differences with a P value ≤
0.05 were considered statistically significant. All laboratory variables, that reached significance in the
univariate analyses were then evaluated in multivariate analyses adjusted for patients’ age, preoperative
clinical condition, treatment modalities, and molecular aberrations. Multivariate binary logistic regression
analyses were performed for 1-YS and Cox-Regression analysis was applied for evaluation of OS. Missing
values were replaced using multiple imputation. Kaplan-Meier curves and log-rank test were used for
survival analysis. 3.1. Patients’ characteristics This cohort comprised 900 patients with newly diagnosed and histologically confirmed GBM. The median
age was 65.1 years (IR 55.8–72.3 years). 524 patients were male (58.2%). Microsurgical resection was
performed in 70.9% of patients and 72.6% of patients received combined radio- and chemotherapy
postoperatively. The median overall survival was 8.93 months (IR 3.5–16.4 months). One-year survival rate
was 41.1%. Detailed patients’ characteristics are summarized in Table 1. 3.2. Electrolytes and kidney function in GBM In this cohort of GBM, electrolyte disorders occurred in 275 (30.6%) of GBM patients, whereas renal
disorders were more frequent, affecting 544 patients (60.4%). In 74 patients (8.2%), more than one
electrolyte parameter was deranged. The most frequent electrolyte disorder was hyperchloremia (13%),
followed by hyponatremia (6.7%). A restricted GFR occurred in 29% of patients, whereas hyperuricemia was
present in 48% of patients. Frequencies of electrolyte and renal disorders are shown in Fig. 1. Associations of each electrolyte disorder with patients’ baseline parameters were tested. Here, frequent
associations with patients’ age, preoperative clinical performance, and previous comorbidities were
detected. For electrolyte disorders, no significant association with renal disorders were found. Detailed
information on associations of electrolyte and renal disorders with the above-mentioned parameters are
presented in Table 2. 3.3.1. Univariate and survival analysis In univariate analysis, there was a significant association between hyponatremia (p < 0.001/p = 0.001),
hypochloremia (p < 0.001/p < 0.001) and hypocalcemia (p < 0.001/p = 0.005) with OS and 1-YS respectively. Patients with hyponatremia, hypochloremia or hypocalcemia had a shorter OS and survived less frequently
one year. All results of univariate analyses are shown in Table 3. Survival analyses using Kaplan-Meier
curves and the log-rank test confirmed poorer survival in GBM patients with hypochloremia (p < 0.001),
hyponatremia (p = 0.026) and hypocalcemia (p < 0.001). The Kaplan-Meier curves are shown in Fig. 2A-C. Page 5/17 Page 5/17 Moreover, the impact of the number of electrolyte disorders on OS was also investigated. Here, a significant
association between the number of electrolyte disorders and OS was found (p < 0.001). This association is
further supported by Kaplan-Meier analysis (log-rank test p < 0.001) as shown in Fig. 2D. 3.4.1. Univariate and survival analyses Patients with an urea serum level > 20 mg/dl showed a significantly shorter median OS than patients with
an urea level ≤ 20 mg/dl (p < 0.001, 7.3 vs. 10.6 months). A GFR < 60 ml/min/1.73 m2 was also associated
with a shorter median OS (p < 0.001, 6.7 vs. 10.2 months). Additionally, patients with a GFR <
60 ml/min/1.73 m2 showed less frequently a survival of more than 1 year (p = 0.001). This association was
also found in patients with a urea serum level > 20 mg/dl (p < 0.001). Altered creatinine levels were not
associated with OS or 1YS. Survival analyses applying the log-rank test and Kaplan-Meier curves revealed a significantly worse survival
in patients with a serum urea level > 20 mg/dl (p < 0.001) and in patients with a GFR < 60 ml/min/1.73 m2
(p < 0.001) (see Supplemental Figure S1). 3.4.2. Multivariate analysis Multivariate analyses did not reveal a serum urea level > 20 mg/dl or a restricted GFR as independent
predictive factors for 1-YS and OS in binary logistic and Cox regression analyses respectively (see
Supplemental Table S1). 3.3.2. Multivariate analysis In multivariate analysis, only hypochloremia was found to be predictive for 1-YS, independently of age, sex,
extent of resection, clinical performance status, postoperative therapy and molecular alterations (adjusted
odds ratio [aOR] = 0.33 [95% CI: 0.13–0.84], p = 0.021). For OS, cox regression analysis showed a
significantly poorer survival in patients with hypochloremia (hazard ratio [HR] = 1.59 [95% CI: 1.16–2.18], p
= 0.004), independently of covariates. Hyponatremia (p = 0.2) and hypocalcemia (p = 0.39) showed no
association with OS in multivariate analysis. The results of multivariate analyses are listed in Table 4. 4. Discussion In cancer patients, electrolyte and acid-base disorders are reported in more than 50% (8). Moreover, kidney
function and electrolyte homeostasis have been linked to patients’ prognosis for several types of cancer (8,
10, 11). This study identified hypochloremia in GBM as an independent prognostic marker for 1-YS and OS,
whereas all other investigated electrolytes and parameters of kidney function failed to display a consistent
relation to patients’ outcome. Chloride is the main anion in plasma and interstitial fluid and carries a significant role in maintaining serum
osmolarity and acid-balance. There are several possible causes for hypochloremia in patients, like chloride Page 6/17 loss via vomiting or diarrhea, use of diuretics, or excess water gain due to infusion of hypotonic solutions. During the last years, chloride serum levels gained increasing attention in the intensive care of critically ill
patients and also in cancer patients (13). In intensive care units, hypochloremia occurs in up to one-third of
all individuals (14–16). This study found hypochloremia in 6.3% of patients with GBM in the preoperative
routine investigation of electrolytes. This study cohort mainly comprises patients that are admitted to
hospital for elective surgery of intracranial lesions, whereas the studies mentioned-above investigated
critically ill patients in intensive care units. This might explain the higher rate of hypochloremia in the
studies that were mainly conducted on patient collectives in intensive care units. Several studies additionally addressed the association between hypochloremia and patients’ outcome. Kimura et al. detected a significantly higher mortality in patients with hypochloremia after elective thoracic
or abdominal surgery (16). Similar results revealed a study of 106,505 adult patients undergoing
noncardiac surgery. Here, patients with preoperative hypochloremia showed a significant increased 90-days
mortality compared to patients with normochloremia. Patients with hypochloremia also had a higher risk of
postoperative acute kidney injury (17). These results are in agreement with several investigations reporting
a poorer outcome for patients with hypochloremia (14, 18–20) whereas Thongprayoon et al. only found
hospital-acquired hyperchloremia in 39,298 patients to be associated with increased in-hospital mortality
(21). There is only sparse evidence for the influence of hypochloremia on the outcome of cancer patients. One recent study retrospectively collected clinical data and electrolytes parameters from all cancer patients
treated over one year. In 25,881 patients, hypochloremia occurred in 24.5% of patients. The authors found a
higher in-hospital mortality in patients with electrolyte disorders compared to patients with normal
electrolytes (8). 4. Discussion In another investigation among 5,089 patients with colorectal cancer, hypochloremia was
associated with a worse overall survival and shorter disease-free survival (9). This study detected
hypochloremia to be an independent prognostic factor for OS and 1-YS in GBM patients and is well in line
with previous results. So far, there are no investigations that specifically address hypochloremia and its
prognostic impact on GBM survival. One recent study analyzed the predictive value of hyponatremia in 200
GBM patients. Similar to the results of this study, the authors could not demonstrate an association
between hyponatremia and patients’ outcome (22). In this cohort of GBM patients, arterial hypertension,
diabetes, a higher age, and a poor preoperative clinical status characterized the cohort of patients with
hypochloremia. This observation is in line with previous studies, that also reported renal dysfunction,
hypertension, and diabetes as risk factors for electrolyte disorders (8). Hypochloremia might be a surrogate marker for patients with a specific risk profile that is associated with a
poorer outcome. Petnak et al. investigated chloride levels of adult patients at discharge and found both
hypochloremia and hyperchloremia to be associated with an increased risk of one-year mortality (23). In
18,825 critically ill adult patients, a fluctuation of chloride serum levels during the first 72 hours after
admission to the intensive care unit was associated with increased 30-day mortality (6). So far, there are no
studies on patients with GBM that included the evaluation of hypochloremia during the course of the
disease and the influence of an early correction of hypochloremia on patients’ outcome. 5. Limitations Page 7/17 Page 7/17 The limitations of this study are mainly due to its retrospective design. The interpretation of the results is
limited due to in part incomplete data, that carry the risk of inaccuracy. Furthermore, treatment strategies in
our cohort were heterogenous and several factors, that might influence patients’ renal function and
electrolytes could not be incorporated in the analysis, e.g. medication at admission, repetitive vomiting, etc. 6. Conclusion Despite the limitations mentioned above, this study is based on a large cohort of GBM patients and their
corresponding serum laboratory parameters at admission. Hypochloremia was identified as an
independent prognostic factor making serum chloride levels a promising preoperative biomarker in GBM. The results of this study will have to be confirmed in a prospective study including multiple centers. Additionally, the role of hypochloremia during the adjuvant therapies and the effect of adjustment of
chloride serum levels on survival should be elucidated. GBM: glioblastoma GBM: glioblastoma MGMT: O6-methylguanin-DNA-methyltransferase gene promotor Ethical approval The study was approved by the Institutional Ethics Committee, University of Essen (15-6504-BO and 15-
6505-BO). As this is an retrospective analysis, consent on participation is not applicable. Abbreviations Abbreviations
GBM: glioblastoma
MGMT: O6-methylguanin-DNA-methyltransferase gene promo
IDH1: isocitrate-dehydrogenase gene 1
OS: overall survival
1-YS: 1- year survival
GFR: glomerular filtration rate
MDRD: Modification of Diet in Renal Disease
KPS: Karnofsky Performance Scale
aOR: adjusted odds ratio
HR: hazard ratio Consent for publication Consent for publication Availability of data The data that support the findings of this study are available from the corresponding author upon
reasonable request. The manuscript does not contain any individual person’s data. The manuscript does not contain any individual person’s data. Authors contributions All authors gave their final approval of the manuscript to be published and agreed to be accountable for al
aspects of the work. Daniela Pierscianek: Conception of the work, Data acquisition, analysis and interpretation, Drafting the
work Daniela Pierscianek: Conception of the work, Data acquisition, analysis and interpretation, Drafting the
work Daniela Pierscianek: Conception of the work, Data acquisition, analysis and interpretation, Drafting the
work
Marvin Darkwah Oppong: Data acquisition and analysis, Drafting the work
Yahya Ahmadipour: Data acquisition, Revising the Manuscript
Laurèl Rauschenbach: Data acquisition, Revising the Manuscript
Anna Michel: Data acquisition, Revising the Manuscript
Sied Kebir: Data acquisition, Revising the Manuscript
Philipp Dammann: Data analysis and interpretation, Revising the Manuscript
Karsten H. Wrede: Data analysis and interpretation, Revising the Manuscript
Martin Glas: Data analysis and interpretation, Revising the Manuscript
Jörg Hense: Data analysis and interpretation, Revising the Manuscript
Christoph Pöttgen: Data analysis and interpretation, Revising the Manuscript
Ulrich Sure: Conception of the work, Data interpretation, Revising the Manuscript
Ramazan Jabbarli: Conception of the work, Data interpretation, Revising the Manuscript
Acknowledgements Marvin Darkwah Oppong: Data acquisition and analysis, Drafting the work Yahya Ahmadipour: Data acquisition, Revising the Manuscript Laurèl Rauschenbach: Data acquisition, Revising the Manuscript Anna Michel: Data acquisition, Revising the Manuscript Sied Kebir: Data acquisition, Revising the Manuscript q
,
g
p
Philipp Dammann: Data analysis and interpretation, Revising the Manuscript
Karsten H. Wrede: Data analysis and interpretation, Revising the Manuscript
Martin Glas: Data analysis and interpretation, Revising the Manuscript
Jörg Hense: Data analysis and interpretation, Revising the Manuscript
Christoph Pöttgen: Data analysis and interpretation, Revising the Manuscript
Ulrich Sure: Conception of the work, Data interpretation, Revising the Manuscript
Ramazan Jabbarli: Conception of the work, Data interpretation, Revising the Manuscript Competing interests The authors state that there are no conflicts of interest, ethical adherence or any financial disclosures. The authors state that there are no conflicts of interest, ethical adherence or any financial disclosures. Acknowledgements not applicable not applicable References 1. Stupp R, Taillibert S, Kanner A, Read W, Steinberg D, Lhermitte B, et al. Effect of Tumor-Treating Fields
Plus Maintenance Temozolomide vs Maintenance Temozolomide Alone on Survival in Patients With
Glioblastoma: A Randomized Clinical Trial. JAMA. 2017;318(23):2306–16. 2. Pierscianek D, Ahmadipour Y, Kaier K, Darkwah Oppong M, Michel A, Kebir S, et al. The SHORT Score
for Preoperative Assessment of the Risk for Short-Term Survival in Glioblastoma. World Neurosurg. 2020. 3. Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, et al. MGMT gene silencing and
benefit from temozolomide in glioblastoma. N Engl J Med. 2005;352(10):997–1003. 4. Nobusawa S, Watanabe T, Kleihues P, Ohgaki H. IDH1 mutations as molecular signature and predictive
factor of secondary glioblastomas. Clin Cancer Res. 2009;15(19):6002–7. 5. Pierscianek D, Ahmadipour Y, Michel A, Chihi M, Oppong MD, Kebir S, et al. Preoperative Survival
Prediction in Patients With Glioblastoma by Routine Inflammatory Laboratory Parameters. Anticancer
Res. 2020;40(2):1161–6. 6. Kim HJ, Oh TK, Song IA, Lee JH. Association between fluctuations in serum chloride levels and 30-day
mortality among critically ill patients: a retrospective analysis. BMC Anesthesiol. 2019;19(1):79. 7. Oh HJ, Kim SJ, Kim YC, Kim EJ, Jung IY, Oh DH, et al. An increased chloride level in hypochloremia is
associated with decreased mortality in patients with severe sepsis or septic shock. Sci Rep. 2017;7(1):15883. 8. Li Y, Chen X, Shen Z, Wang Y, Hu J, Xu J, et al. Electrolyte and acid-base disorders in cancer patients
and its impact on clinical outcomes: evidence from a real-world study in China. Ren Fail. 2020;42(1):234–43. 9. Li Q, Dai W, Jia H, Li Y, Xu Y, Li X, et al. Prognostic Impact of Hypochloremia in Patients With Stage I to
III Colorectal Cancer After Radical Resection. Dis Colon Rectum. 2018;61(11):1273–80. 10. Li W, Chen X, Wang L, Wang Y, Huang C, Wang G, et al. The prognostic effects of hyponatremia and
hyperchloremia on postoperative NSCLC patients. Curr Probl Cancer. 2019;43(5):402–10. 11. Castillo JJ, Glezerman IG, Boklage SH, Chiodo J 3rd, Tidwell BA, Lamerato LE, et al. The occurrence of
hyponatremia and its importance as a prognostic factor in a cross-section of cancer patients. BMC
Cancer. 2016;16:564. 12. Cheungpasitporn W, Thongprayoon C, Qian Q. Dysmagnesemia in Hospitalized Patients: Prevalence
and Prognostic Importance. Mayo Clin Proc. 2015;90(8):1001-10. 12. Cheungpasitporn W, Thongprayoon C, Qian Q. Dysmagnesemia in Hospitalized Patients: Prevalence
and Prognostic Importance. Mayo Clin Proc. 2015;90(8):1001-10. 13. References Bandak G, Kashani KB. Chloride in intensive care units: a key electrolyte. F1000Res. 2017;6:1930. 13. Bandak G, Kashani KB. Chloride in intensive care units: a key electrolyte. F1000Res. 2017;6:1930. 14. Shao M, Li G, Sarvottam K, Wang S, Thongprayoon C, Dong Y, et al. Dyschloremia Is a Risk Factor for
the Development of Acute Kidney Injury in Critically Ill Patients. PloS one. 2016;11(8):e0160322. 14. Shao M, Li G, Sarvottam K, Wang S, Thongprayoon C, Dong Y, et al. Dyschloremia Is a Risk Factor for
the Development of Acute Kidney Injury in Critically Ill Patients. PloS one. 2016;11(8):e0160322. Page 10/17 15. Van Regenmortel N, Verbrugghe W, Van den Wyngaert T, Jorens PG. Impact of chloride and strong ion
difference on ICU and hospital mortality in a mixed intensive care population. Ann Intensive Care. 2016;6(1):91. 16. Kimura S, Matsumoto S, Muto N, Yamanoi T, Higashi T, Nakamura K, et al. Association of serum
chloride concentration with outcomes in postoperative critically ill patients: a retrospective
observational study. J Intensive Care. 2014;2(1):39. 16. Kimura S, Matsumoto S, Muto N, Yamanoi T, Higashi T, Nakamura K, et al. Association of serum
chloride concentration with outcomes in postoperative critically ill patients: a retrospective
observational study. J Intensive Care. 2014;2(1):39. 17. Oh TK, Do SH, Jeon YT, Kim J, Na HS, Hwang JW. Association of Preoperative Serum Chloride Levels
With Mortality and Morbidity After Noncardiac Surgery: A Retrospective Cohort Study. Anesth Analg. 2019;129(6):1494–501. 17. Oh TK, Do SH, Jeon YT, Kim J, Na HS, Hwang JW. Association of Preoperative Serum Chloride Levels
With Mortality and Morbidity After Noncardiac Surgery: A Retrospective Cohort Study. Anesth Analg. 2019;129(6):1494–501. 18. Li Z, Xing C, Li T, Du L, Wang N. Hypochloremia is associated with increased risk of all-cause mortality
in patients in the coronary care unit: A cohort study. J Int Med Res. 2020;48(4):300060520911500. 18. Li Z, Xing C, Li T, Du L, Wang N. Hypochloremia is associated with increased risk of all-cause mortality
in patients in the coronary care unit: A cohort study. J Int Med Res. 2020;48(4):300060520911500. 19. Ter Maaten JM, Damman K, Hanberg JS, Givertz MM, Metra M, O'Connor CM, et al. Hypochloremia,
Diuretic Resistance, and Outcome in Patients With Acute Heart Failure. Circ Heart Fail. 2016;9(8). 19. Ter Maaten JM, Damman K, Hanberg JS, Givertz MM, Metra M, O'Connor CM, et al. Hypochloremia,
Diuretic Resistance, and Outcome in Patients With Acute Heart Failure. Circ Heart Fail. References 2016;9(8). 20. Prins KW, Kalra R, Rose L, Assad TR, Archer SL, Bajaj NS, et al. Hypochloremia Is a Noninvasive
Predictor of Mortality in Pulmonary Arterial Hypertension. J Am Heart Assoc. 2020;9(5):e015221. 20. Prins KW, Kalra R, Rose L, Assad TR, Archer SL, Bajaj NS, et al. Hypochloremia Is a Noninvasive
Predictor of Mortality in Pulmonary Arterial Hypertension. J Am Heart Assoc. 2020;9(5):e015221. 21. Thongprayoon C, Cheungpasitporn W, Petnak T, Mao MA, Chewcharat A, Qureshi F, et al. Hospital-
Acquired Serum Chloride Derangements and Associated In-Hospital Mortality. Medicines (Basel). 2020;7(7). 22. Mrowczynski OD, Bourcier AJ, Liao J, Langan ST, Specht CS, Rizk EB. The predictive potential of
hyponatremia for glioblastoma patient survival. J Neurooncol. 2018;138(1):99–104. 23. Petnak T, Thongprayoon C, Cheungpasitporn W, Bathini T, Vallabhajosyula S, Chewcharat A, et al. Serum Chloride Levels at Hospital Discharge and One-Year Mortality among Hospitalized Patients. Med Sci (Basel). 2020;8(2). mut.: IDH1-Mutation; MGMT-meth.: O6-Methylguanin-DNA-Methyltransferase Abbreviations: IDH1-mut.: IDH1-Mutation; MGMT-meth.: O6-Methylguanin-DNA-Methyltransferase
Promotor-Methylation; KPS: Karnofsky Performance Scale; preop: preoperatively; CTX: Chemotherapy; RTX:
radiotherapy; IR: interquartile range data, Mann-Whitney U test; *: categorical data, fishers’ exact test/chi-square Table 3. Univariate analysis of serum electrolytes and kidney function parameters and patients’ outcome. Abbreviations: KPS: Karnofsky Performance Scale; aHTN: arterial hypertension, DM: Diabetes mellitus; BMI:
Body Mass Index; Na+: Natrium; K+: potassium; Cl-: Chloride; Ca2+: Calcium; GFR: glomerular filtration rate. Tables Table 1. Patients’ characteristics. Page 11/17 Page 11/17 Parameter
Value
Number of patients, n
900
Age in years, median (IR)
65.14 (55.8 – 72.3)
Sex (female), n (%)
376 (41.8 %)
Tumor resection, n (%)
638 (70.9 %)
IDH1-mut. (R132H), n (%)
17 (3.0 %)
MGMT-meth., n (%)
311 (41.3 %)
KPS preop < 80%
239 (28.1 %)
arterial hypertension
452 (54.3%)
diabetes mellitus
153 (18.3%)
hypothyroidism
107 (11.9%)
Overall survival in months, median (IR)
8.93 (3.5 – 16.4)
1-Year survival
361 (41.1 %) Abbreviations: IDH1-mut.: IDH1-Mutation; MGMT-meth.: O6-Methylguanin-DNA-Methyltransferase
Promotor-Methylation; KPS: Karnofsky Performance Scale; preop: preoperatively; CTX: Chemotherapy; RTX:
radiotherapy; IR: interquartile range Table 2. Univariate analyses of electrolyte and renal parameters and patients’ characteristics and
comorbidities. Page 12/17 Page 12/17 Page 12/17 Laboratory
parameter
age#
(in
years)
sex*
KPS#
aHTN*
DM*
Hypo-
thyreodism*
BMI#
(kg/m2)
Hypernatremia
p=0.590
p=0.867
p=0.034
p=0.383
p=0.372
p=0.426
p=0.953
Hyponatremia
p=0.008
p=0.491
p=0.012
p<0.001
p<0.001
p=0.098
p=0.591
Hyperkalemia
p=0.031
p=0.848
p=0.165
p=0.178
p=1.00
p=0.379
p=0.145
Hypokalemia
p<0.001
p=0.254
p=0.368
p=0.001
p=0.076
p=0.024
p=0.077
Hyperchloremia
p=0.343
p=0.017
p=0.993
p=0.011
p=1.00
p=0.002
p=0.093
Hypochloremia
p=0.008
p=0.202
p=0.003
p<0.001
p<0.001
p=1.00
p=0.124
Hypercalcemia
p=0.496
p=0.912
p=0.130
p=0.187
p=0.781
p=0.787
p=0.057
Hypocalcemia
p<0.001
p=0.333
p=0.006
p=0.002
p=0.296
p=0.473
p=0.153
Hyperuricemia
p<0.001
p=0.002
p=0.003
p<0.001
p=0.036
p=0.249
p=0.114
Creatinine
>1.1mg/dl
p<0.001
p<0.001
p=0.207
p<0.001
p=0.001
p=0.047
p=0.007
GFR <
60ml/min/1.73m2
p<0.001
p<0.001
p=0.795
p<0.001
p<0.001
p=0.012
p=0.032
Abbreviations: KPS: Karnofsky Performance Scale; aHTN: arterial hypertension, DM: Diabetes mellitus; BMI:
Body Mass Index; Na+: Natrium; K+: potassium; Cl-: Chloride; Ca2+: Calcium; GFR: glomerular filtration rate. #: continuous data, Mann-Whitney U test; *: categorical data, fishers’ exact test/chi-square Table 3. Univariate analysis of serum electrolytes and kidney function parameters and patients’ outcome. Page 13/17 Page 13/17 Page 13/17 Parameter
Overall survival (median)
1-year survival (%)
Na+ >145mmol/L vs. ≤145 mmol/L
7.1 vs. 8.8 months;
p=0.134
36.8% vs. 40.5%;
p=0.736
Na+ <135mmol/L vs. ≥135 mmol/L
3.5 vs. 9.2 months;
p<0.001
19.3% vs. 41.9%;
p=0.001
Cl- >107mmol/L vs. ≤107 mmol/L
8.9 vs. 8.7 months;
p=0.813
38.7% vs. 40.7%;
p=0.751
Cl- <98mmol/L vs. ≥98 mmol/L
3.5 vs. 9.4 months;
p<0.001
13.2% vs. 42.3%;
p<0.001
K+ >5.0mmol/L vs. ≤5.0 mmol/L
5.7 vs. 8.8 months;
p=0.171
27.6% vs. 40.8%;
p=0.180
K+ <3.5mmol/L vs. ≥3.5 mmol/L
7.0 vs. 8.8 months;
p=0.297
31.6% vs. 40.5%;
p=0.487
Ca2+ >2.65mmol/L vs. ≤2.65 mmol/L
14.2 vs. 8.7 months;
p=0.321
60.0% vs. 39.2%;
p=0.205
Ca2+ <2.08mmol/L vs. ≥2.08 mmol/L
2.7 vs. Tables 9.2 months;
p<0.001
19.0% vs. 40.9%;
p=0.005
Creatinine >1.1mg/dl vs. ≤1.1 mg/dl
8.3 vs. 9.3 months;
p=0.192
36.8% vs. 42.4%;
p=0.121
Urea >20mg/dl vs. ≤20mg/dl
7.3 vs. 10.6 months;
p<0.001
32.4% vs. 47.9%;
p<0.001
GFR <60ml/min/1.73qm vs. ≥60ml/min/1.73qm
6.7 vs. 10.2 months;
p<0.001
30.4% vs. 44.8%;
p=0.001 Abbreviations Table 4. Multivariate analysis for OS and 1-YS using Cox and binary regression analysis respectively. Abbreviations Page 14/17 Page 14/17 Page 14/17 Parameters
OS predictors
1-YS predictors
aHR
95% CI
p-value
aOR
95% CI
p-value
Sex (female)
1.02
0.88 – 1.17
0.816
1.15
0.81 – 1.61
0.438
Age (continuous)
1.03
1.02 – 1.04
<0.001
0.96
0.95 – 0.98
<0.001
Surgical resection
0.45
0.38 – 0.53
<0.001
4.95
3.25 – 7.53
<0.001
KPS <80%
1.47
1.22 – 1.78
<0.001
0.54
0.34 – 0.87
0.012
MGMT (methylated)
0.70
0.59 – 0.83
<0.001
1.76
1.22 – 2.54
0.003
IDH1 (mutated)
0.83
0.59 – 1.16
0.262
2.13
0.80 – 5.62
0.119
RTX/CTX
0.44
0.35 – 0.55
<0.001
5.07
2.71 – 9.48
<0.001
Hyponatremia
1.23
0.89 – 1.69
0.2
0.49
0.23 – 1.05
0.068
Parameters
OS predictors
1-YS predictors
aHR
95% CI
p-value
aOR
95% CI
p-value
Sex (female)
1.02
0.88 – 1.17
0.838
1.13
0.81 – 1.59
0.475
Age (continuous)
1.03
1.03 – 1.03
<0.001
0.96
0.95 – 0.98
<0.001
Surgical resection
0.44
0.38 – 0.52
<0.001
4.89
3.20 – 7.46
<0.001
KPS <80%
1.44
1.20 – 1.71
<0.001
0.55
0.35 – 0.88
0.013
MGMT (methylated)
0.70
0.59 – 0.83
<0.001
1.76
1.22 – 2.56
0.003
IDH1 (mutated)
0.82
0.57 – 1.18
0.271
2.19
0.76 – 6.32
0.132
RTX/CTX
0.45
0.36 – 0.55
<0.001
5.05
2.69 – 9.48
<0.001
Hypochloremia
1.59
1.16 – 2.18
0.004
0.33
0.13 – 0.84
0.021 Parameters
OS predictors
1-YS predictors
aHR
95% CI
p-value
aOR
95% CI
p-value
Sex (female)
1.02
0.88 – 1.17
0.816
1.15
0.81 – 1.61
0.438
Age (continuous)
1.03
1.02 – 1.04
<0.001
0.96
0.95 – 0.98
<0.001
Surgical resection
0.45
0.38 – 0.53
<0.001
4.95
3.25 – 7.53
<0.001
KPS <80%
1.47
1.22 – 1.78
<0.001
0.54
0.34 – 0.87
0.012
MGMT (methylated)
0.70
0.59 – 0.83
<0.001
1.76
1.22 – 2.54
0.003
IDH1 (mutated)
0.83
0.59 – 1.16
0.262
2.13
0.80 – 5.62
0.119
RTX/CTX
0.44
0.35 – 0.55
<0.001
5.07
2.71 – 9.48
<0.001
Hyponatremia
1.23
0.89 – 1.69
0.2
0.49
0.23 – 1.05
0.068 Parameters
OS predictors
1-YS predictors
aHR
95% CI
p-value
aOR
95% CI
p-value
Sex (female)
1.02
0.88 – 1.17
0.838
1.13
0.81 – 1.59
0.475
Age (continuous)
1.03
1.03 – 1.03
<0.001
0.96
0.95 – 0.98
<0.001
Surgical resection
0.44
0.38 – 0.52
<0.001
4.89
3.20 – 7.46
<0.001
KPS <80%
1.44
1.20 – 1.71
<0.001
0.55
0.35 – 0.88
0.013
MGMT (methylated)
0.70
0.59 – 0.83
<0.001
1.76
1.22 – 2.56
0.003
IDH1 (mutated)
0.82
0.57 – 1.18
0.271
2.19
0.76 – 6.32
0.132
RTX/CTX
0.45
0.36 – 0.55
<0.001
5.05
2.69 – 9.48
<0.001
Hypochloremia
1.59
1.16 – 2.18
0.004
0.33
0.13 – 0.84
0.021 Page 15/17 Page 15/17 Parameters
OS predictors
1-YS predictors
aHR
95% CI
p-value
aOR
95% CI
p-value
Sex (female)
1.01
0.88 – 1.17
0.858
1.14
0.81 – 1.61
0.446
Age (continuous)
1.03
1.02 – 1.03
<0.001
0.96
0.95 – 0.98
<0.001
Surgical resection
0.44
0.37 – 0.52
<0.001
4.98
3.26 – 7.61
<0.001
KPS <80%
1.46
1.21 – 1.76
<0.001
0.54
0.34 – 0.87
0.011
MGMT (methylated)
0.70
0.60 – 0.83
<0.001
1.73
1.21 – 2.48
0.003
IDH1 (mutated)
0.83
0.60 – 1.16
0.262
2.07
0.78 – 5.51
0.132
RTX/CTX
0.44
0.35 – 0.54
<0.001
5.23
2.82 – 9.70
<0.001
Hypocalcemia
1.16
0.82 – 1.64
0.388
0.92
0.40 – 2.13
0.847 Abbreviations: aOR: adjusted Odds Ratio; aHR: adjusted Hazard Ratio; 95% CI: 95% Confidence Interval;
KPS: Karnofsky Performance Scale; RTX/CTX: combined Radio- and Chemotherapy; OS: overall survival;
1YS: 1-year survival; Abbreviations: aOR: adjusted Odds Ratio; aHR: adjusted Hazard Ratio; 95% CI: 95% Confidence Interval;
KPS: Karnofsky Performance Scale; RTX/CTX: combined Radio- and Chemotherapy; OS: overall survival;
1YS: 1-year survival; Abbreviations: aOR: adjusted Odds Ratio; aHR: adjusted Hazard Ratio; 95% CI: 95% Confidence Interval;
KPS: Karnofsky Performance Scale; RTX/CTX: combined Radio- and Chemotherapy; OS: overall survival;
1YS: 1-year survival; Abbreviations: aOR: adjusted Odds Ratio; aHR: adjusted Hazard Ratio; 95% CI: 95% Confidence Interval;
KPS: Karnofsky Performance Scale; RTX/CTX: combined Radio- and Chemotherapy; OS: overall survival;
1YS: 1-year survival; Figure 2 Survival analysis using Kaplan-Meier curves for hyponatremia (A), hypochloremia (B) and hypocalcaemia
(C). Figure 2D shows survival analysis for combined electrolyte disorders. Figures Figure 1 Page 16/17 Prevalence of electrolyte and renal disorders in the analyzed GBM cohort. Preoperative serum lab values
were available for: sodium, potassium and creatinine in 860 patients; chloride and urea in 857 patients;
calcium in 717 and glomerular filtration rate (GFR) in 645 patients. Reference ranges with appropriate units
are shown in white bars. The prevalence of aberrations below (light grey bars) and above (dark grey bars)
the range limits is reported in percentages. Prevalence of electrolyte and renal disorders in the analyzed GBM cohort. Preoperative serum lab values
were available for: sodium, potassium and creatinine in 860 patients; chloride and urea in 857 patients;
calcium in 717 and glomerular filtration rate (GFR) in 645 patients. Reference ranges with appropriate units
are shown in white bars. The prevalence of aberrations below (light grey bars) and above (dark grey bars)
the range limits is reported in percentages. Figure 2
Survival analysis using Kaplan-Meier curves for hyponatremia (A), hypochloremia (B) and hypocalcaemia
(C). Figure 2D shows survival analysis for combined electrolyte disorders. Figure 2 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. TableS1.docx FigureS1.docx Page 17/17
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|
English
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Deep Saturation Nonlinearity of 5G Media and Potential Link to Covid-19
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Mohsen Lutephy Mohsen Lutephy Abstract 5G broadband millimeter LFs (low frequencies) are filtered and do not influence
into the cells, but in the nonlinear media, the modulation instability of the fast
underlying carrier wave leads to appear the slowly varying perturbation parasite
envelopes (noises) which is described by nonlinear Schrodinger equation (NLSE). Thus, the 5G pump waves in nonlinearity leads to extremely low frequency elec-
tromagnetic pulse envelopes enable to pass the filters such as the skin, and
disintegrating in the cells to the 5G carrier waves and disordering genome as a
probable origin to organize the corona virus via covering separated part of the
genome with the capsids. A so called physical solution on the modulation instability
of the nonlinear media is the Kuznetsov-Ma breather revealed previously in the
optical fibers and accordingly we have detected here the signature of the
Kuznetsov-Ma breather self-similar solution of the NLSE on the global distribution
pattern of the covid-19 infection and death cases as an agreement between the
theoretical results and observations for covid-19. A possible potential link between
the covid-19 and 5G nonlinear internet media is revealed, verifying that the
covid-19 global patterns of the infection and death cases are statistically significant. Keywords: Nonlinear optics, Radiation health risk, Epidemiology, LF radiobiology,
covid-19, 5G 1. Introduction 5G internet media uses millimeter waves (10–300 GHZ) in which are easily
blocked environmentally and not travel far. Despite previous generations of wire-
less media, the 5G multiple antennas arrange in “phased arrays” [1, 2] that work
together to emit focused, steerable, laser-like beams that track each other causes to
appear nonlinear dispersive media, environmentally revealing modulation instabil-
ities due to nonlinearity which is observable in the optical fibers e.g. [3–5] (there are
many papers to cite here). In the linearity, the plane wave is ever stable and perturbations do not grow
whereas that in the nonlinearity, the perturbation in the background plane wave is
expanded and can produce high amplitude perturbation parasites (noises) via the
modulation instabilities which yields to its relevant wave solutions. y
Bill P. Curry in his so-called graph reported exponential Microwave absorption
in brain tissue, newly confirmed more in the paper “Exposure of Insects to Radio-
Frequency Electromagnetic Fields from 2 to 120 GHz” [6]. However the 5G waves 1 Nonlinear Optics - Nonlinear Nanophotonics and Novel Materials for Nonlinear Optics are affecting the cells in the long-term exposure e.g. [7–14] (the articles published in
the field of the cells at the exposure of the LF EMF is out of capacity all to cite here)
and also bioeffects at exposure of modulated EMF (MEMF) of radiofrequencies of
low intensity e.g. [15] and reference in, it is yet correct that the 5G waves are
blocked by skin and rarely influence in the bodies is for radio entropy. Then it has
been thought that “The 5G Health Hazard That Isn’t” as titled in the New York
Times. It is real also the radio waves become safer at higher frequencies for that
easier to block environmentally and also by skin. But according to the reports, the
cells exposed to extremely low frequencies (ELFs) presented an increase of the
number of cells with high damaged DNA as compared with non-exposed cells. Then
if the 5G media was linear, the 5G electromagnetic waves were almost safe for
healthiness of the species in the earth, but since the 5G media is nonlinear then for
modulation instability of the nonlinear media, the 5G wireless internet produces
extremely low frequency perturbation parasite pulses similar to the perturbation
pulses produced in the fiber optics. 1. Introduction p
p
p
Some properties of nonlinearity are appeared in nonlinear media such as self-
focusing [16, 17] and wave steepening [18]. The nonlinear dispersive media is
described by the nonlinear Schrodinger equation (NLSE) discussed in many papers
and the books e.g. [19] as observed in the deep-water wave propagation [20, 21] and
optical fiber [22, 23]. NLSE is a central model of nonlinear science, applying to hydrodynamics,
plasma physics, molecular biology and optics. NLSE describes the slowly varying
envelope that modulates a fast underlying carrier wave. The perturbation method
on the NLSE yields to the wave-packet envelopes as the parasite for 5G low fre-
quency carrier waves, enable to cross the skin and expose the cells in the field of
extremely low frequencies (ELFs). Thus, the 5G background pump waves can inter
noticeable to the cells and damaging DNA and producing some disorders may be
relevant to the corona virus which is a part of genome covered by the capsids
(capsidal DNA). If this is true it should be an identity between the distribution
patterns of the corona virus infection and death cases and wave pattern of the 5G
perturbation parasite envelopes. Wonderfully we detect here the identity between the covid-19 distribution
pattern of the infection and death cases and the Kuznetsov-Ma breather self-similar
solution of the nonlinear Schrodinger equation (NLSE) which describes the
instability of the radio frequency waves in dispersive nonlinear media. Deep Saturation Nonlinearity of 5G Media and Potential Link to Covid-19
DOI: http://dx.doi.org/10.5772/intechopen.98826 Eq. (1) describes many physical systems, primarily those in which nonlinear
waves propagate in isotropic media [29]. The parameter φ describes the slowly
varying envelope which modulates a fast underlying carrier wave and the nonlinear
term f
φ
j j2
is specific to the physical system.
On the (1 + 1)D NLSE, two particular forms of nonlinearity are Kerr-type and
saturable type that are common in optics [30]. The Kerr-type, where f
φ
j j2
¼ φ
j j2
and the saturable type, where f
φ
j j2
¼ φ
j j2= 1 þ φ
j j2
.
On the (1 + 1)D NLSE, two particular forms of nonlinearity are Kerr-type and
saturable type that are common in optics [30]. The Kerr-type, where f
φ
j j2
¼ φ
j j2
and the saturable type, where f
φ
j j2
¼ φ
j j2= 1 þ φ
j j2
.
Of course where φ
j j2= 1 þ φ
j j2
≈1
1= φ
j j2
, the nonlinearity is called the
“deep saturation nonlinearity” [31] and most of the interesting properties and
energy of the solitons is in the regions where φ
j j2 > > 1 [31].
Of course where φ
j j2= 1 þ φ
j j2
≈1
1= φ
j j2
, the nonlinearity is called the
“deep saturation nonlinearity” [31] and most of the interesting properties and
2 Of course where φ
j j2= 1 þ φ
j j2
≈1
1= φ
j j2
, the nonlinearity is called the
“deep saturation nonlinearity” [31] and most of the interesting properties and
2 energy of the solitons is in the regions where φ
j j2 > > 1 [31]. If φ ξ, τ
ð
Þ is a solution of the (1 + 1)D NLSE in deep saturation nonlinearity, then
a whole family of the solutions is obtained [31] by re-scaling via real parameter ε as φ ξ, τ
ð
Þ ! Deep Saturation Nonlinearity of 5G Media and Potential Link to Covid-19
DOI: http://dx.doi.org/10.5772/intechopen.98826 eit 1ε2
ð
Þε1φ εξ, ε2τ
(2) (2) Then all solutions of the same order of (1 + 1)D deep saturable NLSE are related
each other by rescaling and the solutions are self-similar to one another in their
physical properties, such as intensity, shape, etc. “This is because the natural scale
in the saturable NLSE is visible only in the margins of the intensity profile of the
soliton, and its effect on the shape is tiny” [31]. OntheNonlinearSchrodingerequation,themodulationinstabilityofthebackground
plane wave motivates to create localized breathers [32]. Of course there are various types
ofthenumericalandanalyticalsolutionsdescribingbreathersrelevanttothedefinitionof
the nonlinear term f
φ
j j2
, but qualitatively breathers can be modeled in a specific
solution [33, 34] as the localized pulses on the continuum background plane wave that: the nonlinear term f
φ
j j2
, but qualitatively breathers can be modeled in a specific
solution [33, 34] as the localized pulses on the continuum background plane wave that: φ ¼
2 1 2a
ð
Þ cosh
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8a 1 2a
ð
Þ
p
τ
þ i
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8a 1 2a
ð
Þ
p
sinh
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8a 1 2a
ð
Þ
p
τ
ffiffiffiffiffi
2a
p
cos 2
ffiffiffiffiffiffiffiffiffiffiffiffiffi
1 2a
p
ξ
cosh
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8a 1 2a
ð
Þ
p
τ
"
#
(3) Where a < 1/2, the solution is Akhmediev breather and for a = 1/2 the solution is
peregrine soliton and for a > 1/2 it is Kuznetsov-Ma breather. Ofcourse amplitude of the
background wave has been deleted here for reality that the background plane wave is
without modulation and thus, the envelopes do work as the independent wave packets. without modulation and thus, the envelopes do work as the independent wave packets. First solution of the NLSE was the Kuznetsov-Ma (KM) breather [35, 36] and we
consider also a Kuznetsov-Ma breather type solution for perturbation of the carrier
wave in deep saturation nonlinearity. This solution does show the localized
l
i
f h b
k
d
h
i di
i p
p
p
First solution of the NLSE was the Kuznetsov-Ma (KM) breather [35, 36] and we
consider also a Kuznetsov-Ma breather type solution for perturbation of the carrier
wave in deep saturation nonlinearity. 2.1 Theoretical frameworks The envelope evolution of nonlinear systems is extracted in the form of
nonlinear Schrodinger equation (NLSE) from different methods such as Fourier-
Mode coupling e.g. [24], or Multiple-Scale analysis [25]. The NLS equation in
nonlinear optics was first derived by Kelley in 1965 using a nonlinear electromag-
netic wave Maxwell’s equation introduced by Chiao et al. one year earlier [26, 27]. Furthermore, Karpman and Krushkal in 1969 derived the NLS equation using
Whitham-Lighthill adiabatic approximation [28]. Consider a system described by the normalized nonlinear Schrodinger equation
[(1 + 1)D NLSE]: i ∂φ
∂t þ 1
2
∂2φ
∂x2 þ f
φ
j j2
φ ¼ 0
(1) (1) 2 Deep Saturation Nonlinearity of 5G Media and Potential Link to Covid-19
DOI: http://dx.doi.org/10.5772/intechopen.98826 2.2 The evidences for the theoretical arguments 5G sideband waves are background carrier wave which in the nonlinearity yields
to the relevant instabilities. The description of instabilities in optics as rogue waves
is recent, however, first used in 2007 when shot-to-shot measurements of fiber
supercontinuum (SC) spectra by Solli et al. yielded long-tailed histograms for
intensity fluctuations at long wavelengths [37]. y
g
g
On the modulation instability of the 5G media, however the 5G monochromatic
waves do not directly affect the cells but producing extremely low frequency enve-
lope impulses which are enable to influence into the cells. The scale invariance in
the deep saturation nonlinearity according to the Eq. (2) yields to the answer as the
full family of the self-similar solutions (intensity in the term φ
j j2) that φ
j j2 ¼
X
þ∞
n¼0
εnφ εnξ, ε2nτ
2
(5) (5) Very short period waves are neglected actually and very long waves are trivial in
short time (in the scale of lesser than several years) and then actually we can
consider a solution as a suit of four consequent envelopes from the Eq. (5) in series
of n ¼ 0, 1, 2, 3
f
g. The time τ is free scale mathematically and we can rescale τ þ π=2 ! The time τ is free scale mathematically and we can rescale τ þ π=2 ! τ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8a 2a 1
ð
Þ
p
. We assume arbitrary the frame center at point ξ ¼ 0 and then by th
Eqs (4) (5) we deduce for n
0 1 2 3
f
g an actual answer as follows The time τ is free scale mathematically and we can rescale τ þ π=2 ! τ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8a 2a
1
ð
Þ
p
We assume arbitrary the frame center at point ξ
0 and then τ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8a 2a 1
ð
Þ
p
. We assume arbitrary the frame center at point ξ ¼ 0 and then by the
Eqs. Deep Saturation Nonlinearity of 5G Media and Potential Link to Covid-19
DOI: http://dx.doi.org/10.5772/intechopen.98826 This solution does show the localized
pulsation of the background wave as the periodic noise. p
y
pulsation of the background wave as the periodic noise. The experimental results in the optical fibers e.g. [5] verify that the Kuznetsov-
Ma breather is matched with real term Re φ
f g and in fact the imaginary term Im φ
f g
can be imaginary and then the physical answer is real term of the Eq. (3), that is, φ ¼
2 1 2a
ð
Þ cos
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8a 2a 1
ð
Þ
p
τ
ffiffiffiffiffi
2a
p
cos 2
ffiffiffiffiffiffiffiffiffiffiffiffiffi
1 2a
p
ξ
cos
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8a 2a 1
ð
Þ
p
τ
"
#
(4) (4) Notice that cosh iτ
ð Þ ¼ cos τð Þ and then in the Kuznetsov-Ma breather solution
in which a > 1/2, we have cosh
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8a 1 2a
ð
Þ
p
τ
¼ cos
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8a 2a 1
ð
Þ
p
τ
. Notice that cosh iτ
ð Þ ¼ cos τð Þ and then in the Kuznetsov-Ma breather solution
in which a > 1/2, we have cosh
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8a 1 2a
ð
Þ
p
τ
¼ cos
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8a 2a 1
ð
Þ
p
τ
. However if we use the φ included to both of the imaginary and real terms, the
solution numerically is still near to the Eq. (4) and difference is neglect-able. 3 Nonlinear Optics - Nonlinear Nanophotonics and Novel Materials for Nonlinear Optics 2.2 The evidences for the theoretical arguments (4), (5) we deduce for n ¼ 0, 1, 2, 3
f
g, an actual answer as follows φ
j j2 ¼
ε3 2 2a 1
ð
Þ sin ε6τ
ffiffiffiffiffi
2a
p
sin ε6τ
ð
Þ
þ ε2 2 2a 1
ð
Þ sin ε4τ
ð
Þ
ffiffiffiffiffi
2a
p
sin ε4τ
ð
Þ
þ ε1 2 2a 1
ð
Þ sin ε2τ
ð
Þ
ffiffiffiffiffi
2a
p
sin ε2τ
ð
Þ
"
þ 2 2a 1
ð
Þ sin τð Þ
ffiffiffiffiffi
2a
p
sin τð Þ
#2 (6) Figure 1. Theoretical wave (self-similar corona-like) solution of covid-19 infection pattern for a ¼ 1, ε ¼ 1=4, kI ¼ 1=5
driven by the Eqs. (6), (7). Fi Figure 1. Figure 1. Theoretical wave (self-similar corona-like) solution of covid-19 infection pattern for a ¼ 1, ε ¼ 1=4, kI ¼ 1=5
driven by the Eqs. (6), (7). 4 4 Deep Saturation Nonlinearity of 5G Media and Potential Link to Covid-19
DOI: http://dx.doi.org/10.5772/intechopen.98826 Deep Saturation Nonlinearity of 5G Media and Potential Link to Covid-19
DOI: http://dx.doi.org/10.5772/intechopen.98826 If corona virus infection cases per time symbolized here by nI relates to 5G
wave’s breather amplitude φ
j j2, thus there should be exist a coefficient kI to corre-
late the intensity of 5G perturbation envelopes to the infection of the corona virus as nI ¼ kI φ
j j2
(7) (7) The infection cases per time interval symbolized here with nI in unit K (cases/
time) is drawn on the Eqs. (6), (7) in Figure 1 with the best fit in ε ¼ 1=4, a = 1,
kI = 1/5 (K in vertical axis and time in horizontal axis in the radians scale). The infection cases per time interval symbolized here with nI in unit K (cases/
time) is drawn on the Eqs. (6), (7) in Figure 1 with the best fit in ε ¼ 1=4, a = 1,
kI = 1/5 (K in vertical axis and time in horizontal axis in the radians scale). Transferring the time from radians to the date, the wave pattern matches nicely
with covid-19 infection pattern reported by live such as www.worldometers.info. 2.2 The evidences for the theoretical arguments The domain and time scales of the wave envelopes match with covid-19 infection Transferring the time from radians to the date, the wave pattern matches nicely
with covid-19 infection pattern reported by live such as www.worldometers.info. The domain and time scales of the wave envelopes match with covid-19 infection
pattern as compared in the Figure 2. Figure 2. Covid-19 infection pattern to date (below diagram) compared to 5G perturbation pulses in the time scaled with
radians (above diagram). Figure 2. Covid-19 infection pattern to date (below diagram) compared to 5G perturbation pulses in the time scaled with
radians (above diagram). Figure 2. Covid-19 infection pattern to date (below diagram) compared to 5G perturbation pulses in the time scaled with
radians (above diagram). 5 Nonlinear Optics - Nonlinear Nanophotonics and Novel Materials for Nonlinear Optics The corona virus is transmitted by human to human biologically and thus, the
biologic spreader effect moderates extremes of the physical waves. For example if
the antennas do not work for a short time, still the covid-19 is continued for
moderation of the biological effects. Of course deviation from the physical source
will appear along the time. In reality the 5G internet media includes the antennas
distributed in the earth and thus, the phase of the perturbation envelopes can vary
by changes in the antennas. y
g
By the way via comparing the covid-19 infection diagram with the wave solution
Eq. (6) along the year 2020, still the covid-19 is matched with the Kuznetsov-Ma
wave solution of the NLSE both in the phase and shape of the wave. We have drawn
the pure wave solution (above diagram in the Figure 2.) without moderation of the
extremes and then difference in the intensity between the diagrams of the wave
solution and covid-19 infection pattern is natural. In reality the above diagram in
the Figure 2 which is for Kuznetsov-Ma breather should be moderated to fit in the
intensity with below diagram in the Figure 2 which is for covid-19 infection cases. The 5G injection effect of the parasite pulses in the cells does not depend to the
cases-age but deaths per time interval symbolized here by nD depends to the Figure 3. Covid-19 death cases pattern (below diagram which is copied from online corona-meter web) compared with
the ϕ2 from Eq. (6). Figure 3. Figure 3. Acknowledgements We need to thanks from human right organizations and scientists and doctors
and generally people for their warnings for health risks by EMF which is increasing
every day and have transferred the earth to an electromagnetic bathroom and
nonlinear dangerous media which increases the health risks globally. 2.2 The evidences for the theoretical arguments Covid-19 death cases pattern (below diagram which is copied from online corona-meter web) compared with
the ϕ2 from Eq. (6). 6 6 Deep Saturation Nonlinearity of 5G Media and Potential Link to Covid-19
DOI: http://dx.doi.org/10.5772/intechopen.98826 Deep Saturation Nonlinearity of 5G Media and Potential Link to Covid-19
DOI: http://dx.doi.org/10.5772/intechopen.98826 cases-age. The index nD=nI is larger for elders. The population of elder cases is
decreased proportionally along the time and then the index nD=nI is flattening
rapidly to asymptotic size 0:02. We find numerically a function for damping
effect of the index nD=nI as follows nD=nI ¼ 0:02 þ
‘1
1 þ 0:3τ þ 0:005τ2
(8) (8) An initial shock wave is observed for death cases in onset of the covid-19
pandemic which is flattening asymptotically to the normal size. This is matched
with daily deaths observed in covid-19 (Figure 3). The wireless antennas have potential to transfer the earth to the nonlinear media
as the source of extremely low frequency (ELF) electromagnetic envelopes (Trojan
horse) affecting the cells, disordering the genome and damaging the species which
may be visible in the next generations the more. 3. Conclusions However the 5G broadband electromagnetic pump waves are blocked mainly
and environmentally disable to pass the skin but on the nonlinearity of 5G internet
media as a result of modulation instability we find extremely low frequency per-
turbation parasites as the solution of nonlinear Schrodinger equation (NLSE),
enable to pass the skin and disordering the genome as the potential link between the
5G and covid-19. Accordingly the global patterns of the corona virus infection and
death cases follow the Kuznetsov-Ma type breather solution of the NLSE which is a
perturbation envelope. In detail we find that the global patterns (infection and
death) of the corona virus is matched with deep saturation nonlinearity of 5G
internet broadband media which yields to a self-similarity of the infection and
death cases global patterns of the corona virus. Conflict of interest The authors declare no conflict of interest. 7 References vicinity of mobile phone base station. Electromagnetic Biology and Medicine. 2017;36(3):295-305 [1] Hong W, Jiang ZH, Yu C, et al. Mint:
Multibeam antenna technologies for 5G
wireless communications. IEEE Tr Ant
Prop. 2017;65(12):6231-6249 [1] Hong W, Jiang ZH, Yu C, et al. Mint:
Multibeam antenna technologies for 5G
wireless communications. IEEE Tr Ant
Prop. 2017;65(12):6231-6249 [9] Russell LC. Mint: 5G wireless
telecommunications expansion: Public
health and environmental implications. Environmental Research. 2018;165:
484-495 [9] Russell LC. Mint: 5G wireless
telecommunications expansion: Public
health and environmental implications. Environmental Research. 2018;165:
484-495 [2] Chou H-T. Mint: Design Metodology
for the Multi-Beam phased Array of
antennas with Relatively Arbitrary
Coverage Sector. Conference paper:
2017 11th European Conference on
Antennas and Propagation; Paris,
France. DOI: 10.23919/
EuCAP.2017.7928095 [10] Obajuluwa AO, Akinyemi AJ,
Afolabi OB, et al. Mint: Exposure to
radio-frequency electromagnetic waves
alters acetylcholinesterase gene
expression, exploratory and motor
coordination-linked behaviour in male
rats. Toxicol Rep. 2017;4:530-534 [3] Dudley JM, Genty G, Dias F,
Kibler B, Mint AN. Modulation
instability, Akhmediev Breathers and
continuous wave supercontinuum
generation. Opt. Express. 2009;17:
21497-21508 [11] Kostoff RN, Heroux P, Aschner M,
Tsatsakis A. Mint: Adverse health
effects of 5G mobile networking
technology under real-life conditions. 2020;323, 35-40 [11] Kostoff RN, Heroux P, Aschner M,
Tsatsakis A. Mint: Adverse health
effects of 5G mobile networking
technology under real-life conditions. 2020;323, 35-40 [4] Van Simaeys G, Emplit Ph, &
Haelterman M. Mint: Experimental
demonstration of the Fermi-Pasta-Ulam
recurrence in a modulationally unstable
optical wave. Phys. Rev. Lett. 2001;87,
033902 [12] Mint DCA. Towards 5G
communication systems: are there
health implications? Int J Hyg Environ
Health. 2018;221(3):367-375 [12] Mint DCA. Towards 5G
communication systems: are there
health implications? Int J Hyg Environ
Health. 2018;221(3):367-375 [5] Kibler B, Fatome J, Finot C, Millot G,
Dias F, Genty G, et al. Mint: The
Peregrine soliton in nonlinear fibre
optics. Nature Physics. 2010;6:795 [13] Hardell L, Mint SC. Biological effects
from electromagnetic field exposure and
public exposure standards. Biomed
Pharmacother. 2008;62:104-109 [13] Hardell L, Mint SC. Biological effects
from electromagnetic field exposure and
public exposure standards. Biomed
Pharmacother. 2008;62:104-109 [6] Thielens A, Bell D, Mortimore DB,
Greco MK, Martens L, Mint JW. Exposure of Insects to Radio-Frequency
Electromagnetic Fields from 2 to 120
GHz. Scientific Reports. 2018;8:3924 [14] Eghlidouspour M, Ghanbari A,
Mortazavi S, Mint AH. Author details
Mohsen Lutephy
Islamic Azad University (IAU), Tehran, Iran
*Address all correspondence to: lutephy@gmail.com
© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/3 0) which permits unrestricted use distribution and reproduction in any medium
Nonlinear Optics - Nonlinear Nanophotonics and Novel Materials for Nonlinear Optics Nonlinear Optics - Nonlinear Nanophotonics and Novel Materials for Nonlinear Optics © 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited. 8 Deep Saturation Nonlinearity of 5G Media and Potential Link to Covid-19
DOI: http://dx.doi.org/10.5772/intechopen.98826 Deep Saturation Nonlinearity of 5G Media and Potential Link to Covid-19
DOI: http://dx.doi.org/10.5772/intechopen.98826 Deep Saturation Nonlinearity of 5G Media and Potential Link to Covid-19
DOI: http://dx.doi.org/10.5772/intechopen.98826 [7] Yakymenko I, Tsybulin O, Sidorik E,
Henshel D, Kyrylenko O, Mint KS.
Oxidative mechanisms of biological
activity of low-intensity radiofrequency
radiation. Electromagnetic Biology and
Medicine. 2016;35(2):186-202 [15] Grigoriev Y. Mint: Bioeffcets of
modulated electromagentci fields in the
acute experiments. Annu Russ Natl
Comm Non-Ionising Radiat Protect.
2004:16-73 [12] Mint DCA. Towards 5G
communication systems: are there
health implications? Int J Hyg Environ
Health. 2018;221(3):367-375 References Effects of
radiofrequency exposure emitted from a
GSM mobile phone on proliferation,
differentiation, and apoptosis of neural
stem cells. Anat Cell Biol. 2017;50(2):
115-123 [7] Yakymenko I, Tsybulin O, Sidorik E,
Henshel D, Kyrylenko O, Mint KS. Oxidative mechanisms of biological
activity of low-intensity radiofrequency
radiation. Electromagnetic Biology and
Medicine. 2016;35(2):186-202 [15] Grigoriev Y. Mint: Bioeffcets of
modulated electromagentci fields in the
acute experiments. Annu Russ Natl
Comm Non-Ionising Radiat Protect. 2004:16-73 [8] Zothansiama ZM, Lalramdinpuii M,
Mint JGC. Impact of radiofreqiuency
radiation on DNA damage and
antioxidants in peripheral blood
lymphocytes of humans residing in the [16] Zakharov VE. Zh. Eksp. Teor. Fiz. 62, 1745. Sov. Phys. JETP. 1972;35:908 9 Nonlinear Optics - Nonlinear Nanophotonics and Novel Materials for Nonlinear Optics [17] Hasegawa, A. Phys. Rev. 1970;A 1,
1746 [29] Infeld E and Rowlands G. Mint:
Nonlinear Waves, Solitons, and Chaos. Cambridge University Press,
Cambridge, England. 1990; Chap. 5
(in particular, p. 127. [29] Infeld E and Rowlands G. Mint:
Nonlinear Waves, Solitons, and Chaos. Cambridge University Press,
Cambridge, England. 1990; Chap. 5
(in particular, p. 127. [19] Malomed B. Mint: Nonlinear
Schrodinger equation. In Scott, Alwyn
(ed), Encyclopedia of Nonlinear
Science, New York: Routledge, 2005;
pp. 639-643. [30] Segev M, Stegeman G. Phys. Today
51 (8), 42 (1998); M. Segev. Opt. Quantum Electron. 1988;30:503 [31] Soljacic MM, Segev and Menyuk CR. Mint: Self-similarity and fractals in
soliton-supporting systems. Physical
Review E. 2000; Volume (61), No.2. [20] Benjamin TB, Feir JE. Mint: The
disintegration of Wa-vetrains on deep
water. Part 1. J. Fluid Mech. 1967;27:
417-430 [32] Akhmediev N, Ankiewicz AS. Non-
linear pulses and beams. Chapman &
Hall; 1997 [21] Onorato M, Proment D, Clauss G,
Klein M. Mint: Roque Waves: From
Nonlinear Schrodinger Breather
Solutions to Sea-Keeping Test. PLoS
ONE. 2013;8(2): e54629. DOI:10.1371/
journal.pone.0054629 [33] Akhmediev NN, Mint KVI. Modulation instability and periodic
solutions of the nonlinear Schrödinger
equation. Theor. Math. Phys. 1986;69:
1089-1093 [22] Tai K, Hasegawa A, Tomita A. Mint:
observation of moulational instability in
optical fibers. Phys. Rev. Lett. 1986;56:
135-138 [34] Akhmediev NN, Eleonski VM,
Kulagin NE. Mint: First-oeder exact
solutions of the nonlinear Schrodinger
equation”. Theoritical and Mathematical
Physics. 1987;72 (2): 809-818. DOI:
10.1007/BF01017105 [23] Mint AGP. Nonlinear Fiber Optics. Academic Press; 2013 [35] Mint KE. Solitons in a
parametrically unstable plasma. Sov. Phys. Dokl. 1977;22:507-508 [24] Zakharov VE. L’vov VS, and
Falkovich C. Mint: Kolmogorov spectra
of turbulence Ι. Wave turbulence. References Springer-Verlag; 1992 [36] Ma YCM. The perturbed plane-
wave solutions of the cubic
Schr¨odinger equation. Studies in
Applied Mathematics. 1979;60:43-58 [36] Ma YCM. The perturbed plane-
wave solutions of the cubic
Schr¨odinger equation. Studies in
Applied Mathematics. 1979;60:43-58 [25] Newell AC. Mint: Solitons in
Mathematics and Physics. CBMS-NSF
Regeional Conference. 1985;15:157-163 [26] Chiao RY, Garmire E, Mint TCH. Self-trapping of optical beams. Physical
Review Letters. 1964;13:479 [37] Solli DR, Ropers C, Koonath P,
Mint JB. Optical rogue waves. Nature. 2007;450:1054-1057 [27] Mint KP. Self-focusing of optical
beams. Physical Review Letters. 1965;15:
1005 [28] Karpman VI, Krushkal EM. Mint:
Modulated waves in nonlinear
dispersive media. Soviet Physics JETP. 1969;28:277 10
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Classification of Human Motions Using Micro-Doppler Radar in the Environments with Micro-Motion Interference
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Received: 5 May 2019; Accepted: 4 June 2019; Published: 7 June 2019 Received: 5 May 2019; Accepted: 4 June 2019; Published: 7 June 2019 Abstract: Human motion classification based on micro-Doppler effect has been widely used in various
fields. However, the motion classification performance would be greatly degraded if the wireless
environment has non-target micro-motion interference. In this case, the interference signal aliases with
the signal of target human motions and then generates cross-terms, making the signals hard to be used
to identify target human motions. Existing methods do not consider this non-target micro-motion
interference and have poor resistance to such interference. In this paper, we propose a target human
motion classification system that can work in the scenarios with non-target micro-motion interference. Specifically, we build a continuous wave radar transceiver working in a low-frequency radar band
using the software defined radio equipment Universal Software Radio Peripheral (USRP) N210 to
collect signals. Moreover, we use Empirical Mode Decomposition and S-transform successively to
remove non-target micro-motion interference and improve the time-frequency resolution of the raw
signal. Then, an Energy Aggregation method based on S-method is proposed, which can suppress
cross-terms and background noise. Furthermore, we extract a set of features and classify four human
motions by adopting Bagged Trees. Extensive experiments using the test-bed show that under the
scenarios with non-target micro-motion interference, 97.3% classification accuracy can be achieved. Keywords: human motion classification; non-target micro-motion interference; micro-Doppler effect;
continuous wave radar sensors sensors sensors Sensors 2019, 19, 2598; doi:10.3390/s19112598 www.mdpi.com/journal/sensors Xiaolin Ma 1,*, Running Zhao 1, Xinhua Liu 1
, Hailan Kuang 1
and Mohammed A. A. Al-qaness 2 1
Key Laboratory of Fiber Optical Sensing Technology and Information Processing, Ministry of Education,
and Hubei Key Laboratory of Broadband Wireless Communication and Sensor Networks, School of
Information Engineering, Wuhan University of Technology, Wuhan 430070, China;
zhaorunning@whut.edu.cn (R.Z.); liuxinhua@whut.edu.cn (X.L.); kuanghailan@whut.edu.cn (H.K.)
2
School of Computer Science, Wuhan University, Wuhan 430072, China; alqaness@whu.edu.cn
*
Correspondence: maxiaolin0615@whut.edu.cn; Tel.: +86-27-8729-0335 1
Key Laboratory of Fiber Optical Sensing Technology and Information Processing, Ministry of Education,
and Hubei Key Laboratory of Broadband Wireless Communication and Sensor Networks, School of
Information Engineering, Wuhan University of Technology, Wuhan 430070, China;
zhaorunning@whut.edu.cn (R.Z.); liuxinhua@whut.edu.cn (X.L.); kuanghailan@whut.edu.cn (H.K.)
2
School of Computer Science, Wuhan University, Wuhan 430072, China; alqaness@whu.edu.cn
*
Correspondence: maxiaolin0615@whut.edu.cn; Tel.: +86-27-8729-0335 *
Correspondence: maxiaolin0615@whut.edu.cn; Tel.: +86-27-8729-0335 Classification of Human Motions Using
Micro-Doppler Radar in the Environments
with Micro-Motion Interference Xiaolin Ma 1,*, Running Zhao 1, Xinhua Liu 1
, Hailan Kuang 1
and Mohammed A. A. Al-qaness 2 Xiaolin Ma 1,*, Running Zhao 1, Xinhua Liu 1
, Hailan Kuang 1
and Mohammed A. A. Al-qaness 2 1. Introduction Based on the spectrogram obtained by STFT, they extracted
motion features for classification, in which the accuracy is 91.9%. ,
y
The aforementioned studies have high requirements on the equipment, and most of them use
high-frequency radar band or multistatic radars. As the radar frequency increases, the resolution will
increase, and the resolution of the time-frequency distribution obtained by time-frequency analysis
will also increase. In this case, it is easier to distinguish the motion state of single or multiple people. Additionally, increasing the number of radars can result in richer echo signals of moving human
bodies, so better classification performance can be obtained. However, both increasing the carrier
frequency of radar and increasing the number of radars require high costs on hardware. On the
other hand, some works [16–21] focus on feature extraction. Under the premise of reducing the
radar frequency appropriately, the accuracy of motion states classification can be ensured by selecting
proper features or adopting effective feature extraction methods. Fairchild et al. [16] used S-band
radar and W-band millimeter wave radar to identify four types of motions, namely human walking,
standing, bending, and swinging, in cases of through-wall and non-through-wall environments. In the
work, the Empirical Mode Decomposition (EMD) was used to produce a unique feature vector from
the human micro-Doppler signals, following which a Support Vector Machine (SVM) was used to
classify human motions, and the accuracy rates were 75% and 90%, respectively. Also, there are many
other methods [17–20] that can be used in human motion classification, such as Linear Predictive
Coding (LPC), Singular Value Decomposition, and Hierarchical Image Classification Architecture. In a
previous study [21], researchers used Information-Theoretic (IT) feature selection techniques to identify
essential features and minimize the total number of required features, while maximizing classification
performance. Results show that for signal-noise-ratios over 10 dB and at least 1 s of data, this approach
yields 96% correct classification. However, the echo signals of the human body acquired in the low-frequency radar band
have low time-frequency aggregation and resolution, and high classification accuracy cannot be
guaranteed by the improvement of feature extraction only. Therefore, some researchers improved the
aggregation degree in the time-frequency domain through time-frequency processing and improved the
resolution of the time-frequency distribution to facilitate feature extraction for subsequent classification
procedures. 1. Introduction Human motion detection and classification systems have been widely used due to the increased
demands on social security and surveillance systems. These systems can be implemented by various
techniques [1], such as image-based or video-based techniques, and wireless radar-based techniques. Compared to image-based or video-based techniques, radar-based human motion detection and
classification systems can work in scenarios regardless of light, in smoke and dust conditions, and even
more, some have through-wall capabilities [2,3]. Human motion can cause frequency shift of a radar echo signal, and then produce corresponding
Doppler signatures. Thus, Doppler radars can be used to detect human motion. Chen et al. [4] proposed
the concept that an object or any structures on the object may have mechanical vibrations or rotations,
called micro-motion dynamics. Micro-motion dynamics may induce additional frequency modulations
on the returned radar signal, which generate sidebands about the target’s Doppler frequency, called Sensors 2019, 19, 2598; doi:10.3390/s19112598 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors 2 of 19 Sensors 2019, 19, 2598 the micro-Doppler effect. A micro-Doppler signal can be regarded as a unique signature of a motion
aroused by the human body. Therefore, it can be applied to human motion classification [5–12]. However, as the complexity of the applied environment increases, non-target (i.e., not the target human
body) micro-motion interference may appear. Such an interference signal can alias with the signal of the
target human motion, and then generate cross-terms, which means the collected signals cannot reflect
the real body motion of the target human. Then, systems applied in such scenarios would be much
more complex than those for single human body scenarios without any micro-motion interference. Fioranelli et al. [13] used multiple sets of X-band continuous wave radars to collect the signal
associated with human motion. Then, they extracted features based on Short-Time Fourier Transform
(STFT) and classified the unarmed and potentially armed personnel. The experimental results showed
that the three-node multistatic system has higher classification accuracy than that of the single
monostatic radar. In a previous study [14], researchers used the Frequency Modulated Continuous
Wave (FMCW) radar system with a carrier frequency of 77 GHz to analyze the human micro-Doppler
signatures of one or two persons. In a previous study [15], authors used a continuous wave radar
system with a carrier frequency of 24 GHz to detect three types of human motions: two-arm motion,
one-arm motion, and no-arm motion. 1. Introduction For instance, a Hermite multi-window algorithm based on S-method was proposed in
previous studies [22,23], which used a few first orders of Hermite functions to improve time-frequency
aggregation. Then, they extracted features to classify human gait. In a previous study [24], the authors
used the multiwindow Adaptive S-method (AS-method) to analyze radar echoes collected in indoor
and outdoor non-interference environments. They separated six states of human motions using the
SVM classifier trained by the extracted features. The trained SVM can detect a human body with an
accuracy of 95.4% for the two cases without interference. Although these methods can classify human Sensors 2019, 19, 2598 3 of 19 motions under the low-frequency radar band using the time-frequency analysis, all of the signals were
collected in the environments without non-target interference. If micro-motion interference aroused by
a non-target human body exists, the interference signal would alias with the signal of target human
motion and then generate cross-terms. The existing studies cannot effectively resist such interference
or reduce its influence on the signal of the target human motion. Therefore, the time-frequency
distribution after the time-frequency processing cannot reflect the real body motion of the target human,
and misjudgment is likely to occur at the procedure of classification. In this paper, we propose a target human motion classification system working in the low-frequency
radar band, suitable for non-target micro-motion interference, which can achieve accurate classification
of the target human motions in the scenarios with the interference from non-target micro-motion. Particularly, we build a continuous wave radar transceiver with a carrier frequency of 4.2 GHz for radar
echo data acquisition of human motion in cases of existing non-target micro-motion interference. In the
non-target micro-motion interference scenarios, the interference signal aliases with the signal of target
human motion and then generates cross-terms. In order to achieve accurate classification of human
motion under low-frequency radar bands in the case of micro-motion interference from non-targets,
we design a time-frequency analysis scheme to solve issues on cross-terms and time-frequency resolution. Firstly, we use an Empirical Mode Decomposition (EMD) algorithm reconstructing the collected raw
signal to filter out the non-target micro-motion interference and adopt S-transform to improve the
time-frequency resolution. Then, a new method of Energy Aggregation is obtained by optimizing
the classical S-method, which can suppress cross-terms and background noise. 1. Introduction The resolution of the
time-frequency distribution obtained after the time-frequency analysis scheme above is sufficient for
subsequent feature extraction. Finally, three effective features are extracted, and four motion states are
classified by Bagged Trees [25]. The main contributions in this paper can be summarized as follows: The main contributions in this paper can be summarized as follows: 1. This paper proposes a target human motion classification system that can work in the scenarios
with non-target micro-motion interference. Under the non-target micro-motion interference
caused by a non-target human body, the proposed system can accurately classify four different
motion states, including walking without any arm swinging, walking with one arm swinging,
walking with both arms swinging, and running. 2. This paper designs a time-frequency analysis scheme, which can (1) filter out non-target
micro-motion interference, (2) improve the time-frequency resolution of the collected signal,
and (3) suppress the negative influence of cross-terms and background noise. 3. This paper proposes a new method of Energy Aggregation based on the classical S-method. By using S-transform instead of STFT, and increasing the weight of time-frequency point energy
spectral density, we greatly improve the time-frequency resolution and mitigate the effect caused
by background noise, while maintaining the ability of the S-method in terms of cross-terms
suppression and time-frequency aggregation enhancement. 4. This paper uses the software defined radio equipment Universal Software Radio Peripheral
(USRP) N210 and GNU Radio software tool-kits [26] to construct the continuous wave radar
system. Our experiments show the high performance of the proposed system in terms of its
interference resistance and classification accuracy under various experimental scenarios. 2.1. System Overview In this paper, we propose a target human motion classification system that can work in the scenarios
with non-target micro-motion interference. In the scenarios with non-target micro-motion interference,
the proposed system can accurately classify four different human motions, i.e., walking without any
arm swinging, walking with one arm swinging, walking with both arms swinging, and running. 4 of 19 Sensors 2019, 19, 2598 As shown in Figure 1, the proposed system consists of three parts, as follows: Micro-Motion Signal
Collection, Micro-Doppler Signal Analysis, and Feature Extraction and Motion State Classification. Sensors 2019, 19, x FOR PEER REVIEW
4 of 19
Signal Collection, Micro-Doppler Signal Analysis, and Feature Extraction and Motion State As shown in Figure 1, the proposed system consists of three parts, as follows: Micro-Motion Signal
Collection, Micro-Doppler Signal Analysis, and Feature Extraction and Motion State Classification. e
o
,
,
Signal Collection, Micro-Doppler Signal Analysis, and Feature Extraction and Motion State 1. Micro-Motion Signal Collection: The micro-motion signals are collected by continuous wave
radar transceivers. Such signals are radar echoes and they are represented by discrete
complex signals. The transceivers are built on a test-bed using USRP N210 and GNU
Radio. USRP N210 is a software-defined-radio equipment, and GNU Radio is an open-source
software-defined-radio tool-kit. Classification. 1. Micro-Motion Signal Collection: The micro-motion signals are collected by continuous wave
radar transceivers. Such signals are radar echoes and they are represented by discrete complex
signals. The transceivers are built on a test-bed using USRP N210 and GNU Radio. USRP N210
is a software-defined-radio equipment, and GNU Radio is an open-source software-defined-
di
t
l kit 2. Micro-Doppler Signal Analysis: We use the micro-Doppler signal analysis module performing
time-frequency processing on the raw data, so as to (1) filter out the micro-motion interference
generated by the non-target human body, and then (2) improve the time-frequency resolution,
(3) suppress the cross-terms and mitigate the effect caused by background noise. In this way,
the influence of non-target micro-motion interference on the target human motion can be
reduced significantly. Firstly, we use the EMD algorithm to decompose the raw signal into
multiple time-frequency components and then select the appropriate components for signal
reconstruction, which can filter out the interference. Then, we use S-transform to perform
time-frequency analysis on the reconstructed signal. 2.1. System Overview The frequency resolution of the S-transform
can be automatically adjusted as the frequency changes, so it can increase the time-frequency
resolution of the reconstructed signal. Finally, we use Energy Aggregation to process the signal
after the S-transform to suppress cross-terms and mitigate the effect caused by background noise. The signal processed by the above methods of time-frequency analysis reduces the influence of
non-target micro-motion interference and generates a high-resolution time-frequency distribution
that reflects the real body motion of the target human. radio tool-kit. 2. Micro-Doppler Signal Analysis: We use the micro-Doppler signal analysis module performing
time-frequency processing on the raw data, so as to (1) filter out the micro-motion interference
generated by the non-target human body, and then (2) improve the time-frequency resolution,
(3) suppress the cross-terms and mitigate the effect caused by background noise. In this way, the
influence of non-target micro-motion interference on the target human motion can be reduced
significantly. Firstly, we use the EMD algorithm to decompose the raw signal into multiple time-
frequency components and then select the appropriate components for signal reconstruction,
which can filter out the interference. Then, we use S-transform to perform time-frequency
analysis on the reconstructed signal. The frequency resolution of the S-transform can be
automatically adjusted as the frequency changes, so it can increase the time-frequency resolution
of the reconstructed signal. Finally, we use Energy Aggregation to process the signal after the S-
transform to suppress cross-terms and mitigate the effect caused by background noise. The
signal processed by the above methods of time-frequency analysis reduces the influence of non-
target micro-motion interference and generates a high-resolution time-frequency distribution
that reflects the real body motion of the target human 3. Feature Extraction and Motion State Classification: We extract a set of features, i.e., Doppler
bandwidth, Micro-Doppler bandwidth and Effective arm-swing time difference, from the
time-frequency distribution obtained in the Micro-Doppler Signal Analysis part. Further,
we adopt ensemble learning by combining bagging and the decision tree, and accurately classify
the four motion states, including walking without any arm swinging, walking with one arm
swinging, walking with both arms swinging, and running, under the scenarios with non-target
micro-motion interference. that reflects the real body motion of the target human. 3. 2.1. System Overview Feature Extraction and Motion State Classification: We extract a set of features, i.e., Doppler
bandwidth, Micro-Doppler bandwidth and Effective arm-swing time difference, from the time-
frequency distribution obtained in the Micro-Doppler Signal Analysis part. Further, we adopt
ensemble learning by combining bagging and the decision tree, and accurately classify the four
motion states, including walking without any arm swinging, walking with one arm swinging,
walking with both arms swinging, and running, under the scenarios with non-target micro-
motion interference 3.3 Figure 1. Architecture of the proposed system. Figure 1. Architecture of the proposed system. Figure 1. Architecture of the proposed system. Figure 1. Architecture of the proposed system. 2.2. Micro-Motion Signal Collection
2.2. Micro-Motion Signal Collection 2.2. Micro-Motion Signal Collection
2.2. Micro-Motion Signal Collection Continuous wave radar has a simple structure and low requirements on the equipment. Software radio technology has strong flexibility and has great advantages in terms of being open
Continuous wave radar has a simple structure and low requirements on the equipment. Software
radio technology has strong flexibility and has great advantages in terms of being open source and low 5 of 19 Sensors 2019, 19, 2598 cost. Therefore, we build a continuous-wave radar transceiver using USRP N210 and GNU Radio. Among them, USRP N210 is a software defined radio equipment, and GNU Radio is an open-source
software development toolkit. More details about USRP N210 can be found in the Section 3.1
Experiment Testbed. We use such a transceiver as the front-end sub-system for micro-motion signal
sensing and collection. The main components of the transceiver are shown in Figure 2. Sensors 2019, 19, x FOR PEER REVIEW
5 of 19
Section 3.1 Experiment Testbed. We use such a transceiver as the front-end sub-system for micro-
motion signal sensing and collection. The main components of the transceiver are shown in Figure 2. Figure 2. Main components of the continuous-wave radio transceiver. Figure 2. Main components of the continuous-wave radio transceiver. Figure 2. Main components of the continuous-wave radio transceiver. Figure 2. Main components of the continuous-wave radio transceiver. The continuous wave signal source module generates a digital continuous wave signal. In order
to facilitate the analysis of the signal, the discrete continuous wave signal takes the form of a complex
signal. In the Field Programmable Gate Array (FPGA) chip of the USRP1 motherboard, the complex
signal is up-converted from the digital baseband signal to the intermediate frequency (IF) signal. The
digital IF signal is then converted into an analog IF signal by the Digital-to-Analog Converter (DAC)
module. Finally, the analog IF signal is up-converted to the analog radio frequency (RF) signal
through the RF front end in the USRP1 daughter board, and the antenna completes the transmission
of the electromagnetic wave signal. The continuous wave signal source module generates a digital continuous wave signal. In order
to facilitate the analysis of the signal, the discrete continuous wave signal takes the form of a complex
signal. In the Field Programmable Gate Array (FPGA) chip of the USRP1 motherboard, the complex
signal is up-converted from the digital baseband signal to the intermediate frequency (IF) signal. 2.3. Micro-Doppler Signal Analysis
2.3. Micro-Doppler Signal Analysis The raw data gathered from the micro-motion signal collection module contains non-target
micro-motion interference. Also, we use the low-frequency band radar transceiver to collect the
human echo signals, so the time-frequency resolution of the raw signal is poor and often accompanied
by cross-terms. Thus, this section focuses on removing non-target micro-motion interference,
improving time-frequency signal resolution, suppressing cross-terms, and mitigating the effect
caused by background noise
The raw data gathered from the micro-motion signal collection module contains non-target
micro-motion interference. Also, we use the low-frequency band radar transceiver to collect the human
echo signals, so the time-frequency resolution of the raw signal is poor and often accompanied
by cross-terms. Thus, this section focuses on removing non-target micro-motion interference,
improving time-frequency signal resolution, suppressing cross-terms, and mitigating the effect caused
by background noise. 2.2. Micro-Motion Signal Collection
2.2. Micro-Motion Signal Collection The digital IF signal is then converted into an analog IF signal by the Digital-to-Analog Converter
(DAC) module. Finally, the analog IF signal is up-converted to the analog radio frequency (RF) signal
through the RF front end in the USRP1 daughter board, and the antenna completes the transmission of
the electromagnetic wave signal. of the electromagnetic wave signal. The signal received by the antenna passes through the RF front end, the Analog-to-Digital
Converter (ADC) module and the FPGA module in the USRP2 sequentially, and finally becomes the
digital baseband signal. At the same time, the signal transceiver interface realizes synchronization of
continuous wave radar transmission and reception to remove the delay of the device. The transmit
and receive signals after synchronization are mixed to filter out the high frequency carrier. Eventually, the signal reduces data redundancy by resampling, and obtains an effective signal for
adapting to PC-side data processing. The signal received by the antenna passes through the RF front end, the Analog-to-Digital
Converter (ADC) module and the FPGA module in the USRP2 sequentially, and finally becomes the
digital baseband signal. At the same time, the signal transceiver interface realizes synchronization of
continuous wave radar transmission and reception to remove the delay of the device. The transmit
and receive signals after synchronization are mixed to filter out the high frequency carrier. Eventually,
the signal reduces data redundancy by resampling, and obtains an effective signal for adapting to
PC-side data processing. Eventually, the signal red
adapting to PC-side data
PC-side data processing. 2 3 1 Empirical Mode Decomposition
2.3.1. Empirical Mode Decomposition 2.3.1. Empirical Mode Decomposition
We use Empirical Mode Decomposition to remove non-target micro-motion interference in the
raw data. The EMD is an adaptive time-frequency technique that is well-suited for non-linear and
non-stationary time series. One advantage of EMD is that it does not require any prior knowledge of
the input signal and is not affected by the selection of a kernel function. Therefore, EMD is widely
d i
th fi ld f i
l
i
[27]
We use Empirical Mode Decomposition to remove non-target micro-motion interference in the
raw data. The EMD is an adaptive time-frequency technique that is well-suited for non-linear and
non-stationary time series. One advantage of EMD is that it does not require any prior knowledge of
the input signal and is not affected by the selection of a kernel function. Therefore, EMD is widely
used in the field of signal processing [27]. used in the field of signal processing [27]. EMD decomposes a signal into its Intrinsic Mode Functions (IMFs) based on the time-scale of
the oscillations. Each IMF satisfies the following two conditions: (1) the number of extrema and the
number of zero crossing must be equal or differ at most by one and (2) the mean value must be zero
EMD decomposes a signal into its Intrinsic Mode Functions (IMFs) based on the time-scale of
the oscillations. Each IMF satisfies the following two conditions: (1) the number of extrema and the
number of zero crossing must be equal or differ at most by one, and (2) the mean value must be zero. Sensors 2019, 19, 2598 6 of 19 The faster oscillations modes in the signal will occur in the lower-indexed IMFs, whereas the slower
oscillation modes will appear in the higher-indexed IMFs. These oscillatory modes are components
of the original signal, with the useful feature that each IMF is orthogonal to all of the other IMFs. The orthogonality of the IMFs allows EMD to be used in a number of ways, for example filtering
signals, i.e., we can perform signal filtering by selectively adding some of the IMF signals together
while omitting others. The EMD algorithm consists mainly of a sifting process, which is described next. First, we identify
the extrema of the signal x(t). Using the extrema, the envelope of the minima, denoted as Xmin(t),
and the envelope of the maxima, denoted as Xmax(t), are formed by using cubic spline interpolation. 2 3 1 Empirical Mode Decomposition
2.3.1. Empirical Mode Decomposition Next, the average of the upper and lower envelopes is calculated as: Xenv(t) = Xmax(t) + Xmin(t)
2
,
(1) (1) We subtract the average curve Xenv(t) from the signal x(t) to get the local oscillation mode h1(t): h1(t) = x(t) −Xenv(t),
(2) (2) Here, if h1(t) cannot satisfy the above criteria to be an IMF, we replace h1(t) with x(t) and repeat
the above process until hk(t) satisfies the criteria, where k is the repeating round. If hk(t) is an IMF,
we save it as C1(t): C1(t) = hk(t),
(3) ( )
C1(t) = hk(t),
(3 (3) We subtract C1(t) from the signal x(t) to obtain the residue r1(t), which can be expressed as: We subtract C1(t) from the signal x(t) to obtain the residue r1(t), which can be expressed as: r1(t) = x(t) −C1(t),
(4) (4) If r1(t) is not a monotonic function, we repeat the overall procedures by setting x(t) = rn(t) and
increase n by one, where n is the repeating round. On the contrary, if rn(t) is a monotonic function,
the process is completed. All IMFs and residue component can be expressed as follows: x(t) = C1(t) + C2(t) + . . . + Cn(t) + rn(t),
(5) (5) By observing the time-frequency distribution of the raw data, we can find that the time-scale of
the oscillation caused by non-target micro-motion interference lies between the target human body
swing arm and the target human trunk motion. Therefore, we remove some index IMFs corresponding
to non-target micro-motion interference, and then add together the other index IMFs to reconstruct
the signal. The time-frequency distributions of the raw signal before and after using EMD processing is
shown in Figure 3. As shown in Figure 3a, the interference signal aliases with the signal of target human
motions, making the signals hard to use to identify target human motions. It can be seen obviously
from Figure 3b that after using EMD processing, most of the non-target micro-motion interferences are
filtered out, while other useful components are retained. However, the time-frequency resolution of
time-frequency distribution is poor, which is not convenient for subsequent feature extraction. 7 of 19
bsequent Sensors 2019, 19, 2598
frequency resolu
feature extraction (a)
(b)
Figure 3. (a) Time-frequency distribution of the raw signal before using Empirical Mode
Decomposition (EMD) processing; (b) time-frequency distribution of the raw signal after using EMD
processing. Figure 3. 2 3 1 Empirical Mode Decomposition
2.3.1. Empirical Mode Decomposition (a) Time-frequency distribution of the raw signal before using Empirical Mode Decomposition
(EMD) processing; (b) time-frequency distribution of the raw signal after using EMD processing. 3.2. S-transform (a) (b) (b) (a) Figure 3. (a) Time-frequency distribution of the raw signal before using Empirical Mod
Decomposition (EMD) processing; (b) time-frequency distribution of the raw signal after using EMD
i
Figure 3. (a) Time-frequency distribution of the raw signal before using Empirical Mode Decomposition
(EMD) processing; (b) time-frequency distribution of the raw signal after using EMD processing. 2 S t
f processing.
2.3.2. S-transform However, the effect caused 8 of 19
he time-
used by Sensors 2019, 19, 2598
As shown in
f
l by cross-terms and background noise is so serious that it is hard to separate the effective signal to
extract features. cross-terms and background noise is so serious that it is hard to separate the effective signal to extract
features. Figure 4. Time-frequency distribution of the signal processed by S-transform. Figure 4. Time-frequency distribution of the signal processed by S-transform. Figure 4. Time-frequency distribution of the signal processed by S-transform
Figure 4. Time-frequency distribution of the signal processed by S-transform. processing.
2.3.2. S-transform The S-transform is used to improve signal time-frequency resolution. Extracting pertinent
information from noisy, multi-component, and non-stationary signals with complex backgrounds is a
challenge in the field of signal processing. The Fourier transform can effectively process time-invariant
signals. However, it does not reflect the local time information of the signal, so it has certain limitations
on the analysis of time-variant or non-stationary signals. To solve these problems, many time-frequency
analysis methods begin to process non-stationary signals in the joint time-frequency domain. Since
the micro-Doppler radar echo signal of the human body is a non-stationary signal, we can use the
time-frequency analysis method for human-body micro-Doppler signal analysis. The S-transform is a linear transform with the advantages of Short-Time Fourier Transform
and Wavelet Transform. It uses a variable analyzing window width and preserves the phase
information, which allows its frequency resolution to be adjusted automatically as the frequency
changes, with high time resolution in the high frequency band and low-frequency resolution in the
low-frequency band. Thus, it can overcome the shortcoming of STFT fixed resolution and maintain
multi-resolution characteristics [28]. Derived from the STFT, the standard S-transform ST(t, f) of a time varying signal x(t) is given by ST(t, f) =
+∞
Z
−∞
x(t)ω(t −τ)e−j2πftdt,
(6) (6) ω(t) is a Gaussian window centered at t = 0 and used to extract a signal segment of x(t). It is
d as: where ω(t) is a Gaussian window centered at t = 0 and used to extract a signal segment of x(t). It is
defined as:
f
f2 2 ω(t) =
f
√
2π
e−f2t2
2 ,
(7) (7) (7) A constraint is added to restrict the window width σ = 1/
f
to be a function of the frequency. The window width σ varying inversely with frequency makes the S-transform performing a
multi-resolution analysis on the signal. h
i Then, the S-transform of the discrete signal x[kT] can be defined as ST
h
kT,
n
NT
i
: ST
kT, n
NT
=
N−1
X
m=0
X
m + n
NT
e−2π2m2
n2
e
j2πmk
N ,
(8) (8) where N is the total number of sampling points and T is the time domain sampling interval. As shown in Figure 4, the signal processed by S-transform can effectively improve the
time-frequency resolution of the signal and maintain multi-resolution. 2.3.3. Energy Aggregation based on S-method
2.3.3. Energy Aggregation based on S-method The S-method proposed by Stankovic et al. [29] is widely used in signal processing, which ensures
the time-frequency aggregation of multi-component signals and suppresses cross-terms. However,
S-method has limited improvement in the time-frequency resolution of the signal. Additionally,
when unfiltered non-target micro-motion interference signals appear near the effective signal, which are
difficult to filtered, processing the collected signal with S-method can cause the effective signal to be
flooded by the background noise signal, thereby making the separation of the effective signal from
the collected signal harder. Therefore, we propose a new method based on S-method, named Energy
Aggregation, to solve such problems in S-method and maintain its advantages. The details are as below. Firstly, we briefly summarize the idea of S-method. S-method is proposed to suppress the
cross-terms caused by the Winger-Ville distribution. The S-method distribution SM(t, ω) for the
continuous signal x(t) is defined as: SM(t, ω) =
Z +∞
−∞
P(θ)STFT(t, ω + θ)STFT∗(t, ω −θ)dθ,
(9) (9) where P(θ) is a fixed length window in the frequency domain. Accordingly, S-method distribution for
discrete signals is: SM(n, k) =
L
X
l=−L
P(l)STFT(n, k + l)STFT∗(n, k −l),
(10) (10) where P(l) is a fixed-length window in the frequency domain, and its length is 2L + 1. Regarding the
S-method distribution, when L = 0, the S-method distribution has the distribution characteristics of
the STFT, while if L = N/2, it has the distribution characteristics of the pseudo Winger-Ville. Thus,
selecting an appropriate window length L can simultaneously have the advantages of both, i.e., having
a high time-frequency aggregation and suppressing cross-terms. When the frequency domain window
P(l) is a rectangular window, Equation (10) can be converted into: SM(n, k) =
STFT(n, k)
2 + 2Re
L
X
l=1
STFT(n, k + l)STFT∗(n, k −l),
(11) (11) where
STFT(n, k)
2 is the energy spectral density of the time-frequency point (n, k), and the summation
is the energy sum of those time-frequency points in the frequency domain window P(l) at the same time
point n. The basic idea is to supplement the energy of the time-frequency point by adding the energy
of the points in the frequency domain window to that of point (n, k). 2.3.3. Energy Aggregation based on S-method
2.3.3. Energy Aggregation based on S-method Although the classical S-method
has good performance in processing multi-component signals, there are still some shortcomings that 9 of 19 Sensors 2019, 19, 2598 cannot be ignored when the signal contains unfiltered interference, including: (1) the STFT in the
S-method formula is used to suppress cross-terms. However, its time-frequency resolution is fixed,
meaning that the time-frequency resolution of the S-method is poor; (2) when the classical S-method
processes the background noise, a large amount of unfiltered interference signal energy is added to
the background noise signal. This may likely increase the energy of the background noise signal to
a similar extent of the effective signal energy, and then the effective signal might be flooded by the
processed background noise signal; (3) the calculation in Equation (11) is somewhat complicated. p
g
g
q
p
To overcome the above three shortcomings, we optimize the classical S-method to obtain a new
method: Energy Aggregation. The corresponding improvements are as follows: (1) we replace the STFT
in Equation (11) with the S-transform mentioned in Equation (8). The S-transform can overcome the
fixed resolution issue of STFT, and meanwhile, it can maintain multi-resolution characteristics of STFT. This allows Energy Aggregation to improve the time-frequency resolution of the signal; (2) we introduce
the weight P = 2L + 1 for
STFT(n, k)
2. This can increase the weight of the time-frequency points’ own
energy spectral density in the total energy spectral density and reduce the weight of the supplement
energy spectral density. By this way, even if the unfiltered signal energy is added to the background
noise, the background noise energy is not to a similar extent of the effective signal energy. Therefore,
the difference of energy spectral density between the effective signal and the background noise signal
is increased, which can make the effective signal not be flooded by the background noise signal;
(3) the second part in Equation (11) is replaced by the calculation of energy spectral density in the
proposed Energy Aggregation, as shown in Equation (12) below. This can simplify the calculation. The above improvements enable Energy Aggregation to increase the time-frequency resolution
of the signal and suppress background noise as well, while maintaining the ability of the S-method
in terms of cross-terms suppression and time-frequency aggregation enhancement. 2.4.1. Feature Ext
2.4.1. Feature Extraction After Energy Aggregation, we extract three features in the time-frequency distribution, i.e.,
Doppler Bandwidth, Micro-Doppler Bandwidth, and Effective Arm-Swing Time Difference. The
Doppler bandwidth reflects the change in trunk velocity during human motion. The micro-Doppler
bandwidth reflects the swinging speed of the arm and the swinging amplitude of the arm during the
entire movement. The effective arm-swing time difference reflects the number of swinging arms. As
shown in Figure 6, the time-frequency signals are divided by energy gradients. From that we can
After Energy Aggregation, we extract three features in the time-frequency distribution, i.e., Doppler
Bandwidth, Micro-Doppler Bandwidth, and Effective Arm-Swing Time Difference. The Doppler
bandwidth reflects the change in trunk velocity during human motion. The micro-Doppler bandwidth
reflects the swinging speed of the arm and the swinging amplitude of the arm during the entire
movement. The effective arm-swing time difference reflects the number of swinging arms. As shown
in Figure 6, the time-frequency signals are divided by energy gradients. From that we can directly
get the Doppler bandwidth and micro-Doppler bandwidth. Then, we extract the peak point of the
swing-arm and use the swing-arm matching to obtain the corresponding time and get the effective arm
swing time difference. The three features can be summarized as follows:
Sensors 2019, 19, x FOR PEER REVIEW
10 of 19
directly get the Doppler bandwidth and micro-Doppler bandwidth. Then, we extract the peak point
f h
d
h
h
b
h
d
d
h 1. Doppler Bandwidth: This reflects the change of trunk velocity during the movement of the human
body. When the value of Doppler bandwidth is small, the movement velocity of the trunk is slow. When it is large, the movement velocity of the trunk is faster. of the swing-arm and use the swing-arm matching to obtain the corresponding time and get the
effective arm swing time difference. The three features can be summarized as follows:
1. Doppler Bandwidth: This reflects the change of trunk velocity during the movement of the
human body When the value of Doppler bandwidth is small the movement velocity of the 1. Doppler Bandwidth: This reflects the change of trunk velocity during the movement of the human
body. When the value of Doppler bandwidth is small, the movement velocity of the trunk is slow. When it is large, the movement velocity of the trunk is faster. 2.4.1. Feature Ext
2.4.1. Feature Extraction of the swing-arm and use the swing-arm matching to obtain the corresponding time and get the
effective arm swing time difference. The three features can be summarized as follows:
1. Doppler Bandwidth: This reflects the change of trunk velocity during the movement of the
human body When the value of Doppler bandwidth is small the movement velocity of the 2. Micro-Doppler Bandwidth: The total bandwidth of the effective signal is used as the micro-Doppler
bandwidth, which reflects the situation of the swinging arm. When the value is small, it means
that there is no arm swing. When the value is large, the arm swings. human body. When the value of Doppler bandwidth is small, the movement velocity of the
trunk is slow. When it is large, the movement velocity of the trunk is faster. 2. Micro-Doppler Bandwidth: The total bandwidth of the effective signal is used as the micro-
Doppler bandwidth, which reflects the situation of the swinging arm. When the value is small, 3. Effective Arm-Swing Time Difference: This reflects the time difference between the forward
swinging arm and the backward swinging arm, and the number of swinging arms can be
decided based on this. A larger value represents an arm-swing, and a smaller value represents
two arm-swings. pp
g
g
it means that there is no arm swing. When the value is large, the arm swings. 3. Effective Arm-Swing Time Difference: This reflects the time difference between the forward
swinging arm and the backward swinging arm, and the number of swinging arms can be
decided based on this. A larger value represents an arm-swing, and a smaller value represents
two arm swings 3. Effective Arm-Swing Time Difference: This reflects the time difference between the forward
swinging arm and the backward swinging arm, and the number of swinging arms can be
decided based on this. A larger value represents an arm-swing, and a smaller value represents
two arm-swings. it means that there is no arm swing. When the value is large, the arm swings. 3. Effective Arm-Swing Time Difference: This reflects the time difference between the forward
swinging arm and the backward swinging arm, and the number of swinging arms can be
decided based on this. A larger value represents an arm-swing, and a smaller value represents
two arm swings Figure 6. Three features selected from time-frequency distribution. Figure 6. Three features selected from time-frequency distribution. Figure 6. 2.3.3. Energy Aggregation based on S-method
2.3.3. Energy Aggregation based on S-method The formula for
Energy Aggregation EA(n, k) can be expressed as: EA(n, k) = P ∗
ST(n, k)
2 +
L
X
l=−L
ST(n, k + l)
2,
(12) (12) where, the first part is the Energy Aggregation calculates the energy spectral density of the
time-frequency point (n, k), multiplied by the coefficient P. The second part is the sum of the
energy spectral density of those frequency points on both sides, with k as the axis of symmetry, at the
same time point n. Since the S-transform has been used in Section 2.3.2, we only need to continue to
perform the subsequent calculation of the Energy Aggregation in Equation (12). The results of the two methods, S-method and Energy Aggregation, are shown in the Figure 5. The results show that although the S-method can improve the time-frequency aggregation and suppress
cross-terms, the time-frequency resolution is low and the interference of background noise signals
is aggravated, as shown in Figure 5a. Instead, by using the Energy Aggregation proposed in this
paper, the time-frequency resolution can be effectively improved, while maintaining the ability of the
S-method in terms of cross-terms suppression and time-frequency aggregation enhancement, as shown
in Figure 5b. Furthermore, in Figure 5b, the difference of energy spectral density between the effective
signal and the background noise signal is more obvious. Thus, the Energy Aggregation obtains the
capability of suppressing the background noise and cross-terms, which is more conducive to separating
the effective signal for extracting features in the next step. 10 of 19
nducive Sensors 2019, 19, 2598
obtains th
to separat (a)
(b)
Figure 5. (a) Time-frequency distribution of signal processed by S-method; (b) time-frequency
distribution of signal processed by Energy Aggregation. Figure 5. (a) Time-frequency distribution of signal processed by S-method; (b) time-frequency
distribution of signal processed by Energy Aggregation. 4 F
t
E t
ti
d M ti
St t Cl
ifi
ti (a) (b) (b) (a) Figure 5. (a) Time-frequency distribution of signal processed by S-method; (b) time-frequency
distribution of signal processed by Energy Aggregation. Figure 5. (a) Time-frequency distribution of signal processed by S-method; (b) time-frequency
distribution of signal processed by Energy Aggregation. 3.1. Experimental Testbed
3.1. Experimental Testbed We built an experimental testbed using software defined radio equipment USRP N210. Specifically, a continuous-wave radar system with a carrier frequency centered at 4.2 GHz is
implemented on the USRP N210 device with Sea-based X-band Radar (SBX) daughterboard by using
the GNU Radio, an open-source software development toolkit. In this system, a radar transceiver
contains two USRPs accompanied by a Multiple Input Multiple Output (MIMO) cable for time
synchronization, to implement signal transmission, and for reception, respectively. Here, the SBX
daughterboard provides a 40MHz bandwidth and can work at a variety of bands in the 400–4400
MHz range. Two PCB directional RF antennas complete the RF transceiver, whose frequency band is
850–6500 MHz. Also, the GNU Radio is installed in a laptop computer with a Gigabit Ethernet
network card. The USRP is connected to the computer by a Gigabit high-speed network cable. The
discrete complex signals generated from the USRP are processed in the computer
We built an experimental testbed using software defined radio equipment USRP N210. Specifically,
a continuous-wave radar system with a carrier frequency centered at 4.2 GHz is implemented on
the USRP N210 device with Sea-based X-band Radar (SBX) daughterboard by using the GNU Radio,
an open-source software development toolkit. In this system, a radar transceiver contains two
USRPs accompanied by a Multiple Input Multiple Output (MIMO) cable for time synchronization,
to implement signal transmission, and for reception, respectively. Here, the SBX daughterboard
provides a 40MHz bandwidth and can work at a variety of bands in the 400–4400 MHz range. Two PCB
directional RF antennas complete the RF transceiver, whose frequency band is 850–6500 MHz. Also,
the GNU Radio is installed in a laptop computer with a Gigabit Ethernet network card. The USRP
is connected to the computer by a Gigabit high-speed network cable. The discrete complex signals
generated from the USRP are processed in the computer. discrete complex signals generated from the USRP are processed in the computer. The measurement of the data is performed in an indoor environment with the following
configuration. The transmitting and receiving antennas are placed under the same vertical plane. The
distance between the two PCB antennas is about 10 cm and 1.2 m from the ground. 2.4.1. Feature Ext
2.4.1. Feature Extraction Three features selected from time-frequency distribution. Figure 6. Three features selected from time-frequency distribution. Sensors 2019, 19, 2598 11 of 19 2.4.2. Motion State Classification We take the above three features as inputs. Next, we use the idea of ensemble learning to
combine bagging and the decision tree, and classify the target human motions under non-target
micro-motion interference. First, given a data set containing m samples, we randomly take a sample from the data set into
the sampling set and put the sample back into the initial data set. After randomly sampling m times,
we obtain a sample set containing m samples. Then, we repeat the above steps T times, and we obtain
T sampling sets, with each sampling set containing m samples. Finally, we train the decision tree based
on each sample set to obtain T different decision trees, and then use the simple voting method to obtain
the decision tree with the highest number of votes. The classification result produced by the decision
tree with the highest number of votes is used as the result of Bagged Trees. The flow chart is shown in
the Figure 7. Sensors 2019, 19, x FOR PEER REVIEW
11 of 19 Figure 7. The flow chart of Bagged Trees. Figure 7. The flow chart of Bagged Trees. Figure 7. The flow chart of Bagged Trees. Figure 7. The flow chart of Bagged Trees. We tested this classification method using ten-fold cross-validation. We divided the data set into
ten equal parts, and took nine of them as a training set and used the remainder as a test set. Taking
turns in this way, we finally calculated the accuracy of the classification by calculating the mean of
each correct rate. Related experimental results can be found in Section 3.2. We tested this classification method using ten-fold cross-validation. We divided the data set into
ten equal parts, and took nine of them as a training set and used the remainder as a test set. Taking
turns in this way, we finally calculated the accuracy of the classification by calculating the mean of
each correct rate. Related experimental results can be found in Section 3.2. 3.1. Experimental Testbed
3.1. Experimental Testbed In order to reduce
the multipath effect, we place the PCB antenna in the middle position and the target human body
The measurement of the data is performed in an indoor environment with the following
configuration. The transmitting and receiving antennas are placed under the same vertical plane. The distance between the two PCB antennas is about 10 cm and 1.2 m from the ground. In order to
reduce the multipath effect, we place the PCB antenna in the middle position and the target human 12 of 19 Sensors 2019, 19, 2598 body moves radially toward the antenna. The range of the measurement is between 0 and 5 m. Non-target human bodies are located 1.5 m away from the antenna and 0.5 m away from the target
human. The specific experimental scene is shown in Figure 8. Each acquisition process is 15 s, which is
divided into three 5-seconds segments. The first 5 s is static, the middle 5 s is the effective signal of
the target human motion, and in the last 5 s the target human body remains in motion. We select the
middle 5 s as the valid data segment. We collect continuous-wave radar echo signals generated from 5 human bodies undergoing
different activities, and the signals were collected under the scenarios with non-target micro-motion
interference. Only a single target human body is tested at one time, with the target moving directly
toward the radar. The signals for a target human body undergoing four different activities are collected,
including: (1) walking without any arm swinging (without arm), (2) walking with one arm swinging
(single arm), (3) walking with both arms swinging (both arms), and (4) running. Among them,
the walking without any arm swinging, the walking with one arm swinging, and the walking with both
arms swinging are all maintained at a constant speed, and the running maintains a relatively stable
speed. Considering the non-target micro-motion interference, this paper sets two kinds of interference,
which are divided into: (1) non-target sudden interference, and (2) non-target continuous swing arm
interference. The sudden interference is the sudden swinging or standing for a non-target human
body and continuous swing arm interference is when a non-target human body maintains a uniform
swinging arm during the whole experimental period. Sensors 2019, 19, x FOR PEER REVIEW
12 of 19
continuous swing arm interference. 3.1. Experimental Testbed
3.1. Experimental Testbed The sudden interference is the sudden swinging or standing for
a non-target human body and continuous swing arm interference is when a non-target human body
maintains a uniform swinging arm during the whole experimental period. Figure 8. Experimental scene and settings. Figure 8. Experimental scene and settings. Figure 8. Experimental scene and settings. Figure 8. Experimental scene and settings. 3.2. Results
3.2. Results In the case of non-target sudden interference and non-target continuous swing arm interference,
the filtering of the interference and the high resolution of the time-frequency distribution are critical
to the system. Next, we show the good processing result of the proposed method on the signal
collected by the continuous-wave radar system mentioned above. In addition, the classification
results of the four human motion states can also be seen. In the case of non-target sudden interference and non-target continuous swing arm interference,
the filtering of the interference and the high resolution of the time-frequency distribution are critical to
the system. Next, we show the good processing result of the proposed method on the signal collected
by the continuous-wave radar system mentioned above. In addition, the classification results of the
four human motion states can also be seen. Non-target sudden interference is a weak interference. The non-target human body suddenly
swings or stands for a shorter duration. The non-target continuous swing arm interference is a strong
interference, where the non-target human body maintains a uniform swing arm during the whole
experimental period. Time-frequency distribution of non-target micro-motion interference is shown
in Figure 9. Non-target sudden interference is a weak interference. The non-target human body suddenly
swings or stands for a shorter duration. The non-target continuous swing arm interference is a strong
interference, where the non-target human body maintains a uniform swing arm during the whole
experimental period. Time-frequency distribution of non-target micro-motion interference is shown in
Figure 9. g
Under the non-target micro-motion interference, walking without any arm swinging, walking with
one arm swinging, walking with both arms swinging, and running, the time-frequency distributions of
four motion states processed by the time-frequency analysis scheme are shown in the Figure 10: 13 of 19
e is shown Sensors 2019, 19, 2598
experimental pe
in Figure 9. 13 of 19
e is shown (a)
(b)
Figure 9. (a) Time-frequency distribution of non-target sudden interference; (b) Time-frequency
distribution of non-target continuous swing arm interference. Figure 9. (a) Time-frequency distribution of non-target sudden interference; (b) Time-frequency
distribution of non-target continuous swing arm interference. Sensors 2019, 19, x FOR PEER REVIEW
1 (a)
ors 2019 19 x FOR PEER REVIEW (b)
1 (b) (a)
REVIEW Figure 9. (a) Time-frequency distribution of non-target sudden interference; (b) Time-frequenc
distribution of non-target continuous swing arm interference. Figure 9. 3.2. Results
3.2. Results (a) Time-frequency distribution of non-target sudden interference; (b) Time-frequency
distribution of non-target continuous swing arm interference. nsors 2019, 19, x FOR PEER REVIEW distribution of non target continuous swing arm interference. Under the non-target micro-motion interference, walking without any arm swinging, wal
with one arm swinging, walking with both arms swinging, and running, the time-frequ
distributions of four motion states processed by the time-frequency analysis scheme are shown in
Figure 10:
(a)
(b)
(c)
(d)
Figure 10. (a) Time-frequency distribution of walking with one arm swinging. (b) Time-frequency
distribution of walking with both arms swinging; (c) Time-frequency distribution of running. (d)
Time-frequency distribution of walking without either arm swinging. Figure 10. (a) Time-frequency distribution of walking with one arm swinging. (b) Time-frequency
distribution of walking with both arms swinging; (c) Time-frequency distribution of running. (d) Time-frequency distribution of walking without either arm swinging. nce, walking without any arm swinging, w
ms swinging, and running, the time-freq
time-frequency analysis scheme are shown
(b) Under the non-target micro-motion in
th one arm swinging, walking with b
tributions of four motion states processed
gure 10:
(a) (b) (a) (c) (d) (d) (c) Figure 10. (a) Time-frequency distribution of walking with one arm swinging. (b) Time-frequency
distribution of walking with both arms swinging; (c) Time-frequency distribution of running. (d
Time-frequency distribution of walking without either arm swinging. Figure 10. (a) Time-frequency distribution of walking with one arm swinging. (b) Time-frequency
distribution of walking with both arms swinging; (c) Time-frequency distribution of running. (d) Time-frequency distribution of walking without either arm swinging. The collected signal is mainly divided into two parts. One is the torso with stronger energy, an
the other is the part around the torso with lower energy. The strongest return in each time-frequenc
distribution comes from that of the torso, while the periodic micro-Doppler modulations surroundin
the torso return come from arm movements. It can be seen from the time-frequency distributions
the four motions that after the signal is processed by the time-frequency analysis scheme, th
interference of the non-target micro-motion is reduced, and the background noise and cross-term
The collected signal is mainly divided into two parts. One is the torso with stronger energy, and the
other is the part around the torso with lower energy. 3.2. Results
3.2. Results The strongest return in each time-frequency
distribution comes from that of the torso, while the periodic micro-Doppler modulations surrounding
the torso return come from arm movements. It can be seen from the time-frequency distributions of the
four motions that after the signal is processed by the time-frequency analysis scheme, the interference
of the non-target micro-motion is reduced, and the background noise and cross-terms caused by the Sensors 2019, 19, 2598 14 of 19 interference are suppressed. Therefore, it is possible to distinguish between the swing arm movement
and the trunk movement. At the same time, the time-frequency resolution of the signal is improved,
which can reveal the real body of the target human motion. In Figure 10c, the Doppler frequency is
highest due to the faster movement of the torso during running. In Figure 10d, there is no swing arm in
the walking without any arm swinging, and the micro-Doppler bandwidth is the smallest compared to
the other three types of motion. In Figure 10a,b, the effective arm swing time difference of walking with
one arm swinging is large, but the time difference of walking with both arms swinging is small. Since
the number of swinging arms is different during the two movements, therefore, the time difference of
the swing arm pair is different during a complete swing arm process. After feature extraction, we use
Bagged Trees to classify human motions and verify the validity of the proposed system with ten-fold
cross-validation. The final average classification accuracy rate is 97.3%, and Table 1 give the results of
the corresponding confusion matrix. Table 1. Confusion matrix results of the proposed system. Single Arm
Both Arms
Running
Without Arm
Single Arm
96
3
0
1
Both Arms
3
95
2
0
Running
0
0
100
0
Without Arm
1
1
0
98 Table 1. Confusion matrix results of the proposed system. As can be seen from Table 1, the recognition accuracy rate of running is up to 100%, implying that
non-target micro-motion interference has the least impact on it. The accuracy of walking without any
arm swinging is 98%, and the accuracy of walking with one arm swinging is 96%. The accuracy of
walking with both arms swinging is the lowest, at only 95%. This is because non-target micro-motion
interference interferes with the recognition of the effective swing arm pair, which affects the extraction
of the time difference. 3.2. Results
3.2. Results Thus, the walking with both arms swinging is misjudged as walking with one
arm swinging. For non-target sudden interference cases, such as sudden swings, sudden standing, etc.,
the interference is characterized by non-sustainability and small amplitude. In this case, the target
human motion state classification still has a higher classification accuracy, which is basically the
same as non-target human body continuous swing arm interference. It can be seen that under the
low-frequency radar band in the case of micro-motion interference from non-targets, the proposed
system performs well and has high classification accuracy. Also, it can effectively reduce the impact of
interference on the target human motion. 3.3. Discussion 3.3.1. Comparison with Other Systems Next, we compare the other three human motion state classification systems with the proposed
system in the scenarios with non-target micro-motion interference. In a previous study [30], the authors
used S-band continuous wave radar with a carrier frequency of 9.8 GHz to collect target human motion
data, and classified the three motion states of walking with no bag, walking with one bag held by
one hand, and walking with one bag held by both hands. Firstly, the micro-Doppler signatures are
obtained by performing time-frequency analysis on the radar data. Then, two micro-Doppler features
are extracted from the time-frequency domain. Finally, the one-versus-one support vector machine is
used to realize multi-class classification. We apply the above method to the data obtained in this paper. The results are shown in Table 2. In the case of non-target micro-motion interference, the accuracy rate
of this method for the four movement states of the target human body is 88.5%. Among them, accuracy
results for walking with one arm swinging and walking with both arms swinging were 78% and 81%, Sensors 2019, 19, 2598 15 of 19 respectively. In scenarios with non-target micro-motion interference, the classification accuracy of
these two modes of motion is low, so this system has poor resistance to interference. Table 2. Confusion matrix results of Short-Time Fourier Transform (STFT). Single Arm
Both Arms
Running
Without Arm
Single Arm
78
17
0
5
Both Arms
14
81
0
5
Running
0
0
100
0
Without Arm
5
0
0
95 Table 2. Confusion matrix results of Short-Time Fourier Transform (STFT). In a previous study [24], the authors use Multiwindow AS-method for time-frequency analysis
and extract 12 features to classify seven motion states through SVM based on decision tree. Among
them, running, walking, walking while holding a stick, and boxing while moving forward are four
sports states that are similar to the selected sports types in this paper, so we compare these with this
system. We used the same method to classify the four motion states under non-target micro-motion
interference. As shown in Table 3, the accuracy is 90.8%. The interference has a great influence on
walking with one arm swinging and walking with both arm swinging, and the error rates are 12% and
17%, respectively. Thus, it is easy for misclassification to occur when classifying. Table 3. Confusion matrix results of Multiwindow Adaptive S-method. 3.3.1. Comparison with Other Systems Single Arm
Both Arms
Running
Without Arm
Single Arm
92
8
0
0
Both Arms
12
88
0
0
Running
0
0
100
0
Without Arm
15
2
0
83 Table 3. Confusion matrix results of Multiwindow Adaptive S-method. In a previous study [17], a simple feature extraction method is applied to the motion state
classification, which is Linear Predictive Coding (LPC). The echo signals are processed using LPC
by selecting appropriate sizes for the non-overlapping time window, LPC coefficients, and decision
time frames. The confusion matrix is presented in Table 4. When this method is applied to non-target
continuous swing arm interference, the accuracy rate of the four motion states is 83.3%. Even worse,
the accuracy of walking with one arm swinging and walking with both arms swinging was only 73%
and 77%, respectively. Therefore, the influence of interference on this method is more serious. Table 4. Confusion matrix results of LPC. Single Arm
Both Arms
Running
Without Arm
Single Arm
73
20
0
7
Both Arms
23
77
0
0
Running
0
0
100
0
Without Arm
10
7
0
83 Table 4. Confusion matrix results of LPC. Table 4. Confusion matrix results of LPC. According to the above results, the classification accuracy rate of the system proposed in previous
studies [17,24,30] cannot meet the basic classification requirements in the case of non-target micro-motion
interference. However, the system proposed in this paper can resist interference effectively and still
have high classification accuracy under interference. To further illustrate the performance of the
system, we tested the four motion state classification results in the scenarios without any non-target
micro-motion interference. Additionally, we compare the proposed system with the three systems
mentioned above in the scenarios with or without non-target micro-motion interference. As shown in Figure 11a, the system still has a high accuracy rate for the classification of the
four motion states in the scenarios without non-target micro-motion interference. Compared with Sensors 2019, 19, 2598 16 of 19 the interference situation, the accuracy rate is slightly improved and the overall stability is stable,
indicating that the system has good stability. As shown in Figure 11b, in the scenarios without
non-target micro-motion interference, the accuracy of the proposed system, STFT, multi-window
AS-method, and LPC are 98.3%, 91.8%, 94.2%, and 85.5%, respectively. 3 3 2 The Classification Accuracy of Different Distances
3.3.2. The Classification Accuracy of Different Distances 3.3.2. The Classification Accuracy of Different Distances
The distance between the non-target human body (interference source) and the target human
body or the PCB antenna affects the classification accuracy of the target human motion state. We
conducted a distance experiment to explore this effect. As shown in Figure 12, we define the above
two types of distance, including: (1) the distance between interference source and target, and (2) the
distance between interference source and the PCB antenna. First, we consider the effect of the distance
between interference source and target on the accuracy rate, and set the irrelevant variable, the
distance between interference source and PCB antenna, as unchanged. As shown in Figure 13, when
the distance between interference source and target is 0.5 m, the interference has the greatest
influence on the target human motion. The classification accuracy at this case is 97.3%. As the distance
increases, the influence of interference on the target human motion is reduced, so the accuracy is
slowly increased. When the distance is 1 m, the classification accuracy is 97.5%. However, when the
distance is greater than 1.5m, the interference of non-target micro-motion can be neglected. At this
point the classification accuracy rate is 98 3% which is almost the same as when there is no
The distance between the non-target human body (interference source) and the target human body
or the PCB antenna affects the classification accuracy of the target human motion state. We conducted
a distance experiment to explore this effect. As shown in Figure 12, we define the above two types
of distance, including: (1) the distance between interference source and target, and (2) the distance
between interference source and the PCB antenna. First, we consider the effect of the distance between
interference source and target on the accuracy rate, and set the irrelevant variable, the distance between
interference source and PCB antenna, as unchanged. As shown in Figure 13, when the distance between
interference source and target is 0.5 m, the interference has the greatest influence on the target human
motion. The classification accuracy at this case is 97.3%. As the distance increases, the influence of
interference on the target human motion is reduced, so the accuracy is slowly increased. When the
distance is 1 m, the classification accuracy is 97.5%. However, when the distance is greater than 1.5m,
the interference of non-target micro-motion can be neglected. 3.3.1. Comparison with Other Systems The proposed system has
a high accuracy rate in the presence or absence of non-target micro-motion interference, and it is
slightly improved without interference. However, other systems are more sensitive to interference,
and the classification accuracy is significantly improved without interference, which indicates that the
proposed system has better robustness and stability. Additionally, the computational complexity values of the three compared systems are O(Nlog2N),
O(N + Nlog2N), and O
LP + P2
, and the proposed system has the highest computational complexity,
which is O
N + N2log2N
. Although the proposed method has relatively high computation complexity,
it can effectively resist interference and improve time-frequency resolution, consequently achieving
much better performance, which is more important. Sensors 2019, 19, x FOR PEER REVIEW
16 of 19 (a)
(b)
Figure 11. (a) Comparison of classification rate of the proposed system with or without interference;
(b) comparison of classification rate of different systems with or without interference. Figure 11. (a) Comparison of classification rate of the proposed system with or without interference;
(b) comparison of classification rate of different systems with or without interference. (b) (a) (b) (a) Figure 11. (a) Comparison of classification rate of the proposed system with or without interference;
(b) comparison of classification rate of different systems with or without interference. Figure 11. (a) Comparison of classification rate of the proposed system with or without interference;
(b) comparison of classification rate of different systems with or without interference. 3 3 2 The Classification Accuracy of Different Distances
3.3.2. The Classification Accuracy of Different Distances Classification rate of two types of distances. 3 3 2 The Classification Accuracy of Different Distances
3.3.2. The Classification Accuracy of Different Distances At this point, the classification accuracy
rate is 98.3% which is almost the same as when there is no interference. 17 of 19
re is no Sensors 2019, 19, 2598
point, the classif
i t
f Figure 12. Illustration of two types of distances. Figure 12. Illustration of two types of distances. Figure 12. Illustration of two types of distances. Figure 12. Illustration of two types of distances. Then, we consider the influence of the distance between interference source and PCB antenna
on the accuracy, and set the irrelevant variable, the distance between interference source and target,
as unchanged. As shown in Figure 13, the classification accuracy is the lowest when the distance
Then, we consider the influence of the distance between interference source and PCB antenna
on the accuracy, and set the irrelevant variable, the distance between interference source and target,
as unchanged. As shown in Figure 13, the classification accuracy is the lowest when the distance
between interference source and PCB antenna is 0.5 m, which is 96.3%. As the distance increases, the
intensity of the interference signal in the radar echo signal gradually decreases. Therefore, the influence
of interference on the target human motion is gradually reduced, and the accuracy of the motion
state classification is slowly increased. From the influence of the two interference distances on the
classification accuracy of the target human motion, we find that the classification accuracy slowly
increases and remains stable as the distance increases. This guarantees the credibility of the system
and shows that the system has strong adaptability. Sensors 2019, 19, x FOR PEER REVIEW
17 of 19
between interference source and PCB antenna is 0.5 m, which is 96.3%. As the distance increases, the
intensity of the interference signal in the radar echo signal gradually decreases. Therefore, the
influence of interference on the target human motion is gradually reduced, and the accuracy of the
motion state classification is slowly increased. From the influence of the two interference distances
on the classification accuracy of the target human motion, we find that the classification accuracy
slowly increases and remains stable as the distance increases. This guarantees the credibility of the
system and shows that the system has strong adaptability. Figure 13. Classification rate of two types of distances. Figure 13. Classification rate of two types of distances. Figure 13. Classification rate of two types of distances
Figure 13. 4. Conclusions
4. Conclusions In the scenarios in which there exist surrounding non-target humans, the interference signal
aliases with the signal of target human motion and then generates cross-terms, which make the signal
unable to reflect the true motion of the target human. In this paper, we propose a target human
motion classification system that can work in environments with non-target micro-motion
interference. Under the premise of using low-band radar waves, the proposed system can overcome
the non-target micro-motion interference and accurately classify the target human motions. We use
EMD and S-transform successively to remove non-target micro-motion interference and improve the
time-frequency resolution of the signal. In addition, Energy Aggregation is proposed as a new
method by optimizing the classical S-method, which largely reduces the effects of cross-terms and
background noise. Then, a set of features are extracted from the time-frequency distribution and are
In the scenarios in which there exist surrounding non-target humans, the interference signal
aliases with the signal of target human motion and then generates cross-terms, which make the
signal unable to reflect the true motion of the target human. In this paper, we propose a target
human motion classification system that can work in environments with non-target micro-motion
interference. Under the premise of using low-band radar waves, the proposed system can overcome
the non-target micro-motion interference and accurately classify the target human motions. We use
EMD and S-transform successively to remove non-target micro-motion interference and improve the
time-frequency resolution of the signal. In addition, Energy Aggregation is proposed as a new method
by optimizing the classical S-method, which largely reduces the effects of cross-terms and background
noise. Then, a set of features are extracted from the time-frequency distribution and are fed in to Sensors 2019, 19, 2598 18 of 19 Bagged Trees for motion classification. Our experiments show that the proposed system can achieve a
high accuracy of 97.3% to classify four types of motion states. Author Contributions: X.M. and R.Z. initiated and discussed the research problem; R.Z. performed experiments
and produced figures; X.L. and H.K. checked and ensured the correctness of the experimental results; X.M., R.Z.,
and M.A.A.A. analyzed the data; X.M. and R.Z. wrote the paper. All authors have read and approved the paper. Funding: This work is sponsored by National Natural Science Foundation of China with Grant Nos. 61502361 and
61772088. References 1. Nanzer, J.A. A review of microwave wireless techniques for human presence detection and classification. IEEE Trans. Microw. Theory Tech. 2017, 65, 1780–1794. [CrossRef] 1. Nanzer, J.A. A review of microwave wireless techniques for human presence detection and classification. IEEE Trans. Microw. Theory Tech. 2017, 65, 1780–1794. [CrossRef] 2. Qi, F.; Liang, F.; Lv, H.; Li, C.; Chen, F.; Wang, J. Detection and classification of finer-grained human
activities based on stepped-frequency continuous-wave through-wall radar. Sensors 2016, 16, 885. [CrossRef]
[PubMed] 2. Qi, F.; Liang, F.; Lv, H.; Li, C.; Chen, F.; Wang, J. Detection and classification of finer-grained human
activities based on stepped-frequency continuous-wave through-wall radar. Sensors 2016, 16, 885. [CrossRef]
[PubMed] 3. Chen, Z.; Li, G.; Fioranelli, F.; Griffiths, H. Personnel recognition and gait classification based on multistatic
micro-doppler signatures using deep convolutional neural networks. IEEE Geosci. Remote Sens. Lett. 2018,
15, 669–673. [CrossRef] 3. Chen, Z.; Li, G.; Fioranelli, F.; Griffiths, H. Personnel recognition and gait classification based on multistatic
micro-doppler signatures using deep convolutional neural networks. IEEE Geosci. Remote Sens. Lett. 2018,
15, 669–673. [CrossRef] 4. Chen, V.C. Micro-Doppler effect of micromotion dynamics: A review. In Proceedings of the International
Society for Optical Engineering (SPIE), Orlando, FL, USA, 29 March 2003; pp. 240–249. 4. Chen, V.C. Micro-Doppler effect of micromotion dynamics: A review. In Proceedings of the International
Society for Optical Engineering (SPIE), Orlando, FL, USA, 29 March 2003; pp. 240–249. 5. Yang, Y.; Hou, C.; Lang, Y.; Guan, D.; Huang, D.; Xu, J. Open-set human activity recognition based on
micro-Doppler signatures. Pattern Recognit. 2019, 85, 60–69. [CrossRef] 5. Yang, Y.; Hou, C.; Lang, Y.; Guan, D.; Huang, D.; Xu, J. Open-set human activity recognition based on
micro-Doppler signatures. Pattern Recognit. 2019, 85, 60–69. [CrossRef] 6. Erol, B.; Amin, M.G.; Gurbuz, S.Z. Automatic data-driven frequency-warped cepstral feature design for
micro-Doppler classification. IEEE Trans. Aerosp. Electron. Syst. 2018, 54, 1724–1738. [CrossRef] 6. Erol, B.; Amin, M.G.; Gurbuz, S.Z. Automatic data-driven frequency-warped cepstral feature design for
micro-Doppler classification. IEEE Trans. Aerosp. Electron. Syst. 2018, 54, 1724–1738. [CrossRef] 7. Fioranelli, F.; Ritchie, M.; Griffiths, H. Bistatic human micro-Doppler signatures for classification of indoor
activities. In Proceedings of the 2017 IEEE Radar Conference, Seattle, WA, USA, 8–12 May 2017; pp. 610–615. 8. Seyfioglu, M.S.; Ozbayoglu, A.M.; Gurbuz, S.Z. Deep convolutional autoencoder for radar-based classification
of similar aided and unaided human activities. IEEE Trans. Aerosp. Electron. Syst. 2018, 54, 1709–1723. [CrossRef] 9. 4. Conclusions
4. Conclusions And, we thank Jian Liu (email: jianliu@winlab.rutgers.edu), who comes from the WINLAB, Rutgers
University, New Brunswick, NJ 08902, USA, for giving us valuable comments regarding the original manuscript. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Li, W.; Tan, B.; Piechocki, R. Passive radar for opportunistic monitoring in e-health applications. IEEE J. Transl. Eng. Health Med. 2018, 6, 1–10. [CrossRef] [PubMed] 10. Chen, V. Doppler signatures of radar backscattering from objects with micro-motions. IET Signal Process. 2008, 2, 291–300. [CrossRef] 11. Kim, Y.; Ling, H. Human activity classification based on micro-Doppler signatures using a support vector
machine. IEEE Trans. Geosci. Remote Sens. 2009, 47, 1328–1337. 12. Narayanan, R.M.; Zenaldin, M. Radar micro-Doppler signatures of various human activities. IET Radar
Sonar Navig. 2015, 9, 1205–1215. [CrossRef] 13. Fioranelli, F.; Ritchie, M.; Griffiths, H. Multistatic human micro-Doppler classification of armed/unarmed
personnel. IET Radar Sonar Navig. 2015, 9, 857–865. [CrossRef] 14. Bjorklund, S.; Petersson, H.; Nezirovic, A.; Guldogan, M.B.; Gustafsson, F. Millimeter-wave radar
micro-Doppler signatures of human motion. In Proceedings of the 2011 12th International Radar Symposium
(IRS), Leipzig, Germany, 7–9 September 2011; pp. 167–174. 15. Li, J.; Phung, S.L.; Tivive, F.H.C.; Bouzerdoum, A. Automatic classification of human motions using Doppler
radar. In Proceedings of the 2012 International Joint Conference on Neural Networks (IJCNN), Brisbane,
QLD, Australia, 30 July 2012; pp. 1–6. 16. Fairchild, D.P.; Narayanan, R.M. Classification of human motions using empirical mode decomposition of
human micro-Doppler signatures. IET Radar Sonar Navig. 2014, 8, 425–434. [CrossRef] 17. Javier, R.J.; Kim, Y. Application of linear predictive coding for human activity classification based on
micro-Doppler signatures. IEEE Geosci. Remote Sens. Lett. 2014, 11, 1831–1834. [CrossRef] 18. Fioranelli, F.; Ritchie, M.; Griffiths, H. Performance analysis of centroid and SVD features for personnel
recognition using multistatic micro-Doppler. IEEE Geosci. Remote Sens. Lett. 2016, 13, 725–729. [CrossRef] Sensors 2019, 19, 2598 19 of 19 19. Ritchie, M.; Ash, M.; Chen, Q.; Chetty, K. Through wall radar classification of human micro-Doppler using
singular value decomposition analysis. Sensors 2016, 16, 1401. [CrossRef] [PubMed] 20. Tivive, F.H.C.; Bouzerdoum, A.; Amin, M.G. Automatic human motion classification from Doppler
spectrograms. In Proceedings of the 2010 2nd International Workshop on Cognitive Information Processing,
Elba, Italy, 14–16 June 2010; pp. 237–242. y
pp
21. Tekeli, B.; Gurbuz, S.Z.; Yuksel, M. Information-theoretic feature selection for human micro-Doppler signature
classification. IEEE Trans. Geosci. Remote Sens. 2016, 54, 2749–2762. [CrossRef] 22. Orovic, I.; Stankovic, S.; Thayaparan, T.; Stankovic, L. Multiwindow S-method for instantaneous frequency
estimation and its application in radar signal analysis. IET Signal Process. 2010, 4, 363–370. [CrossRef] 23. Orovi´c, I.; Stankovi´c, S.; Amin, M. References A new approach for classification of human gait based on time-frequency
feature representations. Signal Process. 2011, 91, 1448–1456. [CrossRef] 24. Li, F.; Yang, C.; Xia, Y.; Ma, X.; Zhang, T.; Zhou, Z. An adaptive S-method to analyze micro-Doppler signals
for human activity classification. Sensors 2017, 17, 2769. [CrossRef] 25. Breiman, L. Bagging predictors. Mach. Learn. 1996, 24, 123–140. [CrossRef] 26. Mitola, J.I. Cognitive Radio: An Integrated Agent Architecture for Software Defined Radio. Ph.D. Thesis,
Royal Institute of Technology, Stockholm, Sweden, 2000. 27. Camarena-Martinez, D.;
Valtierra-Rodriguez, M.;
Perez-Ramirez, C.A.;
Amezquita-Sanchez, J.P.;
de Jesus Romero-Troncoso, R.; Garcia-Perez, A. Novel downsampling empirical mode decomposition
approach for power quality analysis. IEEE Trans. Ind. Electron. 2016, 63, 2369–2378. [CrossRef] 28. Stockwell, R.G.; Mansinha, L.; Lowe, R.P. Localization of the complex spectrum: The S transform. IEEE Trans. Signal Process. 1996, 44, 998–1001. [CrossRef] c, L. A method for time-frequency analysis. IEEE Trans 29. Stankovic, L. A method for time-frequency analysis. IEEE Trans. Signal Process. 1994, 42, 225–229. [CrossRef]
30. Yang, L.; Li, G.; Ritchie, M.; Fioranelli, F.; Griffiths, H. Gait classification based on micro-doppler
features. In Proceedings of the 2016 CIE International Conference on Radar (RADAR), Guangzhou,
China, 10–13 October 2016; pp. 1–4. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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https://openalex.org/W2845000172
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https://www.mdpi.com/1422-0067/19/7/1981/pdf?version=1531104132
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English
| null |
Label-Free Quantitative Proteomics Combined with Biological Validation Reveals Activation of Wnt/β-Catenin Pathway Contributing to Trastuzumab Resistance in Gastric Cancer
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International journal of molecular sciences
| 2,018
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cc-by
| 14,520
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Received: 6 June 2018; Accepted: 4 July 2018; Published: 6 July 2018 Abstract: Resistance to trastuzumab, which specifically target HER2-positive breast and gastric cancer,
can develop ultimately in cancer patients. However, the underlying mechanisms of resistance in
gastric cancer have not been fully elucidated. Here, we established trastuzumab-resistant MKN45 and
NCI N87 gastric cancer sublines from their parental cells. The resistant cells exhibited characteristics
of epithelial-mesenchymal transition (EMT) and acquired higher migratory and invasive capacities. To exploit the activated pathways and develop new strategies to overcome trastuzumab resistance,
we investigated MKN45 and MKN45/R cells via label-free quantitative proteomics, and found
pathways that were altered significantly in MKN45/R cells, with the Wnt/β-catenin pathway being
the most significant. We further confirmed the activation of this pathway by detecting its key
molecules in MKN45/R and NCI N87/R cells via Western blot, in which Wnt3A, FZD6, and CTNNB1
increased, whereas GSK-3β decreased, manifesting the activation of the Wnt/β-catenin pathway. Correspondingly, inhibition of Wnt/β-catenin pathway by ICG-001, a specific Wnt/β-catenin
inhibitor, preferentially reduced proliferation and invasion of trastuzumab-resistant cells and reversed
EMT. Concurringly, CTNNB1 knockdown in stable cell lines potently sensitized cells to trastuzumab
and induced more apoptosis. Taken together, our study demonstrates that the Wnt/β-catenin
pathway mediates trastuzumab resistance, and the combination of Wnt/β-catenin inhibitors with
trastuzumab may be an effective treatment option. Keywords:
gastric cancer; trastuzumab resistance; Wnt/β-catenin pathway; EMT; label-free
quantitative proteomics International Journal of
Molecular Sciences International Journal of
Molecular Sciences www.mdpi.com/journal/ijms Label-Free Quantitative Proteomics Combined with
Biological Validation Reveals Activation of
Wnt/β-Catenin Pathway Contributing to Trastuzumab
Resistance in Gastric Cancer Wenhu Liu 1,2,† ID , Jiangbei Yuan 3,†, Zhenzhong Liu 4, Jianwu Zhang 1 and Jinxia Chang 2,*
1
Department of Medicinal Chemistry, Department of Pharmacology, School of Pharmacy, North Sichuan
Medical College, Nanchong 637100, China; wenhuliu@cqu.edu.cn (W.L); jianwuzhang@nsmc.edu.cn (J.Z)
2
Innovative Platform of Basic Medical Sciences, Department of Microbiology and Immunology,
School of Basic Medical Sciences, North Sichuan Medical College, Nanchong 637100, China
3
School of Pharmaceutical Sciences and Innovative Drug Research Center, Chongqing University,
Chongqing 401331, China; yuanjiangbei@cqu.edu.cn e
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1
Department of Medicinal Chemistry, Department of Pharmacology, School of Pharmacy, North Sichuan
Medical College, Nanchong 637100, China; wenhuliu@cqu.edu.cn (W.L); jianwuzhang@nsmc.edu.cn (J.Z)
2
Innovative Platform of Basic Medical Sciences, Department of Microbiology and Immunology,
School of Basic Medical Sciences, North Sichuan Medical College, Nanchong 637100, China
3
School of Pharmaceutical Sciences and Innovative Drug Research Center, Chongqing University,
Chongqing 401331, China; yuanjiangbei@cqu.edu.cn 4
Department of Preventive Medicine, North Sichuan Medical College, Nanchong 637100, China;
liuzhenzhong@nsmc.edu.cn *
Correspondence: jinxiachang@nsmc.edu.cn; Tel.: +86-817-335-2013 †
These two authors contributed equally to this work. 1. Introduction Gastric carcinoma, which is the fourth leading cause of cancer death worldwide, has half of its
incidences detected in East Asian countries, with mortality rates being higher than other countries [1,2]. The search for the adequate and novel treatment has been a constant quest in this regard. Human
epidermal growth factor receptor-2 (HER-2), a transmembrane tyrosine kinase receptor belonging to the
family of epidermal growth factor receptor (EGFR), is an important gastric cancer target encoded by the Int. J. Mol. Sci. 2018, 19, 1981; doi:10.3390/ijms19071981 www.mdpi.com/journal/ijms 2 of 21 Int. J. Mol. Sci. 2018, 19, 1981 ErbB2 gene located on chromosome 17q21 [3,4]. A positive correlation exists, as inferred from numerous
studies, between HER-2 over-expression and cancer cell proliferation, malignancy, metastasis, and
poor outcomes [5–7]. HER-2 over-expression and/or ErbB2 gene amplification (20% of gastric cancer
cases) represents a negative predictor of response to chemotherapy and a positive factor to anti-HER2
agents [4]. Previous studies have confirmed that HER-2 activation can be perceived as a trigger of
multiple cell signal transduction pathways, which promotes aberrant cell proliferation and drug
resistance [8,9]. As a result of rapid advancement in the field of tumor biology, attention has been focused on the
new modality of molecular targeted therapy for advanced cancer [10,11]. Molecular-targeted drugs
such as trastuzumab (Herceptin®), a humanized monoclonal antibody interfering with the extracellular
domain of HER2/neu receptor, has been proved to be beneficial in patients with HER2-positive
advanced gastric and breast cancer in clinical treatment [12,13]. Unfortunately, the acquired resistance
could hinder the effectiveness of trastuzumab [14,15]. In clinical practice, acquired resistance can be a major barrier for antineoplastic agents. Some potential mechanisms of trastuzumab resistance include mutational activation of the
phosphatidylinositide 3-kinase (PI3K)/AKT pathway [16], up-regulation of insulin-like growth factor
receptor (IGFR) and hetero-dimerization of IGFR/HER-2 [17,18], loss of phosphatase and tensin
homolog gene (PTEN) function [19], and accumulation of truncated HER-2 receptor (p95HER-2) [20],
all of which have been verified as principal pathways in breast cancer. Although gastric cancer does
possess some of these pathway modulations, there are some gastric cancer-specific mechanisms too. For instance, over-expression of miR-223 in miR-223/FBXW7 pathway [21], up-regulation of fibroblast
growth factor receptor 3 (FGFR3)/AKT axis [22], activation of β2-adrenergic receptor (β2-AR) signaling,
and loss of HER-2 [23,24] are some of the mechanisms. As opposed to breast cancer, gastric cancer still
lacks extensive research in signaling pathways which mediate acquired trastuzumab resistance. 1. Introduction g
g p
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Mass spectrometry-based proteomics has emerged as a powerful tool for large-scale protein
analysis in biological research [25,26]. Ding et al. have developed a novel technique in recent
years named label-free quantification workflow (Fast-quan) for protein quantification, in which
7000 proteins can be detected and quantified within 12 h of mass spectrometry running time [27]. Here, the trastuzumab-resistant sublines, MKN45/R and NCI N87/R, were obtained by continuous
exposure to increasing doses of trastuzumab up to 80 µg/mL. We proved that there is an association
between acquirement of trastuzumab resistance and EMT. We also performed label-free proteome
profiling of MKN45 and MKN45/R, analyzed differential proteins and explored the corresponding
pathways using bioinformatics techniques. In addition, a series of biological validation were
conducted and the activation of canonical Wnt/β-catenin pathway in both MKN45/R and NCI
N87/R cells was confirmed. Suppression of Wnt/β-catenin signaling by ICG-001 decreased viability
and induced apoptosis of trastuzumab resistant cells in a dose-dependent manner and reversed EMT. Also, knockdown of β-catenin suppressed cell proliferation and enhanced sensitivity to trastuzumab
of resistant cells, implying this pathway to be a possible treatment target for trastuzumab-resistant
gastric carcinoma. 2.1. Establishment of Trastuzumab-Resistant Gastric Cancer Cell Lines We employed Western blot to detect the expression of HER-2 in all six gastric cancer cell lines,
including NCI N87, MKN45, MKN28, BGC823, MGC803, and SGC7901, with a relatively high level
being observed in MKN45 and NCI N87 cells (Figure S1a). To simulate the in vivo mode of resistance,
we treated MKN 45 and NCI N87 cell lines with increasing doses of trastuzumab for five months. Once the drug concentration level reached up to 80 µg/mL, trastuzumab-resistant sublines MKN45/R
and NCI N87/R were then harvested. The IC50 values of MKN45 and MKN45/R cells were 56.48 and
414.52 µg/mL, and that of NCI N87 and NCI N87/R cells were 73.22 and 436.17 µg/mL, respectively 3 of 21
of 20 Int. J. Mol. Sci. 2018, 19, 1981
I t J Mol S i 2018 19 (Figure S1b,c). The resistance index of MKN45/R and NCI N87/R cell lines for trastuzumab were
7.34 and 5.96 respectively, indicating the remarkable resistance of MKN45/R and NCI N87/R cells to
trastuzumab in vitro. Furthermore, we detected cleaved poly ADP-ribose polymerase (PARP) levels in
parental and trastuzumab-resistant cells after trastuzumab treatment (0, 60, 80 µg/mL) by Western
blot; consistent with the inhibition rate of trastuzumab on cell viability, it showed an increase in
a dose-dependent manner, with a more prominent increase observed in parental cells (Figure S1d). respectively (Figure S1b,c). The resistance index of MKN45/R and NCI N87/R cell lines for
trastuzumab were 7.34 and 5.96 respectively, indicating the remarkable resistance of MKN45/R and
NCI N87/R cells to trastuzumab in vitro. Furthermore, we detected cleaved poly ADP-ribose
polymerase (PARP) levels in parental and trastuzumab-resistant cells after trastuzumab treatment (0,
60, 80 μg/mL) by Western blot; consistent with the inhibition rate of trastuzumab on cell viability, it
showed an increase in a dose-dependent manner, with a more prominent increase observed in
l
ll (
S d) To determine if the acquired resistance of the above mentioned sublines was related to HER-2
over-expression, HER-2 level in parental cells and resistant sublines were detected by Western blot
which showed no statistical difference. This implied different mechanisms of acquired resistance other
than HER-2 expression change [28] (Figure S1e). parental cells (Figure S1d). To determine if the acquired resistance of the above mentioned sublines was related to HER-2
over-expression, HER-2 level in parental cells and resistant sublines were detected by Western blot
which showed no statistical difference. 2.1. Establishment of Trastuzumab-Resistant Gastric Cancer Cell Lines This implied different mechanisms of acquired resistance
other than HER-2 expression change [28] (Figure S1e). 2.2. Trastuzumab-Resistant Gastric Cancer Cells Exhibit an EMT-Like Phenotypic Change
2.2. Trastuzumab-Resistant Gastric Cancer Cells Exhibit an EMT-Like Phenotypic Change EMT is of vital importance in many cancer-related biological events, which contributes to invasion,
metastasis, and drug resistance of cancer [29]. When compared with parental cells, MKN45/R and NCI
N87/R cells exhibited an EMT-like phenotype (Figure 1a,b). Immunofluorescence staining of β-tubulin
also revealed the morphological change of NCI N87/R and MNK45/R cells (Figure 1c). Results of
Western blot and immunofluorescence revealed that expression of epithelial marker E-cadherin was
dramatically down-regulated while mesenchymal markers N-cadherin and vimentin were markedly
up-regulated in MKN45/R and NCI N87/R cells when compared with their parental cells (Figure 1d,e). Moreover, EMT-related transcription factors, including snail1 and twist, were significantly up-regulated
in trastuzumab-resistant cells (Figure 1d). These results suggest an EMT phenotypic conversion in
resistant cells. yp
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EMT is of vital importance in many cancer-related biological events, which contributes to invasion,
metastasis, and drug resistance of cancer [29]. When compared with parental cells, MKN45/R and NCI
N87/R cells exhibited an EMT-like phenotype (Figure 1a,b). Immunofluorescence staining of β-tubulin
also revealed the morphological change of NCI N87/R and MNK45/R cells (Figure 1c). Results of
Western blot and immunofluorescence revealed that expression of epithelial marker E-cadherin was
dramatically down-regulated while mesenchymal markers N-cadherin and vimentin were markedly
up-regulated in MKN45/R and NCI N87/R cells when compared with their parental cells (Figure 1d,e). Moreover, EMT-related transcription factors, including snail1 and twist, were significantly
up-regulated in trastuzumab-resistant cells (Figure 1d). These results suggest an EMT phenotypic
conversion in resistant cells. Figure 1. Cont. Figure 1. Cont. 4 of 21
f 20 Int. J. Mol. Sci. 2018, 19, 1981 Figure 1. Trastuzumab resistance is associated with epithelial-mesenchymal transition (EMT) in
gastric cancer cells. (a,b) MKN45/R and NCI N87/R cells showed morphological changes when
compared with their parental cells respectively; (c) Images of cells stained with antibodies against
β-tubulin (red), and nuclei stained with DAPI (blue) (original magnification ×10 for NCI N87 and
NCI N87/R cells, ×20 for MKN45 and MKN45/R cells respectively); (d) Expression of E-cadherin,
N-cadherin, vimentin, snail1, and twist in trastuzumab-resistant cells and their parental cells as
detected by western blot; (e) NCI N87, NCI N87/R, MKN45, and MKN45/R cells were labeled with
antibodies against E-cadherin, N-cadherin, and vimentin. Binding was detected by Alexa Fluor®
594-labeled secondary antibody. Staining of nuclei was done with 1 μg/mL DAPI. Figure 1. Trastuzumab resistance is associated with epithelial-mesenchymal transition (EMT) in gastric
cancer cells. 2.2. Trastuzumab-Resistant Gastric Cancer Cells Exhibit an EMT-Like Phenotypic Change
2.2. Trastuzumab-Resistant Gastric Cancer Cells Exhibit an EMT-Like Phenotypic Change (a,b) MKN45/R and NCI N87/R cells showed morphological changes when compared
with their parental cells respectively; (c) Images of cells stained with antibodies against β-tubulin
(red), and nuclei stained with DAPI (blue) (original magnification ×10 for NCI N87 and NCI N87/R
cells, ×20 for MKN45 and MKN45/R cells respectively); (d) Expression of E-cadherin, N-cadherin,
vimentin, snail1, and twist in trastuzumab-resistant cells and their parental cells as detected by western
blot; (e) NCI N87, NCI N87/R, MKN45, and MKN45/R cells were labeled with antibodies against
E-cadherin, N-cadherin, and vimentin. Binding was detected by Alexa Fluor® 594-labeled secondary
antibody. Staining of nuclei was done with 1 µg/mL DAPI. Figure 1. Trastuzumab resistance is associated with epithelial-mesenchymal transition (EMT) in
gastric cancer cells. (a,b) MKN45/R and NCI N87/R cells showed morphological changes when
compared with their parental cells respectively; (c) Images of cells stained with antibodies against
β-tubulin (red), and nuclei stained with DAPI (blue) (original magnification ×10 for NCI N87 and
NCI N87/R cells, ×20 for MKN45 and MKN45/R cells respectively); (d) Expression of E-cadherin,
N-cadherin, vimentin, snail1, and twist in trastuzumab-resistant cells and their parental cells as
detected by western blot; (e) NCI N87, NCI N87/R, MKN45, and MKN45/R cells were labeled with
antibodies against E-cadherin, N-cadherin, and vimentin. Binding was detected by Alexa Fluor®
594-labeled secondary antibody. Staining of nuclei was done with 1 μg/mL DAPI. Figure 1. Trastuzumab resistance is associated with epithelial-mesenchymal transition (EMT) in gastric
cancer cells. (a,b) MKN45/R and NCI N87/R cells showed morphological changes when compared
with their parental cells respectively; (c) Images of cells stained with antibodies against β-tubulin
(red), and nuclei stained with DAPI (blue) (original magnification ×10 for NCI N87 and NCI N87/R
cells, ×20 for MKN45 and MKN45/R cells respectively); (d) Expression of E-cadherin, N-cadherin,
vimentin, snail1, and twist in trastuzumab-resistant cells and their parental cells as detected by western
blot; (e) NCI N87, NCI N87/R, MKN45, and MKN45/R cells were labeled with antibodies against
E-cadherin, N-cadherin, and vimentin. Binding was detected by Alexa Fluor® 594-labeled secondary
antibody. Staining of nuclei was done with 1 µg/mL DAPI. 2.3. Trastuzumab Resistant Cells Exhibit Increased Capacity of Migration and Proliferation
Concomitant enhancement in cellular migration and proliferation activities is co
2.3. Trastuzumab Resistant Cells Exhibit Increased Capacity of Migration and Proliferation 2.3. Trastuzumab Resistant Cells Exhibit Increased Capacity of Migration and Proliferation
Concomitant enhancement in cellular migration and proliferation activities is com
2.3. Trastuzumab Resistant Cells Exhibit Increased Capacity of Migration and Proliferation g
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with acquisition of resistance by cancer cells. We evaluated migration abilities of the resistant cells
by wound healing assays. In contrast to parental cells, resistant cells showed prominently higher
migration potential evidenced by the rapid and complete wound healing (Figure 2a–d). Moreover,
by means of plate colony formation assay we detected proliferative potency of trastuzumab resistant
cells which formed bigger and multiple colonies than that of parental cells (Figure 2e,f). These data
indicated that trastuzumab-resistant gastric cancer cells acquired higher malignant behaviors than
their parental cells. Concomitant enhancement in cellular migration and proliferation activities is commonly along
with acquisition of resistance by cancer cells. We evaluated migration abilities of the resistant cells
by wound healing assays. In contrast to parental cells, resistant cells showed prominently higher
migration potential evidenced by the rapid and complete wound healing (Figure 2a–d). Moreover,
by means of plate colony formation assay we detected proliferative potency of trastuzumab resistant
cells which formed bigger and multiple colonies than that of parental cells (Figure 2e,f). These data
indicated that trastuzumab-resistant gastric cancer cells acquired higher malignant behaviors than
their parental cells. 5 of 21
5
f 20 Int. J. Mol. Sci. 2018, 19, 1981 J. Mol. Sci. 2018, 19, x
5
Figure 2. Trastuzumab resistant cells exhibit aggressive migration. (a,b) The confluent cell
monolayers were wounded by scraping and the closure of the wounded areas was monitored. The
images of the wounds were taken at different time points; (c,d) Migration activities were analyzed by
measuring the distance cells traveled toward the center of the wound; (e,f) Colony formation assays
were performed and colony numbers were counted and analyzed. All experiments were performed
in triplicate. * p < 0.05, ** p < 0.01 vs. controls. Figure 2. Trastuzumab resistant cells exhibit aggressive migration. (a,b) The confluent cell monolayers
were wounded by scraping and the closure of the wounded areas was monitored. The images of the
wounds were taken at different time points; (c,d) Migration activities were analyzed by measuring the
distance cells traveled toward the center of the wound; (e,f) Colony formation assays were performed
and colony numbers were counted and analyzed. 2.3. Trastuzumab Resistant Cells Exhibit Increased Capacity of Migration and Proliferation
Concomitant enhancement in cellular migration and proliferation activities is co
2.3. Trastuzumab Resistant Cells Exhibit Increased Capacity of Migration and Proliferation All experiments were performed in triplicate
* p < 0.05, ** p < 0.01 vs. controls. Figure 2. Trastuzumab resistant cells exhibit aggressive migration. (a,b) The confluent cell
monolayers were wounded by scraping and the closure of the wounded areas was monitored. The
images of the wounds were taken at different time points; (c,d) Migration activities were analyzed by
measuring the distance cells traveled toward the center of the wound; (e,f) Colony formation assays
were performed and colony numbers were counted and analyzed. All experiments were performed
in triplicate. * p < 0.05, ** p < 0.01 vs. controls. Figure 2. Trastuzumab resistant cells exhibit aggressive migration. (a,b) The confluent cell monolayers
were wounded by scraping and the closure of the wounded areas was monitored. The images of the
wounds were taken at different time points; (c,d) Migration activities were analyzed by measuring the
distance cells traveled toward the center of the wound; (e,f) Colony formation assays were performed
and colony numbers were counted and analyzed. All experiments were performed in triplicate. * p < 0.05, ** p < 0.01 vs. controls. 2.5. Identification of Differentially Expressed Proteins and Enrichment Analysis We performed paired two-tailed student’s t tests (the dataset showed a normal distribution
(Figure S5) to identify differentially expressed proteins with statistical significance. A volcano plot
elucidated differential protein abundance against the corresponding p value obtained from t test
(Figure 3a, Table S3). Compared with MKN45, the quantitative values of 286 proteins (5.24% of the
proteome) displayed a more than twofold increase in MKN45/R cells (p value < 0.05, and marked as red
dots), whereas 211 proteins (3.86% of the proteome) showed more than twofold decrease in MKN45/R
cells (p value < 0.05, and marked as blue dots). The rest of the 4964 proteins (90.90% of the proteome)
were considered as no significant change. The increased and decreased proteins were subjected to
gene ontology (GO) terms enrichment analysis by the WebGestalt (http://www.webgestalt.org). The enrichment analysis of up- or down-regulated proteins in cellular component, biological
process and molecular function were shown in Figure 3b,c respectively. Notably, up-regulated
proteins in MKN45/R cells were annotated as residing in protein complex, Wnt signalosome,
Wnt-Frizzled-LRP5/6 complex and cell-cell adherens junction, whereas down-regulated proteins
in MKN45/R cells were annotated as perinuclear region of cytoplasm, β-catenin destruction
complex, and focal adhesion. Regarding biological processes, up-regulated proteins include those
involved in regulation of β-catenin destruction complex disassembly, Wnt signaling pathway, serine
phosphorylation, and positive regulation of EMT, while down-regulated proteins were mainly
shown as ubiquitin conjugating enzyme activity, protein hetero-dimerization activity and β-catenin
binding. With regard to molecular functions, upregulated proteins are enriched in protein binding,
Wnt-activated receptor activity, Wnt-activated binding, and cadherin binding involved in cell-cell
adhesion, whereas down-regulated proteins function in ubiquitin-dependent protein catabolic process,
antigen processing and presentation, and β-catenin destruction complex assembly, etc. the STRING
database (https://string-db.org/) was then utilized to uncover the relationship between the increased
and decreased proteins. Interactions between them with high confidence (scores > 0.7) were shown
using Cytoscape (v.3.6.0). Red and blue nodes represented up- and down-regulated proteins
respectively, nodes with more than 10 interacting neighbors were displayed in large size while nodes
with 5–10 neighbors were displayed in medium size. We noticed that CTNNB1, AKT1, Wnt3A, and
MAPK represent main hubs formed by up-regulated proteins, and GSK-3β, Axin1/2, and UBE family
proteins represented primary hubs formed by down-regulated ones (Figure 3d). 2.4. Label-Free Proteomic Profiling of MKN45 and MKN45/R Cells
2.4. Label-Free Proteomic Profiling of MKN45 and MKN45/R Cells To explore signal pathways that mediate trastuzumab resistance in trastuzumab-resistant cells,
we implemented label-free quantitative proteomics profiling for a proteome comparison between
MKN45 and MKN45/R cells as according to our previous protocols [30]. Three biological repeats of
these two cell lines were processed respectively. Protein abundance was quantified using intensity
based absolute quantification (iBAQ) and normalized by the fraction of total (FOT) which
represented standardized value of a protein across samples. The FOT was multiplied by 105 for the
ease of presentation. We detected 7645 proteins with at least two unique peptides at 1% peptide
FDR. From this pool, 5996 proteins were identified at 1% protein FDR (Figure S2, Table S1) with 5461
proteins being detected from three out of six experiments which were subsequently selected for
To explore signal pathways that mediate trastuzumab resistance in trastuzumab-resistant cells,
we implemented label-free quantitative proteomics profiling for a proteome comparison between
MKN45 and MKN45/R cells as according to our previous protocols [30]. Three biological repeats of
these two cell lines were processed respectively. Protein abundance was quantified using intensity
based absolute quantification (iBAQ) and normalized by the fraction of total (FOT) which represented
standardized value of a protein across samples. The FOT was multiplied by 105 for the ease of
presentation. We detected 7645 proteins with at least two unique peptides at 1% peptide FDR. From this
pool, 5996 proteins were identified at 1% protein FDR (Figure S2, Table S1) with 5461 proteins being 6 of 21 Int. J. Mol. Sci. 2018, 19, 1981 detected from three out of six experiments which were subsequently selected for bioinformatics
analyses (Figure S2, Table S2). The high correlation (r ≥0.85) of each pair of the three repeats displayed
a good repeatability (Figure S3). A principal component analysis (PCA) revealed that samples of
MKN45 and MKN45/R were completely separated, and three biological repeats of each cell line
were well-clustered (Figure S4). The distribution of these proteins in different ratio range are shown
in Figure S5. 2.5. Identification of Differentially Expressed Proteins and Enrichment Analysis The gray dots are considered as no significant change; (b,c) Gene ontology
(GO) enrichment analysis of upregulated (red bar) and downregulated proteins (blue bar) involved
in cell component, molecular function and biological process; (d) Network analysis of differentially
expressed proteins performed by Cytoscape. Red and blue nodes represent increased and decreased
proteins respectively. Sizes of nodes correspond to numbers of interacting neighbors; (e) WebGestalt
analysis identified major biological pathways in which differential proteins are involved. Each colored
line indicates a different pathway; (f) Heat map visualization of forty differential proteins identified
from several pathways in both MKN45 and MKN45/R cells. The increased and decreased proteins are
represented by range of red and blue intensities, respectively. Figure 3. Bioinformatics analysis of differentially expressed proteins and significantly altered
pathways. (a) Differentially expressed proteins in MKN45 and MKN45/R cells are illustrated by
volcano plot. The mean ratios of three biological repeats (MKN45/R group/MKN45 group) are
plotted in log2 scale (x-axis) against the corresponding −log10 p value (y-axis). The vertical dotted
lines mark twofold change and horizontal dotted lines represent cutoff p value = 0.05. Proteins which
showed fold changes greater than 2 or less than 0.5 and p < 0.05 are considered as up- or
down-regulated and marked in red and blue, respectively. The gray dots are considered as no
significant change; (b,c) Gene ontology (GO) enrichment analysis of upregulated (red bar) and
downregulated proteins (blue bar) involved in cell component, molecular function and biological
process; (d) Network analysis of differentially expressed proteins performed by Cytoscape. Red and
blue nodes represent increased and decreased proteins respectively. Sizes of nodes correspond to
numbers of interacting neighbors; (e) WebGestalt analysis identified major biological pathways in
Figure 3. Bioinformatics analysis of differentially expressed proteins and significantly altered pathways. (a) Differentially expressed proteins in MKN45 and MKN45/R cells are illustrated by volcano plot. The mean ratios of three biological repeats (MKN45/R group/MKN45 group) are plotted in log2 scale
(x-axis) against the corresponding −log10 p value (y-axis). The vertical dotted lines mark twofold
change and horizontal dotted lines represent cutoff p value = 0.05. Proteins which showed fold changes
greater than 2 or less than 0.5 and p < 0.05 are considered as up- or down-regulated and marked in
red and blue, respectively. 2.5. Identification of Differentially Expressed Proteins and Enrichment Analysis Enrichment analysis
displayed that some signaling pathways showed remarkable changes in MKN45/R cells, which are
correlated with cancer metastasis, invasion and drug resistance, with main interaction nodes formed
by components of Wnt/β-catenin (p = 1.10 × 10−11), insulin receptor (p = 2.68 × 10−7), interleukin-17
(p = 2.46 × 10−9), Notch (p = 1.56 × 10−6), Toll like receptor cascades (p = 1.85 × 10−7), MAPK cascades
(p = 7.18 × 10−6), TGF-β (p = 7.74 × 10−6), angiogenesis cascades (p = 3.94 × 10−9), and focal adhesion
(p = 8.14 × 10−4) signaling pathways (Figure 3e). Quantification of these proteins are represented in
heat map (Figure 3f). 7 of 21 Int. J. Mol. Sci. 2018, 19, 1981 J
,
,
Int. J. Mol. Sci. 2018, 19, x
7 of 20
Figure 3. Bioinformatics analysis of differentially expressed proteins and significantly altered
pathways. (a) Differentially expressed proteins in MKN45 and MKN45/R cells are illustrated by
volcano plot. The mean ratios of three biological repeats (MKN45/R group/MKN45 group) are
plotted in log2 scale (x-axis) against the corresponding −log10 p value (y-axis). The vertical dotted
lines mark twofold change and horizontal dotted lines represent cutoff p value = 0.05. Proteins which
showed fold changes greater than 2 or less than 0.5 and p < 0.05 are considered as up- or
down-regulated and marked in red and blue, respectively. The gray dots are considered as no
significant change; (b,c) Gene ontology (GO) enrichment analysis of upregulated (red bar) and
downregulated proteins (blue bar) involved in cell component, molecular function and biological
process; (d) Network analysis of differentially expressed proteins performed by Cytoscape. Red and
blue nodes represent increased and decreased proteins respectively. Sizes of nodes correspond to
numbers of interacting neighbors; (e) WebGestalt analysis identified major biological pathways in
Figure 3. Bioinformatics analysis of differentially expressed proteins and significantly altered pathways. (a) Differentially expressed proteins in MKN45 and MKN45/R cells are illustrated by volcano plot. The mean ratios of three biological repeats (MKN45/R group/MKN45 group) are plotted in log2 scale
(x-axis) against the corresponding −log10 p value (y-axis). The vertical dotted lines mark twofold
change and horizontal dotted lines represent cutoff p value = 0.05. Proteins which showed fold changes
greater than 2 or less than 0.5 and p < 0.05 are considered as up- or down-regulated and marked in
red and blue, respectively. 2.5. Identification of Differentially Expressed Proteins and Enrichment Analysis Following this, we explored the relationship between up- and down- regulated proteins with the
STRING database to display protein-protein interaction network. CTNNB1 represents a primary center
in the network involving differential proteins in Wnt/β-catenin pathway, and nine of them, FZD1/6,
LRP5/6, Wnt3A, GSK-3β, APC and Axin1/2, constitute the center of the network with high interaction
confidence scores (above > 0.9) (Figure S6b). These signaling molecules were demonstrated in an
interactive network of canonical Wnt/β-catenin pathway (Figure S6c). Our results showed that some signaling components exhibited prominent changes in MKN45/R
cells while changes in Wnt/β-catenin pathway molecules attracted our attention. Mass spectrometry
identified twelve key signaling molecules associated with Wnt/β-catenin pathway (Table S4),
including CTNNB1, Axin1/2, GSK-3β, FZD1/6, Wnt3A, LRP5/6, APC, SOX9, and PLCB3 which
displayed prominent changes in protein quantification (Figure 4a). Next, we confirmed the
expression alteration of Wnt3A, FZD6, CTNNB1, and GSK-3β in MKN45/R and NCI N87/R cells via
Western blot (Figure 4b). Consistent with our mass spectrometry data, an increase of Wnt3A, FZD6 and
CTNNB1, and a decrease of GSK-3β was observed in these two resistant cell lines as compared to their
parental cells (Figure 4c,d). In addition, WebGestalt revealed the connection of Wnt/β-catenin signaling
molecules to multiple diseases or pathophysiology of diseases, out of which the foremost nine include
adenomatous polyposis coli, hepatoblastoma, neoplasm invasiveness, fibroma, osteoarthritis,
colonic neoplasms, tumor angiogenesis, neoplasm metastasis, and pancreatic neoplasms (Figure
S6a). Following this, we explored the relationship between up- and down- regulated proteins with
the STRING database to display protein-protein interaction network. CTNNB1 represents a primary
center in the network involving differential proteins in Wnt/β-catenin pathway, and nine of them,
FZD1/6, LRP5/6, Wnt3A, GSK-3β, APC and Axin1/2, constitute the center of the network with high
interaction confidence scores (above > 0.9) (Figure S6b). These signaling molecules were
demonstrated in an interactive network of canonical Wnt/β-catenin pathway (Figure S6c). Figure 4. Validation of differentially expressed proteins in Wnt/β-catenin signaling pathway. (a)
Twelve Wnt/β-catenin pathway components differentially expressed in MKN45/R and MKN45 cells
were measured by mass spectrometry; (b) Expression of some Wnt/β-catenin pathway proteins
detected by western blot. β-actin was used as a loading control; (c,d) Quantification of Western blot
signals in two groups of cell lines. Three independent biological replicates were shown as mean ±
SEM. * p < 0.05, ** p < 0.01 vs. control. Figure 4. Validation of differentially expressed proteins in Wnt/β-catenin signaling pathway. 2.5. Identification of Differentially Expressed Proteins and Enrichment Analysis The gray dots are considered as no significant change; (b,c) Gene ontology
(GO) enrichment analysis of upregulated (red bar) and downregulated proteins (blue bar) involved
in cell component, molecular function and biological process; (d) Network analysis of differentially
expressed proteins performed by Cytoscape. Red and blue nodes represent increased and decreased
proteins respectively. Sizes of nodes correspond to numbers of interacting neighbors; (e) WebGestalt
analysis identified major biological pathways in which differential proteins are involved. Each colored
line indicates a different pathway; (f) Heat map visualization of forty differential proteins identified
from several pathways in both MKN45 and MKN45/R cells. The increased and decreased proteins are
represented by range of red and blue intensities, respectively. Int. J. Mol. Sci. 2018, 19, 1981
MKN45/R cells. The
intensities respective 8 of 21
ue 2.6. Label-Free Quantitative Proteomics Profiling Reveals the Activation of Canonical Wnt/β-Catenin Signaling
Pathway in Trastuzumab-Resistant Cells
2.6. Label-Free Quantitative Proteomics Profiling Reveals the Activation of Canonical Wnt/β-Catenin
Signaling Pathway in Trastuzumab-Resistant Cells 2.6. Label-Free Quantitative Proteomics Profiling Reveals the Activation of Canonical Wnt/β-Catenin Signaling
Pathway in Trastuzumab-Resistant Cells
2.6. Label-Free Quantitative Proteomics Profiling Reveals the Activation of Canonical Wnt/β-Catenin
Signaling Pathway in Trastuzumab-Resistant Cells Label-Free Quantitative Proteomics Profiling Reveals the Activation of Canonical Wnt/β-Catenin Signaling
way in Trastuzumab-Resistant Cells
Label-Free Quantitative Proteomics Profiling Reveals the Activation of Canonical Wnt/β-Catenin
aling Pathway in Trastuzumab-Resistant Cells Our results showed that some signaling components exhibited prominent changes in MKN45/R
cells while changes in Wnt/β-catenin pathway molecules attracted our attention. Mass spectrometry
identified twelve key signaling molecules associated with Wnt/β-catenin pathway (Table S4), including
CTNNB1, Axin1/2, GSK-3β, FZD1/6, Wnt3A, LRP5/6, APC, SOX9, and PLCB3 which displayed
prominent changes in protein quantification (Figure 4a). Next, we confirmed the expression alteration
of Wnt3A, FZD6, CTNNB1, and GSK-3β in MKN45/R and NCI N87/R cells via Western blot
(Figure 4b). Consistent with our mass spectrometry data, an increase of Wnt3A, FZD6 and CTNNB1,
and a decrease of GSK-3β was observed in these two resistant cell lines as compared to their parental
cells (Figure 4c,d). In addition, WebGestalt revealed the connection of Wnt/β-catenin signaling
molecules to multiple diseases or pathophysiology of diseases, out of which the foremost nine
include adenomatous polyposis coli, hepatoblastoma, neoplasm invasiveness, fibroma, osteoarthritis,
colonic neoplasms, tumor angiogenesis, neoplasm metastasis, and pancreatic neoplasms (Figure S6a). 2.5. Identification of Differentially Expressed Proteins and Enrichment Analysis (a) Twelve Wnt/β-catenin pathway components differentially expressed in MKN45/R and MKN45
cells were measured by mass spectrometry; (b) Expression of some Wnt/β-catenin pathway proteins
detected by western blot. β-actin was used as a loading control; (c,d) Quantification of Western blot
signals in two groups of cell lines. Three independent biological replicates were shown as mean ± SEM. * p < 0.05, ** p < 0.01 vs. control. Figure 4. Validation of differentially expressed proteins in Wnt/β-catenin signaling pathway. (a)
Twelve Wnt/β-catenin pathway components differentially expressed in MKN45/R and MKN45 cells
were measured by mass spectrometry; (b) Expression of some Wnt/β-catenin pathway proteins
detected by western blot. β-actin was used as a loading control; (c,d) Quantification of Western blot
signals in two groups of cell lines. Three independent biological replicates were shown as mean ±
SEM. * p < 0.05, ** p < 0.01 vs. control. Figure 4. Validation of differentially expressed proteins in Wnt/β-catenin signaling pathway. (a) Twelve Wnt/β-catenin pathway components differentially expressed in MKN45/R and MKN45
cells were measured by mass spectrometry; (b) Expression of some Wnt/β-catenin pathway proteins
detected by western blot. β-actin was used as a loading control; (c,d) Quantification of Western blot
signals in two groups of cell lines. Three independent biological replicates were shown as mean ± SEM. * p < 0.05, ** p < 0.01 vs. control. Int. J. Mol. Sci. 2018, 19, 1981 9 of 21 2.7. ICG-001 Reduces Viability, Induces Apoptosis and Inhibits β-Catenin Phosphorylation of MKN45/R and
NCI N87/R Cells
Int. J. Mol. Sci. 2018, 19, x
9 of 20 2.7. ICG-001 Reduces Viability, Induces Apoptosis and Inhibits β-Catenin Phosphorylation of MKN45/R and
NCI N87/R Cells
Int. J. Mol. Sci. 2018, 19, x
9 of 20 ICG-001 Reduces Viability, Induces Apoptosis and Inhibits β-Catenin Phosphorylation of MKN45/R and
N87/R Cells
Int J Mol Sci 2018 19 x
9 of 20 2.7. ICG-001 Reduces Viability, Induces Apoptosis and Inhibits β-Catenin Phosphorylation of MKN45/R and
NCI N87/R Cells ICG-001 is known to be a novel selective inhibitor of the canonical Wnt/β-catenin pathway
which targets various cancer cell lines [31,32]. It disrupts the interaction of β-catenin with
T-cell factor/lymphoid enhancer factor (TCF/LEF) by binding to the transcriptional coactivator
cyclic adenosine monophosphate (AMP) response element-binding protein (CBP) and antagonizes
Wnt/β-catenin/TCF-mediated transcription. 2.5. Identification of Differentially Expressed Proteins and Enrichment Analysis To examine the inhibitory effect of ICG-001 on
Wnt/β-catenin pathway in gastric cancer cells, MKN45, MKN45/R, NCI N87, and NCI N87/R
cells were cultured under different concentrations of ICG-001 for 72 h. ICG-001 suppressed MKN45,
MKN45/R, NCI N87, and NCI N87/R cell proliferation in a dose-dependent manner, however,
the effect was more prominent in MKN45/R and NCI N87/R cells at all dosages (Figure 5a,b). It reached statistical significance at 4 µM with cell viabilities of 83.4% and 64.7% in MKN45 and
MKN45/R, and 6 µM with cell viabilities of 81.6% and 66.3% in NCI N87 and NCI N87/R, respectively
(p < 0.05) (Figure 5a,b). In keeping with the inhibition of ICG-001 on cell viability, western blot also
showed a dose-dependent increase of cleaved PARP after ICG-001 treatment, with a more prominent
increase observed in resistant sublines (Figure 5c). These results indicated that trastuzumab-resistant
gastric cancer cells became more sensitive to β-catenin inhibition by ICG-001 than their parental
cells, which coheres with its dependence on the β-catenin pathway for survival. To further confirm
the activation of Wnt/β-catenin pathway which mediates trastuzumab resistance, Western blot was
conducted to detect the phosphorylation of β-catenin, which is a known signaling event in the canonical
Wnt/β-catenin pathway. As anticipated, phosphor β-catenin were higher in MKN45/R and NCI
N87/R cells than their parental cells, and the decrease of phosphor β-catenin in MKN45/R after
ICG-001 treatment was more pronounced between 6 µM and 10 µM (Figure 5d). Similar results were
observed between NCI N87 and NCI N87/R cells (Figure 5d). Moreover, immunofluorescence assay
also confirmed the significant decrease of phosphor β-catenin in MKN45/R and NCI N87/R cells
following treatment with ICG-001 (Figure 5e). To summarize, our results revealed the activation of
canonical Wnt/β-catenin pathway in MKN45/R and NCI N87/R cells which can be preferentially
inhibited by ICG-001. 2.7. ICG-001 Reduces Viability, Induces Apoptosis and Inhibits β-Catenin Phosphorylation of MKN45/R and
NCI N87/R Cells
ICG-001 is known to be a novel selective inhibitor of the canonical Wnt/β-catenin pathway
which targets various cancer cell lines [31,32]. It disrupts the interaction of β-catenin with T-cell
factor/lymphoid enhancer factor (TCF/LEF) by binding to the transcriptional coactivator cyclic
adenosine monophosphate (AMP) response element-binding protein (CBP) and antagonizes
Wnt/β-catenin/TCF-mediated transcription. To examine the inhibitory effect of ICG-001 on
Wnt/β-catenin pathway in gastric cancer cells, MKN45, MKN45/R, NCI N87, and NCI N87/R cells
were cultured under different concentrations of ICG-001 for 72 h. 2.5. Identification of Differentially Expressed Proteins and Enrichment Analysis ICG-001 suppressed MKN45,
MKN45/R, NCI N87, and NCI N87/R cell proliferation in a dose-dependent manner, however, the
effect was more prominent in MKN45/R and NCI N87/R cells at all dosages (Figure 5a,b). It reached
statistical significance at 4 μM with cell viabilities of 83.4% and 64.7% in MKN45 and MKN45/R, and
6 μM with cell viabilities of 81.6% and 66.3% in NCI N87 and NCI N87/R, respectively (p < 0.05)
(Figure 5a,b). In keeping with the inhibition of ICG-001 on cell viability, western blot also showed a
dose-dependent increase of cleaved PARP after ICG-001 treatment, with a more prominent increase
observed in resistant sublines (Figure 5c). These results indicated that trastuzumab-resistant gastric
cancer cells became more sensitive to β-catenin inhibition by ICG-001 than their parental cells, which
coheres with its dependence on the β-catenin pathway for survival. To further confirm the activation
of Wnt/β-catenin pathway which mediates trastuzumab resistance, Western blot was conducted to
detect the phosphorylation of β-catenin, which is a known signaling event in the canonical
Wnt/β-catenin pathway. As anticipated, phosphor β-catenin were higher in MKN45/R and NCI
N87/R cells than their parental cells, and the decrease of phosphor β-catenin in MKN45/R after
ICG-001 treatment was more pronounced between 6 μM and 10 μM (Figure 5d). Similar results were
observed between NCI N87 and NCI N87/R cells (Figure 5d). Moreover, immunofluorescence assay
also confirmed the significant decrease of phosphor β-catenin in MKN45/R and NCI N87/R cells
following treatment with ICG-001 (Figure 5e). To summarize, our results revealed the activation of
canonical Wnt/β-catenin pathway in MKN45/R and NCI N87/R cells which can be preferentially
inhibited by ICG-001. Figure 5. Cont. Figure 5. Cont. 10 of 21 Int. J. Mol. Sci. 2018, 19, 1981 Figure 5. ICG-001 preferentially inhibited cell viability and phosphorylation of β-catenin and
induced apoptosis in trastuzumab resistant cells. (a,b) Cells cultured for 24 h were either treated with
or without ICG-001 at the indicated concentrations or (dimethyl sulfoxide) DMSO, which was set as
control. Cell viability was determined by CCK-8 assay 72 h later. Results expressed as % control
represented the mean of three experiments. * p < 0.05, ** p < 0.01 vs. 2.5. Identification of Differentially Expressed Proteins and Enrichment Analysis controls; (c) Cells exposed to
ICG-001 for 24 h under different concentrations of (0, 6, 8 μM) were analyzed by Western blot for
full-length PARP and cleaved PARP, with β-actin being a loading control; (d) Cells were exposed to
ICG-001 for 24 h at the indicated concentrations and with DMSO as control, and cell lysates were
probed with phosphor- and total antibodies of β-catenin signaling pathway. β-actin was used as a
loading control. The representative images were cropped and shown; (e) MKN45/R and NCI N87/R
cells were treated with ICG-001 (6 μM) for 24 h and DMSO as control which was followed by
collection for immunofluorescence. Nuclear staining was performed using DAPI (blue), and total
β-catenin and phosphor β-catenin were stained by Alexa Fluor® 488-labeled secondary antibody
(green). Slides were imaged using ×20 lens of Leica DMi8 fluorescent microscope. 2.8. ICG-001 Inhibits the Invasion of Trastuzumab-Resistant Cells
Figure 5. ICG-001 preferentially inhibited cell viability and phosphorylation of β-catenin and induced
apoptosis in trastuzumab resistant cells. (a,b) Cells cultured for 24 h were either treated with or without
ICG-001 at the indicated concentrations or (dimethyl sulfoxide) DMSO, which was set as control. Cell viability was determined by CCK-8 assay 72 h later. Results expressed as % control represented the
mean of three experiments. * p < 0.05, ** p < 0.01 vs. controls; (c) Cells exposed to ICG-001 for 24 h under
different concentrations of (0, 6, 8 µM) were analyzed by Western blot for full-length PARP and cleaved
PARP, with β-actin being a loading control; (d) Cells were exposed to ICG-001 for 24 h at the indicated
concentrations and with DMSO as control, and cell lysates were probed with phosphor- and total
antibodies of β-catenin signaling pathway. β-actin was used as a loading control. The representative
images were cropped and shown; (e) MKN45/R and NCI N87/R cells were treated with ICG-001 (6 µM)
for 24 h and DMSO as control which was followed by collection for immunofluorescence. Nuclear
staining was performed using DAPI (blue), and total β-catenin and phosphor β-catenin were stained
by Alexa Fluor® 488-labeled secondary antibody (green). Slides were imaged using ×20 lens of Leica
DMi8 fluorescent microscope. Figure 5. ICG-001 preferentially inhibited cell viability and phosphorylation of β-catenin and
induced apoptosis in trastuzumab resistant cells. 2.5. Identification of Differentially Expressed Proteins and Enrichment Analysis (a,b) Cells cultured for 24 h were either treated with
or without ICG-001 at the indicated concentrations or (dimethyl sulfoxide) DMSO, which was set as
control. Cell viability was determined by CCK-8 assay 72 h later. Results expressed as % control
represented the mean of three experiments. * p < 0.05, ** p < 0.01 vs. controls; (c) Cells exposed to
ICG-001 for 24 h under different concentrations of (0, 6, 8 μM) were analyzed by Western blot for
full-length PARP and cleaved PARP, with β-actin being a loading control; (d) Cells were exposed to
ICG-001 for 24 h at the indicated concentrations and with DMSO as control, and cell lysates were
probed with phosphor- and total antibodies of β-catenin signaling pathway. β-actin was used as a
loading control. The representative images were cropped and shown; (e) MKN45/R and NCI N87/R
cells were treated with ICG-001 (6 μM) for 24 h and DMSO as control which was followed by
collection for immunofluorescence. Nuclear staining was performed using DAPI (blue), and total
β-catenin and phosphor β-catenin were stained by Alexa Fluor® 488-labeled secondary antibody
(green). Slides were imaged using ×20 lens of Leica DMi8 fluorescent microscope. 2.8. ICG-001 Inhibits the Invasion of Trastuzumab-Resistant Cells
Figure 5. ICG-001 preferentially inhibited cell viability and phosphorylation of β-catenin and induced
apoptosis in trastuzumab resistant cells. (a,b) Cells cultured for 24 h were either treated with or without
ICG-001 at the indicated concentrations or (dimethyl sulfoxide) DMSO, which was set as control. Cell viability was determined by CCK-8 assay 72 h later. Results expressed as % control represented the
mean of three experiments. * p < 0.05, ** p < 0.01 vs. controls; (c) Cells exposed to ICG-001 for 24 h under
different concentrations of (0, 6, 8 µM) were analyzed by Western blot for full-length PARP and cleaved
PARP, with β-actin being a loading control; (d) Cells were exposed to ICG-001 for 24 h at the indicated
concentrations and with DMSO as control, and cell lysates were probed with phosphor- and total
antibodies of β-catenin signaling pathway. β-actin was used as a loading control. The representative
images were cropped and shown; (e) MKN45/R and NCI N87/R cells were treated with ICG-001 (6 µM)
for 24 h and DMSO as control which was followed by collection for immunofluorescence. 2.5. Identification of Differentially Expressed Proteins and Enrichment Analysis Nuclear
staining was performed using DAPI (blue), and total β-catenin and phosphor β-catenin were stained
by Alexa Fluor® 488-labeled secondary antibody (green). Slides were imaged using ×20 lens of Leica
DMi8 fluorescent microscope. Cancer cells usually become more invasive after the acq
evaluated how ICG-001 inhibited invasion of MNK45/R and N
2.8. ICG-001 Inhibits the Invasion of Trastuzumab-Resistant Cells (a,b) (Left panels) Representative image of cell invasion (original magnification
100×). (a,b) (Right panels) Quantitative results of invasion assays, the results are expressed as mean
±SEM of three independent experiments. * p < 0.05 vs. controls. N.S = no statistical significance. Figure 6. ICG-001 inhibits invasion of trastuzumab resistant cells. Cells were cultured with
or without ICG-001 under indicated concentrations, and their invasive ability was evaluated by
transwell assay. (a,b) (Left panels) Representative image of cell invasion (original magnification 100×). (a,b) (Right panels) Quantitative results of invasion assays, the results are expressed as mean ±SEM of
three independent experiments. * p < 0.05 vs. controls. N.S = no statistical significance. Figure 6. ICG-001 inhibits invasion of trastuzumab resistant cells. Cells were cultured with or
without ICG-001 under indicated concentrations, and their invasive ability was evaluated by
transwell assay. (a,b) (Left panels) Representative image of cell invasion (original magnification
100×). (a,b) (Right panels) Quantitative results of invasion assays, the results are expressed as mean
±SEM of three independent experiments. * p < 0.05 vs. controls. N.S = no statistical significance. Figure 6. ICG-001 inhibits invasion of trastuzumab resistant cells. Cells were cultured with
or without ICG-001 under indicated concentrations, and their invasive ability was evaluated by
transwell assay. (a,b) (Left panels) Representative image of cell invasion (original magnification 100×). (a,b) (Right panels) Quantitative results of invasion assays, the results are expressed as mean ±SEM of
three independent experiments. * p < 0.05 vs. controls. N.S = no statistical significance. 2.9. Knockdown of β-Catenin in Trastuzumab-Resistant Cells Suppresses Proliferation and Enhances
Trastuzumab Sensitivity
2.9. Knockdown of β-Catenin in Trastuzumab-Resistant Cells Suppresses Proliferation and Enhances
Trastuzumab Sensitivity 2.9. Knockdown of β-Catenin in Trastuzumab-Resistant Cells Suppresses Proliferation and Enhances
Trastuzumab Sensitivity
2.9. Knockdown of β-Catenin in Trastuzumab-Resistant Cells Suppresses Proliferation and Enhances
Trastuzumab Sensitivity To ascertain whether the Wnt/β-catenin pathway is critical for maintainence of trastuzumab
resistance, we established stable cell lines via lentiviral small interfering RNA (siRNA) mediated
β-catenin knockdown. The efficiency of β-catenin knockdown was assessed by real time-polymerase
chain reaction (RT-PCR) and Western blot respectively (Figure 7a,b). We compared the proliferation
activity of cells transfected with scramble control and siRNA targeting β-catenin. Cancer cells usually become more invasive after the acq
evaluated how ICG-001 inhibited invasion of MNK45/R and N
2.8. ICG-001 Inhibits the Invasion of Trastuzumab-Resistant Cells Without ICG-001, more MKN45/R and NCI N87/R cells passed through the polycarbonate
membrane than their parental cells (p < 0.05). However, with ICG-001 treatment, invasion of
MKN45/R and NCI N87/R cells was remarkably inhibited (p < 0.05) while invasion of MKN45 and
NCI N87 cells was not (p > 0.05) (Figure 6a,b). These results indicated that activation of
Wnt/β-catenin pathway facilitates invasion of MKN45/R and NCI N87/R cells in vitro, and ICG-001
can preferentially inhibit the invasion of MKN45/R and NCI N87/R cells. Cancer cells usually become more invasive after the acquisition of drug resistance. Here we
evaluated how ICG-001 inhibited invasion of MNK45/R and NCI N87/R cells by transwell assays. Without ICG-001, more MKN45/R and NCI N87/R cells passed through the polycarbonate membrane
than their parental cells (p < 0.05). However, with ICG-001 treatment, invasion of MKN45/R and
NCI N87/R cells was remarkably inhibited (p < 0.05) while invasion of MKN45 and NCI N87 cells
was not (p > 0.05) (Figure 6a,b). These results indicated that activation of Wnt/β-catenin pathway
facilitates invasion of MKN45/R and NCI N87/R cells in vitro, and ICG-001 can preferentially inhibit
the invasion of MKN45/R and NCI N87/R cells. 11 of 21 Int. J. Mol. Sci. 2018, 19, 1981 Figure 6. ICG-001 inhibits invasion of trastuzumab resistant cells. Cells were cultured with or
without ICG-001 under indicated concentrations, and their invasive ability was evaluated by
transwell assay. (a,b) (Left panels) Representative image of cell invasion (original magnification
100×). (a,b) (Right panels) Quantitative results of invasion assays, the results are expressed as mean
±SEM of three independent experiments. * p < 0.05 vs. controls. N.S = no statistical significance. 9. Knockdown of β-Catenin in Trastuzumab-Resistant Cells Suppresses Proliferation and Enhances
Figure 6. ICG-001 inhibits invasion of trastuzumab resistant cells. Cells were cultured with
or without ICG-001 under indicated concentrations, and their invasive ability was evaluated by
transwell assay. (a,b) (Left panels) Representative image of cell invasion (original magnification 100×). (a,b) (Right panels) Quantitative results of invasion assays, the results are expressed as mean ±SEM of
three independent experiments. * p < 0.05 vs. controls. N.S = no statistical significance. 9 Knockdown of β Catenin in Trastuzumab Resistant Cells Suppresses Proliferation and Enhances Figure 6. ICG-001 inhibits invasion of trastuzumab resistant cells. Cells were cultured with or
without ICG-001 under indicated concentrations, and their invasive ability was evaluated by
transwell assay. Cancer cells usually become more invasive after the acq
evaluated how ICG-001 inhibited invasion of MNK45/R and N
2.8. ICG-001 Inhibits the Invasion of Trastuzumab-Resistant Cells As expected,
knockdown of β-catenin in trastuzumab-resistant MKN45/R and NCI N87/R cells led to a decrease in
proliferation (Figure 7c,d); correspondingly, cell viability decreased after treatment with trastuzumab
(Figure 7e,f), implicating the direct correlation between Wnt/β-catenin pathway and trastuzumab
resistance. Moreover, Annexin V-FITC/PI staining showed 5.9- and 2.8-fold increases in cell
apoptosis upon trastuzumab treatment in MKN45/R siβ-catenin and NCI N87/R siβ-catenin cells
compared to their controls respectively (Figure 7g). These data suggest that knockdown of β-catenin
causes a decrease in cell proliferation, an increase in trastuzumab sensitivity and enhancement in
apoptosis of gastric cancer cells. To ascertain whether the Wnt/β-catenin pathway is critical for maintainence of trastuzumab
resistance, we established stable cell lines via lentiviral small interfering RNA (siRNA) mediated
β-catenin knockdown. The efficiency of β-catenin knockdown was assessed by real time-polymerase
chain reaction (RT-PCR) and Western blot respectively (Figure 7a,b). We compared the proliferation
activity of cells transfected with scramble control and siRNA targeting β-catenin. As expected,
knockdown of β-catenin in trastuzumab-resistant MKN45/R and NCI N87/R cells led to a decrease in
proliferation (Figure 7c,d); correspondingly, cell viability decreased after treatment with trastuzumab
(Figure 7e,f), implicating the direct correlation between Wnt/β-catenin pathway and trastuzumab
resistance. Moreover, Annexin V-FITC/PI staining showed 5.9- and 2.8-fold increases in cell apoptosis
upon trastuzumab treatment in MKN45/R siβ-catenin and NCI N87/R siβ-catenin cells compared
to their controls respectively (Figure 7g). These data suggest that knockdown of β-catenin causes
a decrease in cell proliferation, an increase in trastuzumab sensitivity and enhancement in apoptosis of
gastric cancer cells. 12 of 21 12 of 21 Int. J. Mol. Sci. 2018, 19, 1981 J
,
,
Figure 7. Knockdown of β-catenin enhances the sensitivity of trastuzumab-resistant gastric cancer
cells to trastuzumab (a,b) Evaluation of β-catenin knockdown in trastuzumab-resistant MKN45 and
NCI N87 cells by real time-polymerase chain reaction (RT-PCR) and Western blot respectively; (c,d)
Knockdown of β-catenin via small interfering RNA (siRNA) targeting β-catenin (siβ-catenin)
decreased proliferation of MKN45/R and NCI N87/R cells; (e,f) Knockdown of β-catenin induced a
decrease of living cells after trastuzumab treatment; (g) Detection of cell apoptosis by Annexin V/PI
staining upon treatment with 80 μg/mL trastuzumab for 96 h. All data represent three independent
experiments. * p < 0.05, ** p < 0.01 vs. control. Cancer cells usually become more invasive after the acq
evaluated how ICG-001 inhibited invasion of MNK45/R and N
2.8. ICG-001 Inhibits the Invasion of Trastuzumab-Resistant Cells 2 10 I hibiti
f W t/β C t
i
Si
li
b ICG 001 R
EMT i
T
t
b R i t
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Figure 7. Knockdown of β-catenin enhances the sensitivity of trastuzumab-resistant gastric cancer cells
to trastuzumab (a,b) Evaluation of β-catenin knockdown in trastuzumab-resistant MKN45 and NCI N87
cells by real time-polymerase chain reaction (RT-PCR) and Western blot respectively; (c,d) Knockdown
of β-catenin via small interfering RNA (siRNA) targeting β-catenin (siβ-catenin) decreased proliferation
of MKN45/R and NCI N87/R cells; (e,f) Knockdown of β-catenin induced a decrease of living cells
after trastuzumab treatment; (g) Detection of cell apoptosis by Annexin V/PI staining upon treatment
with 80 µg/mL trastuzumab for 96 h. All data represent three independent experiments. * p < 0.05,
** p < 0.01 vs. control. Figure 7. Knockdown of β-catenin enhances the sensitivity of trastuzumab-resistant gastric cancer
Figure 7. Knockdown of β-catenin enhances the sensitivity of trastuzumab-resistant gastric cancer cells Figure 7. Knockdown of β-catenin enhances the sensitivity of trastuzumab-resistant gastric cancer
cells to trastuzumab (a,b) Evaluation of β-catenin knockdown in trastuzumab-resistant MKN45 and
NCI N87 cells by real time-polymerase chain reaction (RT-PCR) and Western blot respectively; (c,d)
Knockdown of β-catenin via small interfering RNA (siRNA) targeting β-catenin (siβ-catenin)
decreased proliferation of MKN45/R and NCI N87/R cells; (e,f) Knockdown of β-catenin induced a
decrease of living cells after trastuzumab treatment; (g) Detection of cell apoptosis by Annexin V/PI
staining upon treatment with 80 μg/mL trastuzumab for 96 h. All data represent three independent
experiments. * p < 0.05, ** p < 0.01 vs. control. Figure 7. Knockdown of β-catenin enhances the sensitivity of trastuzumab-resistant gastric cancer cells
to trastuzumab (a,b) Evaluation of β-catenin knockdown in trastuzumab-resistant MKN45 and NCI N87
cells by real time-polymerase chain reaction (RT-PCR) and Western blot respectively; (c,d) Knockdown
of β-catenin via small interfering RNA (siRNA) targeting β-catenin (siβ-catenin) decreased proliferation
of MKN45/R and NCI N87/R cells; (e,f) Knockdown of β-catenin induced a decrease of living cells
after trastuzumab treatment; (g) Detection of cell apoptosis by Annexin V/PI staining upon treatment
with 80 µg/mL trastuzumab for 96 h. All data represent three independent experiments. * p < 0.05,
** p < 0.01 vs. control. 3. Discussion
3. Discussion Tratuzumab, which has been used in clinical therapy for 20 years, is one of the most effective
anti-HER2 antibodies in breast and gastric cancer, yet it shows a limited curative effect due to tumor
acquired resistance. Although multiple mechanisms of trastuzumab resistance have been proposed
in breast cancer, it is ambiguous whether similar mechanisms exist in gastric cancer. Therefore,
understanding the molecular mechanisms and identifying the phenotype of trastuzumab resistance
in gastric cancer is of great significance in developing novel therapeutic strategies. Targeted DNA
exome and messenger RNA (mRNA) sequencing has been extensively applied in analyses and
identification of the genome and transcriptome; however genomic, transcriptomic, and proteomic
data was shown to have a poor coherence [33,34]. Without doubt, measurement of protein is more
efficient than detecting DNA and mRNA for mining actionable drug targets. Owing to recent
progress in liquid chromatography coupled to high resolution mass spectrometry instrumentation
and techniques, proteome profiling has become a fairly precise method. Advanced mass
spectrometry-based proteomics in our previous research demonstrated that activated mTOR
signaling contributes to trastuzumab resistance by inducing NCI N87 cells at low dosage (10 μg/mL)
of trastuzumab. In the present study, we acquired a pair of trastuzumab resistant gastric cancer cell
lines (MKN45/R and NCI N87/R) by using a higher concentration of trastuzumab (80 μg/mL). Label-free proteome profiling was employed to identify specific signaling pathways that mediated
trastuzumab resistance using MKN45 and MKN45/R cell lines. Intriguingly, the Wnt/β-catenin
pathway rather than mTOR signaling is the most dominant activated signaling pathway that
mediates trastuzumab resistance. Since MKN45 and NCI N87 were derived from different types of
gastric cancer that possess variant genetic backgrounds, it is possible that they may acquire
resistance via different mechanisms Besides the activation of different pathways may implicate a
Tratuzumab, which has been used in clinical therapy for 20 years, is one of the most effective
anti-HER2 antibodies in breast and gastric cancer, yet it shows a limited curative effect due to tumor
acquired resistance. Although multiple mechanisms of trastuzumab resistance have been proposed
in breast cancer, it is ambiguous whether similar mechanisms exist in gastric cancer. Therefore,
understanding the molecular mechanisms and identifying the phenotype of trastuzumab resistance in
gastric cancer is of great significance in developing novel therapeutic strategies. 2.10. Inhibition of Wnt/β-Catenin Signaling by ICG-001 Reverses EMT in Trastuzumab Resistant Cells
T
i
f
th
i
i ht i t
th
i hibiti
f W t/β
t
i
th
i
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2.10. Inhibition of Wnt/β-Catenin Signaling by ICG-001 Reverses EMT in Trastuzumab Resistant Cel 2.10. Inhibition of Wnt/β-Catenin Signaling by ICG-001 Reverses EMT in Trastuzumab Resistant Cells
To
ai
fu the i
i ht i to the i hibitio
of W t/β ate i
ath
ay i
e ulati
the EMT
2.10. Inhibition of Wnt/β-Catenin Signaling by ICG-001 Reverses EMT in Trastuzumab Resistant Cells To gain further insights into the inhibition of Wnt/β-catenin pathway in regulating the EMT
process, we carried out Western blot to examine the expression of E-cadherin, N-cadherin, and
vimentin which are EMT-related markers. Comparing with the control group, E-cadherin was
significantly up-regulated, while N-cadherin and vimentin were down-regulated in MKN45/R and
NCI N87/R cells after treatment with ICG-001 for 48 h (Figure 8a–c). Our results indicated that
ICG-001 reversed the decrease of E-cadherin, and the increase of N-cadherin and vimentin in
To gain further insights into the inhibition of Wnt/β-catenin pathway in regulating the EMT
process, we carried out Western blot to examine the expression of E-cadherin, N-cadherin, and
vimentin which are EMT-related markers. Comparing with the control group, E-cadherin was
significantly up-regulated, while N-cadherin and vimentin were down-regulated in MKN45/R and
NCI N87/R cells after treatment with ICG-001 for 48 h (Figure 8a–c). Our results indicated that ICG-001
reversed the decrease of E-cadherin, and the increase of N-cadherin and vimentin in MKN45/R and 13 of 21
13 of 20 13 of 21
13 of 20 Int. J. Mol. Sci. 2018, 19, 1981
I
l NCI N87/R cells, manifesting that inhibition of β-catenin signaling by ICG-001 reverses EMT in
trastuzumab-resistant gastric cancer cells. MKN45/R and NCI N87/R cells, manifesting that inhibition of β-catenin signaling by ICG-001
reverses EMT in trastuzumab-resistant gastric cancer cells. Figure 8. Inhibition of Wnt/β-catenin pathway by ICG-001 reverses EMT in trastuzumab resistant
gastric cancer cells. (a) Western blot analysis of E-cadherin, N-cadherin, and vimentin in
trastuzumab resistant cells before and after 48 h ICG-001(6 μΜ) treatment; (b,c) Expression of
E-cadherin, N-cadherin and vimentin were normalized against β-actin. The results are expressed as
mean ± SEM of three independent experiments. ** p < 0.01 vs. control. Figure 8. Inhibition of Wnt/β-catenin pathway by ICG-001 reverses EMT in trastuzumab resistant
gastric cancer cells. (a) Western blot analysis of E-cadherin, N-cadherin, and vimentin in trastuzumab
resistant cells before and after 48 h ICG-001(6 µM) treatment; (b,c) Expression of E-cadherin, N-cadherin
and vimentin were normalized against β-actin. The results are expressed as mean ± SEM of three
independent experiments. 2.10. Inhibition of Wnt/β-Catenin Signaling by ICG-001 Reverses EMT in Trastuzumab Resistant Cells
T
i
f
th
i
i ht i t
th
i hibiti
f W t/β
t
i
th
i
l ti
th
2.10. Inhibition of Wnt/β-Catenin Signaling by ICG-001 Reverses EMT in Trastuzumab Resistant Cel ** p < 0.01 vs. control. Figure 8. Inhibition of Wnt/β-catenin pathway by ICG-001 reverses EMT in trastuzumab resistant
gastric cancer cells. (a) Western blot analysis of E-cadherin, N-cadherin, and vimentin in
trastuzumab resistant cells before and after 48 h ICG-001(6 μΜ) treatment; (b,c) Expression of
E-cadherin, N-cadherin and vimentin were normalized against β-actin. The results are expressed as
mean ± SEM of three independent experiments. ** p < 0.01 vs. control. Figure 8. Inhibition of Wnt/β-catenin pathway by ICG-001 reverses EMT in trastuzumab resistant
gastric cancer cells. (a) Western blot analysis of E-cadherin, N-cadherin, and vimentin in trastuzumab
resistant cells before and after 48 h ICG-001(6 µM) treatment; (b,c) Expression of E-cadherin, N-cadherin
and vimentin were normalized against β-actin. The results are expressed as mean ± SEM of three
independent experiments. ** p < 0.01 vs. control. 3. Discussion
3. Discussion Targeted DNA exome
and messenger RNA (mRNA) sequencing has been extensively applied in analyses and identification
of the genome and transcriptome; however genomic, transcriptomic, and proteomic data was shown
to have a poor coherence [33,34]. Without doubt, measurement of protein is more efficient than
detecting DNA and mRNA for mining actionable drug targets. Owing to recent progress in liquid
chromatography coupled to high resolution mass spectrometry instrumentation and techniques,
proteome profiling has become a fairly precise method. Advanced mass spectrometry-based proteomics
in our previous research demonstrated that activated mTOR signaling contributes to trastuzumab
resistance by inducing NCI N87 cells at low dosage (10 µg/mL) of trastuzumab. In the present study,
we acquired a pair of trastuzumab resistant gastric cancer cell lines (MKN45/R and NCI N87/R) by
using a higher concentration of trastuzumab (80 µg/mL). Label-free proteome profiling was employed
to identify specific signaling pathways that mediated trastuzumab resistance using MKN45 and
MKN45/R cell lines. Intriguingly, the Wnt/β-catenin pathway rather than mTOR signaling is the
most dominant activated signaling pathway that mediates trastuzumab resistance. Since MKN45 and
NCI N87 were derived from different types of gastric cancer that possess variant genetic backgrounds,
it is possible that they may acquire resistance via different mechanisms. Besides, the activation of Int. J. Mol. Sci. 2018, 19, 1981 14 of 21 different pathways may implicate a shift of survival signaling in trastuzumab-resistant gastric cancer
cells at different dosages of trastuzumab. Wnt/β-catenin pathway is known to play an important role in organogenesis and pathogenesis of
a variety of diseases which includes cancer as well [35,36]. Roles of canonical Wnt/β-catenin pathway
in regulating tumor cell properties, such as self-renewal, motility and tumor acquired resistance,
has prompted the research for therapeutic strategies targeting this pathway [37–39]. Dysregulation of
Wnt/β-catenin pathway has been recognized to be associated with the development and progression
of several types of cancers including breast cancer, ovarian cancer, and colorectal cancer [36,40,41]. In breast cancer, co-expression of Wnt ligands, including Wnt3, Wnt4, Wnt5a, and Wnt7a, leads to
the activation of Wnt pathway [42], while in colorectal cancer, mutation or loss function of the
APC gene or protein is more likely to be the reason for Wnt pathway activation [43]. 3. Discussion
3. Discussion Data from
our current study indicated that Wnt3A, FZD6, and CTNNB1 were up-regulated, while GSK-3β
was down-regulated in MKN45/R and NCI N87/R cells when compared to their parental cells,
suggesting the acquirement of trastuzumab resistance through activation of canonical Wnt/β-catenin
pathway in gastric cancer. Furthermore, specific modulation of Wnt/β-catenin pathway by CTNNB1
knockdown reversed trastuzumab resistance, decreased cell proliferation, and increased apoptosis of
trastuzumab-resistant gastric cancer cells, indicating the pivotal role of Wnt/β-catenin pathway in
trastuzumab resistance of gastric cancer cells. EMT has been widely recognized as a crucial process associated with cancer progression and
drug resistance, during which cancer cells go through phenotypic alterations and acquire a higher
potential for metastasis [44–46]. Since changes in cellular behavior are linked to EMT, we further
investigated cell morphology and the expression of key EMT markers, showing an up-regulation
of two mesenchymal markers (N-cadherin and vimentin) and a down-regulation of an epithelial
marker (E-cadherin) in MKN45/R and NCI N87/R cells. Two transcription factors, snail1 and twist,
which have been identified as master regulators of EMT, also showed a remarkable upregulation in the
resistant cells. These data indicate that prolonged treatment of trastuzumab induces the EMT process,
which is involved in trastuzumab resistance of gastric cancer cells. Mounting evidence suggests that Wnt/β-catenin pathway plays an important role in EMT
regulation of various cancers. In breast cancer, Wnt3 over-expression activates Wnt/β-catenin
pathway which leads to trans-activation of EGFR and promotes EMT in trastuzumab-resistant cells [42]. In ovarian cancer, modulation of a single upstream gate-keeper of Wnt signaling, secreted frizzled
related protein 4 (SFRP4), which functions as a tumor suppressor, can activate Wnt signaling and
promote EMT [47]. Our results suggest that over-expression of Wnt3A and FZD6 activates the
Wnt/β-catenin pathway in trastuzumab resistant gastric cancer cells, which is accompanied with
EMT. Moreover, ICG-001, a specific inhibitor of β-catenin signaling, dramatically reduced phosphor
β-catenin and reversed EMT, demonstrating that inhibition of Wnt/β-catenin signaling attenuates
EMT in MKN45/R and NCI N87/R cells, suggesting that Wnt/β-catenin signaling is one of the major
pathways involved in EMT that plays an integral role in trastuzumab-acquired resistance. Our current
study concludes that Wnt/β-catenin pathway could be considered as a target for trastuzumab-resistant
gastric cancer, and Wnt/β-catenin pathway inhibitors coupled with trastuzumab may help conquer
resistance and improve the effects of trastuzumab in gastric cancer. 4.3. Establishment of Stable Trastuzumab Resistant Cell Lines with β-Catenin Knockdown β-catenin lentiviral siRNA plasmid (pGLV5-CTNNB1) and scrambled siRNA plasmid
(pGLV5-NC) were acquired from Vipotion Biotechnology (Guangzhou, China). MKN45/R and NCI
N87/R cell lines with β-catenin knockdown were established by infection of high titer lentiviral
particles from GenePharma (Shanghai, China). Briefly, 293T cells were cultured in 6 cm plates with
antibiotic-free media. When the confluence reached about 70%, cells were co-transfected with 2 µg
packaging plasmid and 10 µg of siRNA plasmid mixed with lipofectamine 2000 (Invitrogen, Carlsbad,
CA, USA) according to the manufacturer’s protocol. After incubation for 48 h, supernatants containing
virus particles were collected by centrifugation. Target cells were transduced with virus particles
by plating 1 × 105 cells/well in a 6-well plate. Knockdown efficiency was assessed by RT-PCR and
Western blot. Stable cell lines with β-catenin knockdown were used in the subsequent study. 3. Discussion
3. Discussion Moreover, pathway enrichment analysis in our study also uncovered some other pathways
relevant to trastuzumab resistance which include insulin receptor, interleukin-17, Notch, Toll-like
receptor cascades, Mitogen-activated protein kinase (MAPK) cascades, TGF-β, angiogenesis cascades,
and focal adhesion pathways. Nevertheless, the underlying mechanisms of these pathways still require
further investigation. 15 of 21 Int. J. Mol. Sci. 2018, 19, 1981 4.4. Resistance Index Assays Cells
were
incubated
in
96-well
plates
at
a
density
of
5
×
103
cells/well
and
cultured for 24 h. Then cells were exposed to trastuzumab under different concentrations. Absorbance (A) was detected using CCK-8 in a microplate reader at a wavelength of
450 nm at regular intervals. Cell viability was measured according to the following formula:
(A(drug–supplemented) −A(blank))/(A(normal) −A(blank)) × 100%. Cell inhibition rate (%) =100% −Cell
viability. The half maximal inhibitory concentration (IC50) was calculated using GraphPad Prism
5.0 software. The resistance index was calculated according to the following formula: Resistance
index = IC50 (resistant cells)/IC50 (parental cells). 4.2. Development of Trastuzumab-Resistant MKN45/R and NCI N87/R Gastric Cancer Cell Lines Cells were cultured in DMEM medium containing 10 µg/mL trastuzumab (Roche, Basel,
Switzerland) for four days. Cells that survived were cultured continuously under increasing
concentration of trastuzumab (10, 20, 40, and 80 µg/mL) in the following five months. Cell proliferation
was evaluated by CCK-8 (Dojindo, Kumamoto, Japan) assay at regular intervals. MKN45 and NCI N87
cells which grew stably in trastuzumab-containing medium (80 µg/mL) were sub-cultured successfully
and therefore named MKN45/R and NCI N87/R, respectively. 4.3. Establishment of Stable Trastuzumab Resistant Cell Lines with β-Catenin Knockdown 4.1. Cell Culture and Reagents Human gastric cancer cell lines BGC823, MKN28, MGC803, SGC7901, and MKN45 were obtained
from the State Key Laboratory of Proteomics (Beijing, China). The NCI N87 cell line was a generous gift
from Academy of Military Medical Sciences (Beijing, China). All cells were maintained in Dulbecco’s
Modified Eagle’s Medium (DMEM) (Gibco, New York, NY, USA) supplemented with 10% fetal bovine
serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin (Gibco) at 37 ◦C with 5% CO2. 4.2. Development of Trastuzumab-Resistant MKN45/R and NCI N87/R Gastric Cancer Cell Lines 4.5. Wound Healing Assays Cells were cultured in 6-well plates. After 24 h of incubation wounds were created by scraping
the cells with a 200 µL pipette tip. Cells were then washed twice with phosphate-buffered saline
(PBS) to clear away debris at the edge of the scratch and subsequently incubated in DMEM with
10% FBS. Microscopic images of the wounds at different time points were captured by the Leica
DMi8 microscope system (Leica Microsystems, Wetzlar, Germany). The scratch assay was performed
in triplicate. Int. J. Mol. Sci. 2018, 19, 1981 16 of 21 16 of 21 4.8. Viability and Apoptosis Assays Cells were seeded at 5 × 103 cells/well into 96-well plates and cultured for 24 h. After treatment
with a range of indicated concentrations of ICG-001(Medchem express, Princeton, NJ, USA) for 72 h,
cell viability was measured using CCK-8 kit. Absorbance was detected at 450 nm with a microplate
reader (Bio-Rad, Hercules, CA, USA). Apoptosis of cells was detected by dual staining with Annexin
V-FITC/PI (CWBIO, #CW2574, Beijing, China) according to the manufacturer’s protocol. Briefly,
cells were cultured for 24 h, and then treated with trastuzumab and harvested 96 h later. Cells were
resuspended in binding buffer and stained with Annexin V-FITC/PI. After staining, cells were analyzed
with the fluorescent activating cell sorting (FACS) Calibur system. All data were represented based on
three independent experiments. 4.7. Immunofluorescence Staining Immunofluorescence assays were done as previously described [30]. Briefly, cells were washed
3 times with cold PBS, and fixed in 10% formaldehyde for 15 min, after which cells were permeabilized
in 0.5% Triton X-100 for 30 min, and blocked in 2.5% bovine serum albumin (BSA) for 0.5 h. Subsequently, cells were incubated with primary antibody at 4 ◦C, then with secondary antibody
conjugated with Alexa Fluor® 594 (Santa Cruz Biotechnology, #sc-516642, Santa Cruz, CA, USA) or 488
(Santa Cruz Biotechnology, #sc-3895, USA) for 30 min at 37 ◦C. Finally, coverslips were counterstained
with DAPI (Beyotime, Haimen, China) to visualize nuclei. Images were captured under Leica DMi8
fluorescence microscope. 4.9. Reverse Transcription Polymerase Chain Reaction (RT-PCR) Total RNA was extracted using Trizol reagent (Invitrogen, USA). The absorbance of RNA in
RNase-free water was evaluated at 230, 260 and 280 nm using a Nanodrop 2000C spectrophotometer
(Thermo, Waltham, MA, USA). The A260/A280 and A260/A230 ratios were maintained within
the range of 1.80–2.10 and >2.0 respectively to ensure mRNA purity. One microgram (1 µg) of
total RNA was treated with DNase I and the complementary DNA was amplified in vitro using
SuperScript First-Strand cDNA Synthesis Kit (Invitrogen, USA). RT-PCR was subsequently performed
using SYBR-Green Master Mix (Applied Biosystems, Foster City, CA, USA). The expression levels
of target mRNAs were normalized to that of the control gene (β-actin). The sequences of primers
used in this study are as follows:
siRNA β-catenin forward:
5′-CCAGGAUGAUCCUAGC
UAUTT-3′,
reverse:
5′-AUAGCUAGGAUCAUCCUGGTT-3′;
scrambled
siRNA
forward:
5′-GGAAGAUAAUCUUUUCUAATT-3′, reverse: 5′-UUAGAAAAGAUUAUCUUCCTT-3′; β-actin
forward: 5′-GGACTTCGAGCAAGAGATGG-3′, reverse: 3′-GACATGCGGTTGT GTCACGA-5′. 4.6. Colony Formation Assays About 5 × 103 cells/well were seeded in 6-well plates and incubated in DMEM containing 3%
FBS for 14 days, Afterwards, cells were washed three times with PBS, fixed with cold methanol for
15 min, and stained with 0.1% crystal violet. Colonies were photographed and counted. The assay was
repeated three times. 4.13. Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) Analysis Peptides were dissolved in solvent A (0.1% formic acid) and analyzed with FUSION mass
spectrometer (Thermo) equipped with an Easy-nLC 1000 nanoflow high-performance liquid
chromatography (HPLC) system (Thermo). Peptides were then separated on a reversed-phase C18
column (pre-column: 3 µm, 120 Å, 2 cm × 100 µm; analytical column: 1.9 µm, 120 Å, 12 cm × 150 µm)
with an increasing gradient of 7–35% mobile phase B (0.1% formic acid in acetonitrile) at a flow
rate of 600 nL/min for 75 min. A precursor scan was executed by scanning from m/z 300 to 1400
with a resolution of 120,000. Ions in each scan under top-speed mode were automatically isolated in
Quadrupole with a 1.6 m/z window and fragmented by higher energy collision-induced dissociation
with normalized collision energy of 35%. Dynamic exclusion was fixed for 18 s. 4.10. Western Blotting Cells were collected and lysed with radio-immunoprecipitation assay (RIPA) lysis buffer
(50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1%sodium dodecyl
sulfate (SDS), Beyotime) containing 1% protease inhibitor. Lysates were centrifuged at 12,000× g
for 10 min at 4 ◦C, and the supernatant was collected for further use. Equal amounts (20–30 µg)
of proteins were denatured in loading buffer and loaded on a gel after which SDS-PAGE was
performed. Resolved bands were transferred to nitrocellulose membranes which were incubated
with anti-HER-2 (Abcam, #16901, Cambridge, UK), anti-E-cadherin (Cell Signaling, #3195, Danvers, 17 of 21 17 of 21 Int. J. Mol. Sci. 2018, 19, 1981 MA, USA), anti-N-cadherin (Cell Signaling, #13116, USA), anti-vimentin (Cell Signaling, #5741, USA),
anti-snail1 (Cell Signaling, #3879, USA), anti-twist (Cell Signaling, #46702, USA), anti-PARP (Cell
Signaling, #9532, USA), anti-β-catenin (Cell Signaling, #8480, USA), anti-phosphor β-catenin(Ser675)
(Cell Signaling, #4176, USA), anti-Wnt3A (Abcam, #19925, UK), anti-FZD6 (Abcam, #98933, UK),
anti-GSK-3β (Cell Signaling, #9315, USA), and β-actin (Cell Signaling, #4970, USA) respectively at
4 ◦C overnight followed by incubation with horseradish peroxidase (HRP)-labeled goat anti-rabbit or
anti-mouse secondary antibodies (ZSGB-BIO, Beijing, China). Finally, the bands were visualized using
enhanced chemi-luminescence reagent (CWBIO, China). The grey values of these target bands were
measured using Image J software and histograms were plotted using GraphPad Prism software 5.0. 4.11. Cell invasion Assays Following the established protocols [30], cell invasion assays were completed using a 24-well
Transwell chamber containing polycarbonate filters with 8 µm pores which were coated with 60 µL 1:6
diluted Matrigel (BD Biosciences, San Jose, CA, USA). DMEM supplemented with 10% FBS was added
to the lower chamber in the presence or absence of ICG-001 or 0.1% DMSO (as control), 3 × 104 (NCI
N87 and NCI N87/R) or 2 × 104 (MKN45 and MKN45/R) cells/well in 200 µL of serum-free DMEM
were added to the cell culture inserts. Cells were cultured for 22 h (for MKN45 and MKN45/R cells)
or 28 h (for NCI N87 and NCI N87/R cells) respectively. The invaded cells passing through the filter
were fixed with 10% formaldehyde, stained with 0.1% crystal violet and counted in six high power
fields randomly selected under a light microscope. 4.12. Protein Extraction and Peptide Separation Protein extraction and peptide separation were accomplished according to standard protocols
as previously described [48,49]. Briefly, cells were lysed with RIPA buffer containing 1% protease
inhibitors for 20 min at 4 ◦C. Protein concentration was determined by Bradford protein assay kit
(Beyotime, China). Proteins extracted from each sample (100 µg) was digested with trypsin according
to the filter-aided sample preparation (FASP) method [50]. Tryptic peptides were separated using C18
column in a pipette tip and eluted with increasing acetonitrile (6%, 9%, 12%, 15%, 18%, 21%, 25%, 30%
and 35%). The nine separations were combined to six fractions, and dried in a vacuum concentrator. 4.13. Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) Analysis 4.14. Protein Identification and Quantification Raw files were searched against human Refseq protein database by Mascot 2.3 (Matrix Science
Inc., Boston, MA, USA) implemented on Proteome Discoverer 1.4 (Thermo Scientific). The mass
tolerances were 20 ppm for precursor and 0.5 Da for product ions. Two missed cleavages were allowed. The search engine set cysteine carbamidomethylation as a fixed modification, N-terminal acetylation,
and oxidation of methionine as variable modifications. The data were accepted at a false discovery rate
(FDR) of 1% at peptide level. Proteins quantification were done as our previous protocol [30,48,49]. Briefly, proteins were quantified using iBAQ approach, FOT was used to evaluate protein abundance, Int. J. Mol. Sci. 2018, 19, 1981 18 of 21 which was defined as a protein’s iBAQ divided by the total iBAQ of all proteins in one sample, and
FOT was multiplied by 105 for the ease of presentation. 4.15. Proteome Data Filtering and Analysis Proteome data filtering was executed based on the following criteria. All keratins were excluded
from our data and the rest of proteins detected in at least three of six experiments were used further
for bioinformatics. Proteins were regarded as significantly changed in abundance if there was a more
than twofold change between paired samples with a p value <0.05 using paired two-tailed student test. GO enrichment analysis was accomplished by WebGestalt. The significance level was set to p < 0.05
and all identified proteins as reference. Network analysis was done by the STRING, the interaction
score was set to high confidence (scores > 0.7) while the networks were displayed by Cytoscape (v3.6.0). Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/7/
1981/s1. plementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/7
/s1 Author Contributions: Project administration, W.L. and J.Y.; Investigation, Z.L. and J.Z.; Supervision, J.C Funding: This research was funded by the Research Fund for Doctoral Program of North Sichuan Medical College
(CBY17-QD05), Project of Sichuan Province Education Department (17ZB0170), Project of Sichuan Science and
Technology department (2016JY0032), and Project for first-class pharmaceutical sciences of North Sichuan Medical
College (2018). Conflicts of Interest: The authors declare no conflict of interest. Abbreviations
EMT
Epithelial-mesenchymal transition
HER-2
Human epidermal growth factor receptor-2
EGFR
Epidermal growth factor receptor
PI3K
Phosphatidylinositide 3-kinase
IGFR
Insulin-like growth factor receptor
PTEN
Phosphatase and tensin homolog gene
FGFR3
Fibroblast growth factor receptor 3
CBP
cyclic AMP response element-binding protein
β2-AR
β2-adrenergic receptor
iBAQ
Intensity based absolute quantification
FOT
Fraction of total
GO
Gene ontology
PCA
Principal component analysis
SFRP4
Secreted frizzled related protein 4
CCK-8
Cell Count kit-8
IC50
Half maximal inhibitory concentration
BSA
Bovine serum albumin
RT–PCR
Reverse transcription polymerase chain reaction
LC-MS/MS
Liquid chromatography–mass spectrometry/mass spectrometry
FDR
False discovery rate
FASP
Filter-aided sample preparation References Trends Pharmacol. Sci. 2014, 35, 41–50. [CrossRef] [PubMed] 12. Meza-Junco, J.; Au, H.J.; Sawyer, M.B. Critical appraisal of trastuzumab in treatment of advanced stomach
cancer. Cancer Manag. Res. 2011, 3, 57–64. [CrossRef] [PubMed] 13. Slamon, D.; Eiermann, W.; Robert, N.; Pienkowski, T.; Martin, M.; Press, M.; Mackey, J.; Glaspy, J.; Chan, A.;
Pawlicki, M.; et al. Adjuvant trastuzumab in HER2–positive breast cancer. N. Engl. J. Med. 2011, 366,
1273–1283. [CrossRef] [PubMed] 14. Nahta, R.; Esteva, F.J. HER2 therapy: Molecular mechanisms of trastuzumab resistance. Breast Cancer Res. 2006, 8, 215. [CrossRef] [PubMed] 15. Lavaud, P.; Andre, F. Strategies to overcome trastuzumab resistance in HER2-over expressing breast cancers:
Focus on new data from clinical trials. BMC Med. 2014, 12, 132. [CrossRef] [PubMed] 16. Chandarlapaty, S.; Sakr, R.A.; Giri, D.; Patil, S.; Heguy, A.; Morrow, M.; Modi, S.; Norton, L.; Rosen, N.;
Hudis, C.; et al. Frequent mutational activation of the PI3K–AKT pathway in trastuzumab–resistant breast
cancer. Clin. Cancer Res. 2012, 18, 6784–6791. [CrossRef] [PubMed] 17. Nahta, R.; Yuan, L.; Zhang, B.; Kobayashi, R.; Esteva, F.J. Insulin–like growth factor–I receptor/human
epidermal growth factor receptor 2 heterodimerization contributes to trastuzumab resistance of breast
cancer cells. Cancer Res. 2005, 65, 11118–11128. [CrossRef] [PubMed] 18. Lu, Y.; Zi, X.; Zhao, Y.; Mascarenhas, D.; Pollak, M. Insulin–like growth factor–I receptor signaling and
resistance to trastuzumab (Herceptin). J. Natl. Cancer Inst. 2001, 93, 1852–1857. [CrossRef] [PubMed] 18. Lu, Y.; Zi, X.; Zhao, Y.; Mascarenhas, D.; Pollak, M. Insulin like growth factor I receptor signaling and
resistance to trastuzumab (Herceptin). J. Natl. Cancer Inst. 2001, 93, 1852–1857. [CrossRef] [PubMed] 19. Nagata, Y.; Lan, K.H.; Zhou, X.; Tan, M.; Esteva, F.J.; Sahin, A.A.; Klos, K.S.; Li, P.; Monia, B.P.;
Nguyen, N.T.; et al. PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN
predicts trastuzumab resistance in patients. Cancer Cell 2004, 6, 117–127. [CrossRef] [PubMed] p
p
20. Arribas, J.; Baselga, J.; Pedersen, K.; Parra–Palau, J.L. p95HER2 and breast cancer. Cancer Res. 2011, 71,
1515–1519. [CrossRef] [PubMed] 21. Eto, K.; Iwatsuki, M.; Watanabe, M.; Ishimoto, T.; Ida, S.; Imamura, Y.; Iwagami, S.; Baba, Y.; Sakamoto, Y.;
Miyamoto, Y.; et al. The sensitivity of gastric cancer to trastuzumab is regulated by the miR-223/FBXW7
pathway. Int. J. Cancer 2015, 136, 1537–1545. [CrossRef] [PubMed] 22. Piro, G.; Carbone, C.; Cataldo, I.; Di Nicolantonio, F.; Giacopuzzi, S.; Aprile, G.; Simionato, F.; Boschi, F.;
Zanotto, M.; Mina, M.M.; et al. References 1. Ferlay, J.; Soerjomataram, I.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.; Parkin, D.M.; Forman, D.; Bray, F. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J. Cancer 2015, 136, E359–E386. [CrossRef] [PubMed] 1. Ferlay, J.; Soerjomataram, I.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.; Parkin, D.M.; Forman, D.; Bray, F. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J. Cancer 2015, 136, E359–E386. [CrossRef] [PubMed] 2. Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics, 2016. CA Cancer J. Clin. 2016, 66, 7–30. [CrossRef]
[PubMed] 19 of 21 Int. J. Mol. Sci. 2018, 19, 1981 19 of 21 3. Gomez-Martín, C.; Lopez-Rios, F.; Aparicio, J.; Barriuso, J.; García-Carbonero, R.; Pazo, R.; Rivera, F.;
Salgado, M.; Salud, A.; Vázquez-Sequeiros, E.; et al. A critical review of HER2-positive gastric cancer
evaluation and treatment: From trastuzumab, and beyond. Cancer Lett. 2014, 35, 30–40. [CrossRef] [PubMed] . Boku, N. HER2-positive gastric cancer. Gastric Cancer 2014, 17, 1–12. [CrossRef] [PubMed] 4. Boku, N. HER2-positive gastric cancer. Gastric Cancer 2014, 17, 1–12. [CrossRef] [PubMed]
5. Appert-Collin, A.; Hubert, P.; Cremel, G.; Bennasroune, A. Role of ErbB receptors in cancer cell migration
and invasion. Front. Pharmacol. 2015, 6, 283. [CrossRef] [PubMed] 5. Appert-Collin, A.; Hubert, P.; Cremel, G.; Bennasroune, A. Role of ErbB receptors in cancer cell migration
and invasion. Front. Pharmacol. 2015, 6, 283. [CrossRef] [PubMed] 6. Minner, S.; Jessen, B.; Stiedenroth, L.; Burandt, E.; Köllermann, J.; Mirlacher, M.; Erbersdobler, A.;
Eichelberg, C.; Fisch, M.; Brümmendorf, T.H.; et al. Low level HER2 overexpression is associated with
rapid tumor cell proliferation and poor prognosis in prostate cancer. Clin. Cancer Res. 2010, 16, 1553–1560. [CrossRef] [PubMed] 7. Rimawi, M.F.; Schiff, R.; Osborne, C.K. Targeting HER2 for the treatment of breast cancer. Annu. Rev. Med. 2015, 66, 111–128. [CrossRef] [PubMed] 8. Roskoski, R., Jr. The ErbB/HER family of protein–tyrosine kinases and cancer. Pharmacol. Res. 2014, 79,
34–74. [CrossRef] [PubMed] 9. Rexer, B.N.; Arteaga, C.L. Intrinsic and acquired resistance to HER2–targeted therapies in HER2 gene–
amplified breast cancer: Mechanisms and clinical implications. Crit. Rev. Oncog. 2012, 17, 1–16. [CrossRef]
[PubMed] 10. Xu, W.; Yang, Z.; Lu, N. Molecular targeted therapy for the treatment of gastric cancer. J. Exp. Clin. Cancer Res. 2016, 35, 1–11. [CrossRef] [PubMed] 11. Huang, M.; Shen, A.; Ding, J.; Geng, M. Molecularly targeted cancer therapy: Some lessons from the past
decade. References An FGFR3 autocrine loop sustains acquired resistance to trastuzumab in
gastric cancer patients. Clin. Cancer Res. 2016, 22, 6164–6175. [CrossRef] [PubMed] 23. Liu, D.; Yang, Z.; Wang, T.; Yang, Z.; Chen, H.; Hu, Y.; Hu, C.; Guo, L.; Deng, Q.; Liu, Y.; et al. β2-AR signaling
controls trastuzumab resistance-dependent pathway. Oncogene 2016, 35, 47–58. [CrossRef] [PubMed] 20 of 21 20 of 21 Int. J. Mol. Sci. 2018, 19, 1981 24. Pietrantonio, F.; Caporale, M.; Morano, F.; Scartozzi, M.; Gloghini, A.; De Vita, F.; Giommoni, E.; Fornaro, L.;
Aprile, G.; Melisi, D.; et al. HER2 loss in HER2-positive gastric or gastroesophageal cancer after trastuzumab
therapy: Implication for further clinical research. Int. J. Cancer 2016, 139, 2859–2864. [CrossRef] [PubMed] 5. Roepstorff, P. Mass spectrometry based proteomics, background, status and future needs. Protein Cell 2
3, 641–647. [CrossRef] [PubMed] 26. Wilhelm, M.; Schlegl, J.; Hahne, H.; Gholami, A.M.; Lieberenz, M.; Savitski, M.M.; Ziegler, E.; Butzmann, L.;
Gessulat, S.; Marx, H.; et al. Mass–spectrometry–based draft of the human proteome. Nature 2014, 509,
582–587. [CrossRef] [PubMed] 27. Ding, C.; Jiang, J.; Wei, J.; Liu, W.; Zhang, W.; Liu, M.; Fu, T.; Lu, T.; Song, L.; Ying, W.; et al. A fast workflow
for identification and quantification of proteomes. Mol. Cell. Proteom. 2013, 12, 2370–2380. [CrossRef]
[PubMed] 28. Zuo, Q.; Liu, J.; Zhang, J.; Wu, M.; Guo, L.; Liao, W. Development of trastuzumab-resistant human gastric
carcinoma cell lines and mechanisms of drug resistance. Sci. Rep. 2015, 5, 11634. [CrossRef] [PubMed] 29. Zheng, X.; Carstens, J.L.; Kim, J.; Scheible, M.; Kaye, J.; Sugimoto, H.; Wu, C.C.; LeBleu, V.S.; Kalluri, R. EMT program is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature 2015,
527, 525–530. [CrossRef] [PubMed] 30. Liu, W.; Chang, J.; Liu, M.; Yuan, J.; Zhang, J.; Qin, J.; Xia, X.; Wang, Y. Quantitative proteomics profiling
reveals activation of mTOR pathway in trastuzumab resistance. Oncotarget 2017, 8, 45793–45806. [CrossRef]
[PubMed] 31. Eguchi, M.; Nguyen, C.; Lee, S.C.; Kahn, M. ICG–001, a novel small molecule regulator of TCF/β–catenin
transcription. Med. Chem. 2005, 1, 467–472. [CrossRef] [PubMed] 32. Bahrami, A.; Amerizadeh, F.; ShahidSales, S.; Khazaei, M.; Ghayour-Mobarhan, M.; Sadeghnia, H.R.;
Maftouh, M.; Hassanian, S.M.; Avan, A. Therapeutic potential of targeting Wnt/β-catenin pathway in
treatment of colorectal cancer: Rational and progress. J. Cell. Biochem. 2017, 119, 1979–1983. [CrossRef]
[PubMed] 33. References Zhang, B.; Wang, J.; Wang, X.; Zhu, J.; Liu, Q.; Shi, Z.; Chambers, M.C.; Zimmerman, L.J.; Shaddox, K.F.;
Kim, S.; et al. Proteogenomic characterization of human colon and rectal cancer. Nature 2014, 513, 382–387. [CrossRef] [PubMed] 34. Nagaraj, N.; Wisniewski, J.R.; Geiger, T.; Cox, J.; Kircher, M.; Kelso, J.; Pääbo, S.; Mann, M. Deep proteome
and transcriptome mapping of a human cancer cell line. Mol. Syst. Biol. 2011, 7, 548. [CrossRef] [PubMed] 35. Krishnamurthy, N.; Kurzrock, R. Targeting the Wnt/beta-catenin pathway in cancer: Update on effectors
and inhibitors. Cancer Treat. Rev. 2018, 62, 50–60. [CrossRef] [PubMed] 36. Yao, H.; Ashihara, E.; Maekawa, T. Targeting the Wnt/β-catenin signaling pathway in human cancers. Expert Opin. Ther. Targets 2011, 15, 873–887. [CrossRef] [PubMed] 37. Cui, J.; Jiang, W.; Wang, S.; Wang, L.; Xie, K. Role of Wnt/β-catenin signaling in drug resistance of pancreatic
cancer. Curr. Pharm. Des. 2012, 18, 2464–2471. [CrossRef] [PubMed] 38. Bilir, B.; Kucuk, O.; Moreno, C.S. Wnt signaling blockage inhibits cell proliferation and migration, and
induces apoptosis in triple–negative breast cancer cells. J. Transl. Med. 2013, 11, 280. [CrossRef] [PubMed] 38. Bilir, B.; Kucuk, O.; Moreno, C.S. Wnt signaling blockage inhibits cell proliferation and migration, and
induces apoptosis in triple–negative breast cancer cells. J. Transl. Med. 2013, 11, 280. [CrossRef] [PubMed]
39. Mohammed, M.K.; Shao, C.; Wang, J.; Wei, Q.; Wang, X.; Collier, Z.; Tang, S.; Liu, H.; Zhang, F.; Huang, J.; 39. Mohammed, M.K.; Shao, C.; Wang, J.; Wei, Q.; Wang, X.; Collier, Z.; Tang, S.; Liu, H.; Zhang, F.; Huang, J.;
et al. Wnt/β–catenin signaling plays an ever–expanding role in stem cell self–renewal, tumorigenesis and
cancer chemoresistance. Genes Dis. 2016, 3, 11–40. [CrossRef] [PubMed] 40. Schade, B.; Lesurf, R.; Sanguin-Gendreau, V.; Bui, T.; Deblois, G.; O’Toole, S.A.; Millar, E.K.; Zardawi, S.J.;
Lopez-Knowles, E.; Sutherland, R.L.; et al. β-Catenin signaling is a critical event in ErbB2-mediated
mammary tumor progression. Cancer Res. 2013, 73, 4474–4487. [CrossRef] [PubMed] 41. Arend, R.C.; Londoño-Joshi, A.I.; Straughn, J.M., Jr.; Buchsbaum, D.J. The Wnt/β-catenin pathway in ovarian
cancer: A review. Gynecol. Oncol. 2013, 131, 772–779. [CrossRef] [PubMed] 42. Wu, Y.; Ginther, C.; Kim, J.; Mosher, N.; Chung, S.; Slamon, D.; Vadgama, J.V. Expression of Wnt3 activates
Wnt/β-catenin pathway and promotes EMT-like phenotype in trastuzumab-resistant HER2-overexpressing
breast cancer cells. Mol. Cancer Res. 2012, 10, 1597–1606. [CrossRef] [PubMed] 43. References Scholer-Dahirel, A.; Schlabach, M.R.; Loo, A.; Bagdasarian, L.; Meyer, R.; Guo, R.; Woolfenden, S.; Yu, K.K.;
Markovits, J.; Killary, K.; et al. Maintenance of adenomatous polyposis coli (APC)-mutant colorectal cancer
is dependent on Wnt/beta-catenin signaling. Proc. Natl. Acad. Sci. USA 2011, 108, 17135–17140. [CrossRef]
[PubMed] Int. J. Mol. Sci. 2018, 19, 1981 21 of 21 44. Wade, C.A.; Kyprianou, N. Profiling prostate cancer therapeutic resistance. Int. J. Mol. Sci. 2018, 19, 904. [CrossRef] [PubMed] 45. Smith, B.N.; Bhowmick, N.A. Role of EMT in metastasis and therapy resistance. J. Clin. Med. 2016, 5, 17. [CrossRef] [PubMed] 46. Oliveras-Ferraros, C.; Corominas-Faja, B.; Cufí, S.; Vazquez-Martin, A.; Martin-Castillo, B.; Iglesias, J.M.;
López-Bonet, E.; Martin, Á.G.; Menendez, J.A. Epithelial–to–mesenchymal transition (EMT) confers primary
resistance to trastuzumab (Herceptin). Cell Cycle 2012, 11, 4020–4032. [CrossRef] [PubMed] 47. Ford, C.E.; Jary, E.; Ma, S.S.; Nixdorf, S.; Heinzelmann-Schwarz, V.A.; Ward, R.L. The Wnt gatekeeper SFRP4
modulates EMT, cell migration and downstream Wnt signalling in serous ovarian cancer cells. PLoS ONE
2013, 8, e54362. [CrossRef] [PubMed] 48. Lai, M.; Liang, L.; Chen, J.; Qiu, N.; Ge, S.; Ji, S.; Shi, T.; Zhen, B.; Liu, M.; Ding, C.; et al. Multidimensional
Proteomics Reveals a Role of UHRF2 in the Regulation of Epithelial-Mesenchymal Transition (EMT). Mol. Cell. Proteom. 2016, 15, 2263–2278. [CrossRef] [PubMed] 49. Ge, S.; Xia, X.; Ding, C.; Zhen, B.; Zhou, Q.; Feng, J.; Yuan, J.; Chen, R.; Li, Y.; Ge, Z.; et al. A proteomic
landscape of diffuse-type gastric cancer. Nat. Commun. 2018, 9, 1012. [CrossRef] [PubMed] 50. Wisniewski, J.R.; Zougman, A.; Nagaraj, N.; Mann, M. Universal sample preparation method for proteome
analysis. Nat. Methods 2009, 6, 359–362. [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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Synthesis, Affinity for Complementary RNA and DNA, and Enzymatic Stability of Triazole-Linked Locked Nucleic Acids (t-LNAs)
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This is an open access article published under a Creative Commons Attribution (CC-BY)
License, which permits unrestricted use, distribution and reproduction in any medium,
provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY)
License, which permits unrestricted use, distribution and reproduction in any medium,
provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY)
License, which permits unrestricted use, distribution and reproduction in any medium,
provided the author and source are cited. ■INTRODUCTION hinder cell uptake of natural DNA. Building on the seminal
work by Wengel,24 we have previously shown16 that
introducing a conformationally restricted locked nucleic acid
(LNA)25−27 next to the triazole linkage (monomers X and Z,
Figure 1) counterbalances the reduction in binding affinity
caused by the triazole, resulting in modified ONs that bind to
their RNA target with affinity similar to unmodified ONs. Interestingly, Watts et al. found in contrast that LNA does not
stabilize duplexes containing a more rigid four-atom triazole
linkage internally.15 Thus, the biocompatible six-atom triazole
linkage and LNA (monomers X and Z, Figure 1) is a promising
combination and warrants further investigation. Antisense oligonucleotides (ASO) are short single-stranded
nucleic acids (typically 15−25 nucleotides in length) that bind
to their RNA target in a sequence-specific manner and
modulate translation (protein synthesis) or RNA splicing.1
Unmodified natural DNA or RNA strands are not optimum
antisense candidates because of their poor serum stability and
modest binding affinity for RNA. Chemically modified nucleic
acids2−4 offer a promising alternative to their unmodified
counterparts and have been extensively studied since the ASO
concept was first introduced.5 Some of these modifications
possess favorable biophysical properties, which is evident from
the recent success in the clinic.6,7 Despite this, ideal antisense
candidates, with the desired characteristics of efficient unaided
cellular uptake, long serum half-life and high RNA binding
affinity and target specificity, remain elusive and there is a need
to explore new chemically modified nucleic acids. In our previous study, we incorporated monomers W−Z into
oligonucleotides by ligating together two short ONs, one with a
5′-azide group and the other with a 3′-propargyl group.16 This
approach was chosen for ease of ON synthesis. However,
multiple incorporations of the triazole linkage into short ONs
and the introduction of the linkage at the ends of ONs is not
practical using this strategy. Here, we report the synthesis of
dinucleoside phosphoramidites containing the triazole linkage
flanked by LNA as suitable building blocks for the efficient and
flexible incorporation of monomers W−Z into ONs using
standard solid phase phosphoramidite DNA synthesis method-
ology. *
S Supporting Information *
S Supporting Information ABSTRACT: Dinucleoside phosphoramidites containing a
triazole internucleotide linkage flanked by locked nucleic acid
(LNA) were synthesized and incorporated into oligonucleo-
tides (ONs). ONs bearing both LNA and triazole at multiple
sites were obtained and their biophysical properties including
enzymatic stability and binding affinity for RNA and DNA
targets were studied. t-LNAs with four incorporations of a
dinucleoside monomer having LNA on either side of the
triazole linkage bind to their RNA target with significantly
higher affinity and greater specificity than unmodified oligonucleotides, and are remarkably stable to nuclease degradation. A
similar but reduced effect on enzymatic stability and binding affinity was noted for LNA only on the 3′-side of the triazole
linkage. Thus, by combining unnatural triazole linkages and LNA in one unit (t-LNA), we produced a promising class of ONs
with reduced anionic charge and potential for antisense applications. ■INTRODUCTION © 2018 American Chemical Society Cite This: ACS Omega 2018, 3, 6976−6987 Synthesis, Affinity for Complementary RNA and DNA, and Enzymatic
Stability of Triazole-Linked Locked Nucleic Acids (t-LNAs)
Pawan Kumar,†,§
Lynda Truong,† Ysobel Ruth Baker,† Afaf Helmy El-Sagheer,†,‡
and Tom Brown*,†
†Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, U.K. ‡Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez University, Suez
43721, Egypt ■INTRODUCTION U: Phosphodiester-linked DNA. V: Phosphodiester-linked LNA. W: Triazole-linked DNA. X: Triazole-linked 3′-LNA. Y: Triazole-linked 5′-LNA. Z: Triazole-linked 3′,5′-LNA. T =
thymin-1-yl, MeC = 5-methylcytosin-1-yl. (B) The general principle explored in this study, where B = nucleobase, S = modified sugar (e.g., LNA), L =
modified DNA backbone (e.g., triazole). Enzymatic degradation of such structures cannot give rise to monomer units that can enter the
mononucleotide triphosphate pool in cells. Hence, the modifications cannot be incorporated into genomic DNA or RNA. benzoic anhydride to obtain protected nucleosides 3 and 4 in
yields of 86 and 92%, respectively. 5′-Azido-LNA thymidine (6)
was obtained from protected nucleoside 529 by removing the
3′-O-benzyl group followed by nucleophilic displacement of 5′-
O-mesylate with azide. 5′-Azidothymidine (7, Scheme 2) was
prepared following a literature procedure.30 hybridization (duplex-forming) properties. Monomer Z stands
out among those tested; ONs carrying four incorporations of Z
are strikingly resistant toward nucleolytic degradation, and
show enhanced binding to RNA targets with improved affinity
and specificity relative to unmodified ONs. Thus, the
combination of LNA and the triazole linkage provides a new
class of potentially significant antisense oligonucleotide
candidates. hybridization (duplex-forming) properties. Monomer Z stands
out among those tested; ONs carrying four incorporations of Z
are strikingly resistant toward nucleolytic degradation, and
show enhanced binding to RNA targets with improved affinity
and specificity relative to unmodified ONs. Thus, the
combination of LNA and the triazole linkage provides a new
class of potentially significant antisense oligonucleotide
candidates. Dinucleosides 8−11, which contain the triazole linkage with
and without LNA, were prepared in good yields (79−87%) by
Cu(I) catalyzed alkyne azide cycloaddition reaction31,32
between nucleosides 3, 4, 6, and 7 (Scheme 2). 3′-O-
Phosphitylation of 8−11 gave phosphoramidites 12−15 as
suitable building blocks for incorporating monomers W−Z,
respectively, into DNA via solid-phase DNA synthesis. For
introducing monomers W−Z at the 3′-end of ONs, dinucleo-
sides 8−11 were loaded onto an activated amino-SynBase resin
(16) using N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) as the coupling reagent. ■RESULTS AND DISCUSSION DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 ■INTRODUCTION Using this approach, 13-mer ONs with 1, 2, or 4
incorporations of the modified monomers W−Z were readily
obtained, enabling us to study their enzymatic stability and p
y
Recently, we have been interested in backbone-modified
oligonucleotides (ONs) with a special focus on triazole-linked
nucleic acids.8 A series of studies over the past decade led us to
a highly versatile and biocompatible six-atom triazole linkage
(monomer W, Figure 1).9−12 This and other triazole
linkages13,14 have been shown to improve the stability of
modified ONs to degradation by DNase enzymes.14−17
Unfortunately, the thermal stability of duplexes carrying triazole
internucleotide linkages is compromised by the modified
backbone.13,15,17−23 This imposes a restriction on the use of
triazole-linked oligonucleotides in antisense applications, even
though they possess the favorable properties of resistance to
enzymatic degradation and the lack of anionic charge that can Received:
May 22, 2018
Accepted:
May 30, 2018
Published: June 27, 2018 triazole-linked oligonucleotides in antisense applications, even
though they possess the favorable properties of resistance to
enzymatic degradation and the lack of anionic charge that can © 2018 American Chemical Society DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 6976 ACS Omega Article Figure 1. (A) Structures of phosphodiester and triazole-linked DNA and LNA backbones investigated in this study. U: Phosphodiester-linked DNA. V: Phosphodiester-linked LNA. W: Triazole-linked DNA. X: Triazole-linked 3′-LNA. Y: Triazole-linked 5′-LNA. Z: Triazole-linked 3′,5′-LNA. T =
thymin-1-yl, MeC = 5-methylcytosin-1-yl. (B) The general principle explored in this study, where B = nucleobase, S = modified sugar (e.g., LNA), L =
modified DNA backbone (e.g., triazole). Enzymatic degradation of such structures cannot give rise to monomer units that can enter the
mononucleotide triphosphate pool in cells. Hence, the modifications cannot be incorporated into genomic DNA or RNA. Figure 1. (A) Structures of phosphodiester and triazole-linked DNA and LNA backbones investigated in this study. U: Phosphodiester-linked DNA. V: Phosphodiester-linked LNA. W: Triazole-linked DNA. X: Triazole-linked 3′-LNA. Y: Triazole-linked 5′-LNA. Z: Triazole-linked 3′,5′-LNA. T =
thymin-1-yl, MeC = 5-methylcytosin-1-yl. (B) The general principle explored in this study, where B = nucleobase, S = modified sugar (e.g., LNA), L =
modified DNA backbone (e.g., triazole). Enzymatic degradation of such structures cannot give rise to monomer units that can enter the
mononucleotide triphosphate pool in cells. Hence, the modifications cannot be incorporated into genomic DNA or RNA. Figure 1. (A) Structures of phosphodiester and triazole-linked DNA and LNA backbones investigated in this study. ■RESULTS AND DISCUSSION Synthesis of Triazole-Linked Dinucleoside Building Blocksa aReagents and conditions: (a) CuSO4, Na ascorbate, tetrahydrofuran (THF)/t-BuOH/H2O (3:1:1), pyridine, room temperature (rt), 8 (80%), 9
(82%), 10 (87%), 11 (79%); (b) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, N,N-diisopropylethylamine (DIPEA), CH2Cl2, rt, 12 (50%),
13 (62%), 14 (76%), 15 (73%), (c) N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 4-dimethylaminopyridine (DMAP),
Et3N, pyridine, 17 (28 μmol/g loading), 18 (26 μmol/g loading), 19 (29 μmol/g loading), and 20 (28 μmol/g loading). aReagents and conditions: (a) CuSO4, Na ascorbate, tetrahydrofuran (THF)/t-BuOH/H2O (3:1:1), pyridine, room temperature (rt), 8 (80%), 9
(82%), 10 (87%), 11 (79%); (b) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, N,N-diisopropylethylamine (DIPEA), CH2Cl2, rt, 12 (50%),
13 (62%), 14 (76%), 15 (73%), (c) N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 4-dimethylaminopyridine (DMAP),
Et3N, pyridine, 17 (28 μmol/g loading), 18 (26 μmol/g loading), 19 (29 μmol/g loading), and 20 (28 μmol/g loading). Table 1. Thermal Melting (Tm) Data for the DNA/RNA Duplexes Table 1. Thermal Melting (Tm) Data for the DNA/RNA Duplexes
Tma (ΔTmb)
ON
ON sequence (5′−3′)
B = MeCpT (U)
MeCLpTL (V)
MeCtT (W)
MeCtTL (X)
MeCLtT (Y)
MeCLtTL (Z)
ON1
5′-CTC ACT ATC TGB
54.8
nd
53.7 (−1.1)
54.6 (−0.2)
54.7 (−0.1)
55.0 (+0.2)
ON2
5′-BCA CTA TCT GCT
54.0
nd
51.5 (−2.5)
55.7 (+1.7)
50.7 (−3.3)
54.8 (+0.8)
ON3
5′-CTC ABA TCT GCT
55.1
66.0 (+10.9)
49.6 (−5.5)
57.1 (+2.0)
49.8 (−5.3)
58.1 (+3.0)
ON4
5′-CTC ACT ATB GCT
54.4
nd
49.4 (−5.0)
55.0 (+0.6)
49.2 (−5.2)
56.9 (+2.5)
ON5
5′-CTC ABA TBG CT
55.7
nd
43.7 (−12.0)
57.0 (+1.3)
44.1 (−11.6)
58.9 (+3.2)
ON6
5′-BCA BAT BGB
56.7
>75
39.4 (−17.3)
57.8 (+1.1)
38.3 (−18.4)
62.3 (+5.6)
aMelting temperatures (Tm) were obtained from the maxima of the first derivatives of the melting curves (A260 vs temperature) recorded in a buffer
containing 10 mM phosphate and 200 mM NaCl at pH 7.0 using 3.0 μM concentrations of each strand. bΔTm = change in Tm for a modified duplex
relative to the unmodified duplex. RNA target: 5′-AGC AGA UAG UGA G. nd = not determined. p = phosphodiester backbone. L = LNA
nucleotide. t = triazole backbone. aMelting temperatures (Tm) were obtained from the maxima of the first derivatives of the melting curves (A260 vs temperature) recorded in a buffer
containing 10 mM phosphate and 200 mM NaCl at pH 7.0 using 3.0 μM concentrations of each strand. bΔTm = change in Tm for a modified duplex
relative to the unmodified duplex. RNA target: 5′-AGC AGA UAG UGA G. DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 ■RESULTS AND DISCUSSION Chemical Synthesis. First, nucleosides with a 3′-O-
propargyl group and 5′-azido group were prepared (Scheme
1). Accordingly, 5′-O-(4,4′-dimethoxytrityl)-3′-O-propargyl-
LNA 5-methylcytosine (1)16 and 5′-O-(4,4′-dimethoxytrityl)-
3′-O-propargyl-5-methylcytosine (2)28 were reacted with Scheme 1. Synthesis of Alkyne and Azide Nucleosidesa
aReagents and conditions: (a) Bz2O, dimethylformamide (DMF): 3
(86%), 4 (92%); (b) (i) Pd(OH)2/C, MeOH, (ii) NaN3, DMF, 64%
over two steps. Scheme 1. Synthesis of Alkyne and Azide Nucleosidesa Scheme 1. Synthesis of Alkyne and Azide Nucleosidesa Hybridization Studies. Hybridization to Complementary
RNA Targets. Having dinucleoside phosphoramidites in hand
allowed multiple incorporations of the modified monomers
internally and at the termini of ONs. Hence, phosphoramidites
12−15 were used to introduce monomers W−Z, respectively,
into chosen ON sequences (Table 1). Six sequences were
prepared: ON1−ON2 with modified monomers at 3′- or 5′-
end, ON3−ON4 bearing modified monomers in the middle
region, ON5 containing two modifications in the middle, and
ON6 incorporating four modified monomers (at the ends and
in the middle). The modified ONs were then mixed with the
complementary RNA sequences in a 10 mM phosphate buffer
containing 200 mM NaCl at pH 7.0 and the melting
temperatures (Tm) of the resulting duplexes were determined
from the UV melting curves (Figure 2). In accordance with the literature, duplexes formed between
triazole-linked DNA and fully complementary RNA strands
were less stable than the unmodifed DNA/RNA duplex
(compare monomer W with U). However, the drop in Tm
caused by the triazole linkage was fully compensated for by
either the introduction of LNA on the 3′-side (monomer X) or aReagents and conditions: (a) Bz2O, dimethylformamide (DMF): 3
(86%), 4 (92%); (b) (i) Pd(OH)2/C, MeOH, (ii) NaN3, DMF, 64%
over two steps. aReagents and conditions: (a) Bz2O, dimethylformamide (DMF): 3
(86%), 4 (92%); (b) (i) Pd(OH)2/C, MeOH, (ii) NaN3, DMF, 64%
over two steps. 6977 DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 ACS Omega Article Scheme 2. Synthesis of Triazole-Linked Dinucleoside Building Blocksa
aReagents and conditions: (a) CuSO
Na ascorbate tetrahydrofuran (THF)/t BuOH/H O (3:1:1) pyridine room temperature (rt) 8 (80%) 9 Scheme 2. Synthesis of Triazole-Linked Dinucleoside Building Blocksa Scheme 2. Synthesis of Triazole-Linked Dinucleoside Building Blocksa Scheme 2. ■RESULTS AND DISCUSSION Importantly, the thermal stability of a
duplex with four incorporations of monomer Z was significantly
higher than that of the unmodifed duplex (compare ON6U
with ON6Z, ΔTm = +5.6 °C), and for monomer X (with LNA
on the 3′-side of the triazole), a small increase in thermal
stability was observed (compare ON6U with ON6X, ΔTm =
+1.1 °C). In contrast, the modified duplexes carrying four
incorporations of monomer W (triazole linkage without LNA)
or Y (LNA on 5′-side of the triazole linkage) were strikingly −5.0 to −5.5 °C, ON3W, ON4W, ON3Y, and ON4Y). In
contrast, one incorporation of monomers X or Z increased the
thermal stability (ΔTm of +0.6 to +3.0 °C, ON3X, ON4X,
ON3Z, and ON4Z), with monomer Z being more effective
than monomer X (compare ON3X with ON3Z and ON4X
with ON4Z). Indeed, monomer Z with LNA sugars on both
sides of the triazole linkage induced an increase of 3.0 °C
(ON3Z) in Tm, which is the largest increase in this series for a
single modification. Importantly, the thermal stability of a
duplex with four incorporations of monomer Z was significantly
higher than that of the unmodifed duplex (compare ON6U
with ON6Z, ΔTm = +5.6 °C), and for monomer X (with LNA
on the 3′-side of the triazole), a small increase in thermal
stability was observed (compare ON6U with ON6X, ΔTm =
+1.1 °C). In contrast, the modified duplexes carrying four
incorporations of monomer W (triazole linkage without LNA)
or Y (LNA on 5′-side of the triazole linkage) were strikingly In summary, the combined hybridization data (Tables 1 and
2) indicate that LNA on the 3′-side of the triazole linkage
maintains its beneficial effects on RNA binding affinity and
specificity, but this is not true for LNA on the 5′-side of the
triazole linkage. Our explanation for this behavior is that LNA Table 2. Mismatch Data for ONs Containing Modified Monomer in the Center (ON3U−ON3Z) Table 2. ■RESULTS AND DISCUSSION DNA/RN
containing four triazoles with no LNA or four triaz
the 5′-side are significantly less stable than the u
whereas duplexes containing LNA on 3′-side of th
displayed thermal stability comparable to un
Importantly, duplexes with LNA on both the 3′-
linkages were the most stable. Y-axis (% hyp
calculated as follows: [(A260 at T °C −A260 at 20 °C
100. Table 2. Mismatch Data for ONs Containin
d
( ′
′)
ACS Omega Figure 2. Representative UV melting curves for ON6 containing four
triazole linkages (where −t−= triazole). These are the most heavily
modified of all the oligonucleotides in Table 1. Additional melting
curves are given in the SI (Figure S1). DNA/RNA hybrid duplexes
containing four triazoles with no LNA or four triazoles with LNA on
the 5′-side are significantly less stable than the unmodified duplex,
whereas duplexes containing LNA on 3′-side of the triazole linkages
displayed thermal stability comparable to unmodified duplex. Importantly, duplexes with LNA on both the 3′- and 5′-side of the
linkages were the most stable. Y-axis (% hyperchromicity) was
calculated as follows: [(A260 at T °C −A260 at 20 °C)/A260 at 20 °C] x
100. ACS Omega Article less stable than the unmodified duplex (compare ON6W or
ON6Y with ON6U, ΔTm = −17.3 and −18.4 °C, respectively). ON6Y with ON6U, ΔTm
17.3 and
18.4 C, respectively). Hybridization to Mismatched RNA Targets. To avoid “off
target” effects that can lead to the inhibition of a wrong mRNA,
ASOs must be able to differentiate between matched and
mismatched RNA targets. ON3 with a backbone modification
at the center was chosen to assess the binding specificity of the
modified monomers. The nucleotides opposite to MeC or T on
either side of the triazole linkage were changed, and the Tm
differences between matched and mismatched duplexes were
recorded (Table 2). In general, monomers W−Z discriminated
between a matched and a single-mismatch target with the
efficiency similar to that of the unmodified base (U). Monomer
W (triazole linkage without LNA) was least effective in sensing
a mismatched nucleotide. Monomer V (LNA without a triazole
linkage) gave a good mismatch discrimination in all the cases
(e.g., compare ON3U with ON3V), which is in agreement with
the literature.25 Monomer X, with LNA on the 3′-side of the
triazole linkage showed the greatest discrimination for a
mismatched nucleotide on the 3′-side of the triazole linkage. aMelting temperatures (Tm) were obtained from the maxima of the first derivatives of the melting curves (A260 vs temperature) recorded in a buffer
containing 10 mM phosphate and 200 mM NaCl at pH 7.0 using 3.0 μM concentrations of each strand. bΔTm = change in Tm for a mismatch duplex
relative to the corresponding matched duplex. p = phosphodiester backbone. L = LNA nucleotide. t = triazole backbone. Matched RNA target: 5′-
AGCAGAUAGUGAG. Mismatch RNA targets: 5′-AGCAGAUAUUGAG (MeC−U mismatch), 5′-AGCAGAUAAUGAG (MeC−A mismatch), 5′-
AGCAGAUCGUGAG (T−C mismatch), 5′-AGCAGAUGGUGAG (T−G mismatch). ■RESULTS AND DISCUSSION However, marginally inferior discrimination was observed for
monomer Y, which carries LNA on the 5′-side of the linkage. Monomer Z, which has LNA on both sides of the triazole
linkage showed improved discrimination for the mismatch
nucleotide on the 3′-side of the triazole linkage (T−C and T−
G mismatch). Monomer Z discriminated against a mismatch
guanine base (T−G mismatch) with greater efficiency than that
of the unmodified ON (compare ON3U with ON3Z). The T−
G mismatch discrimination for Z was also noticeably superior
to that observed for monomers X or V (compare ON3Z with
ON3X or ON3V). The greater relative destabilization of the
T−G mismatch is significant, as this mismatch is generally quite
stable. The ability to discriminate between mispaired
nucleotides was then studied for ONs containing four
incorporations of monomers X and Z (Table S2) and similar
results were observed. Again, significant destabilization of
duplexes containing a T−G mismatch was observed (compare
ON6Z with ON6U). Figure 2. Representative UV melting curves for ON6 containing four
triazole linkages (where −t−= triazole). These are the most heavily
modified of all the oligonucleotides in Table 1. Additional melting
curves are given in the SI (Figure S1). DNA/RNA hybrid duplexes
containing four triazoles with no LNA or four triazoles with LNA on
the 5′-side are significantly less stable than the unmodified duplex,
whereas duplexes containing LNA on 3′-side of the triazole linkages
displayed thermal stability comparable to unmodified duplex. Importantly, duplexes with LNA on both the 3′- and 5′-side of the
linkages were the most stable. Y-axis (% hyperchromicity) was
calculated as follows: [(A260 at T °C −A260 at 20 °C)/A260 at 20 °C] x
100. −5.0 to −5.5 °C, ON3W, ON4W, ON3Y, and ON4Y). In
contrast, one incorporation of monomers X or Z increased the
thermal stability (ΔTm of +0.6 to +3.0 °C, ON3X, ON4X,
ON3Z, and ON4Z), with monomer Z being more effective
than monomer X (compare ON3X with ON3Z and ON4X
with ON4Z). Indeed, monomer Z with LNA sugars on both
sides of the triazole linkage induced an increase of 3.0 °C
(ON3Z) in Tm, which is the largest increase in this series for a
single modification. ■RESULTS AND DISCUSSION nd = not determined. p = phosphodiester backbone. L = LNA
nucleotide. t = triazole backbone. affinity than the unmodified DNA strand (compare ON2U with
ON2W and ON2Y, ΔTm of −2.5 and −3.3 °C, respectively). In
contrast, monomers X and Z with ΔTm of +1.7 and +0.8 °C
(compare ON2U with ON2X and ON2Z), respectively,
induced a slight increase in binding affinity. A larger drop in
thermal stability was observed with incorporation of either W
or Y at the center of modified duplexes (ΔTms in the range of on both sides of the linkage (monomer Z). This observation is
in full agreement with our previous study.16 Furthermore, the
binding affinity of ONs carrying the modified monomers (W−
Z) at the 3′-end (ON1W−ON1Z) was comparable to that of
the unmodified ON (ON1U), suggesting that all the monomers
are well tolerated at this position. ONs bearing monomers W or
Y at the 5′-end bind to their RNA targets with a slightly lower on both sides of the linkage (monomer Z). This observation is
in full agreement with our previous study.16 Furthermore, the
binding affinity of ONs carrying the modified monomers (W−
Z) at the 3′-end (ON1W−ON1Z) was comparable to that of
the unmodified ON (ON1U), suggesting that all the monomers
are well tolerated at this position. ONs bearing monomers W or
Y at the 5′-end bind to their RNA targets with a slightly lower 6978 DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 −5.0 to −5.5 °C, ON3W, ON4W, ON3Y,
contrast, one incorporation of monomers X or
thermal stability (ΔTm of +0.6 to +3.0 °C,
ON3Z, and ON4Z), with monomer Z bein
than monomer X (compare ON3X with ON
with ON4Z). Indeed, monomer Z with LNA
sides of the triazole linkage induced an inc
(ON3Z) in Tm, which is the largest increase in
single modification. Importantly, the therm
duplex with four incorporations of monomer Z
higher than that of the unmodifed duplex (
with ON6Z, ΔTm = +5.6 °C), and for monom
on the 3′-side of the triazole), a small incr
stability was observed (compare ON6U with
+1.1 °C). In contrast, the modified duplex
incorporations of monomer W (triazole linkag
or Y (LNA on 5′-side of the triazole linkage
Figure 2. Representative UV melting curves for ON
triazole linkages (where −t−= triazole). These ar
modified of all the oligonucleotides in Table 1. A
curves are given in the SI (Figure S1). ■RESULTS AND DISCUSSION L = LNA nucleotide. t = triazole backbone. aMelting temperatures (Tm) were obtained from the maxima of the first derivatives of the melting curves (A260 vs temperature) recorded in a buffer
containing 10 mM phosphate and 200 mM NaCl at pH 7.0 using 3.0 μM concentrations of each strand. bΔTm = change in Tm for a modified duplex
relative to the unmodified duplex 5′-CTC ACT ATC TGMeCT. p = phosphodiester backbone. L = LNA nucleotide. t = triazole backbone. Figure 3. CD spectra of duplexes formed between ON6U−ON6Z and complementary RNA. ON6: 5′-BCA BAT BGB (ON6U, B = MeCpT; ON6V,
B = MeCLpTL; ON6W, B = MeCtT; ON6X, B = MeCtTL; ON6Y, B = MeCLtT; ON6Z, B = MeCLtTL). Figure 3. CD spectra of duplexes formed between ON6U−ON6Z and complementary RNA. ON6: 5′-BCA BAT BGB (ON6U, B = MeCpT; ON6V,
B = MeCLpTL; ON6W, B = MeCtT; ON6X, B = MeCtTL; ON6Y, B = MeCLtT; ON6Z, B = MeCLtTL). Figure 3. CD spectra of duplexes formed between ON6U−ON6Z and complementary RNA. ON6: 5′-BCA BAT BG
B = MeCLpTL; ON6W, B = MeCtT; ON6X, B = MeCtTL; ON6Y, B = MeCLtT; ON6Z, B = MeCLtTL). with the DNA/RNA duplexes, monomer Z gave the highest
melting temperatures for all DNA/DNA duplexes. It is not
surprising that the triazole−LNA combination stabilizes the
DNA/RNA duplexes to a greater degree than it does the DNA/
DNA duplexes. This is because the LNA sugar exists in the C3′-
endo conformation, which occurs in A-type helices (DNA/
RNA and RNA/RNA) but is disfavored in DNA/DNA
duplexes in solution.24,34 on the 3′-side of the triazole is attached to a natural
phosphodiester group, but when present on the 5′-side of the
triazole, the LNA sugar is attached to a more flexible triazole
linkage. LNA stabilizes DNA/RNA duplexes by reducing
phosphate backbone flexibility,24,33 which may not be possible
when the LNA sugar is attached directly to the more flexible
modified linkage and/or is close to an unstable mismatched
base pair. p
Hybridization to DNA Targets. Next, modified ONs were
mixed with their DNA targets in a buffer containing 10 mM
phosphate and 200 mM NaCl at pH 7.0 to give DNA/DNA
duplexes and Tms were recorded. These were then compared
with the Tm of the unmodified DNA/DNA duplex and
differences (ΔTms) were determined (Table 3). ■RESULTS AND DISCUSSION Thermal Melting (Tm) Data for the DNA/DNA Duplexes
Tma (ΔTmb)
ON code
ON sequence (5′−3′)
B = MeCpT (U)
MeCtT (W)
MeCtTL (X)
MeCLtT (Y)
MeCLtTL (Z)
ON1
5′-CTC ACT ATC TGB
54.5
53.7 (−0.8)
52.8 (−1.7)
53.1 (−1.4)
53.5 (−1.0)
ON2
5′-BCA CTA TCT GCT
53.8
50.9 (−2.9)
49.1 (−4.7)
49.4 (−4.4)
49.6 (−4.2)
ON3
5′-CTC ABA TCT GCT
54.9
47.5 (−7.4)
51.4 (−3.5)
44.4 (−10.5)
53.4 (−1.5)
ON4
5′-CTC ACT ATB GCT
54.4
49.7 (−4.7)
51.0 (−3.4)
45.0 (−9.4)
52.1 (−2.3)
ON5
5′-CTC ABA TBG CT
55.9
41.0 (−14.9)
48.6 (−7.3)
34.2 (−21.7)
51.4 (−4.5)
ON6
5′-BCA BAT BGB
58.0
37.6 (−20.4)
45.8 (−12.2)
<−30.0
48.3 (−9.7)
aMelting temperatures (Tm) were obtained from the maxima of the first derivatives of the melting curves (A260 vs temperature) recorded in a buffer
containing 10 mM phosphate and 200 mM NaCl at pH 7.0 using 3.0 μM concentrations of each strand. bΔTm = change in Tm for a modified duplex
relative to the unmodified duplex 5′-CTC ACT ATC TGMeCT. p = phosphodiester backbone. L = LNA nucleotide. t = triazole backbone. Figure 3. CD spectra of duplexes formed between ON6U−ON6Z and complementary RNA. ON6: 5′-BCA BAT BGB (ON6U, B = MeCpT; ON6V,
B = MeCLpTL; ON6W, B = MeCtT; ON6X, B = MeCtTL; ON6Y, B = MeCLtT; ON6Z, B = MeCLtTL). ACS Omega
Article Table 3. Thermal Melting (Tm) Data for the DNA/DNA Duplexes Tma (ΔTmb)
ON code
ON sequence (5′−3′)
B = MeCpT (U)
MeCtT (W)
MeCtTL (X)
MeCLtT (Y)
MeCLtTL (Z)
ON1
5′-CTC ACT ATC TGB
54.5
53.7 (−0.8)
52.8 (−1.7)
53.1 (−1.4)
53.5 (−1.0)
ON2
5′-BCA CTA TCT GCT
53.8
50.9 (−2.9)
49.1 (−4.7)
49.4 (−4.4)
49.6 (−4.2)
ON3
5′-CTC ABA TCT GCT
54.9
47.5 (−7.4)
51.4 (−3.5)
44.4 (−10.5)
53.4 (−1.5)
ON4
5′-CTC ACT ATB GCT
54.4
49.7 (−4.7)
51.0 (−3.4)
45.0 (−9.4)
52.1 (−2.3)
ON5
5′-CTC ABA TBG CT
55.9
41.0 (−14.9)
48.6 (−7.3)
34.2 (−21.7)
51.4 (−4.5)
ON6
5′-BCA BAT BGB
58.0
37.6 (−20.4)
45.8 (−12.2)
<−30.0
48.3 (−9.7)
aMelting temperatures (Tm) were obtained from the maxima of the first derivatives of the melting curves (A260 vs temperature) recorded in a buffer
containing 10 mM phosphate and 200 mM NaCl at pH 7.0 using 3.0 μM concentrations of each strand. bΔTm = change in Tm for a modified duplex
relative to the unmodified duplex 5′-CTC ACT ATC TGMeCT. p = phosphodiester backbone. DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 ■RESULTS AND DISCUSSION Mismatch Data for ONs Containing Modified Monomer in the Center (ON3U−ON3Z)
mismatch data against MeC (5′-side of the linkage)
mismatch data against T
(3′-side of the linkage)
Tma
ΔTmb
Tma
ΔTmb
match
mismatch
match
mismatch
ON code
ON sequence (5′−3′)
MeC−G
MeC−U
MeC−A
T−A
T−C
T−G
ON3U
5′-CTC A MeCpT A TCT GCT
55.1
−17.7
−16.1
55.1
−12.3
−3.4
ON3V
5′-CTC A MeCLpTL A TCT GCT
66.0
−18.1
−17.2
66.0
−12.9
−4.6
ON3W
5′-CTC A MeCtT A TCT GCT
49.6
−17.0
−15.2
49.6
−10.7
−2.1
ON3X
5′-CTC A MeCtTL A TCT GCT
57.1
−16.9
−15.5
57.1
−13.8
−5.6
ON3Y
5′-CTC A MeCLtT A TCT GCT
49.8
−17.1
−16.3
49.8
−11.3
−4.2
ON3Z
5′-CTC A MeCLtTL A TCT GCT
58.1
−17.3
−16.8
58.1
−15.2
−6.8
aMelting temperatures (Tm) were obtained from the maxima of the first derivatives of the melting curves (A260 vs temperature) recorded in a buffer
containing 10 mM phosphate and 200 mM NaCl at pH 7.0 using 3.0 μM concentrations of each strand. bΔTm = change in Tm for a mismatch duplex
relative to the corresponding matched duplex. p = phosphodiester backbone. L = LNA nucleotide. t = triazole backbone. Matched RNA target: 5′-
AGCAGAUAGUGAG. Mismatch RNA targets: 5′-AGCAGAUAUUGAG (MeC−U mismatch), 5′-AGCAGAUAAUGAG (MeC−A mismatch), 5′-
AGCAGAUCGUGAG (T−C mismatch), 5′-AGCAGAUGGUGAG (T−G mismatch). aMelting temperatures (Tm) were obtained from the maxima of the first derivatives of the melting curves (A260 vs temperature) recorded in a buffer
containing 10 mM phosphate and 200 mM NaCl at pH 7.0 using 3.0 μM concentrations of each strand. bΔTm = change in Tm for a mismatch duplex
relative to the corresponding matched duplex. p = phosphodiester backbone. L = LNA nucleotide. t = triazole backbone. Matched RNA target: 5′-
AGCAGAUAGUGAG. Mismatch RNA targets: 5′-AGCAGAUAUUGAG (MeC−U mismatch), 5′-AGCAGAUAAUGAG (MeC−A mismatch), 5′-
AGCAGAUCGUGAG (T−C mismatch), 5′-AGCAGAUGGUGAG (T−G mismatch). 6979 DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 ACS Omega Article Article Table 3. ■RESULTS AND DISCUSSION In all the
cases, the thermal stabilities of the modified duplexes were
lower than those of the unmodified duplexes (negative ΔTms). However, as observed for DNA/RNA duplexes, modifications
were better tolerated at the 3′-end relative to other positions
(ΔTms only in the range of −0.8 to −1.7 °C, compare ON1W−
ON1Z with ON1U). A large drop in Tm was observed for
monomer W at internal positions, which is consistent with the
literature (compare ON3W and ON4W with ON3U).19,20
Surprisingly, monomers X and Z were better accommodated at
the center of the duplex compared to the 5′-end, and the
thermal stability of the modified duplex incorporating
monomer Z in the center was similar to that of unmodified
duplex (compare ON3Z with ON3U, drop of only 1.5 °C). As p
Circular Dichroism (CD) Spectroscopy. CD spectra of
the modified duplexes obtained by mixing ON6 (ON6U−
ON6Z, Table 1) with RNA targets were recorded in a buffer
containing 10 mM phosphate and 200 mM NaCl at pH 7.0 and
the results are shown in Figure 3. Neither LNA nor the triazole
linkage significantly alters the global conformation of the DNA/
RNA duplexes (compare ON6V−ON6Z with ON6U). However, subtle shifts in CD maxima near 272 nm were
observed. For instance, the CD spectrum of the duplex
containing LNA but no triazole linkage (monomer V)
displayed a maximum at 263 nm (a hypsochromic shift of 9
nm). For duplexes containing LNA on the 3′-side or both sides
of the triazole linkage (monomers X and Z), the CD maxima
were observed at around 267 nm (a hypsochromic shift of 5
nm), whereas monomer W (triazole linkage but no LNA) or
monomer Y (LNA on the 5′-side of the linkage) did not induce
a noticeable shift in their CD maxima. Interpretation of these
shifts in terms of DNA conformation is not straightforward, but
the changes are modest, and overall geometry of the duplexes 6980 DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 ACS Omega ACS Omega Article Figure 4. Denaturing polyacrylamide gel electrophoresis (PAGE) analysis of ON6U−ON6Z after digestion with phosphodiesterase 1 from C. adamanteus, snake venom. This shows that the introduction of LNA next to the triazole improves stability against nucleases. Digestions were
performed using snake venom in 50 mM Tris buffer containing 10 mM MgCl2 at pH = 9.0. (A) Comparison of nuclease stability of ON6U, ON6V,
and ON6Z. ■RESULTS AND DISCUSSION (B) Comparison of nuclease stability of ON6W, ON6X, and ON6Z. Figure 4. Denaturing polyacrylamide gel electrophoresis (PAGE) analysis of ON6U−ON6Z after digestion with phosphodiesterase 1 from C. adamanteus, snake venom. This shows that the introduction of LNA next to the triazole improves stability against nucleases. Digestions were
performed using snake venom in 50 mM Tris buffer containing 10 mM MgCl2 at pH = 9.0. (A) Comparison of nuclease stability of ON6U, ON6V,
and ON6Z. (B) Comparison of nuclease stability of ON6W, ON6X, and ON6Z. Figure 5. Denaturing PAGE analysis of ON6U−ON6Z after digestion by FBS in DPBS supplemented with 50% FBS. (A) Comparison of enzymatic
stability of ON6U, ON6V, and ON6Z. (B) Comparison of enzymatic stability of ON6V, ON6X, and ON6Z. Figure 5. Denaturing PAGE analysis of ON6U−ON6Z after digestion by FBS in DPBS supplemented with 50% FB
stability of ON6U, ON6V, and ON6Z. (B) Comparison of enzymatic stability of ON6V, ON6X, and ON6Z. carrying LNA−triazole modifications is likely to be similar to
the unmodified DNA/RNA duplex. Nevertheless, an interesting
and significant trend emerges. The hypsochromic shifts
observed for LNA-containing duplexes in Figure 3 are in line
with previously reported data34 and could be attributed to
enhanced A-form-geometry due to the restricted conforma-
tional mobility of LNA. Assuming that flexible triazole linkages
partially (triazole−3′-LNA) or almost totally (5′-LNA−
triazole) eliminate beneficial LNA-driven A-form conforma-
tional changes, the extent of the hypsochromic shift should
decrease in the following order: ON6V (uncompensated LNA
effects) > ON6X and ON6Z (partially compensated LNA
effects) > ON6Y (almost totally compensated LNA effect) >
ON6W and ON6U (no LNA, zero shift). The relative
magnitudes of these hypsochromic shifts are also proportional
to differences in melting temperatures, precisely as would be
expected. carrying LNA−triazole modifications is likely to be similar to
the unmodified DNA/RNA duplex. Nevertheless, an interesting
and significant trend emerges. The hypsochromic shifts
observed for LNA-containing duplexes in Figure 3 are in line
with previously reported data34 and could be attributed to
enhanced A-form-geometry due to the restricted conforma-
tional mobility of LNA. Assuming that flexible triazole linkages
partially (triazole−3′-LNA) or almost totally (5′-LNA−
triazole) eliminate beneficial LNA-driven A-form conforma-
tional changes, the extent of the hypsochromic shift should
decrease in the following order: ON6V (uncompensated LNA
effects) > ON6X and ON6Z (partially compensated LNA
effects) > ON6Y (almost totally compensated LNA effect) >
ON6W and ON6U (no LNA, zero shift). ACS Omega Article shown that oligonucleotides (t-LNAs) that contain the triazole
linkage in Figure 1 flanked by LNA on the 3′- or both sides
(monomers X and Z, respectively) bind to their RNA targets
with higher affinity and specificity than the equivalent
unmodified ONs. In addition, t-LNAs containing multiple
incorporations of monomers X and Z are highly resistant to
nuclease degradation, and there is a synergistic effect on
enzymatic stability from the combined triazole−LNA linkages. Further biochemical and biological studies are required, but so
far the indications are that t-LNAs constitute a promising class
of oligonucleotides with potential antisense and splice-
modulating applications.36 They display extreme stability to
enzymatic degradation, high RNA-binding affinity and RNA
target specificity, and reduced anionic charge. The effects on
cellular uptake of reducing the overall anionic nature of
oligonucleotides by incorporation of LNA−triazole linkages will
be interesting to study, given that oligonucleotide delivery is a
major issue in therapeutic applications. The dinucleotide
phosphoramidite strategy described in this article also allows
for the synthesis of LNA triazoles, which incorporate
phosphorothioate linkages, which might further enhance cell
uptake. It is also possible that such oligonucleotide analogues
will have different toxicological properties than LNA because it
is not possible for any of the LNA−triazole dimers to be
degraded into LNA nucleotides, so they cannot enter the
mononucleotide triphosphate pool and therefore they cannot
be incorporated into genomic DNA or RNA. The results
presented here and previously16 suggest that, in addition to
potential therapeutic applications, modified t-LNAs could find
uses in imaging, diagnostics, and nontherapeutic siRNA
applications. being more stable than ON6X, which, in turn, is more stable
than ON6W. It is surprising that this 3′-exonuclease cleaves
ON6W so readily, as this must require the removal of an entire
triazole-linked dinucleotide from the 3′-end of the oligonucleo-
tide (by digestion of the closest available unmodified
phosphodiester linkage). In contrast, the extreme stability of
ON6Z to snake venom phosphodiesterase shows that there is a
synergistic effect between LNA and triazole, justifying the
modified DNA linkage design on which this study is based. g
g
y
Next, the stability of ON6V−ON6Z in fetal bovine serum
(FBS) was tested and compared with that of the unmodified
ON6U (Figures 5 and S2). FBS contains predominantly
endonucleases, so it has different enzymatic DNA degrading
activity to snake venom, which is a 3′-exonuclease. ■EXPERIMENTAL SECTION All the reagents were purchased from Sigma-Aldrich, Alfa Aesar,
Fisher Scientific, or Link Technologies and used without
further purification. Pyridine (from KOH) and POCl3 were
freshly distilled before use, and THF was obtained using the
MBraun SPS Bench Top solvent purification system (SPS). All
air/moisture-sensitive reactions were carried out under inert
atmosphere (argon) in an oven-dried glassware. Reactions were
monitored by thin-layer chromatography using Merck Kieselgel
60 F24 silica gel plates (0.22 mm thickness, aluminum backed). The compounds were visualized by UV irradiation at 254/265
nm and by staining in p-anisaldehyde solution. Column
chromatography was carried out under pressure using Biotage
SNAP Ultra columns. The columns were primed with CH2Cl2
containing 1% pyridine before use for all DMTr-containing
compounds. 1H and 13C spectra were measured on a Bruker
AVII 500 spectrometer at 500 and 126 MHz, respectively. Chemical shifts are given in parts per million and were
internally referenced to the appropriate residual solvent signal,
all coupling constants (J) are quoted in hertz (Hz). Assignment
of compounds was aided by correlated spectroscopy,
heteronuclear single quantum coherence, heteronuclear multi-
ple bond correlation, and distortionless enhancement by
polarization transfer-135 experiments. The high-resolution
mass spectra were measured on a Bruker 9.4 FT-ICR-MS
mass spectrometer, and the samples were run in MeOH. ACS Omega FBS was
chosen for this study because it will simulate the extracellular
conditions to which a therapeutic oligonucleotide would be
exposed. ONs were incubated with FBS (50%) in Dulbecco’s
phosphate-buffered saline (DPBS). Unmodified DNA (ON6U)
was completely digested within 4 h of incubation, whereas both
ON6Z and ON6V were stable for up to 8 h (Figure 5A). The
observation that LNA enhances the resistance of triazole-linked
ONs against nucleolytic degradation in serum was further
supported by the fact that ON6Z (LNA on both sides of the
triazole linkage) is more stable (visible up to 24 h) than the
ON6X (LNA only on 3′-side of the linkage), and the
observation that ON6W containing triazole linkages without
LNA is less stable (Figure S2). Interestingly, ON6V (eight
LNAs) was more stable in serum than ON6X (four triazole
linkages and four LNAs), whereas ON6V and ON6Z with eight
LNA sugars have similar stability (both stable up to 24 h). This
confirms that LNA is a significant contributor to enzymatic
stability in serum. The triazole linkage (monomer W) is a
mimic of phosphodiester linkage and is biocompatible,35 so it
might be able to bind to serum endonucleases in a fashion
similar to unmodified linkage and allow adjacent phospho-
diesters to be cleaved (ON6X has eight unmodified
phosphodiester linkages). In contrast, LNA has a locked
sugar (3′-endo), which could inhibit the binding of nucleases to
the single-stranded ON. To summarize, t-LNAs with LNA on
either side of the triazole linkage have the highest stability
against nucleolytic degradation, in addition to the strongest
affinity for RNA targets. ■EXPERIMENTAL SECTION ■RESULTS AND DISCUSSION The relative
magnitudes of these hypsochromic shifts are also proportional
to differences in melting temperatures, precisely as would be
expected. Enzymatic Stability of t-LNAs. Oligonucleotides ON6U−
ON6Z (Table 1) were incubated with phosphodiesterase 1
from Crotalus adamanteus, snake venom (3′-exonuclease), in 50
mM Tris buffer containing 10 mM MgCl2 at pH = 9.0 to
determine their resistance to nucleolytic degradation. Aliquots
were taken over different time intervals and analyzed by 20%
polyacrylamide gel electrophoresis (PAGE) (Figure 4). Figure
4A shows a direct comparison between the unmodified DNA
strand (ON6U), an ON containing eight LNAs but no triazole
linkage (ON6V) and an ON with four incorporations of
monomer Z (ON6Z, best candidate from thermal melting
data). It is evident that multiple incorporations of monomer Z
render the modified ON remarkably resistant to degradation by
snake venom. After 3 h, ON6Z is clearly visible, whereas both
ON6U and ON6V are fully degraded. Next, the enzymatic
stability of ON6W, ON6X, and ON6Z was compared (Figure
4B). This shows that the introduction of LNA next to the
triazole improves stability against this nuclease, with ON6Z 6981 DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 ACS Omega ■CONCLUSIONS 13C NMR (126 MHz,
DMSO) δ 158.2, 158.2 (C4/DMTr), 144.5 (C2), 136.7 (C6),
135.2, 134.9, 132.5, 129.8, 129.3, 128.3, 128.0, 127.7, 126.9,
113.4 (DMTr, Ph), 109.2 (C5), 87.1 (C4′), 86.9 (C1′), 85.9
(DMTr), 79.8 (CCH), 78.0 (CCH), 75.9 (C2′), 75.5
(C3′), 71.7 (C5″), 57.9 (CH2), 57.1 (C5′), 55.1 (2 × OCH3),
13.5 (CH3). A small impurity of ethyl acetate and CH2Cl2 as
residual solvent was found. The compound was used as such in
the next step. white foam. Rf (0.5, 7% MeOH in CH2Cl2). ESI HRMS m/z
318.0810 ([M + Na])+, C11H13O5N5Na+ calc. 318.0809. 1H
NMR (500 MHz, DMSO) δ 11.40 (s, 1H, NH), 7.48 (d, J = 1.2
Hz, 1H, H6), 5.88 (bs, 1H, 3′-OH), 5.48 (s, 1H, H1′), 4.20 (s,
1H, H2′), 3.99 (d, J = 14.0 Hz, 1H, H5″), 3.92 (s, 1H, H3′),
3.88 (d, J = 8.0 Hz, 1H, H5″), 3.79 (d, J = 14.0 Hz, 1H, H5′),
3.74 (d, J = 8.0 Hz, 1H, H5′), 1.82 (d, J = 1.2 Hz, 3H, CH3). 13C NMR (126 MHz, DMSO) δ 164.2 (C4), 150.4 (C2), 134.8
(C6), 109.2 (C5), 87.4 (C4′), 87.1 (C1′), 79.5 (C2′), 71.6
(C5″), 70.4 (C3′), 47.9 (C5′), 12.8 (CH3). (
)
(
)
(
)
(
3)
Synthesis of DNA/DNA Triazole Nucleoside (8). Nucleosides 4 (0.50 g, 0.73 mmol) and 7 (175 mg, 0.66
mmol) were dissolved in THF/H2O/t-BuOH (10 mL, 3:1:1, v/
v/v). To this solution was added pyridine (2−3 drops), CuSO4
(1.5 mL, 7.5% aqueous, w/v), and sodium ascorbate (1.7 mL, 1
M aqueous). The reaction mixture was degassed with argon and
stirred at room temperature for 2 h. The reaction was diluted
with EtOAc (100 mL) and washed with H2O (50 mL) followed
by sat. aqueous solution of ethylenediaminetetraacetic acid
(EDTA) (3 × 50 mL). The combined aqueous phase was back
extracted with EtOAc (50 mL) and the combined organic
phases were dried (Na2SO4) and concentrated under reduced
pressure. The residue was purified using column chromatog-
raphy (0−6% MeOH in CH2Cl2) to obtain 8 (0.50 g, 80%) as a
white foam. Rf (0.4, 6% MeOH in CH2Cl2). ESI HRMS m/z
953.3824 ([M + H])+, C51H53O11N8
+ calc. 953.3828. ■CONCLUSIONS 1H NMR (400 MHz, DMSO) δ 12.95 (bs, 1H,
NH), 8.17 (m, 2H, Ph), 7.80 (s, 1H, H6), 7.62−7.58 (m, 1H,
Ph), 7.52−7.48 (m, 2H, Ph), 7.43−7.40 (m, 2H, DMTr), 7.35
(t, J = 7.6 Hz, 2H, DMTr), 7.30−7.24 (m, 5H, DMTr), 6.93 (d,
J = 8.8 Hz, 4H, DMTr), 6.15 (t, J = 6.8 Hz, 1H, H1′), 4.52−
4.50 (m, 1H, H3′), 4.25 (apparent t, J = 2.4 Hz, 2H, CH2C
CH), 4.13−4.12 (m, 1H, H4′), 3.74 (s, 6H, 2 × OCH3), 3.53
(t, J = 2.4 Hz, 1H, CCH), 3.32−3.29 (m, 1H, H5′), 3.23 (dd,
J = 10.4, 2.8 Hz, 1H, H5′), 2.46−2.39 (m, 2H, H2′), 1.81 (s,
3H, CH3). 13C NMR (100 MHz, DMSO) δ 159.2 (C4), 158.7
(DMTr), 145.0 (C2), 138.5 (C6), 137.0, 135.8, 135.6, 133.0,
130.2, 129.8, 128.8, 128.5, 128.1, 127.3, 113.8 (DMTr, Ph),
110.7 (C5), 86.7 (DMTr), 85.6 (C1′), 83.6 (C4′), 80.4 (C
CH), 78.2 (CCH), 78.0 (H3′), 63.9 (C5′), 56.5 (CH2), 55.5
(2 × OCH3), 37.2 (C2′), 13.2 (CH3). Synthesis of DNA/LNA Triazole Nucleoside (9). Nucleosides 4 (237 mg, 0.80 mmol) and 6 (0.60 g, 0.88
mmol) were dissolved in THF/H2O/t-BuOH (10 mL, 3:1:1, v/
v/v). To this solution was added pyridine (2−3 drops), CuSO4
(1.8 mL, 7.5% aqueous, w/v), and sodium ascorbate (2.0 mL, 1
M aqueous). The reaction mixture was degassed with argon and
stirred at room temperature for 2 h. The reaction was diluted
with EtOAc (100 mL) and washed with H2O (50 mL) and sat. aqueous solution of EDTA (3 × 50 mL). The combined
aqueous phase was back extracted with EtOAc (50 mL) and the
combined organic phase was dried (Na2SO4) and concentrated
under reduced pressure. The residue was purified using column
chromatography (0−6% MeOH in CH2Cl2) to obtain 9 (0.65
g, 82%) as a white foam. Rf (0.4, 6% MeOH in CH2Cl2). ESI
HRMS m/z 979.3617 ([M −H])−, C52H51O12N8
−calc. 979.3631. 1H NMR (500 MHz, DMSO) δ 12.96 (s, 1H, NH
(MeC)), 11.35 (s, 1H, NH (T)), 8.21−8.17 (m, 3H, Ph,
triazole-H), 7.82 (s, 1H, H6 (MeC)), 7.60 (m, 1H, Ph), 7.50 (m,
2H, Ph), 7.41−7.39 (m, 2H, DMTr), 7.34 (t, J = 7.8 Hz, 2H, 5′-Azido-5′-deoxythymidine-LNA (6). To a solution of
nucleoside 529 (1.0 g, 2.28 mmol) in MeOH (15 mL) was
added Pd(OH)2/C (20 wt % loading, 200 mg). The mixture
was degassed with argon (5 min) and then with hydrogen (10
min). ■CONCLUSIONS 1H NMR
(500 MHz, DMSO) δ 12.96 (s, 1H, NH (MeC)), 11.32 (s, 1H,
NH (T)), 8.19 (m, 2H, Ph), 8.10 (s, 1H, triazole-H), 7.82 (s,
1H, H6 (MeC)), 7.60 (m, 1H, Ph), 7.50 (m, 2H, Ph), 7.40−7.39
(m, 2H, DMTr), 7.35−7.32 (m, 3H, DMTr, H6 (T)), 7.28−
7.24 (m, 5H, DMTr), 6.93−6.91 (m, 4H, DMTr), 6.18−6.15
(m, 2H, H1′ (T), H1′ (MeC)), 5.52 (d, J = 4.4 Hz, 1H, 3′-OH
(T)), 4.71 (dd, J = 14.2, 4.4 Hz, 1H, H5′ (T)), 4.63−4.56 (m,
3H, H5′ (T), CH2), 4.46−4.44 (m, 1H, H3′ (MeC)), 4.31−4.27
(m, 1H, H3′ (T)), 4.13−4.12 (m, 1H, H4′ (MeC)), 4.10−4.06
(m, 1H, H4′ (T)), 3.76 (s, 6H, 2 × OCH3), 3.31 (dd, J = 9.8,
4.4 Hz, 1H, H5′ (MeC)), 3.23−3.22 (m, 1H, H5′ (MeC)), 2.46−
2.42 (m, 2H, H2′ (MeC)), 2.22−2.17 (m, 1H, H2′ (T)), 2.13−
2.09 (m, 1H, H2′ (T)), 1.79 (dd, J = 0.9 Hz, 3H, CH3 (T)),
1.68 (s, 3H, CH3 (MeC)). 13C NMR (126 MHz, DMSO) δ
178.6, 164.1, 159.2, 158.7, 150.8, 145.1, 144.2, 138.6, 137.1,
136.5, 135.8, 135.6, 133.0, 130.2, 129.9, 128.8, 128.5, 128.1,
127.3, 125.2, 113.8, 110.7, 110.3, 86.6, 85.4, 84.5, 84.4, 83.8,
78.9, 71.2, 64.0, 62.3, 55.5, 51.6, 38.4, 37.4, 13.0, 12.5. p
N6-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O-proparg-
yl-5-methyl-2′-deoxycytidine (4). To a solution of nucleo-
side 228 (1.20 g, 2.07 mmol) in DMF (5 mL) was added
benzoic anhydride (0.93 g, 4.11 mmol). The reaction mixture
was stirred at room temperature for 20 h. The solvent was
removed and residue was taken up in EtOAc (100 mL), washed
with sat. aqueous NaHCO3 (50 mL), brine (2 × 50 mL), dried
(Na2SO4), and concentrated. The crude mixture was purified
using column chromatography (0−50% EtOAc in hexane) to
obtain 4 (1.30 g, 92%) as a white foam. Rf (0.5, 50% EtOAc in
hexane). ESI HRMS m/z 686.2856 ([M + H])+, C41H40O7N3
+
calc. 686.2861. ■CONCLUSIONS The efficient synthesis of dinucleoside phosphoramidites
containing a triazole linkage and LNA sugars has been
achieved. Access to these dinucleoside phosphoramidites
allowed the incorporation of the triazole linkage flanked by
LNA at multiple positions in oligonucleotides, including the
central region and the 3′- and 5′-ends. This was not feasible
with our earlier ligation-based strategy where short ONs were
joined together using click chemistry. There are several
examples of a short triazole linkage being introduced into
oligonucleotides via phosphoramidite monomers, but in all the
cases, the duplexes were greatly destabilized.21−23 There is also
one report of this short triazole being combined with LNA and
added to oligonucleotides by phophoramidite chemistry, but
unlike our triazole, this particular linkage was strongly duplex-
destabilizing despite the presence of LNA.15 Another study
described the synthesis of triazole-linked DNA by phosphor-
amidite chemistry using an inverted version of the triazole unit
employed in this study, and this linkage slightly destabilized
double-stranded DNA.17 In contrast, in this study, we have p
p
N6-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O-proparg-
yl-5-methyl-LNA Cytidine (3). To a solution of nucleoside
116 (0.74 g, 1.21 mmol) in DMF (5 mL) was added benzoic
anhydride (0.41 g, 1.81 mmol). The reaction mixture was
stirred at room temperature for 18 h. The solvent was removed DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 6982 ACS Omega Article Article and the residue was taken up in EtOAc (100 mL), washed with
sat. aqueous NaHCO3 (50 mL), brine (2 × 50 mL), dried
(Na2SO4), and concentrated. The crude mixture was purified
using column chromatography (0−50% EtOAc in hexane) to
obtain 3 (0.75 g, 86%) as a white foam. Rf (0.4, 40% EtOAc in
hexane). ESI HRMS m/z 712.2663 ([M −H])−, C42H38O8N3
−
calc. 712.2664. 1H NMR (500 MHz, DMSO) δ 13.12 (bs, 1H,
NH), 8.18 (m, 2H, Ph), 7.85 (s, 1H, H6), 7.61−7.58 (m, 1H,
Ph), 7.51−7.47 (m, 4H, DMTr, Ph), 7.37−7.33 (m, 6H,
DMTr), 7.28−7.25 (m, 1H, DMTr), 6.95−6.93 (m, 4H,
DMTr), 5.59 (s, 1H, H1′), 4.68 (s, 1H, H2′), 4.39 (s, 1H, H3′),
4.35 (dd, J = 16.0, 2.4 Hz, 1H, CH2CCH), 4.29 (dd, J = 16.0,
2.4 Hz, 1H, CH2CCH), 3.75−3.71 (m, 7H, 2 × OCH3,
H5″), 3.74−3.72 (d, J = 8.0 Hz, 1H, H5″), 3.55 (t, J = 2.4 Hz,
1H, CCH), 3.44 (d, J = 11.1 Hz, 1H, H5′), 3.38 (d, J = 11.1
Hz, 1H, H5′), 1.81 (s, 3H, CH3). ■CONCLUSIONS 1H NMR (500 MHz, DMSO) δ 13.16 (s, 1H, NH
(MeC)), 11.31 (s, 1H, NH (T)), 8.23 (m, 2H, Ph), 8.03 (s, 1H,
triazole-H), 7.85 (s, 1H, H6 (MeC)), 7.61 (m, 1H, Ph), 7.52 (m,
2H, Ph), 7.42−7.42 (m, 2H, DMTr), 7.34−7.23 (m, 8H,
DMTr, H6 (T)), 6.93−6.90 (m, 4H, DMTr), 6.16 (t, J = 7.0
Hz, 1H, H1′ (T)), 5.60 (s, 1H, H1′ (MeC)), 5.50 (d, J = 4.3 Hz,
1H, 3′-OH (T)), 4.72 (d, J = 12.0 Hz, 1H, CH2), 4.68−4.64
(m, 3H, H2′ (MeC), CH2, H5′ (T)), 4.58 (dd, J = 14.3, 7.6 Hz,
1H, H5′ (T)), 4.41 (s, 1H, H3′ (MeC)), 4.29−4.25 (m, 1H, H3′
(T)), 4.07−4.04 (m, 1H, H4′ (T)), 3.75−3.71 (m, 8H, 2 ×
OCH3, 2 × H5″ (MeC)), 3.41 (d, J = 11.2 Hz, 1H, H5′ (MeC)),
3.36−3.31 (m, 1H, H5′ (MeC), merged with H2O signal from
DMSO), 2.21−2.15 (m, 1H, H2′ (T)), 2.12−2.07 (m, 1H, H2′
(T)), 1.86 (s, 3H, CH3, (MeC)), 1.76 (d, J = 1.2 Hz, 3H, CH3
(T)). 13C NMR (126 MHz, DMSO) δ 178.7, 164.1, 159.7,
158.6, 150.8, 145.0, 143.9, 137.4, 137.1, 136.5, 135.7, 135.4,
133.0, 130.2, 129.8, 128.8, 128.5, 128.0, 127.3, 125.2, 113.8,
110.3, 109.9, 87.6, 87.4, 86.3, 84.5, 84.4, 76.6, 76.5, 72.2, 71.2,
63.1, 58.4, 55.5, 51.7, 38.4, 14.0, 12.5. Synthesis of DNA/DNA Triazole Phosphoramidite
(12). Nucleoside 8 (250 mg, 0.26 mmol) was dissolved in
dry CH2Cl2 (5 mL). DIPEA (200 μL, 1.14 mmol) and 2-
cyanoethyl N,N-diisopropylchlorophosphoramidite (120 μL,
0.56 mmol) were added and the reaction mixture was stirred at
room temperature for 2 h. The reaction was diluted with
CH2Cl2 (30 mL) and washed with sat. aqueous KCl (30 mL). The organic phase was dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified using column
chromatography (0−3% MeOH in CH2Cl2) to obtain 12 (150
mg, 50%) as a white foam. Rf (0.4, 3% MeOH in CH2Cl2). ESI
HRMS m/z 1153.4915 ([M + H])+, C60H70O12N10P+ calc. 1153.4906. 31P NMR (162 MHz, CD3CN) δ 148.76, 148.53. (
3
)
Synthesis of DNA/LNA Triazole Phosphoramidite (13). Nucleoside 9 (300 mg, 0.31 mmol) was dissolved in dry
CH2Cl2 (5 mL). DIPEA (0.22 mL, 1.24 mmol) and 2-
cyanoethyl N,N-diisopropylchlorophosphoramidite (0.14 mL,
0.62 mmol) were added and the reaction mixture was stirred at
room temperature for 2 h. The reaction was diluted with
CH2Cl2 (30 mL) and washed with sat. aqueous KCl (30 mL). ■CONCLUSIONS 13C NMR (126 MHz, DMSO) δ 164.1, 158.7, 150.2,
145.1, 144.0, 137.2, 135.6, 135.5, 134.2, 133.0, 130.2, 129.8,
128.8, 128.5, 128.0, 127.3, 126.2, 113.8, 109.1, 87.6, 87.5, 87.1,
86.3, 79.5, 76.6, 76.5, 72.2, 71.7, 70.4, 63.1, 58.5, 55.5, 46.6,
14.0, 12.5. Some quarternary carbons are not seen in the NMR. DMTr), 7.28−7.23 (m, 5H, DMTr), 6.92 (d, J = 8.8 Hz, 4H,
DMTr), 6.60 (d, J = 1.3 Hz, 1H, H6 (T)), 6.16 (t, J = 6.7 Hz,
1H, H1′ (MeC)), 6.08 (d, J = 6.1 Hz, 1H, 3′-OH), 5.40 (s, 1H,
H1′ (T)), 4.99 (d, J = 15.1 Hz, 1H, H5′ (T)), 4.88 (d, J = 15.1
Hz, 1H, H5′ (T)), 4.66 (d, J = 12.0 Hz, 1H, CH2), 4.62 (d, J =
12.0 Hz, 1H, CH2), 4.48−4.45 (m, 1H, H3′ (MeC)), 4.18 (s,
1H, H2′ (T)), 4.14−4.12 (m, 1H, H4′ (MeC)), 4.03 (d, J = 8.0
Hz, 1H, H5″ (T)), 3.79 (d, J = 3.3 Hz, H3′ (T)), 3.74 (s, 6H, 2
× OCH3), 3.59 (d, J = 8.0 Hz, 1H, H5″ (T)), 3.32−3.20 (m,
1H, H5′ (MeC)), 3.25−3.23 (m, 1H, H5′ (MeC)), 2.47−2.41
(m, 2H, H2′ (MeC)), 1.67 (app s, 6H, 2 × CH3 (MeC and T)). 13C NMR (126 MHz, DMSO) δ 164.1, 158.7, 158.6, 150.2,
145.1, 144.3, 135.8, 135.6, 134.1, 133.0, 130.2, 129.8, 128.8,
128.5, 128.1, 127.3, 126.3, 113.8, 109.1, 87.1, 86.6, 86.3, 83.9,
79.5, 79.0, 71.8, 70.4, 64.0, 62.4, 55.5, 46.5, 13.1, 12.5. Synthesis of LNA/DNA Triazole Nucleoside (10). Nucleosides 3 (0.50 g, 0.73 mmol) and 7 (170 mg, 0.66
mmol) were dissolved in THF/H2O/t-BuOH (10 mL, 3:1:1, v/
v/v). To this solution was added pyridine (2−3 drops), CuSO4
(1.5 mL, 7.5% aqueous, w/v), and sodium ascorbate (1.7 mL, 1
M aqueous). The reaction mixture was degassed with argon and
stirred at room temperature for 2 h. The reaction was diluted
with EtOAc (100 mL) and washed with H2O (50 mL) and sat. aqueous solution of EDTA (3 × 50 mL). The combined
aqueous phase was back extracted with EtOAc (50 mL) and the
combined organic phase was dried (Na2SO4) and concentrated
under reduced pressure. The residue was purified using column
chromatography (0−6% MeOH in CH2Cl2) to obtain 10 (0.54
g, 87%) as a white foam. Rf (0.5, 7% MeOH in CH2Cl2). ESI
HRMS m/z 979.3621 ([M −H])−, C52H51O12N8
−calc. 979.3631. ■CONCLUSIONS The organic phase was dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified using column
chromatography (0−3% MeOH in CH2Cl2) to obtain 13 (225
mg, 62%) as a white foam. Rf (0.5, 4% MeOH in CH2Cl2). ESI
HRMS m/z 1181.4861 ([M + H])+, C61H70O13N10P+ calc. 1181.4856. 31P NMR (162 MHz, CD3CN) δ 149.22, 148.86. (
,
3
)
,
Synthesis of LNA/DNA Triazole Phosphoramidite (14). Nucleoside 10 (350 mg, 0.36 mmol) was dissolved in dry
CH2Cl2 (5 mL). DIPEA (250 μL, 1.44 mmol) and 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (180 μL, 0.72 mmol)
were added and the reaction mixture was stirred at room
temperature for 2 h. The reaction was diluted with CH2Cl2 (30
mL) and washed with sat. aqueous KCl (30 mL). The organic
phase was dried (Na2SO4) and concentrated under reduced
pressure. The residue was purified using column chromatog-
raphy (0−3% MeOH in CH2Cl2) to obtain 14 (320 mg, 76%)
as a white foam. Rf (0.5, 5% MeOH in CH2Cl2). ESI HRMS m/
z 1181.4859 ([M + H])+, C61H70O13N10P+ calc. 1181.4856. 31P
NMR (162 MHz, CD3CN) δ 148.69, 148.55. ,
,
,
,
,
,
Synthesis of LNA/LNA Triazole Nucleoside (11). Nucleosides 3 (120 mg, 0.40 mmol) and 6 (0.36 g, 0.50
mmol) were dissolved in THF/H2O/t-BuOH (5 mL, 3:1:1, v/
v/v). To this solution was added pyridine (2−3 drops), CuSO4
(0.9 mL, 7.5% aqueous, w/v), and sodium ascorbate (1.0 mL, 1
M aqueous). The reaction mixture was degassed with argon and
stirred at room temperature for 2 h. The reaction was diluted
with EtOAc (50 mL) and washed with H2O (30 mL) and a sat. aqueous solution of EDTA (3 × 30 mL). The combined
aqueous phase was back extracted with EtOAc (20 mL) and the
combined organic phase was dried (Na2SO4) and concentrated
under reduced pressure. The residue was purified using column
chromatography (0−6% MeOH in CH2Cl2) to obtain 11 (0.32
g, 79%) as a white foam with a small amount of EtOAc as an ,
,
,
,
,
,
Synthesis of LNA/LNA Triazole Nucleoside (11). Nucleosides 3 (120 mg, 0.40 mmol) and 6 (0.36 g, 0.50
mmol) were dissolved in THF/H2O/t-BuOH (5 mL, 3:1:1, v/
v/v). To this solution was added pyridine (2−3 drops), CuSO4
(0.9 mL, 7.5% aqueous, w/v), and sodium ascorbate (1.0 mL, 1
M aqueous). The reaction mixture was degassed with argon and
stirred at room temperature for 2 h. ■CONCLUSIONS The reaction mixture was stirred under hydrogen at room
temperature for 16 h. The catalyst was filtered offand the filter
cake was washed with MeOH (50 mL). The filtrate was
concentrated under reduced pressure and the residue was
dissolved in DMF (10 mL). NaN3 (300 mg, 4.61 mmol) was
added and the reaction mixture was stirred at 90 °C for 2 h. The solvent was removed under reduced pressure and the
residue was purified using column chromatography (0−7%
MeOH in CH2Cl2) to afford nucleoside 6 (0.43 g, 64%) as 6983 DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 ACS Omega ACS Omega Article Article impurity. Rf (0.4, 6% MeOH in CH2Cl2). ESI HRMS m/z
1009.3721 ([M + H])+, C53H53O13N8
+ calc. 1009.3726. 1H
NMR (500 MHz, DMSO) δ 13.16 (bs, 1H, NH (MeC)), 11.33
(s, 1H, NH (T)), 8.19 (m, 2H, Ph), 8.12 (s, 1H, triazole-H),
7.86 (s, 1H, H6 (MeC)), 7.60 (m, 1H, Ph), 7.50 (m, 2H, Ph),
7.43−7.41 (m, 2H, DMTr), 7.34−7.22 (m, 7H, DMTr), 6.92−
6.88 (m, 4H, DMTr), 6.62 (s, 1H, H6 (T)), 6.05 (d, J = 4.2 Hz,
1H, 3′-OH (T)), 5.60 (s, 1H, H1′ (MeC)), 5.40 (s, 1H, H1′
(T)), 4.97 (d, J = 15.2 Hz, 1H, H5′ (T)), 4.82 (d, J = 15.2 Hz,
1H, H5′ (T)), 4.77 (d, J = 12.1 Hz, 1H, CH2), 4.70−4.66 (m,
2H, CH2, H3′ (MeC)), 4.43 (s, 1H, H2′ (MeC)), 4.16 (s, 1H,
H2′ (T)), 3.98 (d, J = 8.0 Hz, 1H, H5″ (T)), 3.80 (d, J = 4.2
Hz, 1H, H3′ (T)), 3.75−3.72 (m, 8H, 2 × OCH3, 2 × H5″
(MeC)), 3.50 (d, J = 8.0 Hz, 1H, H5″ (T)), 3.41−3.36 (m, 2H,
2 × H5′ (MeC)), 1.85 (s, 3H, CH3, (MeC)), 1.61 (s, 3H, CH3
(T)). 13C NMR (126 MHz, DMSO) δ 164.1, 158.7, 150.2,
145.1, 144.0, 137.2, 135.6, 135.5, 134.2, 133.0, 130.2, 129.8,
128.8, 128.5, 128.0, 127.3, 126.2, 113.8, 109.1, 87.6, 87.5, 87.1,
86.3, 79.5, 76.6, 76.5, 72.2, 71.7, 70.4, 63.1, 58.5, 55.5, 46.6,
14.0, 12.5. Some quarternary carbons are not seen in the NMR. ■CONCLUSIONS DMTr), 7.28−7.23 (m, 5H, DMTr), 6.92 (d, J = 8.8 Hz, 4H,
DMTr), 6.60 (d, J = 1.3 Hz, 1H, H6 (T)), 6.16 (t, J = 6.7 Hz,
1H, H1′ (MeC)), 6.08 (d, J = 6.1 Hz, 1H, 3′-OH), 5.40 (s, 1H,
H1′ (T)), 4.99 (d, J = 15.1 Hz, 1H, H5′ (T)), 4.88 (d, J = 15.1
Hz, 1H, H5′ (T)), 4.66 (d, J = 12.0 Hz, 1H, CH2), 4.62 (d, J =
12.0 Hz, 1H, CH2), 4.48−4.45 (m, 1H, H3′ (MeC)), 4.18 (s,
1H, H2′ (T)), 4.14−4.12 (m, 1H, H4′ (MeC)), 4.03 (d, J = 8.0
Hz, 1H, H5″ (T)), 3.79 (d, J = 3.3 Hz, H3′ (T)), 3.74 (s, 6H, 2
× OCH3), 3.59 (d, J = 8.0 Hz, 1H, H5″ (T)), 3.32−3.20 (m,
1H, H5′ (MeC)), 3.25−3.23 (m, 1H, H5′ (MeC)), 2.47−2.41
(m, 2H, H2′ (MeC)), 1.67 (app s, 6H, 2 × CH3 (MeC and T)). 13C NMR (126 MHz, DMSO) δ 164.1, 158.7, 158.6, 150.2,
145.1, 144.3, 135.8, 135.6, 134.1, 133.0, 130.2, 129.8, 128.8,
128.5, 128.1, 127.3, 126.3, 113.8, 109.1, 87.1, 86.6, 86.3, 83.9,
79.5, 79.0, 71.8, 70.4, 64.0, 62.4, 55.5, 46.5, 13.1, 12.5. impurity. Rf (0.4, 6% MeOH in CH2Cl2). ESI HRMS m/z
1009.3721 ([M + H])+, C53H53O13N8
+ calc. 1009.3726. 1H
NMR (500 MHz, DMSO) δ 13.16 (bs, 1H, NH (MeC)), 11.33
(s, 1H, NH (T)), 8.19 (m, 2H, Ph), 8.12 (s, 1H, triazole-H),
7.86 (s, 1H, H6 (MeC)), 7.60 (m, 1H, Ph), 7.50 (m, 2H, Ph),
7.43−7.41 (m, 2H, DMTr), 7.34−7.22 (m, 7H, DMTr), 6.92−
6.88 (m, 4H, DMTr), 6.62 (s, 1H, H6 (T)), 6.05 (d, J = 4.2 Hz,
1H, 3′-OH (T)), 5.60 (s, 1H, H1′ (MeC)), 5.40 (s, 1H, H1′
(T)), 4.97 (d, J = 15.2 Hz, 1H, H5′ (T)), 4.82 (d, J = 15.2 Hz,
1H, H5′ (T)), 4.77 (d, J = 12.1 Hz, 1H, CH2), 4.70−4.66 (m,
2H, CH2, H3′ (MeC)), 4.43 (s, 1H, H2′ (MeC)), 4.16 (s, 1H,
H2′ (T)), 3.98 (d, J = 8.0 Hz, 1H, H5″ (T)), 3.80 (d, J = 4.2
Hz, 1H, H3′ (T)), 3.75−3.72 (m, 8H, 2 × OCH3, 2 × H5″
(MeC)), 3.50 (d, J = 8.0 Hz, 1H, H5″ (T)), 3.41−3.36 (m, 2H,
2 × H5′ (MeC)), 1.85 (s, 3H, CH3, (MeC)), 1.61 (s, 3H, CH3
(T)). ACS Omega Coupling
efficiencies and overall synthesis yields were determined by
the inbuilt automated trityl cation conductivity monitoring
facility and were ≥98.0% in all the cases. The oligonucleotides
were then cleaved from the solid support and protecting groups
from the nucleobase and backbone were removed by exposure
to concentrated aqueous ammonium hydroxide for 60 min at
room temperature followed by heating in a sealed tube for 5 h
at 55 °C. RNA was purchased from Integrated DNA
Technologies BVBA, Leuven, Belgium. g
p
μ
g
Synthesis of Resin Loaded with DNA/LNA Triazole
Nucleoside (18). Activated resin 1628 (800 mg) was soaked in
anhydrous pyridine (1 mL) for 10 min. EDC (0.329 g, 2.12
mmol), DMAP (14 mg, 0.11 mmol), triethylamine (85 μL, 0.61
mmol), and compound 9 (100 mg, 0.102 mmol) were added to
the resin. The reaction vessel was rotated for 20 h at room
temperature, after which pentachlorophenol (49 mg, 0.18
mmol) was added and the vessel was rotated for an additional 3
h. The solvents were removed by filtration, and the support was
washed with pyridine, CH2Cl2, and diethyl ether. Piperdine
(10% in DMF, 2 mL) was added and the vessel was rotated for
5 min at room temperature. The solvent was removed by
filtration and the support was washed with CH2Cl2 and diethyl
ether. Capping reagent (oligonucleotide synthesis grade, acetic
anhydride/pyridine/THF:N-methylimidazole in THF, 1:1, 2
mL) was added and the vessel was rotated at room temperature
for 1 h. The solvent was removed by filtration, and the resin
was washed with pyridine, CH2Cl2, and diethyl ether and dried
under high vacuum overnight. The loading of nucleoside 9 on
the support was determined by cleaving the DMTr group and
was found to be 26 μmol/g. μ
g
Synthesis of Resin Loaded with LNA/DNA Triazole
Nucleoside (19). Activated resin 1628 (300 mg) was soaked in
1 mL of anhydrous pyridine for 10 min. EDC (0.132 g, 0.850
mmol), DMAP (6 mg, 49 μmol), triethylamine (34 μL, 0.24
mmol), and compound 10 (40 mg, 41 μmol) were added to the
resin. The reaction vessel was rotated for 20 h at room
temperature, after which pentachlorophenol (20 mg, 75 μmol)
was added and the vessel was rotated for an additional 3 h. The
solvents were removed by filtration, and the support was
washed with pyridine, CH2Cl2, and diethyl ether. ACS Omega Capping reagent (oligonucleotide synthesis grade, acetic
anhydride/pyridine/THF:N-methylimidazole in THF, 1:1, 2
mL) was added and the vessel was rotated at room temperature
for 1 h. The solvent was removed by filtration and the resin was
washed with pyridine, CH2Cl2, and diethyl ether and dried
under high vacuum overnight. The loading of nucleoside 11 on
the support was determined by cleaving the DMTr group and
was found to be 28 μmol/g. Synthesis of Resin Loaded with DNA/DNA Triazole
Nucleoside (17). Activated resin 1628 (500 mg) was soaked in
anhydrous pyridine (1 mL) for 10 min. EDC (170 mg, 1.09
mmol), DMAP (36 mg, 0.29 mmol), triethylamine (44 μL, 0.32
mmol), and dinucleoside 8 (56 mg, 59 μmol) were added to
the resin. The reaction vessel was rotated for 20 h at room
temperature, after which pentachlorophenol (26 mg, 98 μmol)
was added and the vessel was rotated for an additional 3 h. The
solvents were removed by filtration, and the support was
washed with pyridine, CH2Cl2, and diethyl ether. Piperdine
(10% in DMF, 2 mL) was added and the vessel was rotated for
5 min at room temperature. The solvent was removed by
filtration and the support was washed with CH2Cl2 and diethyl
ether. Capping reagent (oligonucleotide synthesis grade, acetic
anhydride/pyridine/THF:N-methylimidazole in THF, 1:1, 2
mL) was added and the vessel was rotated at room temperature
for 1 h. The solvent was removed by filtration, and the resin
was washed with pyridine, CH2Cl2, and diethyl ether and dried
under high vacuum overnight to obtain 17. The loading of
nucleoside 8 on the support was determined by cleaving the
DMTr group and was found to be 28 μmol/g. μ
g
Synthesis of Oligonucleotides. Standard DNA phosphor-
amidites, solid supports, and reagents were purchased from
Link Technologies and Applied Biosystems. LNA phosphor-
amidites were obtained from Exiqon. Automated solid-phase
synthesis of oligonucleotides (trityl off) was performed on an
Applied Biosystems 394 synthesizer. The synthesis was
performed on 1.0 μm scale involving cycles of acid-catalyzed
detritylation, coupling, capping, and iodine oxidation. Standard
DNA phosphoramidites were coupled for 60 s, whereas
extended coupling time of 10 min was used for the modified
phosphoramidites including LNA phosphoramidites. Modified
phosphoramidites 12, 13, 14, and 15 were used to prepare ONs
with monomers W, X, Y, and Z respectively. ACS Omega ACS Omega Article 0.58 mmol) were added and the reaction mixture was stirred at
room temperature for 2 h. The reaction was diluted with
CH2Cl2 (30 mL) and washed with sat. aqueous KCl (30 mL). The organic phase was dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified using column
chromatography (0−3% MeOH in CH2Cl2) to obtain 15 (245
mg, 73%) as a white foam. Rf (0.5, 4% MeOH in CH2Cl2). ESI
HRMS m/z 1209.4813 ([M + H])+, C62H70O14N10P+ calc. 1209.4805. 31P NMR (162 MHz, CD3CN) δ 149.27, 148.87. filtration and the support was washed with CH2Cl2 and diethyl
ether. Capping reagent (oligonucleotide synthesis grade, acetic
anhydride/pyridine/THF:N-methylimidazole in THF, 1:1, 2
mL) was added and the vessel was rotated at room temperature
for 1 h. The solvent was removed by filtration and the resin was
washed with pyridine, CH2Cl2, and diethyl ether and dried
under high vacuum overnight. The loading of nucleoside 10 on
the support was determined by cleaving the DMTr group and
was found to be 29 μmol/g. filtration and the support was washed with CH2Cl2 and diethyl
ether. Capping reagent (oligonucleotide synthesis grade, acetic
anhydride/pyridine/THF:N-methylimidazole in THF, 1:1, 2
mL) was added and the vessel was rotated at room temperature
for 1 h. The solvent was removed by filtration and the resin was
washed with pyridine, CH2Cl2, and diethyl ether and dried
under high vacuum overnight. The loading of nucleoside 10 on
the support was determined by cleaving the DMTr group and
was found to be 29 μmol/g. Synthesis of Resin Loaded with LNA/LNA Triazole
Nucleoside (20). Activated resin 1628 (250 mg) was soaked in
1 mL of anhydrous pyridine for 10 min. EDC (0.106 g, 0.683
mmol), DMAP (5 mg, 41 μmol), triethylamine (28 μL, 0.20
mmol), and compound 11 (33 mg, 33 μmol) were added to the
resin. The reaction vessel was rotated for 20 h at room
temperature, after which pentachlorophenol (16 mg, 60 μmol)
was added and the vessel was rotated for an additional 3 h. The
solvents were removed by filtration, and the support was
washed with pyridine, CH2Cl2, and diethyl ether. Piperdine
(10% in DMF, 2 mL) was added and the vessel was rotated for
5 min at room temperature. The solvent was removed by
filtration and the support was washed with CH2Cl2 and diethyl
ether. ■CONCLUSIONS The reaction was diluted
with EtOAc (50 mL) and washed with H2O (30 mL) and a sat. aqueous solution of EDTA (3 × 30 mL). The combined
aqueous phase was back extracted with EtOAc (20 mL) and the
combined organic phase was dried (Na2SO4) and concentrated
under reduced pressure. The residue was purified using column
chromatography (0−6% MeOH in CH2Cl2) to obtain 11 (0.32
g, 79%) as a white foam with a small amount of EtOAc as an Synthesis of LNA/LNA Triazole Phosphoramidite (15). Nucleoside 11 (280 mg, 0.28 mmol) was dissolved in dry
CH2Cl2 (5 mL). DIPEA (0.18 mL, 1.03 mmol) and 2-
cyanoethyl N,N-diisopropylchlorophosphoramidite (136 μL, DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 6984 DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 ■REFERENCES Duplexes were prepared by mixing 3 μM of each
oligonucleotide in a 10 mM phosphate buffer containing 200
mM NaCl at pH 7.0 and annealed by heating to 85 °C and then
slowly cooled to 10 °C before recording the CD spectrum. A
CD spectrum of the buffer alone was recorded and subtracted
from the collected data. (7) Stein, C. A.; Castanotto, D. FDA-Approved Oligonucleotide
Therapies in 2017. Mol. Ther. 2017, 25, 1069−1075. (8) El-Sagheer, A. H.; Brown, T. Click nucleic acid ligation:
Applications in biology and nanotechnology. Acc. Chem. Res. 2012,
45, 1258−1267. (9) El-Sagheer, A. H.; Brown, T. Efficient RNA synthesis by in vitro
transcription of a triazole-modified DNA template. Chem. Commun. 2011, 47, 12057−12058. (10) El-Sagheer, A. H.; Sanzone, A. P.; Gao, R.; Tavassoli, A.; Brown,
T. Biocompatible artificial DNA linker that is read through by DNA
polymerases and is functional in E.coli. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 11338−11343. Notes The authors declare no competing financial interest. ORCID oligonucleotides, which were not separable using a gradient of
acetonitrile in TEAB. Buffer A: 0.1 M HAA, pH 7.5; buffer B:
0.1 M TEAB, pH 7.5, with 50% acetonitrile were used. The
elution was monitored by UV absorption between 260 and 298
nm. Pawan Kumar: 0000-0001-7936-4613
Afaf Helmy El-Sagheer: 0000-0001-8706-1292
Tom Brown: 0000-0002-6538-3036 Pawan Kumar: 0000-0001-7936-4613
Afaf Helmy El-Sagheer: 0000-0001-8706-1292 Present Address
§Alnylam Pharmaceuticals, 300 Third Street, Cambridge,
Massachusetts 02142, United States. E-mail: pkumar@
alnylam.com (P.K.). Thermal Denaturation Experiments. UV DNA melting
curves were recorded in a Cary 4000 Scan UV−vis
spectrophotometer using 3 μM of each oligonucleotide in a
10 mM phosphate buffer containing 200 mM NaCl at pH 7.0. The samples were annealed by heating to 85 °C (10 °C/min)
and then slowly cooling to 20 °C (1 °C/min). Six successive
cycles (heating and cooling) were performed at a gradient of 1
°C/min and the change in UV absorbance at 260 nm was
recorded. The melting temperature was calculated from the first
derivative of the melting curve using an in-built software. ■ACKNOWLEDGMENTS Authors acknowledge the generous funding U.K. BBSRC grants
BB/J001694/2 (extending the boundaries of nucleic acid
chemistry). P.K. holds a Marie Sklodowska-Curie Individual
Fellowship from European Commission, L.T. was funded by
the intramural program of the National Heart, Lung and Blood
Institute, NIH, United States, through the NIH-Oxford-
Cambridge Research Scholars Program. Snake Venom Phosphodiesterase Stability. Five nano-
mole of oligonucleotide was dissolved in 100 μL buffer (50 mM
Tris−HCl, 10 mM MgCl2, pH = 9.0). Twenty microliter of this
solution was removed as a control (0 min). To the remaining
solution was added 2 μL aqueous solution of phosphodiesterase
1 from C. adamanteus venom (from Sigma-Aldrich, catalogue
number P3243, 0.45 units, dissolved in 7 mL H2O). The
reaction was incubated at 37 °C and aliquots (20 μL) were
taken at different time intervals, mixed with formamide (20
μL), and stored at −20 °C. The samples were then analyzed by
denaturing 20% polyacrylamide gel electrophoresis. ■ASSOCIATED CONTENT (11) Birts, C. N.; Sanzone, A. P.; El-Sagheer, A. H.; Blaydes, J. P.;
Brown, T.; Tavassoli, A. Transcription of click-linked DNA in human
cells. Angew. Chem., Int. Ed. 2014, 53, 2362−2365. ■REFERENCES (1) Rinaldi, C.; Wood, M. J. A. Antisense oligonucleotides: the next
frontier for treatment of neurological disorders. Nat. Rev. Neurol. 2018,
14, 9−21. (2) Wan, W. B.; Seth, P. P. The Medicinal Chemistry of Therapeutic
Oligonucleotides. J. Med. Chem. 2016, 59, 9645−9667. Serum Stability. Five nanomole of oligonucleotide was
dissolved in 50 μL Dulbecco’s PBS and 50 μL of FBS (Gibco,
standard (sterile-filtered)) was added. The sample was
thoroughly mixed by vortexing before 20 μL of this solution
was removed, mixed with formamide (20 μL), and stored at
−80 °C as a control (0 h). The remaining reaction mixtures
were incubated at 37 °C and aliquots (20 μL) were taken at
different time intervals, mixed with formamide (20 μL), and
stored at −80 °C. The samples were then analyzed by
denaturing 20% polyacrylamide gel. g
(3) Kurreck, J. Antisense technologies. Eur. J. Biochem. 2003, 270,
1628−1644. (4) Deleavey, G. F.; Damha, M. J. Designing Chemically Modified
Oligonucleotides for Targeted Gene Silencing. Chem. Biol. 2012, 19,
937−954. (5) Zamecnik, P. C.; Stephenson, M. L. Inhibition of Rous sarcoma
virus replication and cell transformation by a specific oligodeox-
ynucleotide. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 280−284. (6) Khvorova, A.; Watts, J. K. The chemical evolution of
oligonucleotide therapies of clinical utility. Nat. Biotechnol. 2017, 35,
238−248. CD Spectroscopy. CD spectra (200−340 nm) were
recorded on a Chirscan Plus spectropolarimeter using a quartz
optical cells with a path length of 5.0 mm. The scans were
performed at 10 °C using a step size of 0.5 nm, a time per point
of 1.0 s, and a bandwidth of 2 nm; the average of four scans is
presented. Duplexes were prepared by mixing 3 μM of each
oligonucleotide in a 10 mM phosphate buffer containing 200
mM NaCl at pH 7.0 and annealed by heating to 85 °C and then
slowly cooled to 10 °C before recording the CD spectrum. A
CD spectrum of the buffer alone was recorded and subtracted
from the collected data. CD Spectroscopy. CD spectra (200−340 nm) were
recorded on a Chirscan Plus spectropolarimeter using a quartz
optical cells with a path length of 5.0 mm. The scans were
performed at 10 °C using a step size of 0.5 nm, a time per point
of 1.0 s, and a bandwidth of 2 nm; the average of four scans is
presented. ACS Omega Piperdine
(10% in DMF, 2 mL) was added and the vessel was rotated for
5 min at room temperature. The solvent was removed by g
g
Purification of Oligonucleotides. The fully deprotected
oligonucleotides were then purified by reverse-phase high-
performance liquid chromatography (HPLC) on a Gilson
system using a Luna 10 μm C8(2) 100 Å pore Phenomenex
column (250 × 10 mm2) with a gradient of acetonitrile in
triethylammonium bicarbonate (TEAB) over 20 min at a flow
rate of 4 mL/min. Buffer A: 0.1 M TEAB, pH 7.5; buffer B: 0.1
M TEAB, pH 7.5, with 50% acetonitrile were used. Elution was
monitored by UV absorption between 260 and 295 nm. A
gradient of acetonitrile in hexylammonium acetate (HAA) over
20 min at a flow rate of 4 mL/min was used to purify DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 6985 Article ACS Omega *
S
Supporting Information The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsomega.8b01086. (12) Shivalingam, A.; Tyburn, A. E. S.; El-Sagheer, A. H.; Brown, T. Molecular Requirements of High-Fidelity Replication-Competent
DNA Backbones for Orthogonal Chemical Ligation. J. Am. Chem. Soc. 2017, 139, 1575−1583. Mass spectrometry data for modified oligonucleotides,
additional oligonucleotide duplex melting data, additional
oligonucleotide enzymatic digestion studies, HPLC
analysis of modified oligonucleotides, NMR spectra of
compounds (PDF) (13) Isobe, H.; Fujino, T.; Yamazaki, N.; Guillot-Nieckowski, M.;
Nakamura, E. Triazole-linked analogue of deoxyribonucleic acid
(TLDNA): Design, synthesis, and double-strand formation with natural
DNA. Org. Lett. 2008, 10, 3729−3732. (14) Isobe, H.; Fujino, T. Triazole-linked analogues of DNA and
RNA (TLDNA and TLRNA): Synthesis and functions. Chem. Rec. 2014, 14, 41−51. ■AUTHOR INFORMATION
Corresponding Author V.; Pozmogova,
G E S
th i
h
t i ti
d i
it
ti it
f th
bi y
O. F.; Smirnov, I. P.; Grokhovsky, S. L.; Aseychev, A. V.; Pozmogova, G. E. Synthesis, characterization and in vitro activity of thrombin-
binding DNA aptamers with triazole internucleotide linkages. Eur. J. Med. Chem. 2013, 67, 90−97. (19) Madhuri, V.; Kumar, V. A. Design and synthesis of
dephosphono DNA analogues containing 1,2,3-triazole linker and
their UV-melting studies with DNA/RNA. Nucleosides, Nucleotides
Nucleic Acids 2012, 31, 97−111. (20) Dallmann, A.; El-Sagheer, A. H.; Dehmel, L.; Mügge, C.;
Griesinger, C.; Ernsting, N. P.; Brown, T. Structure and dynamics of
triazole-linked DNA: Biocompatibility explained. Chem. - Eur. J. 2011,
17, 14714−14717. (21) Varizhuk, A.; Chizhov, A.; Smirnov, I.; Kaluzhny, D.; Florentiev,
V. Triazole-Linked Oligonucleotides with Mixed-Base Sequences:
Synthesis and Hybridization Properties. Eur. J. Org. Chem. 2012, 2012,
2173−2179. (22) Varizhuk, A.; Chizhov, A.; Florentiev, V. Synthesis and
hybridization data of oligonucleotide analogs with triazole internucleo-
tide linkages, potential antiviral and antitumor agents. Bioorg. Chem. 2011, 39, 127−131. (23) Fujino, T.; Kogashi, K.; Okada, K.; Mattarella, M.; Suzuki, T.;
Yasumoto, K.; Sogawa, K.; Isobe, H. Chimeric RNA Oligonucleotides
with Triazole and Phosphate Linkages: Synthesis and RNA
Interference. Chem. - Asian J. 2015, 10, 2683−2688. (24) Vester, B.; Wengel, J. LNA (Locked Nucleic Acid): High-
Affinity Targeting of Complementary RNA and DNA. Biochemistry
2004, 43, 13233−13241. (25) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. LNA (locked
nucleic acids): Synthesis and high-affinity nucleic acid recognition. Chem. Commun. 1998, 4, 455−456. (26) Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J. I.; Morio, K. I.; Doi,
T.; Imanishi, T. Stability and structural features of the duplexes
containing nucleoside analogues with a fixed N-type conformation, 2′-
O,4′-C- methyleneribonucleosides. Tetrahedron Lett. 1998, 39, 5401−
5404. (27) Kaur, H.; Babu, B. R.; Maiti, S. Perspectives on Chemistry and
Therapeutic Applications of Locked Nucleic Acid (LNA). Chem. Rev. 2007, 107, 4672−4697. (28) El-Sagheer, A. H.; Brown, T. New strategy for the synthesis of
chemically modified RNA constructs exemplified by hairpin and
hammerhead ribozymes. Proc. Natl. Acad. Sci. U.S.A. 2010, 107,
15329−15334. (29) Koshkin, A. A.; Fensholdt, J.; Pfundheller, H. M.; Lomholt, C. A
simplified and efficient route to 2′-O, 4′-C-methylene-linked bicyclic
ribonucleosides (locked nucleic acid). J. Org. Chem. 2001, 66, 8504−
8512. (30) Said, H.; Guttroff, C.; El-Sagheer, A. H.; Richert, C. DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 ■AUTHOR INFORMATION
Corresponding Author Corresponding Author (15) Sharma, V. K.; Singh, S. K.; Krishnamurthy, P. M.; Alterman, J. F.; Haraszti, R. A.; Khvorova, A.; Prasad, A. K.; Watts, J. K. Synthesis *E-mail: tom.brown@chem.ox.ac.uk. 6986 DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 ACS Omega Article dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 2002, 67, 3057−3064. dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 2002, 67, 3057−3064. and biological properties of triazole-linked locked nucleic acid. Chem. Commun. 2017, 53, 8906−8909. (33) Owczarzy, R.; You, Y.; Groth, C. L.; Tataurov, A. V. Stability (16) Kumar, P.; El-Sagheer, A. H.; Truong, L.; Brown, T. Locked
nucleic acid (LNA) enhances binding affinity of triazole-linked DNA
towards RNA. Chem. Commun. 2017, 53, 8910−8913. (16) Kumar, P.; El-Sagheer, A. H.; Truong, L.; Brown, T. Locked
(
) (33) Owczarzy, R.; You, Y.; Groth, C. L.; Tataurov, A. V. Stability
and Mismatch Discrimination of Locked Nucleic Acid−DNA
Duplexes. Biochemistry 2011, 50, 9352−9367. (
)
y,
;
,
;
,
;
,
y
and Mismatch Discrimination of Locked Nucleic Acid−DNA
Duplexes. Biochemistry 2011, 50, 9352−9367. (17) Varizhuk, A. M.; Kaluzhny, D. N.; Novikov, R. A.; Chizhov, A. O.; Smirnov, I. P.; Chuvilin, A. N.; Tatarinova, O. N.; Fisunov, G. Y.;
Pozmogova, G. E.; Florentiev, V. L. Synthesis of Triazole-Linked
Oligonucleotides with High Affinity to DNA Complements and an
Analysis of Their Compatibility with Biosystems. J. Org. Chem. 2013,
78, 5964−5969. (34) Bondensgaard, K.; Petersen, M.; Singh, S. K.; Rajwanshi, V. K.;
Kumar, R.; Wengel, J.; Jacobsen, J. P. Structural Studies of LNA:RNA
Duplexes by NMR: Conformations and Implications for RNase H
Activity. Chem. - Eur. J. 2000, 6, 2687−2695. y
(35) Sanzone, A. P.; El-Sagheer, A. H.; Brown, T.; Tavassoli, A. Assessing the biocompatibility of click-linked DNA in Escherichia coli. Nucleic Acids Res. 2012, 40, 10567−10575. (18) Varizhuk, A. M.; Tsvetkov, V. B.; Tatarinova, O. N.; Kaluzhny, (18) Varizhuk, A. M.; Tsvetkov, V. B.; Tatarinova, O. N.; Kaluzhny,
D. N.; Florentiev, V. L.; Timofeev, E. N.; Shchyolkina, A. K.; Borisova, (36) Havens, M. A.; Hastings, M. L. Splice-switching antisense
oligonucleotides as therapeutic drugs. Nucleic Acids Res. 2016, 44,
6549−6563. D. N.; Florentiev, V. L.; Timofeev, E. N.; Shchyolkina, A. K.; Borisova, D. N.;
o e t ev, V. L.;
o eev,
. N.; S c yo k a,
. K.; Bo sova,
O. F.; Smirnov, I. P.; Grokhovsky, S. L.; Aseychev, A. ■AUTHOR INFORMATION
Corresponding Author Two-Step
Synthesis of a 5′-Azidothymidine Building Block for the Assembly of
Oligonucleotides for Triazole-Forming Ligations. Synlett 2012, 23,
2923−2926. (31) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A
stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regiose-
lective “ligation” of azides and terminal alkynes. Angew. Chem., Int. Ed. 2002, 41, 2596−2599. (32) Tornøe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on
solid phase: [1,2,3]-Triazoles by regiospecific copper(I)-catalyzed 1,3- DOI: 10.1021/acsomega.8b01086
ACS Omega 2018, 3, 6976−6987 6987
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Is there a relation between carpal tunnel syndrome and trigger finger?
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© 2015 El-Hadidi; 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/4.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. 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. Results Some studies hypothesize that surgery for one entity
will cause the other or that the carpal tunnel syndrome is
associated with the appearance of trigger digits as a sepa-
rate entity. We obtained information from 565 patients who had
carpal tunnel syndrome that was treated surgically by
open release. 103 patients were males (18.23%); of those;
the affected side was right 37 (36%), left 20 (19%) and,
bilateral 46 (45%). 462 patients were females (81.77%);
of those; the affected side was right 108 (23%), left 64
(14%), and bilateral 290 (63%). The etiology of both TF and CTS is unknown. It is
possible that there is a common etiology present in
many cases, and the relationship to diabetes may help
shed light on this. The Average age for male patients was (55.8 years),
with range of (22-89 years).The average age for female
patients was (53.4 years), with range of (18 – 91 years). 113 patients were diabetic (20%): of those; 28 (18%)
were males, and 85 (82%) were females. The Average age for male patients was (55.8 years),
with range of (22-89 years).The average age for female
patients was (53.4 years), with range of (18 – 91 years). The goal of this study is to quantify the rate of trigger
fingers after open carpal tunnel release. 113 patients were diabetic (20%): of those; 28 (18%)
were males, and 85 (82%) were females. Is there a relation between carpal tunnel
syndrome and trigger finger? Shaher El-Hadidi From 10th Congress of the Asia-Pacific Federation of Societies of Surgery fo the Hand and the 6th Congress
of Asia-Pacific Federation of Societies of Hand Therapists
Kuala Lumpur, Malaysia. 2-4 October 2014 The diagnosis was made on the basis of a standard
history and physical exam performed by the authors. Introduction Idiopathic carpal tunnel syndrome is the most common
upper limb entrapment neuropathy. CTS was defined by the presence of at least two of the
following: numbness or tingling in the median nerve dis-
tribution, diminished median nerve motor function, or a
positive Tinel’s or Phalen’s test. All CTS patients had
nerve conduction tests (NCTs) and electromyography
(EMG) consistent with CTS. The prevalence in the general population is 2.7%. Trigger digit is also a common clinical disorder, which
is characterized by painful locking of the involved digit. Both disorders are important causes of occupational
absenteeism and disability, and often coexist in the same
hand. Cases of TF were defined by a history and/or presence
of catching, clicking or locking and tenderness over the
A1 pulley. Trigger finger and carpal tunnel syndrome together are
in all probability the most common conditions treated by
a hand surgeon. Previous studies have suggested a signifi-
cant concurrence rate between these conditions, but the
implications of this have not been fully explored. Demographic data was obtained from the patients. El-Hadidi BMC Proceedings 2015, 9(Suppl 3):A65
http://www.biomedcentral.com/1753-6561/9/S3/A65 El-Hadidi BMC Proceedings 2015, 9(Suppl 3):A65
http://www.biomedcentral.com/1753-6561/9/S3/A65 MEETING ABSTRACT Open Access Conclusion CTS and TF commonly occur together at presentation
though the symptoms of one condition will be more
prominent. Trigger finger after open carpal tunnel
release is possible to occur. Our results support a com-
mon local mechanism that may be unrelated to the pre-
sence of diabetes. Published: 19 May 2015
doi:10.1186/1753-6561-9-S3-A65
Cite this article as: El-Hadidi: Is there a relation between carpal tunnel
syndrome and trigger finger? BMC Proceedings 2015 9(Suppl 3):A65. Published: 19 May 2015 Diabetes Mellitus was observed in 11 patients in
whom triggering occurred after open carpal tunnel
release (18.6%). doi:10.1186/1753-6561-9-S3-A65
Cite this article as: El-Hadidi: Is there a relation between carpal tunnel
syndrome and trigger finger? BMC Proceedings 2015 9(Suppl 3):A65. doi:10.1186/1753-6561-9-S3-A65
Cite this article as: El-Hadidi: Is there a relation between carpal tunnel
syndrome and trigger finger? BMC Proceedings 2015 9(Suppl 3):A65. Materials and methods Triggering at presentation occurred in 73 patients
(13%); 19 were males and 54 were females. A sampling of 565 consecutive cases was obtained from
January 2002 to June 2011 using a retrospective and
observational study. These were the patients who were
operated upon for carpal tunnel syndrome by open
release of transverse carpal ligament. All the surgeries
were performed under local anesthesia infiltration in
most cases and under general anesthesia in some cases at
the Jordan University Hospital. The average duration of symptoms pre-op was 10.77
months, with range of 1 month.-72 months. The average
duration between releases in cases of bilateral CTS was
1.61 months., with range of 1-6 months. Triggering after open carpal tunnel release occurred in
59 patients (10.44%); 9 were males, and 50 were females. Jordan University Hospital, Amman, 11942, Jordan Jordan University Hospital, Amman, 11942, Jordan El-Hadidi BMC Proceedings 2015, 9(Suppl 3):A65
http://www.biomedcentral.com/1753-6561/9/S3/A65 Page 2 of 2 Page 2 of 2 Table 1. Trigger fingers after open CTS release and their
distribution
Triggering post CTS release:59
Male:9
Female:50
1-Thumb:15
0
15
2-Index:23
5
18
3-Middle:17
2
15
4-Ring:3
1
2
5-Little:1
1
0 Table 1. Trigger fingers after open CTS release and their
distribution Discussion The etiology of CTS is multi-factorial. Patients with
idiopathic carpal tunnel syndrome are predisposed to
the development of trigger digit. The incidence in var-
ious reports ranges from 0.2% to 22%. However, in most
of these studies, trigger digit was only evaluated at the
time of the first visit to the hospital for carpal tunnel
syndrome. Trigger digit occurring after carpal tunnel release was
examined in the retrospective study by Hombal et al. He
reported an incidence of 22% within one year of carpal
tunnel release. Hayashi et al suggest that carpal tunnel
release surgery is a risk factor for the onset of trigger
digit. Surgery may change the environment inside and near
the carpal tunnel. Biomechanically, the flexor tendons at
the wrist are known to displace anteriorly after division
of the transverse carpal ligament. When this ‘‘bowstring-
ing” occurs, the angle of attack of the flexor tendon
against the A1 pulley increases, which may cause a
greater frictional and compressive force between the
flexor tendons and the A1 pulley. This increased force at the tendon-pulley interface
may lead to trigger digit. Fibrocartilagenoumetaplasia is
known to develop at the site of increased compressive
forces on connective tissues, and has been reported at
the friction surface of the A1 pulley taken from a
patient with trigger digit. Surgery may accelerate the development of trigger
digit when carpal tunnel syndrome is mild to moderate. In severe disease, other factors as yet unknown, such as
hypertrophy of the flexor tenosynovium, may mask the
effect of surgery. Submit your next manuscript to BioMed Central
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Submit your manuscript at
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and take full advantage of: Submit your next manuscript to BioMed Central
and take full advantage of: Mackinnon has observed that after CTS surgery a pos-
sible trigger finger pathology, that had not been present
prior to surgery, can occur. In our study, we found that trigger finger after open
carpal tunnel syndrome occurred in 59 patients
(10.44%), diabetes mellitus was observed in 11 patients
only (18.6%).
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Intrusion Detection in Heterogeneous Networks of Resource-Limited Things
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Journal of Telecommunications and Information Technology
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1. Introduction The terms like computer or network are becoming less clear
as the technology advances. No more than ten years ago,
most of the nodes in the Internet were stationary computers
with a wired connection. On the server side of the network
this is still an accurate depiction at least of the physical
setup, although admittedly more and more inaccurate on the
logical side, as virtualization advances and the additional
cloud layer isolates the servers from their hardware. Detection of compromised nodes is most complicated in
this resource- and energy-limited part of the heterogeneous
network, where the extra load introduced by the detection
mechanisms becomes too large. The tradeoffbetween hav-
ing an insecure network or expending energy and resources
on protective measures could be eliminated by introducing
more powerful nodes dealing with this task. Unfortunately,
providing sufficient network coverage would require many
such nodes, effectively multiplying the system cost beyond
sensible limits. On the client side, the network changed completely. Most
of new devices are wireless. Also, the name “device” is
quite appropriate, as more and more of them do not look
like traditional computers (even if that’s what they essen-
tially are). The trend is not only directed at mobility of
computing, as in case of laptops, smartphones, tablets,
etc., but also towards expanding the computational abili-
ties of other things, leading to ideas such as smart home or
smart city. In this paper a workaround limiting the cost of detection
nodes is proposed by allowing each detector to monitor
multiple locations through mobility. The side effect of this approach is that the network is be-
coming full of devices with at least one of the following
limitations: battery power, meaning that energy conserva-
tion becomes crucial factor, or limited computing power,
due to lowering costs, lowering energy consumption or pre-
venting heating. These limitations, the fact that many of
the devices (especially smart things) are designed by com-
panies with little experience in computing and the thing
status, meaning that users are unlikely to participate in in-
stallation of updates (so either the things will never be up-
dated or will have a fully automated update mechanism,
creating a tempting target for attacks) lead to a rather dif-
ficult situation from the security standpoint. 1. Introduction While most
of the things are seen as not worth attacking, the situation
becomes worse when the entire heterogeneous network is
seen as a single system. Unsafe devices are points of entry The paper starts with a discussion of major threats classes
to such networks of small devices in Section 2. Section 3
provides an overview of the approaches toward securing
such networks. Section 4 presents authors idea of a mo-
bile intrusion detection system (IDS). A short conclusion
is given in Section 5. Intrusion Detection
in Heterogeneous Networks
of Resource-Limited Things
Adam Kozakiewicz, Krzysztof Lasota, and Michał Marks
Research and Academic Computer Network (NASK), Warsaw, Poland Paper Paper Abstract—The paper discusses the threats to networks of
resource-limited things such as wireless sensors and the dif-
ferent mechanisms used to deal with them. A novel approach
to threat detection is proposed. MOTHON is a movement-
assisted threat detection system using mobility to enhance
a global threat assessment and provide a separate physical
secure channel to deliver collected information. to the network, threatening other resources. They can also
be used in orchestrated attacks, e.g. providing multiple con-
sistent but false data streams leading to wrong decisions. With diminishing isolation, security of things becomes
crucial. The paper focuses on the client side of such a heteroge-
neous network – the (logically) local network of things,
using multi-hop ad-hoc connections if transmission range
is too short. The energy and power limitations, specialized
hardware, wireless communication and minimal manual
configuration characterizing most of smart things are also
typical in wireless sensor networks (WSNs), making them
a proto-example of a network of things. Many of the re-
sults of research in WSNs security are therefore almost
directly applicable to other devices. The main difference
is that a single WSN is usually rather homogeneous, while
in case of the Internet of Things (IoT) the devices may be
completely different in both hardware and software. Keywords—client honeypot, Internet of Things, intrusion detec-
tion, wireless sensor network. 3.1. Intrusion Prevention the network), target layer and attack goals (communication
obstruction, data capture, modification or data fabrication). The first layer of defense is provided by protection mea-
sures aimed at preventing successful penetration. In case of
wireless networks of resource-limited devices this layer is
unfortunately not as strong as in wired computer networks. Since the medium is freely accessible, the prevention must
be applied at every point in the network. However, ap-
plication of advanced mechanisms is made difficult by the
limited computing resources and the need to preserve en-
ergy. Still, some steps have been made towards provision
of important information security protections. Attacks in physical layer are hard to prevent, as neither
the electromagnetic medium nor the sensors themselves are
(usually) physically protected. Active jamming attacks are
therefore effective, although rather easy to detect. Passive
sniffing is effective unless encryption is used. Physical at-
tacks on nodes are possible even without any tools (destruc-
tion or theft of nodes). More advanced physical attacks are
dangerous to the network as a whole, because of the virtu-
ally unlimited possibility of tampering with the hardware
and programming (e.g. using JTAG interface). Proper application of cryptographic techniques can provide
privacy, authentication and data integrity. Unfortunately,
software implementations require a lot of operations, low-
ering battery life. Hardware support reduces this impact
and is available in radio modules implementing the IEEE
802.15.4 standard [4]. An unfortunate limitation of this so-
lution is the use of a single symmetric key. A lot of work to-
wards introducing cryptographic protections to higher lay-
ers and enabling efficient and secure key distribution has
been performed in recent years, including e.g. TinySec [5],
MiniSec [6], ContikiSec [7], ZigBee, LEAP/LEAP+ [8]. Attacks on the data layer are more limited, usually focusing
on flooding the medium with messages, or using standard
violations such as long frames to cause collisions. Attacks in the network layer are potentially very effective,
but made more difficult by the fact that routing in such
networks is not part of the standard and may be done us-
ing a variety of algorithms. Attacks in this layer usually
focus on affecting the routing decisions in order to either
obstruct communication as such, or to maximize the effec-
tiveness of the limited number of malicious nodes in the
network. 3.1. Intrusion Prevention In the first case, providing false information in
the path building phase or sending many unnecessary path
queries are simple and quite effective sensor-class attacks,
causing additional unnecessary communication and com-
putation by network nodes, draining batteries. The second
group of attacks uses advertising great connection quality
(or other methods) in order to direct as much of the net-
work’s traffic as possible through a malicious node. Then,
after routing is established, the node can be used to monitor
the traffic (sniffing) or obstruct it, either by blackholing the
communication or by selective forwarding increasing loss
frequency. Another protective measure, most effective not in preven-
tion of attacks, but in network protection against already
malicious nodes, is trust management [9]. Due to limited
memory in network nodes the most practical approach is
reputation based. An example of its application to sensor
networks can be found in [10]. 3.2. Intrusion Detection Once a successful attack has been performed, the network
might be operating with one or more malicious nodes. This
constant threat to information security is often more danger-
ous than the initial attack. Detection of misbehaving nodes
allows proper mitigation techniques to be applied, including
blacklisting the node and routing around it or even phys-
ically removing it from the network (if possible). Many
methods have been proposed to detect malicious nodes. Most of them have a common problem – secure delivery
of detection information to the base station or secure prop-
agation of information between non-malicious nodes. If
a single malicious node is detected, it might not be the only
one. If another one is in path of the warning message, it
can easily render the detection system powerless. Therefore
IDS alerts require either a separate secondary channel for
propagation, or effective protection if the primary channel
is used (separate path, encryption, etc). Finally, attacks in the higher layers (transport – applica-
tion) are also possible and potentially most useful in case
of targeted attacks. The range of possibilities is too wide
to describe here. As simple examples consider attacks on
time synchronization algorithms, node location or key dis-
tribution. An attack in this layer, conducted with a deep
understanding of the goals and implementation of the net-
work, can turn the network into an extremely dangerous
misinformation tool. 2. Threats to the Network There are many possible modes of attack against a sensor-
like network of things [1]–[3]. In general, they can be
grouped depending on several factors, such as the activity
of the attacker (active or passive), computing power (sensor
class or laptop class), location (logically inside or outside 10 Intrusion Detection in Heterogeneous Networks of Resource-Limited Things Intrusion Detection in Heterogeneous Networks of Resource-Limited Things 4.1. MOTHON System Overview The authors have proposed a novel approach to threat mon-
itoring in WSN. In presented threat detection system one or
several mobile sensors implementing threat detection func-
tionality are forced to move to desired directions. As it is
presented in Fig. 1, due to the ability to change the location
of a sensor node, information about security events can be
passed directly to the network sink (IIIa – after completion
of all tasks, IIIb – after threat detection) or indirectly via
other nodes from another area of the monitored network. All of the previously described approaches are generally
passive – they either base detection on received traffic only
or (in case of the server honeypot) respond to messages,
but never initiate communication as part of the detection
activity. An active alternative is a client honeypot – a node,
which sends messages in order to verify whether they are
properly forwarded to the base station or other target. The
actual detection is performed at the receiving node, where
any changes to the message or variation from the normal
loss rate can be easily identified. The mechanism requires
that both the sender and the receiver agree on the nature of
the testing message or sequence of messages. This can be
done through predefinition or by using a secondary chan-
nel for transmission of this information. Note that prede-
fined messages are easier to learn and avoid at malicious
nodes. II
I IIIa
IIIb
mobile sensor
sensor
sink
data synchronization
route
threat detection, route modification
Fig. 1. The concept of movement-assisted threat monitoring: I –
task order phase, II – performing actions phase, III – reporting
phase (a – after completion of all tasks, b – after threat detection). II
I IIIa
IIIb
mobile sensor
sensor
sink
data synchronization
route
threat detection, route modification Fig. 1. The concept of movement-assisted threat monitoring: I –
task order phase, II – performing actions phase, III – reporting
phase (a – after completion of all tasks, b – after threat detection). 3. Security Measures Due to the limited computing power of nodes and their
need to conserve energy, any security measure that requires
a lot of computing activity on part of the network nodes
is a mixed blessing. Another problem is the broadcast-
based, self-organizing dynamic nature of such networks –
even if not mobile, they must reorganize to allow for node
malfunctions, etc. There are no natural policy enforcement
points apart from the base station – any node in the network
may be routed around. These problems result in a reduced
choice of security solutions for networks of things. The simplest form of detection of malicious behavior is the
watchdog mechanism [11], using the shared medium aspect
of wireless networks. The node sending a message can
monitor the medium to verify whether the receiving node
forwarded it correctly. There are many variations to this
mechanism in the literature. A different, more sophisticated
approach is using more advanced, rule-based intrusion de-
tection systems [12], capable of detecting many different
kinds of attacks. Both approaches require modification of 11 Adam Kozakiewicz, Krzysztof Lasota, and Michał Marks Finally, threat monitoring can be successfully supported by
controlled geographic migration of sensors that have loco-
motion. A mobility of sensors is leveraged recently for
many WSN applications. Using mobile platforms to assist
sensor placement in a working space can significantly en-
hance the capability to monitor the data and detect attacks. all or selected nodes. A standard watchdog mechanism
only detects malfunctioning neighbors, so it must be active
in most of the nodes to cover the entire network. An IDS
may be somewhat effective even if only implemented at the
base station, but delivers less information. One more approach, often used in classical networks, is
a server honeypot – a service existing only as a target for
attacks. This approach might be applicable to WSNs by
emulating a node on a more powerful device, waiting for
messages modifying its state in illegal ways. This detec-
tion method would be effective against previously unknown
types of attacks as long as the identity of the honeypot node
remains secret. 4. Movement-assisted Threat Monitoring
in WSN Taking into account limited resources of sensors, collec-
tion and analysis of data concerned with network security
are usually performed in a periodic manner and carried
out by selected devices implementing threat monitoring ca-
pabilities. However, in general, it is possible to extend
the functionality of all nodes and to implement permanent
monitoring. Regardless of the selected monitoring scheme,
a common objective of all security systems is establishing
a safe and reliable communication channel for exchanging
security information i.e. reporting, alerting, control traffic,
etc. between nodes. Therefore the communication can be
organized in several ways: The MOTHON (MObile THreat mONitoring for WSN)
system implements the concept depicted in Fig. 1. It is
composed of three components: a mobile platform (MP),
threat monitoring sensor (TMS) and management station
(MS) responsible for controlling of MP and TMSs (see
Fig. 2). It is assumed that the mobile platform can carry
one or several TMS sensors. All these sensors can be
placed in any locations in a workspace. The MOTHON operates in three stages. The management
station initiates the first stage. The aim of the this stage
is to synchronize and gather data about a given network
(topology, characteristics of nodes, etc.) and define moni-
toring mode and plan. Passive and active modes of threat
detection are considered. Next, all data related to deci-
sions done by MS are transferred to a MP and TMS sensor
(or sensors) dedicated to threat monitoring, and the system
switches to the second stage. • by utilizing the transmission channels already set up
to propagate data in the system, • by creating channels using disjoint logical connec-
tions within existing networks, Threat monitoring sensor is carried to the desired destina-
tion by mobile platform (MP). After placing at the tar-
get location TMS starts to perform assigned tasks con-
cerning the threats detection. The third stage relates to
re-synchronization of information between TMS and MS
which can occur in two cases – after completion of all • by adding extra nodes to create a separate sensor
network which shares the same transmission medium, • by equipping sensor nodes with additional hardware
modules (Wi-Fi, GSM, etc.)
that can be used to
establish an additional communication channel. 4. Movement-assisted Threat Monitoring
in WSN 12 Intrusion Detection in Heterogeneous Networks of Resource-Limited Things Intrusion Detection in Heterogeneous Networks of Resource-Limited Things USB
USB
USB
USB
TelosB
TMSs
Raspberry PI RJ45
RJ45
Lego Mindstorms NTX 2.0
Management
application [APP]
Mobile platform [MP]
Management station [MS]
(PC/laptop)
Fig. 2. MOTHON prototype architecture. • using existing communication channel via other
nodes from another area of the monitored network. In both cases MP can leave the TMS (with default network
software) to avoid the occurrence of “temporary” nodes
in the system. Moreover one mobile platform can carry
multiple TMS sensors and place them in different locations
in workspace. 5. Conclusion Starting with a review of threats and security measures
applicable to wireless networks of resource-limited things,
a new approach, introducing mobility as a way of over-
coming the limitations of existing methods has been pre-
sented. 4.3. MOTHON Prototype Architecture The prototype system composed of three elements is pre-
sented in Fig. 2. The management application provided
by MS is a console tool is written in C++. TMSs Mobile platform consists of two hardware components:
Lego Mindstorms NTX 2.0 and Raspberry Pi single board
microcomputer. Moreover it is expected, that MP will
be equipped with GPS module or other localization sys-
tem [13], [14]. Fulfilling all the tasks assigned by management application
requires from this platform significantly greater capabilities
in terms of processing power and energy resources. The
key software components of MP are: Fig. 2. MOTHON prototype architecture. • Control module – the main module, responsible for
communication with all other modules and creating
the logic of solution based on information obtained
from the management station. tasks or after threat detection. TMS transfers collected in-
formation, e.g. detected threats, additional statistics about
traversed route, etc. to MS. It can be implemented in two
ways: • Mobility module – responsible for motion trajectory
planning movement and speed calculation. • all data gathered by TMS are stored in MP, which
transfers this data to MS directly – just after threat
detection or after completion of all tasks, • Threat analysis module – used for data gathered
from TMSs modules analysis and threat detection. • Threat analysis module – used for data gathered
from TMSs modules analysis and threat detection. TMS is implemented over TelosB platform using Con-
tiki OS. Eventually, to complete the system, an automation
process enabling wireless communication between MP an
MS must be added. • using existing communication channel via other
nodes from another area of the monitored network. 4.2. Detection Methods MOTHON can employ either active or passive methods for
threat detection. Data analyses can be carried out either
on-line by the TMS and mobile platform, or post factum
by the mobile platform and management station. Mobility of a threat detection sensor should improve over-
all security state of monitored networks without any need
to perform their reconfiguration or upgrade. The approach
can be used in existing networks without any modifications
to installed devices. The implementation details, such as
means of mobility, depend on the target network – obvi-
ously a different solution is appropriate inside a building
than in case of a network of oceanic drones. Passive methods, which are based on analysis of informa-
tion received from neighbors (IDS) or data sniffed from
a shared medium (watchdog IDS), can keep a copy of the
observed traffic for further analysis. This is not effective
approach in case of threat monitoring with static nodes, but
can be very valuable in case of mobile platform returning
to sink from time to time. Moreover, simultaneous moni-
toring of the communication channel from several locations
in the workspace can ease analysis by allowing detection
of hidden and exposed nodes problems. Various methods of threat detection in MOTHON are cur-
rently under development. In future work authors plan to
conduct experiments in testbed network to show the effec-
tiveness of detection against different kinds of attacks. In contrast to passive methods, active solutions are not lim-
ited only to verification of individual sensor actions (cor-
rect operation of protocols, transmitted information, etc.). Active methods provide tools for verification of the network
operation as a whole, e.g. verification the service packet for-
warding over the network to the sink by sending a specific
content, at the specific time and from specific place. [1] H. K. Kalita and A. Kar, “Wireless sensor networks security analy-
sis”, Int. J. of Next Gener. Netw., vol. 1, no. 1, pp. 1–9, 2009.
[2] Z. S. Bojkovic, B. M. Bakmaz, and M. R. Bakmaz, “Security issues
in wireless sensor networks”, NAUN Int. J. Commun., vol. 2, no. 1,
pp. 106–115, 2008. References 4, pp. 500–528, 2006. [9] A. Felkner, “How the Role-based trust management can be applied to
wireless sensor networks”, J. Telecommun. Inform. Technol., no. 4,
pp. 70–77, 2012. [10] G. Saurabh, L. K. Balzano, and M. B. Srivastava, “Reputation-based
framework for high integrity sensor networks”, ACM Trans. on Sen-
sor Netw., vol 4, no. 3, 2008. [11] F. Hu, J. Ziobro, J. Tillett, and N. K. Sharma, “Secure wireless
sensor networks: problems and solutions”, J. Syst., Cybernet. and
Inform., vol. 1, no. 4, pp. 90–100, 2003. [12] G. Huo, X. Wang, “DIDS: A dynamic model of intrusion detection
system in wireless sensor networks”, in Proc. IEEE Int. Conf. on
Inform. Autom. ICIA 2008, 2008, ZhangJiaJie, China, pp. 374–378. E-mail: krzysztof.lasota@nask.pl
Research and Academic Computer Network (NASK)
Wawozowa st 18
02-796 Warsaw, Poland [13] M. Marks, E. Niewiadomska-Szynkiewicz, and J. Kolodziej, “An
integrated software framework for localization in wireless sensor
network”, Comput. and Inform., vol. 33, no. 2, pp. 369–386,
2014. [14] M. Marks, E. Niewiadomska-Szynkiewicz, and J. Kolodziej, “High
performance wireless sensor network localization system”, Int. J. Ad
Hoc and Ubiquitous Comput., vol. 17, no. 32, pp. 122–133, 2014. Michał Marks received M.Sc. (2007) in Computer Science
and Ph.D. (2015) in Automa-
tion and Robotics from the War-
saw University of Technology. Since 2007 with Research and
Academic Computer Network
(NASK). The author and co-
author of over 30 journal and
conference papers. His research
area focuses on wireless sensor
networks, global optimization, distributed computation in
CPU and GPU clusters, decision support and machine
learning. Michał Marks received M.Sc. (2007) in Computer Science
and Ph.D. (2015) in Automa-
tion and Robotics from the War-
saw University of Technology. Since 2007 with Research and
Academic Computer Network
(NASK). The author and co-
author of over 30 journal and
conference papers. His research
area focuses on wireless sensor
networks, global optimization, distributed computation in
CPU and GPU clusters, decision support and machine
learning. E-mail: michal.marks@nask.pl
Research and Academic Computer Network (NASK)
Wawozowa st 18
02-796 Warsaw, Poland Adam
Kozakiewicz
got
his
M.Sc. in Information Technol-
ogy and Ph.D. in Telecommu-
nications at the Faculty of Elec-
tronics and Information Tech-
nology of Warsaw University
of Technology (WUT), Poland. References [1] H. K. Kalita and A. Kar, “Wireless sensor networks security analy-
sis”, Int. J. of Next Gener. Netw., vol. 1, no. 1, pp. 1–9, 2009. [2] Z. S. Bojkovic, B. M. Bakmaz, and M. R. Bakmaz, “Security issues
in wireless sensor networks”, NAUN Int. J. Commun., vol. 2, no. 1,
pp. 106–115, 2008. 13 Adam Kozakiewicz, Krzysztof Lasota, and Michał Marks tion, optimization methods and network traffic modelin
and control. E-mail: adam.kozakiewicz@nask.pl
Research and Academic Computer Network (NASK)
Wawozowa st 18
02-796 Warsaw, Poland tion, optimization methods and network traffic modeling
and control. [3] I. Butun, S. D. Morgera, and R. Sankar, “A survey of intrusion detec-
tion systems in wireless sensor networks”, IEEE Commun. Surveys
& Tutorials, vol. 16, no. 1, pp. 266–282, 2014. E-mail: adam.kozakiewicz@nask.pl
Research and Academic Computer Network (NASK)
Wawozowa st 18
02-796 Warsaw, Poland [4] IEEE standard for part 15.4: IEEE Std. 802.15.4, IEEE, New York,
Oct. 2011. [5] C. Karlof, N. Sastry, and D. Wagner, “TinySec: A link layer security
architecture for wireless sensor networks”, in Proc. 2nd ACM Conf. on Embedded Netw. Sensor Syst. SenSys 2004, Baltimore, Maryland,
USA, 2004, pp. 162–175. [6] M. Luk, G. Mezzour, A. Perrig, and V. Gligor, “MiniSec: a secure
sensor network communication architecture”, in Proc. 6th Int. Conf. on Inform. Process. in Sensor Netw. IPSN’07, Cambridge, MA, USA,
2007. Krzysztof Lasota works as Re-
search Associate at Network
and Information Security Meth-
ods Team in the Research Divi-
sion of NASK. He received his
B.Sc. in Telecommunications
(2010) and M.Sc. in Telecom-
munications (2011) from War-
saw University of Technology,
Faculty of Electronics and In-
formation Technology and is
currently a Ph.D. student there. He participated in security-
related projects at NASK, including FISHA, HoneySpider
Network and Secure workstation for special applications. Currently he participates in the project “The system of
secure IP communication provision for the power system
management”. Furthermore, his research aims at devel-
oping new methods for threat detection in wireless sensor
networks. [7] L. Casado and P. Tsigas, “ContikiSec: A secure network layer for
wireless sensor networks under the Contiki operating system”, in
Proc. 14th Nordic Conf. on Secure IT Syst.: Identity and Privacy in
the Internet Age NordSec 2009, Oslo, Norway, 2009, pp. 133–147. [8] S. Zhu, S. Setia, and S. Jajodia, “LEAP+: Efficient security mech-
anisms for large-scale distributed sensor networks”, ACM Trans. on
Sensor Netw., vol. 2, no. References Currently he works at NASK
as Assistant Professor and Man-
ager of the Network and Infor-
mation Security Methods Team,
also as part-time Assistant Professor at the Institute of
Control and Computation Engineering at the WUT. His
main scientific interests include security of information
systems (especially industrial networks), parallel computa- E-mail: michal.marks@nask.pl
Research and Academic Computer Network (NASK)
Wawozowa st 18
02-796 Warsaw, Poland 14
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Evolution of NMDA receptor cytoplasmic interaction domains: implications for organisation of synaptic signalling complexes
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BioMed Central BioMed Central BioMed Central Research article Address: 1Genes to Cognition Program, Wellcome Trust Sanger Institute, Cambridge, UK and 2Institute for Science and Technology in Medicine,
School of Medicine Keele University Staffordshire UK Address: 1Genes to Cognition Program, Wellcome Trust Sanger Institute, Cambridge, UK and 2Institute for Science and Technology in Medicine,
School of Medicine, Keele University, Staffordshire, UK Email: Tomás J Ryan - tr1@sanger.ac.uk; Richard D Emes - r.d.emes@hfac.keele.ac.uk; Seth GN Grant* - sg3@sanger.ac.uk;
Noboru H Komiyama - nhk@sanger ac uk Email: Tomás J Ryan - tr1@sanger.ac.uk; Richard D Emes - r.d.emes@hfac.keele.ac.uk; Seth GN Grant* - sg3@sanger.ac.uk;
Noboru H Komiyama - nhk@sanger.ac.uk Email: Tomás J Ryan - tr1@sanger.ac.uk; Richard D Emes - r.d.emes@hfac.keele.ac.uk; Seth GN Grant* - sg3@san
Noboru H Komiyama - nhk@sanger.ac.uk * Corresponding author †Equal contributors * Corresponding author †Equal contributors Received: 17 August 2007
Accepted: 15 January 2008 This article is available from: http://www.biomedcentral.com/1471-2202/9/6 © 2008 Ryan 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. Open
Research article
Evolution of NMDA receptor cytoplasmic interaction domains:
implications for organisation of synaptic signalling complexes
Tomás J Ryan†1, Richard D Emes†1,2, Seth GN Grant*1 and
Noboru H Komiyama1 Open Access Background Knockout (KO) of NR2A results in a deficiency in
both spatial learning and LTP, confirming its essential
function in the hippocampus [13]. NR2B shows the most
severe phenotype of neonatal death at P0 due to lack of
suckling response as well as abnormal brainstem trigemi-
nal nucleus formation, showing its importance during
development of the forebrain [14]. NR2C KO causes a
decrease in NMDA receptor mediated currents in the gran-
ular cells of the cerebellum, and NR2A/NR2C double KOs
result in impaired motor coordination [15,16]. Thus it is
clear that since NR2 gene duplication each paralogue
acquired discrete physiological functions and discrete
developmental properties, both spatially and temporally. NR2A and NR2C are exclusively postnatal. Phenotypes
observed after in vivo mutation of each NR2 subunit in
mice reflect the brain regions they are expressed in [10-
12]. Knockout (KO) of NR2A results in a deficiency in
both spatial learning and LTP, confirming its essential
function in the hippocampus [13]. NR2B shows the most
severe phenotype of neonatal death at P0 due to lack of
suckling response as well as abnormal brainstem trigemi-
nal nucleus formation, showing its importance during
development of the forebrain [14]. NR2C KO causes a
decrease in NMDA receptor mediated currents in the gran-
ular cells of the cerebellum, and NR2A/NR2C double KOs
result in impaired motor coordination [15,16]. Thus it is
clear that since NR2 gene duplication each paralogue
acquired discrete physiological functions and discrete
developmental properties, both spatially and temporally. Vertebrates contain four NR2 subunits with sequence sim-
ilarity that suggests they are paralogues. In contrast a sin-
gle NR2 orthologue is detectable in the invertebrate
lineage, supporting the view that the four NR2 subunits
seen in vertebrates are paralogues and arose from gene
duplication events. Each NR2 paralogue has distinct spa-
tial and temporal expression patterns [10,11]. NR2A and
NR2B are expressed throughout the forebrain, including
the hippocampus. NR2C is restricted to the cerebellum
and NR2D is found predominantly in the midbrain. Developmentally, NR2B is expressed both embryonically
and postnatally. NR2D is primarily embryonic while The object of interest here is the intracellular 'tail' of the
NR2 subunits, which lies in the C-terminal domain. Our
impetus arises from its functional significance, originally
demonstrated in knockin mouse models where the C-ter-
mini of NR2A, NR2B, and NR2C were deleted [17]. Background g
One of the most striking evolutionary adaptations of
mammals is their capacity for learning and memory [1]. Such higher cognitive abilities are generally attributed to
increased brain size and elaborations of cortical structure
[2]. However the contribution that evolution of molecular
complexity of the synapse has made to cognitive proper-
ties of higher organisms has yet to be fully assessed [3]. The N-methyl-D-aspartate (NMDA) receptor is a class of
glutamate gated transmembrane ion channel that func-
tions at the postsynaptic membrane of excitatory synapses
[4]. When opened, Ca2+ influx through the NMDA recep-
tor into the postsynaptic cell activates second messengers
responsible for long lasting synaptic plasticity. Genetic
and pharmacological studies have shown that NMDA
receptors are necessary for normal spatial learning and its
physiological correlate of long term potentiation (LTP)
[4,5]. In addition, NMDA receptors have been shown to
be essential for activity dependant brain development, in
particular in the somatosensory and visual cortices in
adults [6]. Model of N
Figure 1 Model of NR2 Subunit
Figure 1
Model of NR2 Subunit. 1.A: Schematic of vertebrate
NR2A or NR2B subunits. Note the long intracellular C-ter-
minal domain and relative positioning of particular interacting
proteins. PSD-95 is a scaffolding protein, while CaMKII and
P13K are kinases that phosphorylate the NMDA receptor. 1.B: Schematic of invertebrate NR2. Note the significantly
shorter intracellular C-terminus. Each NMDA receptor is a tetramer formed by two het-
erodimers of an obligatory NR1 subunit and one of four
possible NR2 subunits (NR2A, NR2B, NR2C, NR2D) [4]. Each subunit consists of an extracellular N-terminal
domain with three transmembrane regions (M1, M3, and
M4) and one re-entrant loop (M2) that form the channel
pore, and an intracellular C-terminal domain (Fig. 1A). The N-terminal domains contain the ligand binding site,
while the C-terminal domain interacts with intracellular
signalling messengers including MAGUK (membrane
associated guanylate kinase binding) proteins via a PDZ
binding domain at its C-terminus. Binary protein-protein
interaction studies have identified a range of scaffold pro-
teins and enzymes that bind to the C-terminal domain
[7], and proteomic studies have found that these proteins
are assembled into 1–2 MDa size macromolecular signal-
ling complexes known as NRC (NMDA Receptor Com-
plexes) or MASC (MAGUK Associated Signalling
Complexes) (Figure 1) [8,9]. NR2A and NR2C are exclusively postnatal. Phenotypes
observed after in vivo mutation of each NR2 subunit in
mice reflect the brain regions they are expressed in [10-
12]. Abstract Background: Glutamate gated postsynaptic receptors in the central nervous system (CNS) are
essential for environmentally stimulated behaviours including learning and memory in both
invertebrates and vertebrates. Though their genetics, biochemistry, physiology, and role in
behaviour have been intensely studied in vitro and in vivo, their molecular evolution and structural
aspects remain poorly understood. To understand how these receptors have evolved different
physiological requirements we have investigated the molecular evolution of glutamate gated
receptors and ion channels, in particular the N-methyl-D-aspartate (NMDA) receptor, which is
essential for higher cognitive function. Studies of rodent NMDA receptors show that the C-
terminal intracellular domain forms a signalling complex with enzymes and scaffold proteins, which
is important for neuronal and behavioural plasticity Results: The vertebrate NMDA receptor was found to have subunits with C-terminal domains up
to 500 amino acids longer than invertebrates. This extension was specific to the NR2 subunit and
occurred before the duplication and subsequent divergence of NR2 in the vertebrate lineage. The
shorter invertebrate C-terminus lacked vertebrate protein interaction motifs involved with
forming a signaling complex although the terminal PDZ interaction domain was conserved. The
vertebrate NR2 C-terminal domain was predicted to be intrinsically disordered but with a
conserved secondary structure. Conclusion: We highlight an evolutionary adaptation specific to vertebrate NMDA receptor NR2
subunits. Using in silico methods we find that evolution has shaped the NMDA receptor C-terminus
into an unstructured but modular intracellular domain that parallels the expansion in complexity of
an NMDA receptor signalling complex in the vertebrate lineage. We propose the NR2 C-terminus
has evolved to be a natively unstructured yet flexible hub organising postsynaptic signalling. The
evolution of the NR2 C-terminus and its associated signalling complex may contribute to species
differences in behaviour and in particular cognitive function. Page 1 of 14
(page number not for citation purposes) BMC Neuroscience 2008, 9:6 http://www.biomedcentral.com/1471-2202/9/6 Model of NR2 Subunit
Figure 1
Model of NR2 Subunit. 1.A: Schematic of vertebrate
NR2A or NR2B subunits. Note the long intracellular C-ter-
minal domain and relative positioning of particular interacting
proteins. PSD-95 is a scaffolding protein, while CaMKII and
P13K are kinases that phosphorylate the NMDA receptor. 1.B: Schematic of invertebrate NR2. Note the significantly
shorter intracellular C-terminus. Page 2 of 14
(page number not for citation purposes) Background The C-
terminal deletions resulted in behavioural and electro-
physiological phenotypes identical to the null mutants,
but without interfering with NMDA receptor channel for-
mation and gating. Thus the intracellular signalling capac-
ity of NR2 is indispensable for activity dependant NMDA
receptor function. Interestingly, recent research suggests
that differences in the intracellular signalling capacities of Page 2 of 14
(page number not for citation purposes) Page 2 of 14
(page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/6 http://www.biomedcentral.com/1471-2202/9/6 BMC Neuroscience 2008, 9:6 http://www.biomedcentral.com/1471-2202/9/6 http://www.biomedcentral.com/1471-2202/9/6 NR2 subunits may account for their contrasting physio-
logical roles in induction of synaptic plasticity (LTP) and
in neurotoxicity [18,19]. These studies have been strongly
disputed due to lack of subunit specificity of NMDA
receptor pharmacological antagonists [20,21]. Neverthe-
less, NR2 subunit specific interaction and phosphoryla-
tion sites have been identified and imply that NR2
subunit specific properties may me due to C-terminal var-
iation [22]. The profound phenotypic effect of the C-ter-
minal deletions may well be due to protein-protein
interactions between the C-terminal domain and postsyn-
aptic signalling molecules. We examined the intracellular
domain of the NR2 sub-units using in silico methods and
infer striking evolutionary adaptations with unique struc-
tural properties. http://www.biomedcentral.com/1471-2202/9/6 BMC Neuroscience 2008, 9:6 domain surrounding an internal cassette (Fig. 3). The
most similar NR2 paralogues in pairwise comparisons are
NR2A and NR2B, which show 29% identity in their C-ter-
minal domains. Although this is a significantly high sim-
ilarity, it is relatively low in comparison to the N-terminal
regions. When compared on a Dotplot it is clear that their
C-terminal domains differ profoundly relative to their N-
terminal domains (Fig. 4). This indicates that since dupli-
cation in the vertebrate lineage, the NR2 subunits have
retained common ancestral structure in their extracellular
and tramsmembrane domains but diversified in their
intracellular domains, suggesting specific functional
adaptations in intracellular signalling properties for each
paralogue. troglydytes and H. sapiens) (Fig. 5). From this alignment it
was strikingly apparent that vertebrate C-terminal
domains were substantially longer than those of inverte-
brates. It was also clear that, although there is much diver-
sity between NR2 paralogues within a species within the
central portion of the NR2 C-terminus, there were many
regions of conservation between species for each NR2 sub-
unit (Fig. 5). Strikingly, the few amino acids that are con-
served between the four paralogues lie in short 'islands' at
the very beginning of the C-terminus (at G-I/M-YSC motif
at the beginning of the terminal exon) and at the very end
where the PDZ binding domain lies. This suggests that
these regions act as necessary 'sockets', one for the trans-
membrane domain of the NMDA receptor upstream, and
the other for interacting with PDZ containing proteins
downstream [23]. In order to investigate the evolution of the ancestral C-ter-
minal domain of NR2, alignments were performed with
sequences of various species representative of metazoan
clades including the NR2 protein sequences of inverte-
brates (C. elegans, D. melanogaster,); and the NR2A protein
sequences of non-mammalian chordates (D. rerio, G. gal-
lus, X. Tropicalias); and mammals (M. domestica, C. famil-
iaris, B. Taurus, R. norvegicus, M. musculus, M. mulata, P. When the lengths of the NR2 C-terminal regions (defined
by the amino acid sequence starting downstream of the
last predicted transmembrane amino acid) were com-
pared across the above species, as well as S. mansoni, A. aegypti, G. aculeatus, C. porcellus, and O. latipes, it was
noted that the NR2 of all sequenced invertebrate species NR2 Subunit C-terminal (intracellular) Domain Alignment (Mouse)
Figure 3
NR2 Subunit C-terminal (intracellular) Domain Alignment (Mouse). Molecular evolution of NR2 We assessed the similarity of the mouse NR2 subunit
amino acid sequences by multiple sequence alignment. It
was clear that all four NR2 paralogues show a very high
degree of similarity in their extracellular and transmem-
brane domains, most significantly NR2A and NR2B with
69% identity (Fig. 2). Such a high degree of conservation
implies that they are indeed of common origin, and share
the same transmembrane topology. Therefore the NR2
paralogues have remained under purifying selection since
duplication of the ancestral NR2 and are likely to retain
common function in their extracellular and transmem-
brane domains. In contrast to these domains, the C-terminal intracellular
domains of the NR2 paralogues have diverged signifi-
cantly, with only 9 residues conserved between the four
paralogues, primarily at the extremities of the C-terminal R2 Subunit Extracellular and Transmembrane Domain Alignment (Mouse)
igure 2
R2 Subunit Extracellular and Transmembrane Domain Alignment (Mouse). 100% consensus between sequence
hown by black shading. Transmembrane domains (M1, M3, and M4) and re-entry loop (M2) are overlined. M1
M2
M3
M4 NR2 Subunit Extracellular and Transmembrane Domain Alignment (Mouse)
Figure 2
NR2 Subunit Extracellular and Transmembrane Domain Alignment (Mouse). 100% consensus between sequences
shown by black shading. Transmembrane domains (M1, M3, and M4) and re-entry loop (M2) are overlined. Page 3 of 14
(page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/6 http://www.biomedcentral.com/1471-2202/9/6 100% consensus between sequences shown by
black shading. PDZ binding domain indicated by red arrow. PDZ Binding Domain (intracellular) Domain Alignment (Mouse)
nal (intracellular) Domain Alignment (Mouse). 100% consensus between sequences shown by
ng domain indicated by red arrow. NR2 Subunit C terminal (intracellular) Domain Alignment (Mouse)
Figure 3
NR2 Subunit C-terminal (intracellular) Domain Alignment (Mouse). 100% consensus between sequences shown by
black shading. PDZ binding domain indicated by red arrow. Page 4 of 14
(page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/6 Page 5 of 14
(page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/6 BMC Neuroscience 2008, 9:6 Dotplot of NR2A and NR2B (mouse)
Figure 4
Dotplot of NR2A and NR2B (mouse). N-terminal extra-
cellular domain and membrane spanning domain show high
similarity. The C-terminal tail region has very low sequence
similarity. C-terminal domain begins at AA position 866. terminal domain was run in BLASTn and tBLASTn
searches against all available genomic sequences, ESTs,
and non-redundant databases. In addition the amino acid
sequence of the four mouse NR2 C-terminal domains
were used to search the Pfam database for any domain
that might elucidate its origin or function [25]. No hits of
significance were found in either case. Exhaustive searches
of mouse NR2 C-termini with invertebrate genomes could
not identify any homologous gene or similar sequence. Since BLAST searches of invertebrate genomes using the
mouse C-terminal NR2 protein sequence failed to find a
similar region, we inspected the NR2 invertebrate
genomic locus more closely. We had observed that the
NR2 C-terminus was noted to have conserved exonic
structure throughout vertebrates, with the vast majority of
the NR2 C-termini being encoded for in the terminal
exon. This conservation of genomic structure was not seen
in invertebrates, where the genomic sequence of the NR2
C-terminus is interrupted by introns in D. melanogaster, C. elegans, and S. mansoni. In the case of D. melanogaster there
are two introns seperating the NR2 C-terminal domain
nucleotide sequence of 114 and 2857 nucleotides in
length, respectively (Fig. 7). When the nucleotide
sequence of mouse NR2B terminal exon was aligned with
the 2857 nucleotide intron of D. melanogaster NR2 C-ter-
mini, 41.6% identity was found by conducting a local
Smith-Waterman alignment (additional file 1). Although
this is not a strong result at the nucleotide level it may
imply that the sequences have a common origin. A similar
result (42.1%) was obtained using the corresponding NR2
C-terminal intronic sequence of S. mansoni (data not
shown). Dotplot of
Figure 4 Dotplot of
Figure 4 p
(
)
g
Dotplot of NR2A and NR2B (mouse). N-terminal extra-
cellular domain and membrane spanning domain show high
similarity. The C-terminal tail region has very low sequence
similarity. C-terminal domain begins at AA position 866. p
(
)
g
Dotplot of NR2A and NR2B (mouse). N-terminal extra-
cellular domain and membrane spanning domain show high
similarity. The C-terminal tail region has very low sequence
similarity. C-terminal domain begins at AA position 866. are substantially shorter by up to >400 amino acids than
vertebrate NR2A/B and >200 amino acids for NR2C/D,
highlighting a stark contrast in the intracellular domain of
NMDA receptors between vertebrates and invertebrates
(Fig. 1B, Fig. 6A). The invertebrate NR2 C-terminal
domain ranges from 89 residues in C. elegans NR2 to 133
residues in D. melanogaster NR2, whereas the vertebrates
NR2A/B 'tails' range from 581 in M. mulatta NR2A to 754
residues in Oryzias latipes (Medaka) NR2B. Within the ver-
tebrate clade the length of NR2 C-termini does not signif-
icantly vary with the exception of NR2B in Teleosts, which
is predicted from genomic sequence to carry twenty inser-
tions. Teleosts have two copies of each NR subunit, most
likely as a consequence of the whole genome duplication
that occurred in that lineage [24], and which may have
allowed for relaxation of constraint on the second NR2B
copy. Structural aspects of NR2 C-terminal domain evolution Structural aspects of NR2 C-terminal domain evolution
Since the C-terminal domain of NR2 functions by attach-
ing to, and organising intracellular adaptor and signalling
molecules it is possible that the diversity between NR2
paralogues is manifest at the level of protein structure. The
amino acid composition of each subunit was calculated
and it was noted that, when compared to all other constit-
uent amino acids, NR2A and NR2B were enriched for ser-
ine, 13.2% and 12.2%, respectively, while NR2C and
NR2D were substantially enriched for proline at 14.9%
and 21.8%, respectively. It has been reported that an
amino acid composition of >6.91% serine or >12.07%
proline is indicative of intrinsically disordered proteins
[26]. We therefore used a graphic web server FoldIndex to
examine the four mouse NR2 paralogues to further predict
protein folding propensity (Fig. 8, additional file 2) [27]. A large portion of N-terminal extracellular part shows a
relatively high probability for folding, four peaks of very
high probability correspond to the four transmembrane
segments of the NR2s. In contrast to the extracellular and
transmembrane domains the entire C-terminal cytoplas- Since the NMDA receptor is comprised of NR1 and NR2
subunits, we next asked if NR1 also showed marked differ-
ences in length between invertebrates and vertebrates. This was not the case (Fig. 6B) and we therefore inspected
a range of other glutamate receptor subunits including
ionotropic AMPA receptors (e.g. GluR1) and metabo-
tropic receptors (e.g. mGluR1). In mammals these recep-
tor subunits are known to link via their C-termini to
adaptor proteins forming complexes bringing together
these different classes of glutamate receptors. Our results
show that an elongated vertebrate C-terminal domain is
unique to the NR2 glutamate receptor subunit (Fig. 6B). To search for the origin of the vertebrate NR2 C-terminus,
the nucleotide sequence of each mouse NR2 subunit C- Page 5 of 14
(page number not for citation purposes) Page 5 of 14
(page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/6 BMC Neuroscience 2008, 9:6 R2A C-terminal Domain Across Species
NR2A C-terminal Domain Across Species. 80% consensus between sequences shown by bla
sites indicated by blue arrows. Vertebrate PDZ binding domain indicated by red arrow. Invertebr
(T-V/N-L) is present at the C-terminus of the Fly and Worm sequences. Alignment of NR2A C-terminal Domain Across Species
Figure 5
Alignment of NR2A C-terminal Domain Across Species. 80% consensus between sequences shown by black shading. Phosphorylation sites indicated by blue arrows. Page 6 of 14
(page number not for citation purposes) Structural aspects of NR2 C-terminal domain evolution Vertebrate PDZ binding domain indicated by red arrow. Invertebrate PDZ
binding domain (T-V/N-L) is present at the C-terminus of the Fly and Worm sequences. http://www.biomedcentral.com/1471-2202/9/6 BMC Neuroscience 2008, 9:6 Comparison C-termini Length Across Species
Figure 6
Comparison C-termini Length Across Species. 6.A: Comparison of NR2A and NR2B C-termini Length Across Repre-
sentative Species. Lengths in amino acid number were calculated from the position of the most distal transmembrane domain,
as predicted by TMHMM. The longer NR2 can be seen to be Vertebrate Specific. 6.B: Species Comparison of Glutamate
Receptor C-terminal Domain Length. Lengths in amino acid number were calculated from the position of the most distal trans-
membrane domain, as predicted by TMHMM. The vertebrate/invertebrate contrast in length of the C-terminal region is spe-
cific to NR2. Compariso
Figure 6 Compariso
Figure 6 Comparison C-termini Length Across Species
Figure 6
Comparison C-termini Length Across Species. 6.A: Comparison of NR2A and NR2B C-termini Length Across Repre-
sentative Species. Lengths in amino acid number were calculated from the position of the most distal transmembrane domain,
as predicted by TMHMM. The longer NR2 can be seen to be Vertebrate Specific. 6.B: Species Comparison of Glutamate
Receptor C-terminal Domain Length. Lengths in amino acid number were calculated from the position of the most distal trans-
membrane domain, as predicted by TMHMM. The vertebrate/invertebrate contrast in length of the C-terminal region is spe-
cific to NR2. mic tail was predicted to be intrinsically unfolded for all
four NR2s. This indicates that the intracellular tail of
NMDA receptors may under some conditions remain
unfolded. primary sequence identity relative to the rest of the pro-
tein (29%, Fig. 4), they exhibit strikingly similar second-
ary structure, where the relative positioning of alpha
helices and beta sheets is conserved (Fig. 9). PSIPRED was
also applied to NR2C and NR2D but their secondary
structure predictions showed similarity neither to each
other nor NR2A or NR2B (additional file 3). Since the NR2 C-terminus is known to bind directly to
various interacting proteins, it might be expected that it
could adopt a secondary or tertiary structure, and it is of
interest to compare the structure of the different NR2 sub-
units. In the absence of 3D structure we applied the
PSIPRED Protein Structure Prediction Server to compare
the computationally predicted secondary structure of
NR2A and NR2B amino acid sequences (Fig. 9) [28]. Sur-
prisingly, though NR2A and NR2B C-termini show low http://www.biomedcentral.com/1471-2202/9/6 http://www.biomedcentral.com/1471-2202/9/6 BMC Neuroscience 2008, 9:6 Comparison of the Amino Acid Translation of the C-terminal
exon of Mouse NR2B with the corresponding region from
Drosophila
Figure 7
Comparison of the Amino Acid Translation of the C-
terminal exon of Mouse NR2B with the correspond-
ing region from Drosophila. CaMKII phosphorylation site
on mouse NR2B indicated by blue arrow. PDZ binding motifs
indicated by red arrows. PDZ
Binding
Motif
..ESDV
GIYSC.. CaMKII
Binding
Motif
616 Amino Acids
Mouse NR2B C-terminus
GKSLT.. ..TVL
-Intron-
PDZ
Binding
Motif
111 Amino Acids
2857 Nucleotides
Drosophila NR2 C-terminus subunits or other glutamate receptors including iono-
tropic AMPA (Glur1, Glur2) and metabotropic receptors
(mGluR1, mGluR5). The C-termini of all the other men-
tioned glutamate receptors do not exhibit comparable
extensions between invertebrates and vertebrates. Thus
the vertebrate/invertebrate contrast is specific to NR2. All
of these glutamate receptor subtypes are involved with
synaptic plasticity and are linked via C-terminal domains
to intracellular adaptor proteins. Mouse NR2B C-terminus The ~5 fold difference in NR2 length between vertebrates
and invertebrates may in principle result from either a ver-
tebrate specific elongation or an invertebrate specific trun-
cation. Since only two clades are available for comparison
here (invertebrates and vertebrates) it is not possible to
distinguish between the ancestral and the derived state. The most parsimonious explanation for the NR2 length
difference is a loss of function in the common ancestor of
invertebrates, since a single random mutation introducing
a stop codon into the NR2 of a common ancestor of inver-
tebrates would be sufficient to truncate the NR2 C-termi-
nus. However this is an unlikely mechanism for
shortening of the C-terminus since both the invertebrate
and each of the vertebrate NR2 subunits have a type I PDZ
binding domain at their carboxyl termini [30,31]. We
noted that the NR2 C-terminus has a conserved exonic
structure throughout vertebrates, with the vast majority of
the NR2 C-termini being encoded for in the terminal
exon, which is in contrast to invertebrates where it is
coded by multiple exons (and the intron/exon junctions
do not show conservation within invertebrates). Interest-
ingly, the principal intron of the D. melanogaster NR2 C-
terminus bears some similarity (42%) to the terminal
exon of mouse NR2B suggesting that NR2 may have been
internally truncated due to intron gain in a common
ancestor of invertebrates. Alternatively the similarity seen
in the D. http://www.biomedcentral.com/1471-2202/9/6 melanogaster intron may imply a vertebrate spe-
cific elongation perhaps due to the intron becoming cod-
ing, but this may be less likely due to the presence of in-
frame stop codons in an intron. The ancestral state of NR2
may be revealed by further genome sequencing of more
diverse and complex invertebrates. If Lophotrochozoans
were found with the elongated NR2 version, then a C-ter-
minal truncation in the Ecdysozoan clade would be the
most parsimonious explanation [32], and if the shorter
form was unambiguously identified in a Urochordate
genome then a vertebrate specific elongation would be
supported [33]. Compariso
exon of Mo
Drosophila
Figure 7 Comparison of the Amino Acid Translation of the C-
terminal exon of Mouse NR2B with the correspond-
ing region from Drosophila. CaMKII phosphorylation site
on mouse NR2B indicated by blue arrow. PDZ binding motifs
indicated by red arrows. found in vertebrates differ in size due to variation in the
length of their C-terminal domains, the overall level of
conservation between them has not been addressed [4]. We found that there is a profound level of diversity
between the NR2 paralogues, specifically in their C-termi-
nal intracellular domains. In contrast their N-terminal
extracellular regions are highly conserved. Generation of a
phylogenetic tree using the conserved central part of the
NMDA receptor produces a topology grouping with the
NR2A/NR2B subunits in one clade, and the NR2C/NR2D
paralogues in another (additional file 4). The C-termini
alignment reinforces this grouping since NR2A and NR2B
have clearly longer C-termini while NR2C and NR2D are
shorter. Additionally, NR2A and NR2B C-termini show a
significant degree of conservation (29%) and are serine
rich, while NR2C and NR2D (19% identical) are highly
enriched for proline. The two sub groups imply that two
separate duplications of NR2 occurred at the base of the
vertebrate lineage, with the first giving rise to proto-
NR2A/B and proto-NR2C/D paralogues. These have sub-
sequently duplicated and diverged, giving rise to the four
NR2 paralogues found in extant vertebrates. Both rounds
of duplication occurred following divergence from the
urochordates, and are likely a consequence of the whole
genome duplication that took place in early chordate evo-
lution [29]. Discussion
Evolutionary features of the NMDA receptor intracellular
C-terminal domain We used bioinformatic tools to examine the evolutionary
origins of the NR2 intracellular domain. Though it has
previously been observed that the four NR2 paralogues Page 7 of 14
(page number not for citation purposes) Page 7 of 14
(page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/6 Evolution of signalling complexes and behavior f
g
g
p
In comparison with invertebrates, the more diverse family
of vertebrate NR2 subunits and their longer NR2 C-ter-
mini could have significant implications on the intracel-
lular signalling capacity of the NMDA receptor channel. At
the anatomical level, the duplication of the elongated Based on multiple species alignments of NR2 orthologues
we show that vertebrate NR2s are consistently longer than
invertebrate NR2s, and that this is due to difference in C-
terminal domain length. We next asked if this vertebrate
specific intracellular extension was a feature of the NR1 Page 8 of 14
(page number not for citation purposes) BMC Neuroscience 2008, 9:6 http://www.biomedcentral.com/1471-2202/9/6 FoldIndex Comparison of NR2 C-terminal Domain Folding Propensity
Figure 8
FoldIndex Comparison of NR2 C-terminal Domain Folding Propensity. Plot of AA sequence of N-terminus (N) to
C-terminus (C) of mouse NR2A and NR2B against probability of folding. Green areas are predicted to be intrinsically folded,
red areas are predicted to be intrinsically unfolded. Transmembrane regions (M1, M3, and M4) and re-entry loop (M2) are
boxed. p
g
p
y
g
FoldIndex Comparison of NR2 C-terminal Domain Folding Propensity. Plot of AA sequence of N-terminus (N) to
C-terminus (C) of mouse NR2A and NR2B against probability of folding. Green areas are predicted to be intrinsically folded,
red areas are predicted to be intrinsically unfolded. Transmembrane regions (M1, M3, and M4) and re-entry loop (M2) are
boxed. NR2 in the vertebrate common ancestor has allowed for
each paralogue to diverge and establish distinct but over-
lapping anatomical and developmental roles in the mam-
malian brain by subfunctionalization of gene duplicates
[10,34]. At the level of signal transduction complexes, we
propose that the longer vertebrate C-terminus allowed a
greater NMDA receptor dependant signalling complexity
to evolve in vertebrates. brates and have an orthologue in invertebrates. Moreover,
of the 186 mammalian NRC/MASC proteins there are 91
identifiable orthologues in the Drosophila melanogaster
genome (data not shown). These observations suggest
that many vertebrate NMDA receptor interactions with
cytoplasmic signalling proteins are not important to the
physiology and behaviour of invertebrates. NR2 in the vertebrate common ancestor has allowed for
each paralogue to diverge and establish distinct but over-
lapping anatomical and developmental roles in the mam-
malian brain by subfunctionalization of gene duplicates
[10,34]. Page 9 of 14
(page number not for citation purposes) Evolution of signalling complexes and behavior At the level of signal transduction complexes, we
propose that the longer vertebrate C-terminus allowed a
greater NMDA receptor dependant signalling complexity
to evolve in vertebrates. The expansion of the NR2 gene family has been utilised in
the mammalian brain to produce distinct functional roles. Studies of synaptic plasticity at the CA3-CA1 synapses of
the hippocampus, using mice lacking these subunits or
with subunit specific pharmacological antagonists, reveal
differences in synaptic plasticity [12]. As mentioned
above, the subunits are expressed at different times and
locations during development of the nervous system
implying that their specific signalling functions have been
exploited to serve roles in organisation of the anatomi-
cally complex mammalian brain. The mammalian
MAGUK family of proteins, which bind to the PDZ bind-
ing domain at the C-terminus of NR2, shows a similar
evolutionary pattern of duplication followed by subfunc-
tionalisation, with diverse expression and function It is established that the mammalian NMDA receptor is in
close contact with plethora of proteins that form the NRC/
MASC (Fig. 1) [8,35]. It is reasonable to predict that the
invertebrate NR2 C-terminus would bind to a smaller set
of postsynaptic proteins: invertebrate NR2 is far shorter
and has only one identifiable interaction motif (the PDZ
binding domain). If an elongated NR2 intracellular
domain did exist in a common ancestor of invertebrates,
then we predict that those proteins within the NRC/MASC
that bind mammalian NR2 C-terminus would be found in
invertebrates. Indeed, CaMKII [36], alpha-actinin 2 [37],
PLC-gamma [38], Rack1 [39], Grit [40], Clathrin Adapter
Protein 2 [41], and the MAGUKs [30] are all molecules
that directly interact with the NMDA receptor in verte- Page 9 of 14
(page number not for citation purposes) Page 9 of 14
(page number not for citation purposes) BMC Neuroscience 2008, 9:6 http://www.biomedcentral.com/1471-2202/9/6 PSIPRED Secondary Structure Predictions of Mouse NR2A & NR2B C-terminal Domains
Figure 9
PSIPRED Secondary Structure Predictions of Mouse NR2A & NR2B C-terminal Domains. Green barrels repre-
sent alpha helices while yellow arrows signify beta sheets. Prediction confidence represented by blue bars. Structural motifs
occurring in equivalent locations in NR2A and NR2B are boxed in red. NR2B C-terminal Domains
R2A & NR2B C-terminal Domains. Green bar mains. Green barrels repre-
blue bars. Structural motifs y
g
PSIPRED Secondary Structure Predictions of Mouse NR2A & NR2B C-terminal Domains. Green barrels repre-
sent alpha helices while yellow arrows signify beta sheets. Evolution of signalling complexes and behavior PSD-95 and CaMKII might act as cytoplasmic 'lig-
ands' for the NMDA receptor, in an analogous manner to
extracellular ligands such as Zn+, H+, and glutamate [46],
and have allosteric effects on the the conformational state
of NR2. Consistent with this, the interaction of PSD-95
with NR2B is known to modulate NMDA receptor chan-
nel function [49]. By binding to the NR2 C-terminal
domain, these cytoplasmic ligands may in turn control
the overall function of NMDA receptors (or NMDA recep-
tor complex) as a signalling apparatus. This 'structural switch' model implies an evolutionary
selection on the unfolded domain. An alternative expla-
nation is that there is no structural constraint and there-
fore nucleotides are free to be substituted rapidly by
neutral evolution without any structural implications. We
consider this scenario unlikely for the following reasons. Amino acid sequences for the C-terminal domain in each
particular NR2 paralogues are well conserved between ver-
tebrates, implying that since divergence of the NR2 para-
logues each subunit has acquired structures that
underwent purifying selection in vertebrates. Further-
more, all (~10) known phosphorylation sites and protein-
protein interaction sites when mapped onto sequence
alignments for each of NR2A and NR2B were found to be
conserved throughout mammals. These interaction sites
would require a certain extent of structural constraint, and
so their conservation implies a structural framework to
the NR2 C-terminal domain. Finally there is no explana-
tion why the predicted secondary structures for NR2A and
NR2B C-terminal domains have remained structurally
similar since they have diverged, given that they exhibit
such low similarity at the primary sequence level. The
above arguments taken together suggest that a functional
adaptation occurred in vertebrate evolution that accounts
for NR2 C-terminal structure. Within vertebrate evolution
there is evidence of further evolutionary adaptation of
NR2A, which was reported to be under significant positive
selection in primates when compared to rodents [50]. Evolution of signalling complexes and behavior Prediction confidence represented by blue bars. Structural motifs
occurring in equivalent locations in NR2A and NR2B are boxed in red. Page 10 of 14
(page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/6 http://www.biomedcentral.com/1471-2202/9/6 BMC Neuroscience 2008, 9:6 between the four paralogues (PSD-95, PSD-93, SAP-102,
and SAP97). The duplication of MAGUKs in vertebrates
complements the NR2 duplication, resulting in poten-
tially sixteen binary configurations of NR2-MAGUK inter-
actions, substantially increasing the level of NMDA
receptor interaction complexity in vertebrates. The specif-
icity of these interactions has been shown to be crucial to
cognitive function in mammals, as SAP-102 deficient
mice show mild learning defects and impairments in strat-
egy choice during behavioural assays [42], whereas PSD-
95 knockouts show more severe learning defects [43]. These mammalian gene specific roles in cognitive func-
tion are not found in Drosophila since the single Dlg gene
is required for viability and the phenotype of loss of Dro-
sophila NR2 is unknown [44]. Together these studies
make a strong case that the vertebrate combinations of
NR2 and MAGUK paralogues that form key components
of synaptic signalling complexes contribute to the reper-
toire of cognitive behaviours. This model implies that
invertebrates do not have the same signalling diversity
and thus behavioural repertoire, which is consistent with
comparative studies of learning [1]. Our model that evo-
lution of behaviour is impacted by evolution of complex-
ity of signalling complexes adds to the standard model
that points to the numbers of neurons as the determinant
of behavioural evolution. Importantly, the elongated NR2
together with the vertebrate specific NR2 duplication
would have preceded the anatomical enlargement of
brain size associated with cognitive capacities of higher
animals [2]. binding to CaMKII induces an alternative conformation
"B". PSD-95 and CaMKII might act as cytoplasmic 'lig-
ands' for the NMDA receptor, in an analogous manner to
extracellular ligands such as Zn+, H+, and glutamate [46],
and have allosteric effects on the the conformational state
of NR2. Consistent with this, the interaction of PSD-95
with NR2B is known to modulate NMDA receptor chan-
nel function [49]. By binding to the NR2 C-terminal
domain, these cytoplasmic ligands may in turn control
the overall function of NMDA receptors (or NMDA recep-
tor complex) as a signalling apparatus. binding to CaMKII induces an alternative conformation
"B". Authors' contributions Conceived and designed study: TJR, RDE, SGNG, NHK. Performed the analysis: TJR, RDE, NHK. Wrote the paper:
TJR, RDE, SGNG, NHK. All authors read and approved of
the final manuscript. Sequence data & alignments q
g
We obtained protein sequences for all genes from
Ensembl Version 36. Homologues of mouse sequences
were obtained using Ensembl BioMart [51]. Schistosoma
mansoni nucleotide sequences were obtained from the
Wellcome Trust Sanger Institute Pathogen Sequencing
Unit's datababse [52,53]. Multiple sequence alignments
we carried out using ClustalW [54]. Alignments were
shaded using GeneDoc [55]. Where multiple transcripts
are predicted, only a single transcript is aligned. Dotplots
were generated using the Dotmatcher program in the
EMBOSS package [56]. For comparing the length of C-ter-
minal regions of multiple species, the C-terminus of each
protein sequence was defined as the amino acid sequence
distal of the last transmembrane residue as predicted by
TMHMM [57,58]. [http://www.biomedcentral.com/content/supplementary/1471-
2202-9-6-S1.TXT] Additional file 3 PSIPRED Secondary Structure Predictions of Mouse NR2C & NR2D C-
terminal Domains. Green barrels represent alpha helices while yellow
arrows signify beta sheets. Prediction confidence represented by blue bars. Click here for file To generate Nucleotide alignments to find the best region
of similarity, the Smith-Waterman algorithm Water with
an EBLOSUM62 matrix was applied [56]. Phlyogentic tree
was constructed using PhyML [59]. [http://www.biomedcentral.com/content/supplementary/1471-
2202-9-6-S3.JPEG] Protein interaction data Unrooted Phylogenetic Tree of NR2 Subunits. Human (Hs), mouse
(Mm), rat (Rm), zebrafish (Dr), ciona (Ci), Drosophila (Dm), C. ele-
gans (Ce). Bootstrap values are shown at branch points. Click here for file Protein interaction data for each NR2 subunit in mam-
mals were obtained from the Human Protein Reference
Database [7]. [http://www.biomedcentral.com/content/supplementary/1471-
2202-9-6-S4.pdf] Additional file 2 FoldIndex Comparison of NR2 C-terminal Domain Folding Propensity. Plot of AA sequence of N-terminus (N) to C-terminus (C) of mouse
NR2C and NR2D against probability of folding. Green areas are pre-
dicted to be intrinsically folded, red areas are predicted to be intrinsically
unfolded. Click here for file Click here for file [http://www.biomedcentral.com/content/supplementary/1471-
2202-9-6-S2.ppt] Conclusion NMDA receptors display a vertebrate specific elongation
of the intracellular C-terminus of the NR2 subunit. This
extension is unique among ionotropic glutamate recep-
tors to the NMDA receptor NR2 subunits. Significant
diversity in the NR2 C-terminus exists at the sequence
level between the four NR2 paralogues in vertebrates,
though each individual paralogue is highly conserved
amongst non-teleost vertebrates. In contrast to the extra-
cellular and transmembrane domains, each NR2 C-termi-
nus is predicted to be unfolded in its native state. NR2A
and NR2B C-termini are predicted to be highly similar at
the level of secondary structure though they are a poorly
conserved at the primary sequence level. We postulate that
evolution of this vertebrate specific C-terminal domain of
NR2 has resulted in an unfolded but conserved modular
structure that may have contributed to the evolution and
organisation of postsynaptic signalling complexes in ver-
tebrates. http://www.biomedcentral.com/1471-2202/9/6 BMC Neuroscience 2008, 9:6 gous swaps, and trans-species chimeras to study the
molecular evolution of NMDA receptor signalling in vivo. expression sequence tag databases in Genbank by
BLASTn, tBLASTn, and discontiguous megablast similarity
searches, with low complexity masking [60]. To search for
conserved domains, the protein sequence of the intracel-
lular tail of each mouse NR2 subunit was searched against
the Pfam database using the default parameters [25]. Structure predictions Protein folding predictions were made using the FoldIn-
dex graphic web server [27]. Prediction of secondary struc-
ture was made using PSIPRED Protein Structure
Prediction System [28]. Additional file 1 Smith-Waterman Alignment of Mouse NR2B C-terminal Exon Nucle-
otide Sequence against D. melanogaster C-terminal 2857 Nucleotide
Intron. Mouse exon sequence named 'NR2B', D. melanogaster intron
sequence named 'intron'. Click here for file NR2 C-terminal dynamics The analysis of the C-terminus structure suggests a
dynamic switching function of NR2 where the domain
alternates between unfolded and folded states, and that
this modulates interactions with other proteins. The
observed high composition of both serine and proline is
a characteristic of intrinsically disordered proteins [26],
which are natively unfolded and have been associated
with 'hub' proteins in protein-protein interaction net-
works [45]. The adoption of a tertiary conformation, nec-
essary for formation of a signalling complex, may occur
following binding to intracellular adaptors, for example
the PDZ domain containing proteins and CaMKII. This
may be analogous to a mechanism suggested for Kv chan-
nels, where a PDZ binding domain preceded by an intrin-
sically disordered intracellular C-terminus facilitates
structural flexibility as well as robust interaction with the
scaffold protein [46,47]. Since intrinsically unfolded pro-
teins undergo a conformational change from unfolded to
folded when bound to another protein [48], the NR2 C-
terminal domain should be able to form multiple confor-
mations depending on various intracellular binding part-
ners e.g. binding to PSD-95 induces the cytoplasmic
domain to fold into a particular 3D conformation "A" and To test these evolutionary models it will be necessary to
characterise NMDA receptor associated proteins of an
invertebrate model in a comparable manner to that
achieved in mice [8,35]. Detailed experimental study on
the structure of the C-terminal domains using methods
such as X-ray crystallography and nuclear magnetic reso-
nance spectroscopy will be essential to test our structural
predictions [47], as well as to further understand how the
NMDA receptor's intracellular domain structure mediates
its role in the functional organization of the NRC. More
broadly, gene targeting should be employed to generate
new mouse models of the NR2 C-terminus with point
mutations of intracellular ligand interaction sites, paralo- Page 11 of 14
(page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/6 http://www.biomedcentral.com/1471-2202/9/6 References f
28. McGuffin LJ, Bryson K, Jones DT: The PSIPRED protein struc-
ture prediction server. Bioinformatics 2000, 16:404-405. p
29. McLysaght A, Hokamp K, Wolfe KH: Extensive genomic duplica-
tion during early chordate evolution. Nat Genet 2002,
31:200-204. 30. Kornau HC, Schenker LT, Kennedy MB, Seeburg PH: Domain inter-
action between NMDA receptor subunits and the postsynap-
tic density protein PSD-95. Science 1995, 269:1737-1740. p
8. Husi H, Ward MA, Choudhary JS, Blackstock WP, Grant SG: Pro-
teomic analysis of NMDA receptor-adhesion protein signal-
ing complexes. Nat Neurosci 2000, 3:661-669. y p
31. Xia S, Miyashita T, Fu TF, Lin WY, Wu CL, Pyzocha L, Lin IR, Saitoe
M, Tully T, Chiang AS: NMDA receptors mediate olfactory
learning and memory in Drosophila. Curr Biol 2005, 15:603-615. 32
i g
p
,
9. Husi H, Grant SG: Isolation of 2000-kDa complexes of N-
methyl-D-aspartate receptor and postsynaptic density 95
from mouse brain. J Neurochem 2001, 77(1):281-91. g
y
p
32. Tessmar-Raible K, Arendt D: Emerging systems: between verte-
brates and arthropods, the Lophotrochozoa. Curr Opin Genet
Dev 2003, 13:331-340. J
( )
10. Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH: Devel-
opmental and regional expression in the rat brain and func-
tional properties of four NMDA receptors. Neuron 1994,
12:529-540. 33. Satoh N: The ascidian tadpole larva: comparative molecular
development and genomics. Nat Rev Genet 2003, 4:285-295. 33. Satoh N: The ascidian tadpole larva: comparative molecular
development and genomics. Nat Rev Genet 2003, 4:285-295. 34. Li WH, Yang J, Gu X: Expression divergence between duplicate
genes. Trends Genet 2005, 21:602-607. 11. Watanabe M, Inoue Y, Sakimura K, Mishina M: Developmental
changes in distribution of NMDA receptor channel subunit
mRNAs. Neuroreport 1992, 3:1138-1140. p
g
34. Li WH, Yang J, Gu X: Expression divergence between duplicate
genes. Trends Genet 2005, 21:602-607. 35
C ll
MO Y
L C b MP H
H C
I Bl
k
k WP 35. Collins MO, Yu L, Coba MP, Husi H, Campuzano I, Blackstock WP,
Choudhary JS, Grant SG: Proteomic analysis of in vivo phospho-
rylated synaptic proteins. J Biol Chem 2005, 280:5972-5982. p
12. Sprengel R, Single FN: Mice with genetically modified NMDA
and AMPA receptors. Ann N Y Acad Sci 1999, 868:494-501. 13. References 1. Moore BR: The evolution of learning. Biol Rev Camb Philos Soc
2004, 79:301-335. 21. von Engelhardt J, Coserea I, Pawlak V, Fuchs EC, Kohr G, Seeburg PH,
Monyer H: Excitotoxicity in vitro by NR2A- and NR2B-con-
taining NMDA receptors. Neuropharmacology 2007, 53:10-17. 2. Gilbert SL, Dobyns WB, Lahn BT: Genetic links between brain
development and brain evolution. Nat Rev Genet 2005,
6:581-590. g
p
p
gy
22. Chen BS, Roche KW: Regulation of NMDA receptors by phos-
phorylation. Neuropharmacology 2007. 3. Pocklington AJ, Cumiskey M, Armstrong JD, Grant SG: The pro-
teomes of neurotransmitter receptor complexes form mod-
ular networks with distributed functionality underlying
plasticity and behaviour. Mol Syst Biol 2006, 2:. 2006.0023. Epub
2006 Jan 17 23. Nourry C, Grant SG, Borg J-P: PDZ domain proteins: plug and
play! Sci STKE 2003, 2003:RE7. p
y
24. Ito H, Ishikawa Y, Yoshimoto M, Yamamoto N: Diversity of brain
morphology in teleosts: brain and ecological niche. Brain
Behav Evol 2007, 69:76-86. J
4. Cull-Candy S, Brickley S, Farrant M: NMDA receptor subunits:
diversity, development and disease. Curr Opin Neurobiol 2001,
11:327-335. 25. Finn RD, Mistry J, Schuster-Bockler B, Griffiths-Jones S, Hollich V,
Lassmann T, Moxon S, Marshall M, Khanna A, Durbin R, Eddy SR, Son-
nhammer EL, Bateman A: Pfam: clans, web tools and services. Nucleic Acids Res 2006, 34:D247-51. 5. Bear MF, Malenka RC: Synaptic plasticity: LTP and LTD. Curr
Opin Neurobiol 1994, 4:389-399. 26. Serdyuk IN: Structured proteins and proteins with intrinsic
disorder. Molecular Biology 2006, 41:262. 6. Dickson KS, Kind PC: NMDA receptors: neural map designers
and refiners? Curr Biol 2003, 13:R920-2. 27. Prilusky J, Felder CE, Zeev-Ben-Mordehai T, Rydberg EH, Man O,
Beckmann JS, Silman I, Sussman JL: FoldIndex: a simple tool to
predict whether a given protein sequence is intrinsically
unfolded. Bioinformatics 2005, 21:3435-3438. ,
7. Mishra GR, Suresh M, Kumaran K, Kannabiran N, Suresh S, Bala P, Shi-
vakumar K, Anuradha N, Reddy R, Raghavan TM, Menon S, Hanuman-
thu G, Gupta M, Upendran S, Gupta S, Mahesh M, Jacob B, Mathew P,
Chatterjee P, Arun KS, Sharma S, Chandrika KN, Deshpande N, Pal-
vankar K, Raghavnath R, Krishnakanth R, Karathia H, Rekha B, Nayak
R, Vishnupriya G, Kumar HG, Nagini M, Kumar GS, Jose R, Deepthi
P, Mohan SS, Gandhi TK, Harsha HC, Deshpande KS, Sarker M,
Prasad TS, Pandey A: Human protein reference database – 2006
update. Nucleic Acids Res 2006, 34:D411-4. Similarity searches The nucleotide sequence of the intracellular tail of each
mouse NR2 subunit were searched against non-redundant
nucleotide collection, whole genome shotgun reads, and Page 12 of 14
(page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/6 BMC Neuroscience 2008, 9:6 References Sakimura K, Kutsuwada T, Ito I, Manabe T, Takayama C, Kushiya E,
Yagi T, Aizawa S, Inoue Y, Sugiyama H: Reduced hippocampal LTP
and spatial learning in mice lacking NMDA receptor epsilon
1 subunit. Nature 1995, 373:151-155. y
y
p
p
J
36. Leonard AS, Lim IA, Hemsworth DE, Horne MC, Hell JW: Calcium/
calmodulin-dependent protein kinase II is associated with
the N-methyl-D-aspartate receptor. Proc Natl Acad Sci USA
1999, 96:3239-3244. 14. Kutsuwada T, Sakimura K, Manabe T, Takayama C, Katakura N, Kush-
iya E, Natsume R, Watanabe M, Inoue Y, Yagi T, Aizawa S, Arakawa
M, Takahashi T, Nakamura Y, Mori H, Mishina M: Impairment of
suckling response, trigeminal neuronal pattern formation,
and hippocampal LTD in NMDA receptor epsilon 2 subunit
mutant mice. Neuron 1996, 16:333-344. 37. Wyszynski M, Lin J, Rao A, Nigh E, Beggs AH, Craig AM, Sheng M:
Competitive binding of alpha-actinin and calmodulin to the
NMDA receptor. Nature 1997, 385:439-442. 38. Gurd JW, Bissoon N: The N-methyl-D-aspartate receptor sub-
units NR2A and NR2B bind to the SH2 domains of phosphol-
ipase C-gamma. J Neurochem 1997, 69:623-630. 15. Ebralidze AK, Rossi DJ, Tonegawa S, Slater NT: Modification of
NMDA receptor channels and synaptic transmission by tar-
geted disruption of the NR2C gene. J Neurosci 1996,
16:5014-5025. p
g
J
39. Yaka R, Thornton C, Vagts AJ, Phamluong K, Bonci A, Ron D: NMDA
receptor function is regulated by the inhibitory scaffolding
protein, RACK1. Proc Natl Acad Sci USA 2002, 99:5710-5715. p
40. Nakazawa T, Watabe AM, Tezuka T, Kiyama Y, Fukaya M, Arima-
Yoshida F, Horai R, Sudo K, Ebine K, Delawary M, Goto J, Umemori
H, Tezuka T, Iwakura Y, Watanabe M, Yamamoto T, Manabe T:
p250GAP, a novel brain-enriched GTPase-activating protein
for Rho family GTPases, is involved in the N-methyl-d-aspar-
tate receptor signaling. Mol Biol Cell 2003, 14:2921-2934. 16. Kadotani H, Hirano T, Masugi M, Nakamura K, Nakao K, Katsuki M,
Nakanishi S: Motor discoordination results from combined
gene disruption of the NMDA receptor NR2A and NR2C
subunits, but not from single disruption of the NR2A or
NR2C subunit. J Neurosci 1996, 16:7859-7867. 17. Sprengel R, Suchanek B, Amico C, Brusa R, Burnashev N, Rozov A,
Hvalby O, Jensen V, Paulsen O, Andersen P, Kim JJ, Thompson RF,
Sun W, Webster LC, Grant SG, Eilers J, Konnerth A, Li J, McNamara
JO, Seeburg PH: Importance of the intracellular domain of p
g
g
41. Acknowledgements NR2 subunits for NMDA receptor function in vivo. Cell 1998,
92:279-289. NR2 subunits for NMDA receptor function in vivo. Cell 1998,
92:279-289. The authors thank Dr. R. A. W. Frank and Dr. J. D. Armstrong for com-
ments on the manuscript, Dr. M. Berriman for useful discussions and Miss
J.V. Turner for editorial assistance. TJR was supported by a Wellcome
Trust PhD Fellowship. RDE was supported by a Medical Research Council
UK Bioinformatics Training Fellowship. SGNG and NHK were supported
by the Genes to Cognition Program at the Wellcome Trust Sanger Insti-
tute. 18. 18. Liu L, Wong TP, Pozza MF, Lingenhoehl K, Wang Y, Sheng M, Auber-
son YP, Wang YT: Role of NMDA receptor subtypes in govern-
ing the direction of hippocampal synaptic plasticity. Science
2004, 304:1021-1024. 19. 19. Liu Y, Wong TP, Aarts M, Rooyakkers A, Liu L, Lai TW, Wu DC, Lu
J, Tymianski M, Craig AM, Wang YT: NMDA receptor subunits
have differential roles in mediating excitotoxic neuronal
death both in vitro and in vivo. J Neurosci 2007, 27:2846-2857. 20. Berberich S, Jensen V, Hvalby O, Seeburg PH, Kohr G: The role of
NMDAR subtypes and charge transfer during hippocampal
LTP induction. Neuropharmacology 2007, 52:77-86. Page 13 of 14
(page number not for citation purposes) References Roche KW, Standley S, McCallum J, Dune Ly C, Ehlers MD, Wenthold
RJ: Molecular determinants of NMDA receptor internaliza-
tion. Nat Neurosci 2001, 4:794-802. Page 13 of 14
(page number not for citation purposes) BMC Neuroscience 2008, 9:6 http://www.biomedcentral.com/1471-2202/9/6 http://www.biomedcentral.com/1471-2202/9/6 42. Cuthbert PC, Stanford LE, Coba MP, Ainge JA, Fink AE, Opazo P, Del-
gado JY, Komiyama NH, O'Dell TJ, Grant SG: Synapse-associated
protein 102/dlgh3 couples the NMDA receptor to specific
plasticity pathways and learning strategies. J Neurosci 2007,
27:2673-2682. 43. Migaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM,
Makhinson M, He Y, Ramsay MF, Morris RG, Morrison JH, O'Dell TJ,
Grant SG: Enhanced long-term potentiation and impaired
learning in mice with mutant postsynaptic density-95 pro-
tein. Nature 1998, 396:433-439. ,
44. Woods DF, Bryant PJ: Molecular cloning of the lethal(1)discs
large-1 oncogene of Drosophila. Dev Biol 1989, 134:222-235. 45. Haynes C, Oldfield CJ, Ji F, Klitgord N, Cusick ME, Radivojac P, Uver-
sky VN, Vidal M, Iakoucheva LM: Intrinsic disorder is a common
feature of hub proteins from four eukaryotic interactomes. PLoS Comput Biol 2006, 2:e100. p
46. Magidovich E, Fleishman SJ, Yifrach O: Intrinsically disordered C-
terminal segments of voltage-activated potassium channels:
a possible fishing rod-like mechanism for channel binding to
scaffold proteins. Bioinformatics 2006, 22:1546-1550. p
47. Magidovich E, Orr I, Fass D, Abdu U, Yifrach O: Intrinsic disorder
in the C-terminal domain of the Shaker voltage-activated K+
channel modulates its interaction with scaffold proteins. Proc
Natl Acad Sci USA 2007, 104:13022-13027. 48. Fink AL: Natively unfolded proteins. Curr Opin Struct Biol 2005,
15:35-41. 49. Yamada Y, Chochi Y, Takamiya K, Sobue K, Inui M: Modulation of
the channel activity of the epsilon2/zeta1-subtype N-methyl
D-aspartate receptor by PSD-95. J Biol Chem 1999,
274:6647-6652. 50. Dorus S, Vallender EJ, Evans PD, Anderson JR, Gilbert SL, Mahowald
M, Wyckoff GJ, Malcom CM, Lahn BT: Accelerated evolution of
nervous system genes in the origin of Homo sapiens. Cell
2004, 119:1027-1040. 51. Kasprzyk A, Keefe D, Smedley D, London D, Spooner W, Melsopp C,
Hammond M, Rocca-Serra P, Cox T, Birney E: EnsMart: a generic
system for fast and flexible access to biological data. Genome
Res 2004, 14:160-169. 52. Hertz-Fowler C, Peacock CS, Wood V, Aslett M, Kerhornou A,
Mooney P, Tivey A, Berriman M, Hall N, Rutherford K, Parkhill J, Ivens
AC, Rajandream MA, Barrell B: GeneDB: a resource for prokary-
otic and eukaryotic organisms. References Nucleic Acids Res 2004,
32:D339-43. 53. Wellcome Trust Sanger Institute Pathogen Sequencing
Unit's database [http://www.genedb.org] 54. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving
the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties
and weight matrix choice. Nucleic Acids Res 1994, 22:4673-4680. g
55. Nicholas B, Karl B, Nicholas P, Hugh B Jr: GeneDoc: a tool for edit-
ing and annotating multiple sequence alignments. . g
g
p
q
g
56. Rice P, Longden I, Bleasby A: EMBOSS: the European Molecular
Biology Open Software Suite. Trends Genet 2000, 16:276-277. g
p
57. Moller S, Croning MD, Apweiler R: Evaluation of methods for the
prediction of membrane spanning regions. Bioinformatics 2001,
17:646-653. 58. TMHMM [http://www.cbs.dtu.dk/services/TMHMM/]
59
G i d
S L
hi
F D
P G
l O PHYML O li 58. TMHMM [http://www.cbs.dtu.dk/services/TMHMM/] 59. Guindon S, Lethiec F, Duroux P, Gascuel O: PHYML Online – a
web server for fast maximum likelihood-based phylogenetic
inference. Nucleic Acids Res 2005, 33:W557-9. Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
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yours — you keep the copyright
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BioMedcentral
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(page number not for citation purposes) Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
BioMedcentral Publish with BioMed Central and every
scientist can read your work free of charge 60. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lip-
man DJ: Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs. Nucleic Acids Res 1997,
25:3389-3402.
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TURIZM FAOLIYATINI XALQARO HUQUQIY TARTIBGA SOLISH MASALALARI
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Zenodo (CERN European Organization for Nuclear Research)
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Email: Qobilbekqahramonov@gmail.com Annotatsiya: Ushbu maqolada bugungi kundagi globallashuv sharoitida xalqaro
turizm natijasida yuzaga keladigan huquqiy munosabatlarni tartibga solishning dolzarb
masalalari va ularga oid xalqaro miqyosda tartibga solish usullari va normalari,
shuningdek, ko’rsatilayotgan turistik xizmatlarning xalqaro xususiy huquqning
ob’yekti sifatidagi ko’rinishi va tavsifi, hamda, mazkur sohadagi ahamiyat kasb etgan
jihatlari yoritilgan. Kalit so‘zlari: Turizm, turistik xizmat, milliy turizm, xalqaro turizm, xalqaro
turizm institutlar va tashkilotlari, Butunjahon turizm tashkiloti (UNWTO), turistik
shartnomalar, xalqaro huquqiy tartib. TURIZM FAOLIYATINI XALQARO HUQUQIY TARTIBGA SOLISH
MASALALARI. Qaxramonov Qobilbek Qodirali o‘g‘li
Toshkent davlat yuridik universiteti talabasi
Tel: (+998 91) 9692818
Email: Qobilbekqahramonov@gmail.com Qaxramonov Qobilbek Qodirali o‘g‘li
Toshkent davlat yuridik universiteti talabasi
Tel: (+998 91) 9692818 ВОПРОСЫ МЕЖДУНАРОДНО-ПРАВОВОГО РЕГУЛИРОВАНИЯ
ТУРИСТИЧЕСКОЙ ДЕЯТЕЛЬНОСТИ. Аннотация:
В
данной
статье
рассмотрены
актуальные
вопросы
регулирования правоотношений, возникающих вследствие международного
туризма в современных условиях глобализации, а также методы и нормы
регулирования на международном уровне, а также появление и описание
предоставляемых туристских услуг как объекта международного частного права
и освещены важные аспекты в этой области. WWW.HUMOSCIENCE.COM Ключевые слова: Туризм, туристические услуги, национальный туризм,
международный
туризм,
международные
туристические
учреждения
и
организации, Всемирная туристская организация (ЮНВТО), туристские
контракты, международный правопорядок. WWW.HUMOSCIENCE.COM 734 ko’rsatishni tushunishimiz mumkin. Biroq shu o’rinda savol yuzaga keladi: Turizm
xizmati deganda o’zi aslida nimani yoki qanday xizmatlarni tushunishimiz kerak? Turizm, ya’ni sayohat xizmatlari sektori turli xildagi yetkazib beruvchilar, turistik
mahsulotlar, masofaviy marketing tashkilotlari, turoperatorlar va sayyohlik agentlari
va boshqalar o'rtasidagi munosabatlarning murakkab bir tarmog'i hisoblanadi. WWW.HUMOSCIENCE.COM
va boshqalar o rtasidagi munosabatlarning murakkab bir tarmog i hisoblanadi. “Turizm to’g’risida”gi O’zbekiston Respublikasi Qonunida “turistik xizmat”
atamasi xususidagi ta’rif quyidagicha: “turistik xizmatlar — joylashtirish,
ovqatlantirish, transport, ekskursiya va maslahat xizmati ko„rsatish bo„yicha
xizmatlar, shuningdek turist va ekskursantning ehtiyojlarini qanoatlantirishga
qaratilgan xizmatlar”.[2] Umumlashtiradigan bo’lsak, turistik xizmat deganda,
mijozga, agar u o’z xohishiga ko’ra mustaqil sayohatchi bo’lmasa, xizmatlar
ko’rsatuvchi tashkilot yoki shaxs tomonidan sayohat davomida shartnoma doirasida
ko’rsatilishi lozim bo’lgan servis xizmatlar tushunilishi aniqroq va tog’ri yondashuv
bo’ladi. Turistik xizmatlar negizida 2 taraf (turizm tashkiloti va mijoz-xizmat
ko’rsatuvchi) o’rtasida kelishilgan shartnoma yotadi. Agar bir davlatda joylashgan tur
agentligi boshqa bir davlat fuqarosiga yoki bir guruh chet elliklarga xizmat ko’rsatish
yuzasidan shartnoma imzolansa, ushbu tomonlar o’rtasida tuziladigan shartnoma bu
xalqaro huquqiy faoliyatning ob’yekti sifatida qaraladi. Bu shartnomaga nisbatan
qo’llaniladigan xalqaro huquq turistik agentlik joylashgan hududning qonuni bilan
himoyalangan hisoblanadi. Ushbu munosabatlarga xos tegishli jihatlar xalqaro huquqiy
faoliyatga tegishli normativ hujjatlar va normalari bilan tartibga solinadi. Xususan,
Turistlarni himoya qilish bo’yicha xalqaro kodeksi ham turistlarni ma’lum bir davlat
hududida bo’lgan vaqtda ularga nisbatan ma’lum joy qonunini qo’llash bilan
xavfsizliklarini ta’minlashga doir muhim jihatlarni ko’rsatadi. Haqiqatdan ham,
turistik xizmatlar va turizm, umuman olganda, turizm sohasi xalqaro darajada huquqiy
hujjatlar bilan tartibga solingan sohalardan biri hisoblanadi. Bu soha bo’yicha butun
jahonda yirik bir tashkilot faoliyat ko’rsatadi. Butunjahon Turizm Tashkiloti
(UNWTO) Birlashgan Millatlar Tashkilotining qo’shimcha organi bo’lib, uning asosiy
vazifasi ma’suliyatli hisoblanib, barqaror va hamma uchun ochiq turizmni targ'ib WWW HUMOSCIENCE COM
“Turizm to’g’risida”gi O’zbekiston Respublikasi Qonunida “turistik xizmat”
atamasi xususidagi ta’rif quyidagicha: “turistik xizmatlar — joylashtirish,
ovqatlantirish, transport, ekskursiya va maslahat xizmati ko„rsatish bo„yicha
xizmatlar, shuningdek turist va ekskursantning ehtiyojlarini qanoatlantirishga
qaratilgan xizmatlar”.[2] Umumlashtiradigan bo’lsak, turistik xizmat deganda,
mijozga, agar u o’z xohishiga ko’ra mustaqil sayohatchi bo’lmasa, xizmatlar
ko’rsatuvchi tashkilot yoki shaxs tomonidan sayohat davomida shartnoma doirasida
ko’rsatilishi lozim bo’lgan servis xizmatlar tushunilishi aniqroq va tog’ri yondashuv
bo’ladi. Turistik xizmatlar negizida 2 taraf (turizm tashkiloti va mijoz-xizmat
ko’rsatuvchi) o’rtasida kelishilgan shartnoma yotadi. Agar bir davlatda joylashgan tur
agentligi boshqa bir davlat fuqarosiga yoki bir guruh chet elliklarga xizmat ko’rsatish
yuzasidan shartnoma imzolansa, ushbu tomonlar o’rtasida tuziladigan shartnoma bu
xalqaro huquqiy faoliyatning ob’yekti sifatida qaraladi. ISSUES OF INTERNATIONAL LEGAL REGULATION OF TOURISM
ACTIVITIES. Abstract: In this article, current issues of regulation of legal relations arising as
a result of international tourism in today's globalization conditions and methods and
norms of regulation at the international level, as well as the appearance and description
of the provided tourist services as an object of international private law, and the
important aspects in this field are highlighted. Key words: Tourism, tourist service, national tourism, international tourism,
international tourism institutions and organizations, World Tourism Organization
(UNWTO), tourist contracts, international legal order. WWW.HUMOSCIENCE.COM
Avvalosi, “Turizm” tushunchasiga umumiy bir ta’rif keltirsak. Jahonning ko’zga
ko’rinarli va mashhur olimlari, shu soha mutaxassislari tomonidan ushbu tushunchaga
turli ta’rif, fikr va yondashuvlar keltirilgan. Xususan, Buyuk Britaniyadagi Turizm
instituti turizm tushunchasiga quyidagi ta'rifni beradi: “Turizm - bu odamlarning ular
odatda yashaydigan va ishlaydigan joylardan boshqa belgilangan joylarga vaqtincha
qisqa muddatga tashrif buyurishi: u har qanday maqsaddagi sayohatni o'z ichiga oladi,
bir kunlik tashriflar yoki ekskursiyalarni ham”. [1] Umuman olganda, o’zi yashab
turgan muqim joyidan tashqariga chiqish sayohat hisoblanadi, deyishimiz ham
mumkin. Jamiyatdagi mavjud boshqa ijtimoiy munosabatlar kabi turizm ham o’z
navbatida, huquqiy tartibga solingan, ya’ni ma’lum bir huquqiy me’yorlar bilan
belgilab qo’yilgan hamda mavjud va ma’lum yuridik asoslarga ega sohadir. Xalqaro
turizm sohasidagi xizmatlarning bevosita huquqiy tabiatiga baho beradigan bo’lsak,
xizmat ko’rsatish shartnomasi asosida, ma’lum bir haq evaziga mijozga turistik xizmat 735 WWW.HUMOSCIENCE.COM
737
qiluvchi agentlikdir. [3] Bu tashkilot turizmni umumjahon miqyosida keng targ’ib
qilish, uning xalqaro darajada huquqiy asoslarini tashkil etuvchi hujjatlar qabul qilish
va davlatlararo ushbu qonun-qoidalarga rioya qilinishini monitoring qilish kabi
vazifalardan tashkil topgan. Yana bu kabi xalqaro normalar sirasiga Manila Jahon
turizmi to’g’risidagi deklaratsiyani (1980) [4], Gaga turizm to’g’risidagi deklaratsiyani
(1989) [5], Turistlarni himoya qilish xalqaro kodekni [6], Xalqaro eko-turizmni keng
miqyosda targ’ib qilish to’g’risidagi konvensiya va shu kabi ko’plab normalarni
sanashimiz mumkin. Yuqoridagi sanab o’tilgan xalqaro normalar ushbu sohaning
muammoli va munozarali deb hisoblangan jihatlar yuzasidan bo’lgan qo’shimcha
savollarga o’rin qoldirmaslik maqsadida tuzilgan. Birlashgan Millatlar Tashkilotining
Turizm bo’yicha ma’sul organiga a’zo davlatlar har yili sohaga doir hisobotlarni
berishlari lozim. Hisobotlarda mamlakatlar turizm salohiyatidagi madaniy, iqtisodiy,
moliyaviy o’zgarishlar, sayyohlarning xalqaro huquqiy himoyasini ta’minlashga oid
chora-tadbirlar, xususiy va davlat sektoriga oid turistik xizmatlarni ko’rsatish,
tashkilotlarining faoliyatlariga oid ijobiy o’zgarish, xizmatlar ko’rsatishda erishilgan
yutuqlar va shu kabilar haqida atroflicha to’liq bayonotlarini topshirilishi ko’zda
tutilgan. Aytib o’tish joizki, xalqaro huquqiy faoliyatini tartibga solish, ya’ni ushbu
sohaga oid qonun qoidalar va talablarni belgilash, tegishlicha huquqiy tartibni joriy
qilishni har bir davlat o’zining ichki huquqiy tizimi bilan tartibga soladi va tashkil
etilishini ta’minlaydi. Buni Yurtimiz misolida olib qarasak, soha to’g’risidagi
O’zbekiston Respublikasi qonunlari, Prezident va ijro etuvchi hokimiyat Vazirlar
mahkamasining qarorlari bilan ushbu siyosatning izchilligi ta’minlangan. Biroq,
xalqaro turizm faoliyati bu huquqning boshqa xalqaro darajada tartibga solingan
tarmoqlari kabi barcha huquq tizimlaridagi umumiy jihatlarni biriktirgan va ma’lum bi
imtiyozlar ba’zida esa cheklovlar bilan boyitilgan yuqori tizim hisoblanadi. Davlatlar
esa ushbu tizim mexanizmlarini bog’lab turuvchi ajralmas komponent rolini bajaradi. Bugungi kunda butun dunyoda sayohat va turizm qonun-qoidalarini birlashtirish
jarayoni mavjuddir. Bu jahon turizmini yaxlit bir tizim shaklida dunyo tomonidan
qabul qilinishini ta’minlash uchun muhim hisoblanadi. Yurtimizda turizm sohasini qiluvchi agentlikdir. [3] Bu tashkilot turizmni umumjahon miqyosida keng targ’ib
qilish, uning xalqaro darajada huquqiy asoslarini tashkil etuvchi hujjatlar qabul qilish
va davlatlararo ushbu qonun-qoidalarga rioya qilinishini monitoring qilish kabi
vazifalardan tashkil topgan. Yana bu kabi xalqaro normalar sirasiga Manila Jahon
turizmi to’g’risidagi deklaratsiyani (1980) [4], Gaga turizm to’g’risidagi deklaratsiyani
(1989) [5], Turistlarni himoya qilish xalqaro kodekni [6], Xalqaro eko-turizmni keng
miqyosda targ’ib qilish to’g’risidagi konvensiya va shu kabi ko’plab normalarni
sanashimiz mumkin. Yuqoridagi sanab o’tilgan xalqaro normalar ushbu sohaning
muammoli va munozarali deb hisoblangan jihatlar yuzasidan bo’lgan qo’shimcha
savollarga o’rin qoldirmaslik maqsadida tuzilgan. Birlashgan Millatlar Tashkilotining
Turizm bo’yicha ma’sul organiga a’zo davlatlar har yili sohaga doir hisobotlarni
berishlari lozim. Bu shartnomaga nisbatan
qo’llaniladigan xalqaro huquq turistik agentlik joylashgan hududning qonuni bilan
himoyalangan hisoblanadi. Ushbu munosabatlarga xos tegishli jihatlar xalqaro huquqiy
faoliyatga tegishli normativ hujjatlar va normalari bilan tartibga solinadi. Xususan,
Turistlarni himoya qilish bo’yicha xalqaro kodeksi ham turistlarni ma’lum bir davlat
hududida bo’lgan vaqtda ularga nisbatan ma’lum joy qonunini qo’llash bilan
xavfsizliklarini ta’minlashga doir muhim jihatlarni ko’rsatadi. Haqiqatdan ham,
turistik xizmatlar va turizm, umuman olganda, turizm sohasi xalqaro darajada huquqiy
hujjatlar bilan tartibga solingan sohalardan biri hisoblanadi. Bu soha bo’yicha butun
jahonda yirik bir tashkilot faoliyat ko’rsatadi. Butunjahon Turizm Tashkiloti
(UNWTO) Birlashgan Millatlar Tashkilotining qo’shimcha organi bo’lib, uning asosiy
vazifasi ma’suliyatli hisoblanib, barqaror va hamma uchun ochiq turizmni targ'ib 736 Hisobotlarda mamlakatlar turizm salohiyatidagi madaniy, iqtisodiy,
moliyaviy o’zgarishlar, sayyohlarning xalqaro huquqiy himoyasini ta’minlashga oid
chora-tadbirlar, xususiy va davlat sektoriga oid turistik xizmatlarni ko’rsatish,
tashkilotlarining faoliyatlariga oid ijobiy o’zgarish, xizmatlar ko’rsatishda erishilgan
yutuqlar va shu kabilar haqida atroflicha to’liq bayonotlarini topshirilishi ko’zda
tutilgan. Aytib o’tish joizki, xalqaro huquqiy faoliyatini tartibga solish, ya’ni ushbu
sohaga oid qonun qoidalar va talablarni belgilash, tegishlicha huquqiy tartibni joriy
qilishni har bir davlat o’zining ichki huquqiy tizimi bilan tartibga soladi va tashkil
etilishini ta’minlaydi. Buni Yurtimiz misolida olib qarasak, soha to’g’risidagi
O’zbekiston Respublikasi qonunlari, Prezident va ijro etuvchi hokimiyat Vazirlar
mahkamasining qarorlari bilan ushbu siyosatning izchilligi ta’minlangan. Biroq,
xalqaro turizm faoliyati bu huquqning boshqa xalqaro darajada tartibga solingan
tarmoqlari kabi barcha huquq tizimlaridagi umumiy jihatlarni biriktirgan va ma’lum bi
imtiyozlar ba’zida esa cheklovlar bilan boyitilgan yuqori tizim hisoblanadi. Davlatlar
esa ushbu tizim mexanizmlarini bog’lab turuvchi ajralmas komponent rolini bajaradi. Bugungi kunda butun dunyoda sayohat va turizm qonun-qoidalarini birlashtirish 737 tartibga solishga taaluqli bir qator qonun hujjatlari qabul qilingan. Shulardan eng
asosiylari sifatida “Turizm to’g’risida”gi O’zbekiston Respublikasi Qonuni[7] ,
"O’zbekiston Respublikasida turizm sohasini yanada rivojlantirish chora-tadbirlari
to’g’risida"gi O’zbekiston Respublikasi Prezidentining farmoni[8] , "O’zbekiston
Respublikasida turizmni jadal rivojlantirishga oid qo’shimcha chora-tadbirlari
to’g’risida"gi
O’zbekiston
Respublikasi
Prezidenti
qarori[9],
O’zbekiston
Respublikasi Vazirlar Mahkamasining “O’zbekiston Respublikasida turizm sohasini
tiklash va rivojlantirish uchun qulay shart-sharoitlarni yaratish chora-tadbirlari
to’g’risida”gi qarori[10], xalqaro turizmni rivojlantirishga oid O’zbekiston
Respublikasi Prezidentining 2018-yil 6-fevraldagi PQ-3509-son “Kirish turizmini
rivojlantirish chora-tadbirlari to’g’risida"gi qarori[11]kabi hujjatlarni misol qilib
keltira olamiz. Deputatlarimiz shu yilning 9-oktyabr kuni boʻlib oʻtgan Qonunchilik
palatasining majlisida yangi tahrirdagi “Turizm toʻgʻrisida”gi qonun loyihasini koʻrib
chiqdilar va bu xususida qator muhokamalarni olib bordilar. Hozirgi paytda,
mamlakatimiz iqtisodiyotining muhim tarmoqlaridan biri sifatida sanalib borilayotgan
turizmni jadal rivojlantirish uchun har taraflama mukammal va pishiq boʻlgan huquqiy
asos zarur. 19 yil avval qabul qilingan “Turizm toʻgʻrisida”gi Oʻzbekiston
Respublikasi Qonuni hozirgi davr talablarini bajara olmayotgani asli haqiqat. Birinchi
navbatda deputatlar eʼtiboriga havola etilgan “Turizm toʻgʻrisida”gi Oʻzbekiston
Respublikasi qonunini yangilash loyihasi esa, deputatlarimizning taʼkidlashlaricha,
yurtimizda zamonaviy, yuqori samarali va raqobatbardosh turizm tarmogʻini
shakllantirish uchun huquqiy sharoitlar yaratishga xizmat qiladi, -deb hisoblashmoqda
[12]. WWW.HUMOSCIENCE.COM
Yangi qonun loyihasida mamlakatda tashqi va ichki turizmni rivojlantirish uchun
tashkiliy sharoitlar yaratish, turizm sohasidagi faoliyatlarni va munosabatlarni huquqiy
tartibga solishning barqarorligi, aniqligi va moslashuvchanligini taʼminlash, turizm
sohasidagi mavjud sub’yektlar faoliyatining shaffofligini taʼminlovchi huquqiy
mexanizmlarni shakllantirish hisobiga barcha darajadagi budjetlarga turizmdan
keladigan daromadlarni oshirish, qonunlarimizni turizm sohasidagi munosabatlarni 738 tartibga soluvchi xalqaro huquqiy normativ hujjatlar bilan uygʻunligini taʼminlash
borasidagi normalar aks ettirilmoqda. tartibga soluvchi xalqaro huquqiy normativ hujjatlar bilan uygʻunligini taʼminlash
borasidagi normalar aks ettirilmoqda. Deputatlarimiz tomonidan qayd ta’kidlanganidek, yangi qonun loyihasining qabul
qilinishi bilan turizm faoliyati sub’yektlarining huquqiy tartibga solingan holati
aniqlashtiriladi, davlat organining turizmni davlat tomonidan tartibga solish sohasidagi
vakolatlari aniq belgilanadi. Bunda esa, oʻz navbatida, hududlarning turizm
salohiyatini yanada mustahkamlash, mahalliy budjetlarning daromad qismini
oshirishga olib keladi. Yurtimizda yaratilgan ichki qonunchilik doirasida chet
elliklarning davlatda bo’lib turgan paytlaridagi huquqiy himoyasi va erkin harakat
qilishi ta’minlanadi. Aynan shu holatda, ular bilan aloqalar doirasida vujudga
keladigan munosabatlar xalqaro doiradagi ommaviy huquqning belgilarini o’zida aks
ettiradi. O’zbekiston turizm sohasida bir nechta dunyo davlatlari bilan xalqaro
hamkorlikni allaqachon yo’lqa qo’ygan. Ulardan biri 2006-yilda O’zbekiston
Respublikasining “O’zbekturizm” Milliy kompaniyasi bilan Qirg’iz Respublikasining
sanoat, savdo va turizm vazirligi o’rtasida imzolangan “Turizm sohasida hamkorlik
to’g’risida”gi Bitim [13] bu ikki davlatga fuqarolarining qo’shni davlatga erkin kirib
chiqishi, ularning himoyasi va huquqlarining ta’minlashini haqidagi masalalarni
kelishib olishdi. Bu kabi xalqaro hamkorlikka yana bir misol sifatida O’zbekiston
Respublikasi Hukumati bilan Fransiya Respublikasi Hukumati o’rtasida imzolangan
“Turizm sohasida hamkorlik to’g’risida”gi bitimni [14] ko’rsatishimiz mumkin. Bu
bitimga asosan, turistik sohadagi xususiy va davlat sayyohlik sektorlari uchun yangi
loyihalarni eko-turizm, turizmdagi yangi texnologiyalar, madaniy turizm va
turistlarning xavsizligini ta’minlash amalga oshirish ko’zda tutildi. Turizm sohasidagi xalqaro huquqiy faoliayatni tartibga solishning o'ziga xosligi
munosabatlarning keng doirasi bilan bog'liq. Bu munosabatlarga sayohatlar, sayyohlik
va dam olishni tashkil etish jarayonlaridagi ishtirokchi shaxslar kiradi. Mavjud bu
munosabatlar shu qadar xilma-xilki, ular huquqiy tartibga solishning ma'lum bir
murakkablikni keltirib chiqaradi. WWW.HUMOSCIENCE.COM 739 Har qanday holatda “sayyohlik agentligi – davlat”, “turistik – davlat”, “turist –
sayyohlik agentligi” tomonlar o‘rtasidagi munosabatlar tegishli qonun hujjatlari bilan
tartibga solinadi.[15] Bu kabi qonunning normativ hujjatlari ushbu tomonlar o'rtasidagi
munosabatlarning har bir elementini to'liq shaklda qamrab olishi kerak. Ko’pchilik turistik xizmatlar bozorini davlat tomonidan tartibga solish faoliyatlari
mavjud bo'lgan mamlakatlarda ikkita model qo'llaniladi - maxsus davlat organlari yoki
tartibga solish ko'p tarmoqli organlar tomonidan amalga oshiriladi. Bunga misol tariqasida, bir necha mamlakatlarda davlat institutlari qanday
ishlashini quyidagi misollardan ko’rib chiqaylik: 1.Avstriyada turizm sohasi Iqtisodiyot vazirligi tomonidan nazorat qilinadi. Shtatning sayyohlik imtiyozlari dunyoning 26 ta davlatida o'z vakolatxonalariga ega
bo'lgan Avstriya milliy turizm byurosi orqali e'lon qilinadi. 2.Buyuk Britaniya turizm tizimi Madaniyat, tomoshalar va sport vazirligi
tomonidan boshqaradi, u esa bevosita turizm uchun ma’sul hisoblangan organ,
Britaniya Nouristik Authority (BTA) ga bo'ysunadi. Bu tashkilot Buyuk Britaniyaga
xorijiy sayyohlar oqimini jalb qilish va ichki turizmni rivojlantirish bilan shug'ullanadi. 2.Buyuk Britaniya turizm tizimi Madaniyat, tomoshalar va sport vazirligi
tomonidan boshqaradi, u esa bevosita turizm uchun ma’sul hisoblangan organ,
Britaniya Nouristik Authority (BTA) ga bo'ysunadi. Bu tashkilot Buyuk Britaniyaga
xorijiy sayyohlar oqimini jalb qilish va ichki turizmni rivojlantirish bilan shug'ullanadi. 3.Germaniyada esa turizm biznesini tashkil etish bilan Iqtisodiyot vazirligi Milliy
turizm qo'mitasi shug'ullanadi, u Germaniyada turizm mahsulotlarini ilgari surish va
mamlakatga turistlar oqimini ko'paytirish uchun ma’suldir. Bu qoʻmita vakolatxonalari
dunyoning 27 davlatida faoliyat yuritadi. 4.Indoneziyada esa sayyohlar huquqlarini himoya qilish borasida keng
vakolatlarga ega bo'lgan turizm bo'yicha maxsus bo'lim mavjud. Shu sababli,
mamlakatda turistik biznesning barcha korxonalari ustidan nazorat va nazoratni amalga
oshiradigan turistik alohida politsiyasi mavjud. WWW.HUMOSCIENCE.COM
5.Italiyada esa 1983 yilda o’zidayoq turizm va mehmonxona sanoatini
rivojlantirish va takomillashtirishga qaratilgan qonun qabul qilindi. Ushbu qonun
turizm biznesining hududiy miqyosdagi asosiy boshqaruv organlari va ularni faoliyat
yuritish tartibini belgilaydi. Shuningdek, bu qonun mehmonxonalar xo‘jaligining
tasnifini belgilaydi, turistik byurolarga, transport va jamoat birlashmalariga turizm 740 sohasini xalqaro faoliyatini tartibga solish bilan shug‘ullanishga ruxsat berilgan qator
shartlarni belgilab beradi. 6. Fransiya esa Turizm to'g'risidagi qonun turizm xizmati turlarni sotishni
ta'minlaydigan shartlarni belgilaydi. Shu bilan birga, mazkur qonunga bo‘ysunuvchi
yuridik va jismoniy shaxslar doirasi tashkil etilgan bo’lib, turistik xizmatlar deb
hisoblangan xizmatlar ro‘yxati belgilandi. 6. Fransiya esa Turizm to'g'risidagi qonun turizm xizmati turlarni sotishni
ta'minlaydigan shartlarni belgilaydi. Shu bilan birga, mazkur qonunga bo‘ysunuvchi
yuridik va jismoniy shaxslar doirasi tashkil etilgan bo’lib, turistik xizmatlar deb
hisoblangan xizmatlar ro‘yxati belgilandi. 7.Finlyandiyada esa turistik faoliyat “Turlarni sotish huquqiga ega bo‘lgan
sub’yektlar to‘g‘risida”gi va “Turlar va turistik faoliyatni amalga oshirish
to‘g‘risida”gi qonunlar bilan tartibga solinadi. Bunga sabab esa Finlyandiyaning
Yevropa Ittifoqiga qo‘shilishi va Finlyandiya qonunlarini Yevropa Ittifoqi talablariga
muvofiqlashtirish zarurati bo’lgan. 8. Amerika Qo'shma Shtatlarida esa 1961-yilni o’zidayoq AQShning birinchi
milliy xalqaro turizm qonuni qabul qilingan. Ushbu qonundan AQSh Federal Savdo
Departamenti tarkibida faoliyat yuritgan Sayohat va Turizm Boshqarmasini (TTA)
yaratish nazarda tutilgan edi. Ammo vaqt o'tgach, 1981-yilda «Milliy turizm siyosati
to'g'risida»gi qonun qabul qilindi. Bunga qo’shimcha tarzda 1992-yil may oyida
"Turizm siyosati va eksportni rivojlantirish to'g'risida" gi qonun qabul qilindi. AQShning har bir shtatida turizmni rivojlantirish uchun ma’sul bo’lgan tegishli xizmat
mavjuddir. 9.Shveytsariya esa turizm bilan bog'liq barcha masalalarni o’zining eng
yirik milliy reklama agentligi bo'lgan Turizmning markaziy idorasi (CTC) bilan hal
qiladi.[16] O’zbekiston Respublikasida esa Turizmni rivojlantirish davlat qo’mitasi
O’zbekiston Respublikasida turizm sohasida davlat nazoratini amalga oshiruvchi
hamda ijro etuvchi hokimiyat organi sifatida xizmat qiladi. Xalqaro turistik faoliyatni
davlat nazorati shundan iboratki, Yurtimizda turizm iqtisodiyotning ustivor
tarmoqlaridan biri bo’lib hisoblanadi. Ichki va tashqi turizm, shuningdek, ijtimoiy va
madaniy turizmni qo’llab-quvvatlash va rivojlantirish davlat nazoratining ustivor
yo’nalishlari sifatida e’tirof qilingan. Turistik faoliyatni davlat tomonidan qilinadigan
nazorati quyidagicha amalga oshiriladi: WWW.HUMOSCIENCE.COM 741 bitimlar va shartnomalar tuzishning ob’yekti sifatida baholanadi hamda katta ahamiyat
kasb etadi. Bu borada amalga oshiraladigan xalqaro huquqiy faoliyatni tartibga solish
munosabatlarning negizida turuvchi xalqaro huquqiy normalarni amalda qo’llash
jarayonida, ayniqsa, muhim sanalib, xizmatlarni tashkil etishda ajralmas omil bo’lib
xizmat qiladi. • turizm sohasidagi munosabatlarni tartibga solish va takomillashtirishga
qaratilgan me’yoriy va huquqiy hujjatlarni yaratish; • turistik mahsulotni ichki va jahon turizm bozorlariga olib chiqishda yordam
berish; • turistik faoliyatni standartlashtirish va litsenziyalashtirishni amalga oshirish,
turistik mahsulotini sertifikatsiyadan o’tkazish; • O’zbekiston hududida mamlakatga kirish, chiqish va istiqomat qilish qoidalarini
o’rnatish; • turizmni rivojlantirish bo’yicha davlat dasturlarini yaratish va amalga oshirish
uchun to’g’ri byudjet mablag’larini ajratish; • turistlarning huquq va manfaatlarini himoya qilish, ularning xavfsizligini
ta’minlash; turizm faoliyatini kadrlar bilan ta’minlashni qo’llab-quvvatlash va turizm
sohasida ilmiy tadqiqotlarni rivojlantirish va boshqalardan iborat [17]. O’zbekiston hududida sayyohlik sohasini hozirgi zamon talablaridan bo’lgan
innovatsiya va raqamlashtirish orqali o’zgartirish zaruriyati mavjud. Egallanmagan
imkoniyat va imtiyoz sifatida endi rivojlanib kelayotgan agro va etno turizm kabi bozor
segmentlarini rivojlantirish lozimdir. Turizmning rivojlantirishning jahon amaliyoti
shuni ko’rsatadiki, mazkur mavjud industriyani yuqori daromadli sohaga aylantirish
uchun davlat hamda xususiy sektordagi barcha ishtirokchilarning sa’yi-harakatlarini
birlashtirish va mustahkamlash zarur. Bu borada, hukumatning roli xalqaro,
hukumatlararo va xususiy sektor darajalarida turizm sohasidagi rivojlanish siyosatini
moslashtirish hamda muvofiqlashtirish, shuningdek, rejalashtirishni ta’minlashdir. Mavjud mazkur muammolarni tezkor va sifatli yechish hamda xalqaro huquqiy
faoliyatlarni tartibga solishga doir bo’lgan qo’shimcha chora-tadbirlarni amalga
oshirish keyingi davrda O’zbekistonda turizm imkoniyatlarini rivojlantirishda muhim
rol o’ynaydi. WWW.HUMOSCIENCE.COM
Xulosa o’rnida shuni ta’kidlashimiz mumkinki, turizm va turizmga oid xizmatlar
davlatlarning ichki huquqida asosiy o’rin tutishi bilan bir qatorda, dunyoviy huquqiy
faoliyat doirasida, ayniqa, xalqaro huquqiy faoliyatda, hamda, xalqaro iqtisodiy 742 WWW.HUMOSCIENCE.COM Foydalanilgan adabiyotlar ro’yxati 1)
“Turizm to’g’risida”gi O’zbekiston Respublikasi Qonuni. 2019. 2)
O’zbekiston
Respublikasida
turizmni
jadal
rivojlantirishga
o
qo’shimcha chora-tadbirlar to’g’risida”gi O’zbekiston Respublikasi Prezidentini
f
i 2019
il 5
PF 5611 1)
“Turizm to’g’risida”gi O’zbekiston Respublikasi Qonuni. 2019. 2)
O’zbekiston
Respublikasida
turizmni
jadal
rivojlantirishga 1)
“Turizm to’g’risida”gi O’zbekiston Respublikasi Qonuni. 2019. 1)
“Turizm to’g’risida”gi O’zbekiston Respublikasi Qonuni. 2019. 1)
“Turizm to’g’risida”gi O’zbekiston Respublikasi Qonuni. 2019. 2)
O’zbekiston
Respublikasida
turizmni
jadal
rivojlantirishga
oid
qo’shimcha chora-tadbirlar to’g’risida”gi O’zbekiston Respublikasi Prezidentining
farmoni. 2019- yil 5-yanvar. PF-5611-son. 2) 3)
“O’zbekiston Respublikasi Turizmni rivojlantirish davlat qo’mitasi
faoliyatini tashkil etish to’g’risida” O’zbekiston Respublikasi Prezidentining qarori. 2016-yil 2- dekabr. PQ-2666-son. 4)
“O’zbekiston Respublikasi Turizmni rivojlantirish davlat qo’mitasining
faoliyatini yanada takomillashtirish chora-tadbirlari to’grisida”gi O’zbekiston
Respublikasi Prezidentining qarori. 2018-yil 6-fevral. PQ-3510-son. 5)
Travel and Tourism Contracts: Design of Sustainable Tourism Systems,
Sara Landini. Antezza, 2013. p-7. 5)
Travel and Tourism Contracts: Design of Sustainable Tourism Systems,
Sara Landini. Antezza, 2013. p-7. 6)
Manila decloration on world tourism.1980
7)
United Nations World Tourism Organization (UNWTO). 8)
The Hague decloration on tourism. 1989
9)
International Code for the Protection of Tourists. UNWTO. 10)
“Turizm to’g’risida”gi O’zbekiston Respublikasi Qonuni. 20.10.2019. 11)
"O‘zbekiston Respublikasida turizm sohasini yanada rivojlantirish chora- “Turizm to’g’risida”gi O’zbekiston Respublikasi Qonuni. 20.10.2019. 11)
"O‘zbekiston Respublikasida turizm sohasini yanada rivojlantirish chora-
tadbirlari to‘g‘risida"gi PF-5781-son. 13.08.2020. 12)
"O‘zbekiston
Respublikasida
turizmni
jadal
rivojlantirishga
oid
qo‘shimcha chora-tadbirlar to‘g‘risida"gi PQ-5611- son. 05.01.2019. WWW.HUMOSCIENCE.COM 743 13)
“O’zbekiston Respublikasida turizm sohasini tiklash va rivojlantirish
uchun qulay shart-sharoitlarni yaratish chora-tadbirlari to’g’risida”gi Vazirlar
Mahkamasi qarori. 433-son. 10.07.2020. 13)
O zbekiston Respublikasida turizm sohasini tiklash va rivojlantirish
uchun qulay shart-sharoitlarni yaratish chora-tadbirlari to’g’risida”gi Vazirlar
Mahkamasi qarori. 433-son. 10.07.2020. 14)
Quyi palataning matbuot xizmati: https://parliament.gov.uz
15)
“Kirish turizmini rivojlantirish chora-tadbirlari to‘g‘risida"gi O‘zbekiston
Respublikasi Prezidentining 2018-yil 6-fevraldagi PQ-3509-son qarori. 16)
O‘zbekiston Respublikasining “O‘zbekturizm” Milliy kompaniyasi bilan
Qirg‘iz Respublikasining sanoat, savdo va turizm vazirligi o‘rtasida imzolangan
“Turizm sohasida hamkorlik to’g’risida”gi Bitim. 03.10.2006. (11-son). 17)
“Development of Tourism Act”. 1969. 18)
O‘zbekiston Respublikasi Hukumati bilan Fransiya Respublikasi
Hukumati o‘rtasida imzolangan “Turizm sohasida hamkorlik to’g’risida”gi bitim. 18)
O zbekiston Respublikasi Hukumati bilan Fransiya Respublikasi
Hukumati o‘rtasida imzolangan “Turizm sohasida hamkorlik to’g’risida”gi bitim. WWW.HUMOSCIENCE.COM 744
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https://openalex.org/W3082753900
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https://hal.science/hal-03103329/file/1482.full.pdf
|
English
| null |
Phospholipase pPLAIIIα Increases Germination Rate and Resistance to Turnip Crinkle Virus when Overexpressed
|
Plant physiology
| 2,020
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cc-by
| 14,651
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To cite this version: Jin Hoon Jang, Ngoc Quy Nguyen, Bertrand Légeret, Fred Beisson, Yu-Jin Kim, et al.. Phospholipase
pPLAIIIα Increases Germination Rate and Resistance to Turnip Crinkle Virus when Overexpressed. Plant Physiology, 2020, 184 (3), pp.1482-1498. 10.1104/pp.20.00630. hal-03103329 Distributed under a Creative Commons Attribution 4.0 International License Jin Hoon Jang,a Ngoc Quy Nguyen,a Bertrand Légeret,b Fred Beisson,b Yu-Jin Kim,c Hee-Jung Sim,d,e and
Ok Ran Leea,2,3 aDepartment of Applied Plant Science, College of Agriculture and Life Science, Chonnam National University,
Gwangju 61186, Republic of Korea
b aDepartment of Applied Plant Science, College of Agriculture and Life Science, Chonnam National University,
Gwangju 61186, Republic of Korea
bBiosciences and Biotechnologies Institute of Aix-Marseille, Commissariat à l’Énergie Atomique et aux Énergies
Alternatives, Centre National de la Recherche Scientifique and Aix-Marseille University, Commissariat à
l’Énergie Atomique et aux Énergies Alternatives Cadarache, 13108 Saint-Paul-lez-Durance, France
cDepartment of Life Science and Environmental Biochemistry, Pusan National University, Miryang, 50463,
Republic of Korea
dG
D
t
t
f E
i
t l T
i
l
d Ch
i t
K
I
tit t
f T
i
l
Ji j
i dGyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology, Jinju-si,
52834, Republic of Korea nome Engineering, Institute for Basic Science, Daejeon 34126, Republic of Korea Center for Genome Engineering, Institute for Basic Science, Daejeon 34126, Republic ORCID IDs: 0000-0003-0306-5283 (J.H.J.); 0000-0001-9995-7387 (F.B.); 0000-0003-2562-615X (Y.-J.K.); 0000-0002-5205-0080 (H.-J.S.);
0000 0002 6928 5863 (O R L ) ORCID IDs: 0000-0003-0306-5283 (J.H.J.); 0000-0001-9995-7387 (F.B.); 0000-0003-2562-615X (Y.-J.K.); 0000-0002-5205-0080 (H.-J.S.);
0000-0002-6928-5863 (O.R.L.). Patatin-related phospholipase As (pPLAs) are major hydrolases acting on acyl-lipids and play important roles in various plant
developmental processes. pPLAIII group members, which lack a canonical catalytic Ser motif, have been less studied than other
pPLAs. We report here the characterization of pPLAIIIa in Arabidopsis (Arabidopsis thaliana) based on the biochemical and
physiological characterization of pPLAIIIa knockouts, complementants, and overexpressors, as well as heterologous expression
of the protein. In vitro activity assays on the purified recombinant protein showed that despite lack of canonical phospholipase
motifs, pPLAIIIa had a phospholipase A activity on a wide variety of phospholipids. Overexpression of pPLAIIIa in Arabidopsis
resulted in a decrease in many lipid molecular species, but the composition in major lipid classes was not affected. Fluorescence
tagging indicated that pPLAIIIa localizes to the plasma membrane. Although Arabidopsis pplaIIIa knockout mutants showed
some phenotypes comparable to other pPLAIIIs, such as reduced trichome length and increased hypocotyl length, control of seed
size and germination were identified as distinctive pPLAIIIa-mediated functions. Expression of some PLD genes was strongly
reduced in the pplaIIIa mutants. Overexpression of pPLAIIIa caused increased resistance to turnip crinkle virus, which associated
with a 2-fold higher salicylic acid/jasmonic acid ratio and an increased expression of the defense gene pathogenesis-related protein1. HAL Id: hal-03103329
https://hal.science/hal-03103329v1
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abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1482 Plant Physiology, November 2020, Vol. 184, pp. 1482–1498, www.plantphysiol.org 2020 American Society of Plant Biologists. All Rights Reserved. https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. 1482 Plant Physiology, November 2020, Vol. 184, pp. 1482–1498, www.plantphysiol.org 2020 American Society of Plant Biologists. All Rights Reserved.
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. Phospholipase pPLAIIIa Increases Germination Rate and
Resistance to Turnip Crinkle Virus
when Overexpressed1[OPEN] Jin Hoon Jang,a Ngoc Quy Nguyen,a Bertrand Légeret,b Fred Beisson,b Yu-Jin Kim,c Hee-Jung Sim,d,e and
Ok Ran Leea,2,3 Jin Hoon Jang,a Ngoc Quy Nguyen,a Bertrand Légeret,b Fred Beisson,b Yu-Jin Kim
Ok Ran Leea,2,3 020, Vol. 184, pp. 1482–1498, www.plantphysiol.org 2020 American Society of Plant Biologists. All Rights Reserved. https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. RESULTS Despite Lacking Canonical Motifs, pPLAIIIa has Retained
Lipase Activity Arabidopsis pPLAIIIa (At2g39220) was represented
by a single gene encoding a protein of 499 amino acids
with a predicted pI of 6.24 and a molecular mass of 54.5
kD. Like all Arabidopsis pPLAIII proteins, pPLAIIIa
lacked the Ser of the putative Ser-Asp catalytic dyad
because Ser present in the canonical GxSxG motif was
replaced with G (Fig. 1A). However, the second residue
of the putative catalytic Ser-Asp dyad, Asp, was present
in the DGG motif. In addition, it can be noted that the
phosphate or anion binding element DSGGXXG was
not completely conserved in the pPLAIIIa protein be-
cause the second Gly was replaced with Ser. y
p
Arabidopsis pPLAIIIa thus lacked the canonical
phospholipase motif for the catalytic Ser found in other
characterized pPLAIIIs and one could question whether
this protein really possessed phospholipase activity. The recombinant pPLAIIIa protein His-tagged at the
C-terminal end was therefore expressed in Escherichia
coli and purified (Supplemental Fig. S1). In vitro en-
zymatic assays showed that the His-tagged pPLAIIIa
had an acyl-ester hydrolase activity on each of the four
major Arabidopsis phospholipid classes, with a slightly
higher activity on phosphatidic acid (PA) than on phos-
phatidylcholine (PC), phosphatidylethanolamine (PE), or
phosphatidylglycerol (PG; Fig. 1C). Taken together, these results show that pPLAIIIa is a
noncanonical phospholipase A that hydrolyzes various
phospholipids in vitro. y
y
To further shed light on the function of pPLAIIIa, we
studied the activity of the recombinant protein in vitro, 1This work was supported by the National Research Foundation of
Korea, the Ministry of Science, Information and Communication
Technology, and Future Planning (Basic Science Research Program
grant no. 2019R1A2C1004140), the New Breeding Technologies
Development Program of the Rural Development Administration,
Republic of Korea (project no. PJ01532502), the European Regional
Development Fund, the Région Sud, the French Ministry of Research,
and the Commissariat à l’Énergie Atomique et aux Énergies
Alternatives. Jin Hoon Jang,a Ngoc Quy Nguyen,a Bertrand Légeret,b Fred Beisson,b Yu-Jin Kim,c Hee-Jung Sim,d,e and
Ok Ran Leea,2,3 In
Arabidopsis, pPLAIIId is involved in plant response to
auxin (Labusch et al., 2013). Moreover, in rice (Oryza
sativa), OspPLAIIIa overexpression and knockout (KO)
have an opposite effect on the expression of the growth
repressor SLENDER1 in the gibberellin signaling path-
way (Liu et al., 2015). Overexpressors (OE) of pPLAIIIs
display similar stunted growth patterns with additional
functions, such as reduced cellulose content in pPLAIIIb-
OE (Li et al., 2011) or lignin in PgpPLAIIIb-OE and
pPLAIIIa-OE (Jang et al., 2019; Jang and Lee, 2020a,
2020b), and increased seed oil in pPLAIIId-OE (Li et al.,
2013, 2015). Activation tagging of pPLAIIId (STURDY;
Huang et al., 2001) also results in decreased longitudinal
cell elongation and stunted growth, as observed in
pPLAIIIb-OE. The recessive rice mutant dep3 with
OspPLAIIId deficiency displays a dense and erect phe-
notype with short, wide epidermal cells (Qiao et al.,
2011). A comparative proteomic analysis of pPLAIIId-
OE and wild type has shown that one protein signifi-
cantly differs between the OE and wild-type line, and
it was identified as MICROTUBULE-ASSOCIATED
PROTEIN18 (Zheng et al., 2014). Many physiological
functions have been thus associated with pPLAIIIs in
Arabidopsis or other plants, but the molecular path-
ways involved mostly remain to be elucidated. characterized overexpression and KO mutants in Ara-
bidopsis at physiological and molecular levels, and
performed lipidomic analyses. Jin Hoon Jang,a Ngoc Quy Nguyen,a Bertrand Légeret,b Fred Beisson,b Yu-Jin Kim,c Hee-Jung Sim,d,e and
Ok Ran Leea,2,3 These results therefore show that pPLAIIIa has functions that overlap with those of other pPLAIIIs but also distinctive functions, such
as the control of seed germination. This study also provides new insights into the pathways downstream of pPLAIIIa. Lipases are a diverse group of hydrolases that break
down acyl-lipids. Most of them hydrolyze the carboxyl
ester bond between a fatty acid and the glycerol backbone,
but lipase sequences diverge widely, and even the loose
GXSXG esterase consensus for the catalytic Ser is not al-
ways present. Based on their preferred substrate, in vitro
lipases are usually classified into triacylglycerol lipases,
phospholipases, galactolipases, and others. However,
many lipases often act in vitro on a variety of lipid classes,
and the determination of their physiological role is
not trivial. Genome analysis of Arabidopsis (Arabidopsis
thaliana) has indicated that there are as many genes
annotated as potentially involved in lipid breakdown
as there are involved in lipid biosynthesis (Li-Beisson
et al., 2013). Elucidation of the cellular function of the
many putative plant lipases is thus a major challenge. cellular biology, such as signal transduction, cell growth
regulation, and membrane remodeling in response to
environmental stresses and lipid metabolism (Wang,
2001; Meijer and Munnik, 2003; Ryu, 2004; Matos and
Pham-Thi, 2009; Scherer et al., 2010). Phospholipases of
A-type (PLA) hydrolyze the carboxyl ester bond specif-
ically at the sn-1 or sn-2 position of glycerophospholipids
or in some cases at both positions. Plant PLA2 families
are classified into two groups: low-molecular–weight
PLA2s (PLA2a, PLA2b, PLA2g, and PLA2d) and patatin-
related PLAs (pPLAs), the latter of which are homologous
to the potato (Solanum tuberosum) tuber storage protein
patatin. The pPLAs act on glycerogalactolipids as well
as glycerophospholipids to release free fatty acids (FFAs)
and the corresponding lysolipids (Lee et al., 2010; Scherer
et al., 2010). y p
p
p
j
g
By hydrolyzing membrane phosphoglycerolipids,
phospholipases participate in many aspects of plant )
In Arabidopsis, the 10 members of the pPLA family
have been classified into three groups based on gene 1482 Plant Physiology, November 2020, Vol. 184, pp. 1482–1498, www.plantphysiol.org 2020 American Society of https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. 2Author for contact: mpizlee@jnu.ac.kr.
3 Jin Hoon Jang,a Ngoc Quy Nguyen,a Bertrand Légeret,b Fred Beisson,b Yu-Jin Kim,c Hee-Jung Sim,d,e and
Ok Ran Leea,2,3 pPLAIIIa Affects Germination Rate structure and amino acid sequence similarity: pPLAI,
pPLAII (pPLAIIa, pPLAIIb, pPLAIIg, pPLAIId, and
pPLAII«), and pPLAIII (pPLAIIIa, pPLAIIIb, pPLAIIIg,
and pPLAIIId; Holk et al., 2002; Scherer et al., 2010). Both pPLAI and pPLAIIs are involved in plant responses
to pathogens, auxin signaling, and phosphate defi-
ciency. Although the recently characterized pPLAIIIs
lack the canonical catalytic Ser-containing motif GXSXG
(Holk et al., 2002; Scherer et al., 2010), some pPLAIIId
and pPLAIIIb possess a lipase activity with broad sub-
strate specificity (Li et al., 2011, 2013; Lin et al., 2011). In
Arabidopsis, pPLAIIId is involved in plant response to
auxin (Labusch et al., 2013). Moreover, in rice (Oryza
sativa), OspPLAIIIa overexpression and knockout (KO)
have an opposite effect on the expression of the growth
repressor SLENDER1 in the gibberellin signaling path-
way (Liu et al., 2015). Overexpressors (OE) of pPLAIIIs
display similar stunted growth patterns with additional
functions, such as reduced cellulose content in pPLAIIIb-
OE (Li et al., 2011) or lignin in PgpPLAIIIb-OE and
pPLAIIIa-OE (Jang et al., 2019; Jang and Lee, 2020a,
2020b), and increased seed oil in pPLAIIId-OE (Li et al.,
2013, 2015). Activation tagging of pPLAIIId (STURDY;
Huang et al., 2001) also results in decreased longitudinal
cell elongation and stunted growth, as observed in
pPLAIIIb-OE. The recessive rice mutant dep3 with
OspPLAIIId deficiency displays a dense and erect phe-
notype with short, wide epidermal cells (Qiao et al.,
2011). A comparative proteomic analysis of pPLAIIId-
OE and wild type has shown that one protein signifi-
cantly differs between the OE and wild-type line, and
it was identified as MICROTUBULE-ASSOCIATED
PROTEIN18 (Zheng et al., 2014). Many physiological
functions have been thus associated with pPLAIIIs in
Arabidopsis or other plants, but the molecular path-
ways involved mostly remain to be elucidated. f
h
h d l
h
h f
f structure and amino acid sequence similarity: pPLAI,
pPLAII (pPLAIIa, pPLAIIb, pPLAIIg, pPLAIId, and
pPLAII«), and pPLAIII (pPLAIIIa, pPLAIIIb, pPLAIIIg,
and pPLAIIId; Holk et al., 2002; Scherer et al., 2010). Both pPLAI and pPLAIIs are involved in plant responses
to pathogens, auxin signaling, and phosphate defi-
ciency. Although the recently characterized pPLAIIIs
lack the canonical catalytic Ser-containing motif GXSXG
(Holk et al., 2002; Scherer et al., 2010), some pPLAIIId
and pPLAIIIb possess a lipase activity with broad sub-
strate specificity (Li et al., 2011, 2013; Lin et al., 2011). Plant Physiol. Vol. 184, 2020 3Senior author. Jang et al. Strong expression was also observed
in the hydathodes of young leaves and trichomes
(Fig. 2J). Overall, GUS expression was observed in all
organs, with more restriction in the vasculature. PropPLAIIIa::GUS was expressed highly in hypocotyls
elongating in the darkness compared with those grown
in the light (Fig. 2H). Cross-sectional images of stems
indicate strong expression of pPLAIIIa in the xylem and
phloem (Fig. 2I). Strong expression was also observed
in the hydathodes of young leaves and trichomes
(Fig. 2J). Overall, GUS expression was observed in all
organs, with more restriction in the vasculature. Jang et al. Jang et al. PropPLAIIIa::GUS was expressed highly in hypocotyls
elongating in the darkness compared with those grown
in the light (Fig. 2H). Cross-sectional images of stems
indicate strong expression of pPLAIIIa in the xylem and
phloem (Fig. 2I). Strong expression was also observed
in the hydathodes of young leaves and trichomes
(Fig. 2J). Overall, GUS expression was observed in all
organs, with more restriction in the vasculature. KO, OE, and Subcellular Localization of
Arabidopsis pPLAIIIa
(Fig. 3E) showed that pPLAIIIa was overexpressed
very strongly in line 6 (440-fold), line 7 (420-fold),
and line 13 (800-fold), and moderately in line 8 (5-fold). No transcripts were detected for two transfer DNA
insertion lines in pPLAIIIa, indicating that they were
KO mutants (Fig. 3E). Transcript expression levels
corresponded to protein levels (Fig. 3B). Comple-
mentation lines (pPLAIIIa-COM) generated by cross-
ing each OE line in the KO line 1 background
(SAIL830G12) still displayed overexpression of pPLAIIIa
but compromised where line 7 overexpressed 87-fold
and line 13, 94-fold. This complementation result
could be caused by overexpressing using the 35S
Figure 1. Distinctive conserved motifs of
pPLAIIIa and its lipase activity. A, Align-
ment of pPLAIIIa with other pPLAIIIs and
two pPLAIIs from Arabidopsis. Amino
acid sequences were analyzed using the
pairwise sequence alignment program
ClustalW (http://www.clustal.org/clustal2/). Multiple sequence alignment was per-
formed using the program BioEdit (v.7.1.9;
https://bioedit.software.informer.com/7.1/). B, Immunoblot analysis of recombinant
pPLAIIIa protein using anti-63 His tag
antibody on E. coli purified recombinant
proteins. C, In vitro enzymatic assay of
recombinant pPLAIIIa protein using a NEFA
kit. pPLAIIIa fusion protein and 0.5 mM of
each substrate (16:0-18:1 PA, PC, PE, and
PG) were incubated at 30°C for 60 min. Data
represent the mean 6 SE of four independent
biological replicates. Data were analyzed
by one-way ANOVA. Means with different
lowercase letters represent significantly dif-
ferent (P , 0.05), according to Scheffe’s test. Figure 1. Distinctive conserved motifs of
pPLAIIIa and its lipase activity. A, Align-
ment of pPLAIIIa with other pPLAIIIs and
two pPLAIIs from Arabidopsis. Amino
acid sequences were analyzed using the
pairwise sequence alignment program
ClustalW (http://www.clustal.org/clustal2/). Multiple sequence alignment was per-
formed using the program BioEdit (v.7.1.9;
https://bioedit.software.informer.com/7.1/). B, Immunoblot analysis of recombinant
pPLAIIIa protein using anti-63 His tag
antibody on E. coli purified recombinant
proteins. C, In vitro enzymatic assay of
recombinant pPLAIIIa protein using a NEFA
kit. https://plantphysiol.org
Downloaded on March 11, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. Jang et al. This complementation result
could be caused by overexpressing using the 35S
promoter. Interestingly, transcripts for the closely
related pPLAIIIb (72% identity with pPLAIIIa),
pPLAIIIg (56%), and pPLAIIId (34%; Supplemental
Fig. S2) were slightly modulated by the expression
level of pPLAIIIa (Supplemental Fig. S3). In seedling
stages (Fig. 3F), the hypocotyl length of the pplaIIIa
mutants was longer than that of controls, but OE
lines were shorter than controls (Fig. 3G). The root
lengths were only shorter in two of the OE lines and
unaltered in the KO mutant lines (Fig. 3H). Both OE
lines 6 and 7 could be considered as ectopic lines
with similar levels of expression, and OE line 13 was
the most highly expressing line. However, OE line 8
was the most moderate line that was perfectly com-
plemented in KO line 1 (Fig. 3, G and H). From the
seedling stages, all OE lines displayed stunted and
dwarf phenotypes with thicker cotyledons (Fig. 3F), PropPLAIIIa::GUS was expressed highly in hypocotyls
elongating in the darkness compared with those grown
in the light (Fig. 2H). Cross-sectional images of stems
indicate strong expression of pPLAIIIa in the xylem and
phloem (Fig. 2I). Strong expression was also observed
in the hydathodes of young leaves and trichomes
(Fig. 2J). Overall, GUS expression was observed in all
organs, with more restriction in the vasculature. (Fig. 3E) showed that pPLAIIIa was overexpressed
very strongly in line 6 (440-fold), line 7 (420-fold),
and line 13 (800-fold), and moderately in line 8 (5-fold). No transcripts were detected for two transfer DNA
insertion lines in pPLAIIIa, indicating that they were
KO mutants (Fig. 3E). Transcript expression levels
corresponded to protein levels (Fig. 3B). Comple-
mentation lines (pPLAIIIa-COM) generated by cross-
ing each OE line in the KO line 1 background
(SAIL830G12) still displayed overexpression of pPLAIIIa
but compromised where line 7 overexpressed 87-fold
and line 13, 94-fold. This complementation result
could be caused by overexpressing using the 35S
promoter. Interestingly, transcripts for the closely
related pPLAIIIb (72% identity with pPLAIIIa),
pPLAIIIg (56%), and pPLAIIId (34%; Supplemental
Fig. S2) were slightly modulated by the expression
level of pPLAIIIa (Supplemental Fig. S3). In seedling
stages (Fig 3F) the hypocotyl length of the pplaIIIa PropPLAIIIa::GUS was expressed highly in hypocotyls
elongating in the darkness compared with those grown
in the light (Fig. 2H). Cross-sectional images of stems
indicate strong expression of pPLAIIIa in the xylem and
phloem (Fig. 2I). Jang et al. pPLAIIIa fusion protein and 0.5 mM of
each substrate (16:0-18:1 PA, PC, PE, and
PG) were incubated at 30°C for 60 min. Data
represent the mean 6 SE of four independent
biological replicates. Data were analyzed
by one-way ANOVA. Means with different
lowercase letters represent significantly dif-
ferent (P , 0.05), according to Scheffe’s test. (Fig. 3E) showed that pPLAIIIa was overexpressed
very strongly in line 6 (440-fold), line 7 (420-fold),
and line 13 (800-fold), and moderately in line 8 (5-fold). No transcripts were detected for two transfer DNA
insertion lines in pPLAIIIa, indicating that they were
KO mutants (Fig. 3E). Transcript expression levels
corresponded to protein levels (Fig. 3B). Comple-
mentation lines (pPLAIIIa-COM) generated by cross-
ing each OE line in the KO line 1 background
(SAIL830G12) still displayed overexpression of pPLAIIIa
but compromised where line 7 overexpressed 87-fold
and line 13, 94-fold. This complementation result
could be caused by overexpressing using the 35S
promoter. Interestingly, transcripts for the closely
related pPLAIIIb (72% identity with pPLAIIIa),
pPLAIIIg (56%), and pPLAIIId (34%; Supplemental
Fig. S2) were slightly modulated by the expression
level of pPLAIIIa (Supplemental Fig. S3). In seedling
stages (Fig. 3F), the hypocotyl length of the pplaIIIa
mutants was longer than that of controls, but OE
lines were shorter than controls (Fig. 3G). The root
lengths were only shorter in two of the OE lines and
unaltered in the KO mutant lines (Fig. 3H). Both OE
lines 6 and 7 could be considered as ectopic lines
with similar levels of expression, and OE line 13 was
the most highly expressing line. However, OE line 8
was the most moderate line that was perfectly com-
plemented in KO line 1 (Fig. 3, G and H). From the
seedling stages, all OE lines displayed stunted and
dwarf phenotypes with thicker cotyledons (Fig. 3F), (Fig. 3E) showed that pPLAIIIa was overexpressed
very strongly in line 6 (440-fold), line 7 (420-fold),
and line 13 (800-fold), and moderately in line 8 (5-fold). No transcripts were detected for two transfer DNA
insertion lines in pPLAIIIa, indicating that they were
KO mutants (Fig. 3E). Transcript expression levels
corresponded to protein levels (Fig. 3B). Comple-
mentation lines (pPLAIIIa-COM) generated by cross-
ing each OE line in the KO line 1 background
(SAIL830G12) still displayed overexpression of pPLAIIIa
but compromised where line 7 overexpressed 87-fold
and line 13, 94-fold. Spatial and Temporal Expression Patterns of pPLAIIIa To determine the expression pattern of pPLAIIIa in
Arabidopsis, we generated PropPLAIIIa::GUS trans-
formants using 2,087 bp upstream from ATG with 15-
bp coding sequence (total 2,105 bp). The GUS reporter
gene was expressed in most organs including inflores-
cences, flowers, siliques, stems, and leaves (Fig. 2) but
displayed further distinct spatial or temporal expres-
sion patterns compared to other pPLAIIIs promoters
(Dong et al., 2014). pPLAIIIa was highly expressed in
roots, with greater restriction in the vasculature and
meristematic zones of the lateral roots (Fig. 2, A and
C–F). In germinating seeds, GUS activity was observed
in embryo cotyledons and vasculature of roots during
testa rupture and radical emergence (Fig. 2, C and D). Vasculature expression in sepals and petals was also
observed in whole flower organs (Fig. 2B). In cotyle-
dons and true leaves, stomata expression was restricted
in the inner wall of the guard cell region (Fig. 2G). The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy de-
scribed in the Instructions for Authors (www.plantphysiol.org) is: Ok
Ran Lee (mpizlee@jnu.ac.kr). O.R.L. conceived the project and designed the experiments; J.H.J.,
N.Q.N., and Y.-J.K. performed the experiments, except for the lipido-
mics experiment, which was performed by B.L., and hormone anal-
ysis, which was performed by H.-J.S., O.R.L., and F.B.; J.H.J. analyzed
the data and wrote the article. [OPEN]Articles can be viewed without a subscription. [OPEN]Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.20.00630 www.plantphysiol.org/cgi/doi/10.1104/pp.20.00630 Plant Physiol. Vol. 184, 2020 1483 (Fig. 3E) showed that pPLAIIIa was overexpressed
very strongly in line 6 (440-fold), line 7 (420-fold),
and line 13 (800-fold), and moderately in line 8 (5-fold). No transcripts were detected for two transfer DNA
insertion lines in pPLAIIIa, indicating that they were
KO mutants (Fig. 3E). Transcript expression levels
corresponded to protein levels (Fig. 3B). Comple-
mentation lines (pPLAIIIa-COM) generated by cross-
ing each OE line in the KO line 1 background
(SAIL830G12) still displayed overexpression of pPLAIIIa
but compromised where line 7 overexpressed 87-fold
and line 13, 94-fold. This complementation result
could be caused by overexpressing using the 35S
promoter. Interestingly, transcripts for the closely
related pPLAIIIb (72% identity with pPLAIIIa),
pPLAIIIg (56%), and pPLAIIId (34%; Supplemental
Fig. S2) were slightly modulated by the expression
level of pPLAIIIa (Supplemental Fig. S3). In seedling
stages (Fig. Spatial and Temporal Expression Patterns of pPLAIIIa 3F), the hypocotyl length of the pplaIIIa
mutants was longer than that of controls, but OE
lines were shorter than controls (Fig. 3G). The root
lengths were only shorter in two of the OE lines and
unaltered in the KO mutant lines (Fig. 3H). Both OE
lines 6 and 7 could be considered as ectopic lines
with similar levels of expression, and OE line 13 was
the most highly expressing line. However, OE line 8
was the most moderate line that was perfectly com-
plemented in KO line 1 (Fig. 3, G and H). From the
seedling stages, all OE lines displayed stunted and
dwarf phenotypes with thicker cotyledons (Fig. 3F), KO, OE, and Subcellular Localization of
Arabidopsis pPLAIIIa To study the function of pPLAIIIa, we performed
gain- and loss-of-function experiments in Arabidopsis. We first isolated homozygous transfer DNA–insertion
mutants for pPLAIIIa (Fig. 3A). In addition, the full-length
genomic DNA sequence of Arabidopsis pPLAIIIa was
overexpressed in Arabidopsis under the control of the
35S promoter with yellow fluorescence protein (YFP)-
or monomeric red fluorescent protein (mRFP)-tagging
at the C-terminal end. Immunoblotting using mRFP
antibody detected expected size bands only visible in
OE lines but not in KO lines (Fig. 3B). Both C-terminal
YFP and mRFP tagging showed that pPLAIIIa local-
ized to plasma membranes (PMs) in root cells (Fig. 3, C
and D). The fluorescence signal of PM was separated
from the cell wall, indicating its signal is not wall-
associated (Fig. 3D). Quantification of pPLAIIIa tran-
script levels in four independent homozygous OE lines 1484 Plant Physiol. Vol. 184, 2020 https://plantphysiol.org
Downloaded on March 11, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. pPLAIIIa Affects Germination Rate Figure 2. Spatial and temporal gene expression patterns of pPLAIIIa in Arabidopsis. Histochemical analysis of GUS expression
harboring PropPLAIIIa::GUS at different developmental stages. A to J, GUS expression in a fully grown plant (A), a floral organ (B),
a 1-d-old seedling (C), 2-d-old seedlings (D), 4-d-old seedlings (E), the vasculature of the roots and the meristematic zone of the
lateral root (F), the inner cell wall of a guard cell (G), the light- and dark-dependent expression in 6-d-old seedlings (H), the
vasculature of the apical and basal stem (I), and the maturecauline leaf (J). All imageswere brightness-adjusted. Scale bars 5 100 mm. Figure 2. Spatial and temporal gene expression patterns of pPLAIIIa in Arabidopsis. Histochemical analysis of GUS expression
harboring PropPLAIIIa::GUS at different developmental stages. A to J, GUS expression in a fully grown plant (A), a floral organ (B),
a 1-d-old seedling (C), 2-d-old seedlings (D), 4-d-old seedlings (E), the vasculature of the roots and the meristematic zone of the
lateral root (F), the inner cell wall of a guard cell (G), the light- and dark-dependent expression in 6-d-old seedlings (H), the
vasculature of the apical and basal stem (I), and the maturecauline leaf (J). All imageswere brightness-adjusted. Scale bars 5 100 mm. which were also confirmed in the subsequent growth
stages. https://plantphysiol.org
Downloaded on March 11, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. KO, OE, and Subcellular Localization of
Arabidopsis pPLAIIIa mutant and two strong pPLAIIIa-OE lines, and 115
molecular species of acyl-lipids (including phospho-
lipids, galactolipids, and sulfolipids) were analyzed by
liquid chromatography tandem mass spectrometry
(LC-MS/MS; Fig. 4; Supplemental Figs. S4 and S5). Plant Physiol. Vol. 184, 2020 Lipidomics Reveal a Wide Range of Acyl-Lipids Are
Decreased in pPLAIIIa-OE on a Seedling-Weight Basis This
included major phospholipid classes, such as the
mostly extraplastidial PC and PE and the plastidial/
mitochondrial PG, the minor phospholipid class PA,
and some purely plastidial lipid classes such as the
galactolipid monogalactosyldiacylglycerol (MGDG)
and digalactosyldiacylglycerol (DGDG), and the sul-
folipid class sulfoquinovosyldiacylglycerol (SQDG). For plastidial lipids, both “prokaryotic” species (C18/
C16, i.e. C34) and “eukaryotic” species (C18/C18, i.e. C36) were affected (Fig. 4C). The reduction was almost
evenly distributed among all glycerolipid classes, and
the lipid class composition was thus not significantly
affected in the two OE lines (Supplemental Fig. S5). In
the OE lines, a reduction was seen in most of the major
molecular species (Fig. 4C) and the minor ones also
(Supplemental Fig. S4). The PA class appeared to be
particularly impacted with almost all molecular species
of PA reduced by 30% to 50% (Fig. 4C). Lysolipids were
not significantly changed, except for the lysoMGDG
and lysoPG species, which were significantly reduced
in both OE lines. No accumulation of a particular FFA
species (Fig. 4C) or of total FFAs (Fig. 4B) was measured
in the OE lines. p
(
pp
g
)
At the cellular level, when pPLAIIIa was overex-
pressed, longitudinal elongation patterns from cells
were reduced as observed in stems (Fig. 5H) and
flowers (Fig. 5F; Supplemental Fig. S7A); conversely,
stem cells expanded transversely (Fig. 5I). However, the
number of cells per stem cross section was similar in
wild-type, KO, OE, and COM lines (Fig. 5J). Here, OE
line 8 was again solely complemented in the KO back-
ground, and OE line 13 displayed partial complemen-
tation in stem thickness (Fig. 5I). The effect of pPLAIIIa
overexpression on cell size and shape was also ob-
served on trichomes (Supplemental Fig. S7, B and C)
and pollen grains (Fig. 5K). Trichomes are predomi-
nantly three-branched on the adaxial surfaces of the
rosette and cauline leaves of wild type (Higginson et al.,
2003). The overall trichome size was strongly reduced
in pPLAIIIa-OE (Supplemental Fig. S7, B and C), and a
greater number of two-branched trichomes were ob-
served compared with that in control (Supplemental
Fig. S7B). While overexpression of pPLAIIIa had a
strong effect on the sizes of many organs, silencing of
the gene did not produce drastic effects in Arabidopsis
plants. However, some differences occurred in KO
mutants compared with Col-0, including an increase in
length of hypocotyls (Fig. Lipidomics Reveal a Wide Range of Acyl-Lipids Are
Decreased in pPLAIIIa-OE on a Seedling-Weight Basis 3, F and G) and petioles
(Supplemental Fig. S6). This observation suggests the
possible involvement of pPLAIIIa in hormonal growth
control by auxin, ethylene, and gibberellin that is
essential for hypocotyl and petiole elongation (de Wit
et al., 2016; Yang and Li, 2017) in wild-type plants. In conclusion, lipidomic analyses show that total
lipid amount per FW and the composition in glycer-
olipid molecular species remained unchanged in the
pplaIIIa KO mutant. In OE lines, FFAs did not accu-
mulate, and composition in glycerolipid classes was not
significantly changed in either of the OE lines. Lipidomics Reveal a Wide Range of Acyl-Lipids Are
Decreased in pPLAIIIa-OE on a Seedling-Weight Basis Lipidomics Reveal a Wide Range of Acyl-Lipids Are
Decreased in pPLAIIIa-OE on a Seedling-Weight Basis g
pp
g
In the pplaIIIa KO mutant, analysis of the lipid mo-
lecular species did not reveal significant differences,
including in the phospholipid species potentially lo-
calized to the PM (Fig. 4C; Supplemental Fig. S4). In OE
lines, total acyl-lipids were reduced ;25% on a seedling To determine which lipid molecular species may be
affected in the knockout or overexpressor plants, to-
tal lipids were extracted from seedlings of a pplaIIIa 1485 Plant Physiol. Vol. 184, 2020 https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. Jang et al. Fi
KO OE
d
l
i
li
f
PLAIII
i A
bid
i
A Th
f
DNA i
i
i
i
A
bid
i Figure 3. KO, OE, and complementation lines for pPLAIIIa in Arabidopsis. A, The transfer DNA insertion sites in two Arabidopsis
pplallla KO mutants and a diagram showing the OE construct of pPLAIIIa (pPLAIIIa-OE) under the control of the 35S promoter
with YFP or mRFP fusion at the C terminus. B, Immunoblotting of mRFP-tagged pPLAIIIa from Col-0, vector control, KO, and OE
plants. After SDS-PAGE separation, protein was immunoblotted with anti-mRFPantibody. C, Subcellular localization of pPLAIIIa-
YFP in the PM merged with FM4-64. Scale bars 5 10 mm. D, Plasmolysis of root epidermal cells of the pPLAIIIa-mRFP with 0.2 M
of NaCl for 1 min. Scale bars 5 10 mm. E, Transcript levels of pPLAIIIa genes in the controls, KO, OE, and complementation
(COM) lines as measured by RT-qPCR. n 5 3. F, Phenotypes of 4-d-old seedlings. Scale bar 5 1 cm. G and H, The hypocotyl length 1486 Plant Physiol. Vol. 184, 2020 https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. pPLAIIIa Affects Germination Rate (Fig. 5, F and G), hypocotyls (Fig. 3G), roots (Fig. 3H),
and petioles (Supplemental Fig. S6). fresh weight (FW) basis in both lines compared to
the vector control (Fig. 4A). Significant decreases
were observed in many lipid classes (Fig. 4B). Figure 3. (Continued.)
(G) and root length (H) of the controls, for the KO, OE, and COM lines. n 5 19 to 43. In E, G, and H, data represent the mean 6 SE
(SE) of independent replicates. Asterisks indicate significant difference compared with the controls using Student’s t test (*P , 0.05
and **P , 0.01). Col-0 and an empty vector line were used as control for mutant and OE lines, respectively. Plant Physiol. Vol. 184, 2020 pPLAIIIa Expression Level Affects the Size and Shape of
Many Cells and Organs Transversely expanded cell morphology with altered
longitudinal
cell
elongation
has
been
previously
reported after the overexpression of pPLAIIIb and
pPLAIIId (Huang et al., 2001; Li et al., 2011, 2013; Dong
et al., 2014). We thus investigated the possible role of
pPLAIIIa in Arabidopsis growth and development by
performing a detailed phenotypic analysis of several
organs in OE and KO lines for pPLAIIIa. Overall, OE
plants were, on average, 9 cm shorter in strongly
expressing lines compared with the controls (Fig. 5A). The size of the rosette leaves decreased, and the number
of leaves decreased with increasing pPLAIIIa expres-
sion (Fig. 5, B and C). In three strong pPLAIIIa-OE lines
(lines 6, 7, and 13), leaves were 1.7-fold thicker and
contained more water (3%) on average (Fig. 5, D and E). A reduction in organ size in pPLAIIIa-OE lines was also
clearly seen in other organs such as flowers and siliques An exception to the reduction in organ size in OE
lines occurred in seeds, which increased in width
without alteration in length (Fig. 6, A and B). Consis-
tently, seed weight increased 23% in pPLAIIIa-OE (line
13) and decreased 10% in KO mutants (Fig. 6C), thus
showing that pPLAIIIa is involved in seed development
in wild-type plants. In highly overexpressing OE lines,
it was clear that the increased seed weights were at the
expense of seed number per silique (Fig. 6D). p
p
q
g
The fact that seed weights decreased in KO mutants
prompted us to check seed germination rates (Zhong
et al., 2016) and kinetics. After 20 h in the light, KO
mutants showed 19% lower germination rates than the
control, and the OE line showed a 10% greater germi-
nation rate on average (Fig. 6E). However, all KO seeds
germinated by 30 h, which indicated that the germi-
nation rate was not affected, but that germination was Figure 3. (Continued.)
(G) and root length (H) of the controls, for the KO, OE, and COM lines. n 5 19 to 43. In E, G, and H, data represent the mean 6 SE
(SE) of independent replicates. Asterisks indicate significant difference compared with the controls using Student’s t test (*P , 0.05
and **P , 0.01). Col-0 and an empty vector line were used as control for mutant and OE lines, respectively. 1487 Plant Physiol. Vol. 184, 2020 Plant Physiol. Vol. 184, 2020 https://plantphysiol.org
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Many Cells and Organs - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. Figure 4. Total lipid content and abundance of lipid molecular species in OE and KO lines. A to C, Total lipid content (A), total
content of each lipid class (B), and major lipid molecular species content (C) in OE and KO lines. Each molecular species of acyl-
lipid was identified and quantified using UPLC-MS/MS in the KO mutant and OE lines compared with the controls. Values were
normalized by FW of rosette leaves analyzed. Data represent the mean 6 SE of three (controls) to six (KO and OEs) independent
biological replicates. Asterisks indicate significant differences using Student’s t test (*P , 0.05 and **P , 0.01) compared with the
controls. Molecular species are indicated as the total number of carbon atoms in acyl chains: the total number of double bonds. 1488
Plant Physiol. Vol. 184, 2020
Jang et al. Jang et al. Figure 4. Total lipid content and abundance of lipid molecular species in OE and KO lines. A to C, Total lipid content (A), total
content of each lipid class (B), and major lipid molecular species content (C) in OE and KO lines. Each molecular species of acyl-
lipid was identified and quantified using UPLC-MS/MS in the KO mutant and OE lines compared with the controls. Values were
normalized by FW of rosette leaves analyzed. Data represent the mean 6 SE of three (controls) to six (KO and OEs) independent
biological replicates. Asterisks indicate significant differences using Student’s t test (*P , 0.05 and **P , 0.01) compared with the
controls. Molecular species are indicated as the total number of carbon atoms in acyl chains: the total number of double bonds. 1488
Plant Physiol. Vol. 184, 2020 Figure 4. Total lipid content and abundance of lipid molecular species in OE and KO lines. A to C, Total lipid content (A), total
content of each lipid class (B), and major lipid molecular species content (C) in OE and KO lines. Each molecular species of acyl-
lipid was identified and quantified using UPLC-MS/MS in the KO mutant and OE lines compared with the controls. Values were
normalized by FW of rosette leaves analyzed. https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. https://plantphysiol.org
Downloaded on March 11, 2021. - Published by pPLAIIIa Expression Level Affects the Size and Shape of
Many Cells and Organs Data represent the mean 6 SE of three (controls) to six (KO and OEs) independent
biological replicates. Asterisks indicate significant differences using Student’s t test (*P , 0.05 and **P , 0.01) compared with the
controls. Molecular species are indicated as the total number of carbon atoms in acyl chains: the total number of double bonds. 1488 Plant Physiol. Vol. 184, 2020 pPLAIIIa Affects Germination Rate (DAB) staining (Fig. 8B). DAB is oxidized by H2O2 in the
presence of heme-containing proteins to generate a dark-
brown precipitate (Thordal-Christensen et al., 1997). In
OE lines, leaves were greener in 8-week-old plants, when
the leaves of wild-type plants had already become
chlorotic (Fig. 8A). All plant leaves were green up to 28 d,
but the levels of H2O2 were lower when pPLAIIIa was
highly expressed, as observed in the OE lines compared
with the wild type (Fig. 8B) after DAB staining. To
confirm that the delayed senescence was caused by
pPLAIIIa, we analyzed the expression of a representa-
tive downstream gene, SENESCENCE-ASSOCIATED
GENE12 (SAG12), which encodes a Cys protease
(Lohman et al., 1994) upregulated during senescence. Expression of SAG12 was 10-fold lower in four strong
OE lines (Fig. 8C), which shows that high expression of
pPLAIIIa retards senescence. By contrast, expression of
SAG13, which may be induced by stress or cell death,
was only slightly increased in two of the four OE lines
analyzed (Fig. 8D). delayed compared to that in wild type. Treatment with
gibberellic acid (GA)3 did not alter the germination
substantially (Fig. 6F). Because antagonism between
abscisic acid (ABA) and GA plays a key role in con-
trolling seed germination (Koornneef et al., 2002), we
quantified ABA in dry seeds. ABA content increased in
the mutant compared to the wild type (Fig. 6G). Alto-
gether, the data suggest that the KO of pPLAIIIa delays
the initial germination rate by changing endogenous
GA and ABA biosynthesis. Transcript Levels of Ethylene and GA Biosynthesis Genes
Increased during pPLAIIIa-OE Seed Imbibition The antagonistic effects of ethylene and ABA in the
regulation of seed germination have also been exten-
sively studied (for review, see Corbineau et al., 2014). Increased ethylene production is associated with an
accumulation
of
ACC
oxidase
(ACO)
transcripts
(Corbineau et al., 2014). ACO1 and ACO2 are the major
ACOs involved in ethylene synthesis. pPLAIIIa Expression Level Affects the Size and Shape of
Many Cells and Organs ACO1, ACO2,
and ACO4, which exhibit ACO activity (Gómez-Lim
et al., 1993; Linkies et al., 2009; Park et al., 2018), were
all upregulated in OE lines, whereas ACO1 and ACO4
expression decreased in KO lines (Fig. 7A). Numerous
data also suggest that ethylene stimulates seed germi-
nation by affecting GA biosynthesis (Corbineau et al.,
2014). To uncover a possible role of bioactive GA bi-
osynthesis, we analyzed expression of four GA oxi-
dase genes. GA20ox and GA3ox encode enzymes that
catalyze bioactive gibberellin biosynthesis, whereas
GA2ox1 and GA2ox2 are involved in the conversion
of bioactive gibberellins into an inactive form. Two
GA2oxs were upregulated 1.4-fold in KO lines, whereas
GA20ox1 was upregulated 1.3-fold and GA2ox2 was
downregulated 0.6-fold in the OE lines (Fig. 7B). These
data explain that the faster germination in OE lines
might be due to more active forms of GA and increased
levels of ethylene. OE of pPLAIIIa Confers Turnip Crinkle Virus Resistance
by Altering Salicylic Acid and Jasmonic Acid Contents OE of phospholipase activity by SOBER1 reduces PA
levels and suppresses plant immunity to the bacterial
effector AvrBsT (Kirik and Mudgett, 2009). However, a
plant immunity study focusing on virus relative to the
level of PA has not been previously reported. Consid-
ering that plant viruses are pathogens associated with
major threats, resistance to turnip crinkle virus (TCV) in
Arabidopsis, which is one of the few manipulative
plant-virus systems, was tested in mutant and OE lines
(Fig. 9). Formation of the hypersensitive response (HR)
was visible in TCV-inoculated plant leaves 3-d post-
inoculation (DPI; Fig. 9, A and B) and in inflores-
cences at 12 DPI (Supplemental Fig. S8A), with the most
severe effects in the KO mutant. Only highly expressing
pPLAIIIa-OE (line 13) displayed an intact inflores-
cence (Fig. 9B). These phenotypes were consistent
with the corresponding patterns of TCV gene expres-
sion, whereby the relative gene expression dramatically
decreased in OE line 13 and increased in the KO mutant
(Fig. 9C; Supplemental Fig. S8B). After TCV infection,
several defense genes, such as pathogenesis-related
protein1 (PR1), PR2, and PR5, are expressed, and sali-
cylic acid (SA) accumulates (Kachroo et al., 2000). To
determine whether this mechanism could be involved
in higher virus resistance of pPLAIIIa-OE, the gene ex-
pression patterns of two representative SA and jas-
monic acid (JA) pathway genes were analyzed using
leaves at 3 DPI (Fig. 9, D and E). pPLAIIIa Expression Level Affects the Size and Shape of
Many Cells and Organs PR1 was upregulated in
both OE lines, whereas PDF1.2 expression significantly Figure 4. (Continued.)
Dashes (e.g. 18:3-1 and 18:3-2) indicate two species with the same mass and fragmentation pattern but with different retention
times. For MGMG, DGDG, SQDG, PG, PC, and PE classes, only the three or four major species are shown. Minor species for
these lipid classes are shown in Supplemental Figure S4. Plant Physiol. Vol. 184, 2020 OE of pPLAIIIa Delayed Senescence by Reducing ROS Suppression of the PA-generating PLDa1 leads to
reduced superoxide synthesis, and addition of exoge-
nous PA to leaves promotes reactive oxygen species
(ROS) production (Sang et al., 2001). Besides, in several
plant systems, minor lipid PA content has been studied
in relation to stress responses (Wang et al., 2006)
through the modulation of ROS (Hong et al., 2008). Therefore, we decided to monitor hydrogen peroxide
(H2O2) levels in 8-week-old mutant and OE lines
(Fig. 8A) using 3,3-diaminobenzidine tetrahydrochloride Figure 4. (Continued.)
Dashes (e.g. 18:3-1 and 18:3-2) indicate two species with the same mass and fragmentation pattern but with different retention
times. For MGMG, DGDG, SQDG, PG, PC, and PE classes, only the three or four major species are shown. Minor species for
these lipid classes are shown in Supplemental Figure S4. 1489 Plant Physiol. Vol. 184, 2020 https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. Jang et al. Figure 5. Overexpression of pPLAIIIa alters the size and shape of many cells and organs. A and C to E, Statistical analysis of plant
height (A), leaf surface area (C), leaf thickness (D), and leaf water content (E) in 4-week-old plants. Mean 6 SE of three independent
replicates. Asterisks indicate significant difference using Student’s t test (*P , 0.05 and **P , 0.01) compared with the controls. B,
The aerial part of each 4-week-old plant with all individual leaves. The leaves are arranged from cotyledons (left) to the youngest
leaves (right). Scale bars 5 1 cm. F, Floral organs and siliques in the pPLAIIIa-OE line (13) and Col-0. Scale bars 5 0.2 mm (top),
2 mm (middle), and 5 mm (lower). G, Flower in the Col-0 and OE lines. Scale bars 5 500 mm. H, Stems in the Col-0 and OE lines. Scale bars 5 500 mm (upper) and 100 mm (lower). I, The area (millimeters squared) of cross-sectioned stems. J, Cell number of
each sectioned stems. Data represent the mean 6 SD of 10 independent replicates. Asterisks indicate significant difference using
Student’s t test (**P , 0.01) compared with the controls. K, Pollen structures in Col-0, KO, and OE lines. Scale bars 5 10 mm. All surface images were captured using a low-vacuum scanning electron microscope (model no. JSM-IT300; JEOL Korea) at a
10.8-mm working distance and 20.0 kV. Jang et al. Figure 5. OE of pPLAIIIa Delayed Senescence by Reducing ROS Overexpression of pPLAIIIa alters the size and shape of many cells and organs. A and C to E, Statistical analysis of plant
height (A), leaf surface area (C), leaf thickness (D), and leaf water content (E) in 4-week-old plants. Mean 6 SE of three independent
replicates. Asterisks indicate significant difference using Student’s t test (*P , 0.05 and **P , 0.01) compared with the controls. B,
The aerial part of each 4-week-old plant with all individual leaves. The leaves are arranged from cotyledons (left) to the youngest
leaves (right). Scale bars 5 1 cm. F, Floral organs and siliques in the pPLAIIIa-OE line (13) and Col-0. Scale bars 5 0.2 mm (top),
2 mm (middle), and 5 mm (lower). G, Flower in the Col-0 and OE lines. Scale bars 5 500 mm. H, Stems in the Col-0 and OE lines. Scale bars 5 500 mm (upper) and 100 mm (lower). I, The area (millimeters squared) of cross-sectioned stems. J, Cell number of
each sectioned stems. Data represent the mean 6 SD of 10 independent replicates. Asterisks indicate significant difference using
Student’s t test (**P , 0.01) compared with the controls. K, Pollen structures in Col-0, KO, and OE lines. Scale bars 5 10 mm. All surface images were captured using a low-vacuum scanning electron microscope (model no. JSM-IT300; JEOL Korea) at a
10.8-mm working distance and 20.0 kV. 1490 Plant Physiol. Vol. 184, 2020 https://plantphysiol.org
Downloaded on March 11, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. Figure 6. pPLAIIIa expression associates with increased germination rate and seed size. A and B, Mature seeds (A) and size (B) of
control, KO, OE, and COM lines. Scale bar 5 1 mm. Average n 5 50 for seed size. C, The hundred-seed weight of control, KO,
and OE lines. Average n 5 10. D, Seed number per silique (n 5 10). E and F, Germination rates of control, KO, and OE lines after
20-h germination under light conditions. n 5 120. F, The germination rate after treatment with 1 mM of GA3. G, ABA content was
analyzed using UPLC. Mean 6 SE (SE) of three independent replicates. Asterisks indicate significant difference using Student’s t test
(*P , 0.05 and **P , 0.) compared with the controls. 1/2 MS, One-half stregth Murashige and Skoog. pPLAIIIa Affects Germination Rate pPLAIIIa Affects Germination Rate Figure 6. Jang et al. Jang et al. Figure 7. The mRNA levels of ethylene and GA
biosynthesis genes in pPLAIIIa-OE lines. A and
B, Gene expression patterns of ACOs (A) and of
gibberellin oxidases (GA2ox1, GA2ox2, GA3ox1,
and GA20ox1; B) from control, KO, and OE seeds
imbibed for 20 h. Mean 6 SE (SE) of three inde-
pendent replicates. Asterisks indicate significant
difference using Student’s t test (*P , 0.05 and
**P , 0.01) compared with the controls. ncrease in PR1 gene
d OE lines may be
agonistic plant hor-
the KO mutant and
the controls (Fig. 9G). Thus, differential cellular levels of
SA and JA seem to be coordinately involved in TCV
resistance due to the function of pPLAIIIa. A
nd
of
1,
ds
e-
nt
nd Figure 7. The mRNA levels of ethylene and GA
biosynthesis genes in pPLAIIIa-OE lines. A and
B, Gene expression patterns of ACOs (A) and of
gibberellin oxidases (GA2ox1, GA2ox2, GA3ox1,
and GA20ox1; B) from control, KO, and OE seeds
imbibed for 20 h. Mean 6 SE (SE) of three inde-
pendent replicates. Asterisks indicate significant
difference using Student’s t test (*P , 0.05 and
**P , 0.01) compared with the controls. ease in PR1 gene
E lines may be
nistic plant hor-
KO mutant and
the controls (Fig. 9G). Thus, differential cellular levels of
SA and JA seem to be coordinately involved in TCV
resistance due to the function of pPLAIIIa. the controls (Fig. 9G). Thus, differential cellular levels of
SA and JA seem to be coordinately involved in TCV
resistance due to the function of pPLAIIIa. decreased in OE line 13. The 2-fold increase in PR1 gene
expression in the KO mutant and OE lines may be
explained by two endogenous antagonistic plant hor-
mones. SA content was higher in the KO mutant and
OE line 13 compared with the controls (Fig. 9F). This
finding indicates that both SA level and PR1 gene ex-
pression are modulated by the threshold level of rela-
tive pPLAIIIa expression or activity in response to
developmental and environmental cues. However, the
endogenous JA level was two times higher in the mu-
tant and 25% lower in OE lines compared with that in OE of pPLAIIIa Delayed Senescence by Reducing ROS pPLAIIIa expression associates with increased germination rate and seed size. A and B, Mature seeds (A) and size (B) of
control, KO, OE, and COM lines. Scale bar 5 1 mm. Average n 5 50 for seed size. C, The hundred-seed weight of control, KO,
and OE lines. Average n 5 10. D, Seed number per silique (n 5 10). E and F, Germination rates of control, KO, and OE lines after
20-h germination under light conditions. n 5 120. F, The germination rate after treatment with 1 mM of GA3. G, ABA content was
analyzed using UPLC. Mean 6 SE (SE) of three independent replicates. Asterisks indicate significant difference using Student’s t test
(*P , 0.05 and **P , 0.) compared with the controls. 1/2 MS, One-half stregth Murashige and Skoog. 1491 Plant Physiol. Vol. 184, 2020 https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. Figure 7. The mRNA levels of ethylene and GA
biosynthesis genes in pPLAIIIa-OE lines. A and
B, Gene expression patterns of ACOs (A) and of
gibberellin oxidases (GA2ox1, GA2ox2, GA3ox1,
and GA20ox1; B) from control, KO, and OE seeds
imbibed for 20 h. Mean 6 SE (SE) of three inde-
pendent replicates. Asterisks indicate significant
difference using Student’s t test (*P , 0.05 and
**P , 0.01) compared with the controls. DISCUSSION While the molecular and biochemical functions of
pPLAIIIb and pPLAIIId have been characterized in Ara-
bidopsis (Li et al., 2011, 2013), pPLAIIIa has only been
studied in rice (Liu et al., 2015). Here, we characterized Figure 8. pPLAIIIa-OE lines remain green longer with reduced H2O2. A, Eight-week-old plants of the control, KO, and pPLAIIIa-
OE lines. Scale bar 5 5 cm. B, The sixth or seventh leaves were detached from 4-week-old plants and used for DAB staining. Scale
bars 5 1 cm. C and D, Relative gene expression patterns of two senescence marker genes, SAG12 (C) and SAG13 (D). Mean 6 SE
(SE) of three independent replicates. Asterisks indicate significant difference using Student’s t test (*P , 0.05 and **P , 0.01)
compared with the controls. 92
Plant Physiol. Vol. 184, 2020
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Copyright (c) 2020 American Society of Plant Biologists All rights reserved Figure 8. pPLAIIIa-OE lines remain green longer with reduced H2O2. A, Eight-week-old plants of the control, KO, and pPLAIIIa-
OE lines. Scale bar 5 5 cm. B, The sixth or seventh leaves were detached from 4-week-old plants and used for DAB staining. Scale
bars 5 1 cm. C and D, Relative gene expression patterns of two senescence marker genes, SAG12 (C) and SAG13 (D). Mean 6 SE
(SE) of three independent replicates. Asterisks indicate significant difference using Student’s t test (*P , 0.05 and **P , 0.01)
compared with the controls. 1492 Plant Physiol. Vol. 184, 2020 https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. pPLAIIIa Affects Germination Rate Figure 9. Increased SA in pPLAIIIa-OE enhances TCV resistance by changing transcript levels of PR1 and PDF1.2. A, Inflores-
cences of each line 12 DPI of mock and TCV. B, Magnified images of TCVinfected plants from each line. Scale bars 5 2 cm. C to E,
The relative gene expression of TCV, PR1, and PDF1.2 in the inoculated leaves of TCV- and mock-infected plants. The inoculated
leaves were monitored for the presence or absence of the HR, and RNAwas extracted from inoculated leaves on 0 (1 h), 1, 2, and 3
DPI. Expression of PR1 and PDF1.2 was analyzed from the leaves at 1 DPI only. DISCUSSION F and G, Hormone contents of SA and JA as
measured by UPLC in rosette leaves of 5-week-old plants in control, KO, and OE lines. C to G, Four-week-old seedlings were
used for the analysis. Mean 6 SE (SE) of three independent replicates. Asterisks indicate significant difference using Student’s t test
(*P , 0.05 and **P , 0.01) compared with the controls. pPLAIIIa Affects Germination Rate Figure 9. Increased SA in pPLAIIIa-OE enhances TCV resistance by changing transcript levels of PR1 and PDF1.2. A, Inflores-
cences of each line 12 DPI of mock and TCV. B, Magnified images of TCVinfected plants from each line. Scale bars 5 2 cm. C to E,
The relative gene expression of TCV, PR1, and PDF1.2 in the inoculated leaves of TCV- and mock-infected plants. The inoculated
leaves were monitored for the presence or absence of the HR, and RNAwas extracted from inoculated leaves on 0 (1 h), 1, 2, and 3
DPI. Expression of PR1 and PDF1.2 was analyzed from the leaves at 1 DPI only. F and G, Hormone contents of SA and JA as
measured by UPLC in rosette leaves of 5-week-old plants in control, KO, and OE lines. C to G, Four-week-old seedlings were
used for the analysis. Mean 6 SE (SE) of three independent replicates. Asterisks indicate significant difference using Student’s t test
(*P , 0.05 and **P , 0.01) compared with the controls. 1493 Plant Physiol. Vol. 184, 2020 https://plantphysiol.org
Downloaded on March 11, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. Jang et al. directly on phospholipid molecular species and indi-
rectly on plastidial lipid species such as galactolipids,
sulfolipids, or PG. Concerning the major plastidial
lipids MGDG and DGDG, it should be noted that in OE
lines the decrease in their molecular species impacts
similarly both prokaryotic and eukaryotic species (Fig. 4C;
Supplemental Fig. S4) and does not affect the mol %
fraction (Supplemental Fig. S5) of these classes. It is
thus likely that the pPLAIIIa activity in extraplastidial
membranes of OEs has an indirect effect on the overall
content in chloroplast membranes, but does not com-
promise lipid homeostasis in these organelles. pPLAIIIa KO and OE lines in Arabidopsis at the bio-
chemical, morphological, and physiological levels. https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. DISCUSSION Phe-
notypes that confirm and extend those reported in rice
were observed, such as reduced longitudinal growth
and enlarged seed width and a strong relative decrease
in the contents of PA as well as most other polar glyc-
erolipid classes. But we also provide evidence for
the association of pPLAIIIa with new processes, such as
seed germination and virus resistance, and we give new
molecular insights into pPLAIIIa signaling via regulation
of expression of PLD genes. p
p
g
In vitro assays on the partially purified pPLAIIIa
showed that the protein hydrolyzed various phospho-
lipids, with a slightly higher activity on PA (Fig. 1B). This seems to be consistent with a decrease in all
phospholipid classes with a stronger reduction in PA
molecular species in OE lines (Fig. 4C). Measurement of
total phospholipase activities in plant extracts did not
yield further insight into pPLAIIIa in vivo activity, as
no difference could be detected between wild-type and
OE lines using four different phospholipid substrates
(Supplemental Fig. S9). This result was possibly due to
highly active phospholipases other than pPLAIIIa, or to
the presence of inhibitors of pPLAIIIa activity in the
extract. Lipid Substrates of pPLAIIIa In Vivo OE of Arabidopsis pPLAIIIb increases all lipid spe-
cies
analyzed:
phospholipids,
including
PE,
PC,
phosphatidylinositol (PI), phosphatidylserin, PA, and
PG; and galactolipids, including MGDG and DGDG
(Li et al., 2011). Similarly, overexpression of Arabi-
dopsis pPLAIIId also tends to increase the levels of lipid
species, including seed lipid reserves (Dong et al., 2014;
Li et al., 2015). These counterintuitive results showing
increased lipid content in plants overexpressing lipid-
degrading enzymes may be explained by the fact that
pPLAIIIb may normally act in acyl editing mechanisms
involved in lipid biosynthesis. Acyl editing mecha-
nisms involve the removal by phospholipases of spe-
cific fatty acids from membrane phospholipids (for
example, PC) to yield lysophospholipids and a pool of
acyl-CoAs enriched in specific fatty acids that are used
for synthesis of other lipids such as storage lipids (Bates
et al., 2012). Increasing the pool of acyl editing-involved
lysophospholipids through overexpression of specific
phospholipases may create an imbalance in membrane
lipid composition. This imbalance may in turn trigger
an increased flux of de novo fatty acid and membrane
lipid synthesis and eventually result in an overall ac-
cumulation of lipids. In vitro activity assays and lipidomic analyses show
that pPLAIIIa protein is therefore probably a nonspe-
cific phospholipase A acting on a variety of phospho-
lipids in the PM, with a possible slight preference
for PA. PA Levels and PLD Expression PA accumulates at significant levels at the PM in
Arabidopsis (Platre et al., 2018). The possibility that PA
is one of the phospholipid classes targeted by pPLAIIIa
in wild-type plants is supported by the slight preference
of pPLAIIIa for PA in in vitro assays (Fig. 1C) and the
strong reduction of almost all PA lipid species observed
in Arabidopsis pPLAIIIa-OE (Fig. 4). These results on
Arabidopsis and the previous observation in rice that a
preferential decrease in PA occurs when OspPLAIIIa is
overexpressed (Liu et al., 2015) thus support the view
that PA is one of the possible substrates of pPLAIIIa in
wild-type plants of both organisms. However, it should
be stressed that hydrolysis of other phospholipid clas-
ses by Arabidopsis pPLAIIIa in vivo cannot be ruled
out, because lipid species from other phospholipid
classes are also reduced in OE lines (Fig. 4). p
By contrast, Arabidopsis pPLAIIIa-OE displayed re-
duced levels of many molecular lipid species compared
to wild type (Fig. 4; Supplemental Fig. S4). Thus, it
seems that pPLAIIIa overexpression does not act on the
same mechanisms that are altered by pPLAIIIb and
pPLAIIId overexpression (possibly acyl editing cycles,
as discussed before). This idea is consistent with the fact
that lysophospholipids, which are key players in acyl
editing mechanisms, accumulate in pPLAIIIb-OE and
pPLAIIId-OE but not in pPLAIIIa-OE. Interestingly,
despite the decrease in many lipid species in the
OE lines, FFAs did not accumulate in these lines
compared to wild type, which showed that the FA
breakdown machinery was not overwhelmed by the
overexpression of pPLAIIIa. Therefore, the phenotypes
observed in the OE lines are not likely caused by an ex-
cess of FFAs. Some of these phenotypes may result from
the decrease in one or several of the many molecular
lipid species significantly affected in both OE lines (Fig. 4;
Supplemental Fig. S4). The fact that the Arabidopsis
protein localizes to the PM suggests that pPLAIIIa acts g
Intriguingly, no increases in PA molecular species
were detected in pplaIIIa mutants in rice (Liu et al., 2015)
or Arabidopsis (Fig. 4). Because PA is mostly generated
by the activation of PLD (Sang et al., 2001), we checked
the expression of several PLD isoforms in the Arabi-
dopsis pplaIIIa KO mutants. Interestingly, the expres-
sion of the major PLD isoforms PLDa1, PLDz1, and
PLDz2 decreased significantly compared to the controls
in the mutant seedlings (Supplemental Fig. S10). Role of pPLAIIIa in Cell Elongation and Plant Growth OE of pPLAIIIb results in shorter leaves, petioles,
hypocotyls, primary roots, and root hairs compared
with the wild type (Li et al., 2011), which is partly
reminiscent of the phenotypic characteristics of
pPLAIIIa-OE in Arabidopsis (Figs. 3, F–H, and 5;
Supplemental Figs. S5 and S6). Conversely, the re-
cessive rice mutant dep3 defective in OspPLAIIId
displays a taller plant stature (Qiao et al., 2011). Generally, short and stunted morphology is observed
in pPLAIIId-OE in camelina (Camelina sativa; Li et al.,
2015), pPLAIIId-OE in Arabidopsis and Brassica napus
(Dong et al., 2014), pPLAIIIb-OE in Arabidopsis (Li
et al., 2011), OSAG78-OE (OnpPLAIIId-OE) in Arabi-
dopsis (Lin et al., 2011), OspPLAIII-OE in rice (Liu
et al., 2015), and PgpPLAIIIb in Arabidopsis and
poplar (Populus alba 3 Populus glandulosa; Jang et al.,
2019; Jang and Lee, 2020a). Because pPLAIIIb-OE
and pPLAIIId-OE show accumulation of lysolipids and
FFAs, which have deleterious effects on cell membranes,
it is tempting to think that the stunted phenotypes of
pPLAIII-OEs are caused by the accumulation of pPLAIII
products, which are deleterious to cellular activities. However, our data show clearly that pPLAIIIa-OE have
stunted phenotypes in the absence of accumulation of
FFAs or lysolipids. These results thus suggest that
pPLAIIIs, at least pPLAIIIa, are involved in plant growth
and development through the modulation of minor
membrane lipid species (such as PA for pPLAIIIa) rather
than via large changes in FFAs. Modified PA levels by
pPLAIIIa may indirectly regulate cell elongation and
plant growth, for example by affecting subcellular lo-
calization of regulatory proteins (Yao et al., 2013). Reduced PA and JA Levels May Explain the Low Level of
ROS in pPLAIIIa-OE Characteristic features of leaf senescence are the or-
dered disassembly of the altered photosynthetic appa-
ratus and the loss of chlorophyll (Yoshida 2003). Expression of a well-studied senescence-response gene,
SAG12, markedly decreased in all pPLAIIIa-OE lines
(Fig. 8C); the higher the expression of pPLAIIIa, the
greater the reduction in SAG12 transcripts. Suppression
of PLDa leads to reduced superoxide synthesis via
reduction in the minor phospholipid class PA (Sang
et al., 2001). In addition, the formation of PA leads to
the production of other lipid messengers, such as JA
(Wang et al., 2000). Thus, reduced content of PA
(Fig. 4C) and JA (Fig. 9G) due to the overexpression of
pPLAIIIa could be one possible reason for the reduc-
tion in H2O2. https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. PA Levels and PLD Expression Taken 1494 Plant Physiol. Vol. 184, 2020 Plant Physiol. Vol. 184, 2020 Plant Physiol. Vol. 184, 2020 pPLAIIIa Affects Germination Rate together, these data thus suggest that pPLAIIIa hy-
drolyzes PA in Arabidopsis wild-type plants and that
absence of this activity in the pplaIIIa KO results in
decreased expression of several PLD genes to possibly
maintain PA levels in the PM. Other PA-producing
pathways, such as DAG phosphorylation, could also
be involved and may warrant further investigation. However, in OE lines, the situation is clearly more
complex than a simple counterbalance of PA levels by
modulation of PLD expression. Indeed, in OEs there
was an increase in expression of PLD genes only in the
stronger OE line, while other ones showed even a de-
crease in PLD expression. Complexity of the response
observed in OEs may be enhanced by the fact that
pPLAIIIa is also active on other PM phospholipids than
PA,
which
may
activate
membrane
homeostasis
mechanisms and interfere with lipid signaling. of pPLAIIIa reduced the level of JA content (Fig. 9G). Thus, it will be interesting to further investigate how
pPLAIIIa-mediated lowered JA affects the trichome
branching patterning. Role of pPLAIIIa in Seed Germination Role of pPLAIIIa in Seed Germination Overexpression of rice pPLAIIIa in rice (Liu et al.,
2015) and Arabidopsis pPLAIIId in camelina (Li et al.,
2015) thickened seed widths, but the length of seeds
was reduced or not changed significantly. The recessive
rice mutant dep3, where part of the pPLAIIId gene is
deleted, shows smaller and rounder seeds but greater
grain yields than control (Qiao et al., 2011). Seed sizes
and weights can affect germination, but no study has
yet been reported from pPLA gene families. Here, we
show that alteration of seed morphology and weight
can delay germination among pPLAs (Fig. 6). The
importance of the plant hormone GA in promoting
seed germination is well known, and ABA can act
antagonistically. Ethylene also regulates germination
and dormancy of many species via complex hormo-
nal signaling networks (Corbineau et al., 2014). The knockout pplaIIIa mutant contained more ABA
and increased gene expression of GA2ox1, which is
involved in GA inactivation (Fig. 7B). The over-
expression of pPLAIIIa displayed more transcripts of
GA oxidases, which catalyze bioactive GA (Fig. 7B). Ethylene stimulates seed germination by affecting
GA biosynthesis or signaling. Thus, the alternation of
pPLAIIIa delays initial germination speed, possibly
by modulating active and nonactive forms of GA,
and increasing ethylene biosynthesis. Lipid Extraction and Lipidomic Analysis Total lipids were extracted using the following hot isopropanol method. Briefly, 3-week–old Arabidopsis rosettes were cut, immediately weighed, and
placed in 2 mL of boiling isopropanol (85°C) containing 0.01% (w/v) butylated
hydroxytoluene. After 10-min heating, samples were cooled down to room
temperature, and 1 mg of each internal standard was added (PE 17:0/17:0,
MGDG 18:0/18:0, and TAG 17:0/17:0/17:0). Samples were then ground for
1 min using an Ultra-Turrax T25 apparatus (IKA Labortechnik), and 3 mL of
methyl tert-butyl ether was added. After vortexing for 30 s, 1 mL of water was
added and the mixture was shaken vigorously for 30 min and allowed to phase-
separate by centrifugation at 3,000g for 2 min. The uppermost (organic) phase
was collected, and 1 mL of methyl tert-butyl ether was added to the remaining
lower phase. The mixture was shaken for 30 s and allowed to phase-separate by
centrifugation. The upper phase was then combined with the previous organic
phase, and the solvent of the total lipid extract was evaporated to dryness under
a gentle stream of nitrogen gas. The total lipid extract was resuspended in
200 mL of acetonitrile/isopropanol/ammonium formate (65:30:5, v/v/v, final
concentration of ammonium formate was 10 mM) and kept at 220°C until use. Quality control (QC) samples were prepared for data quantification by pooling
aliquots of the lipid extract samples from Col-0 plants to make a QC stock so-
lution. QC samples were evaporated to dryness under a gentle stream of
nitrogen gas and resuspended in 200 mL of the same acetonitrile/isopropanol/
ammonium formate mixture used for samples. A QC sample contained, in
addition to the three internal standards coming from samples, the following
quantification standards added in the molar proportions: PG (17:0/17:0), 4; PA
(17:0/17:0), 1; PE (17:0/17:0) 2; PC (17:0/17:0), 4; PI (17:0/14:1), 1; MGDG
(18:0/16:0), 28; DGDG (18:0/16:0), 12; SQDG (16:0/18:3), 2; LysoPG (17:1), 1;
LysoPE (17:1), 1; LysoPC (17:0), 1; and FFA (17:0), 1 (purity of standards was
determined before use by LC-MS/MS analysis). Samples and QC samples were
subjected to ultra-performance liquid chromatography (UPLC)-MS/MS anal-
yses using an Ultimate RS 3000 UPLC system (Dionex) connected to a quad-
rupole-time-of-flight 5600 mass spectrometer (AB Sciex). Samples were run in
negative mode. Lipids were separated using a C8 2.1 3 150 mm, 2.6-mm column
(Kinetex) and a binary gradient of solution A (60v:40v water/acetonitrile) and
solution B (90v:10v isopropanol/acetonitrile). Observation of Reporter Gene Expression Fluorescencewasobservedbyconfocallaserscanningmicroscopy(modelno. TCS SP5 AOBS/Tandem; Leica). YFP and mRFP were detected using 514/.530
nm, and 543/560-nm to 615-nm excitation/emission filter sets, respectively. The images were acquired at the Korea Basic Science Institute, Gwangju. Korea. Some evidence suggests that at least one member of
the pPLAI and pPLAII subclasses is involved in plant
defenses. pplaI mutants are more sensitive to Botrytis
cinerea infection without altering JA levels (Yang et al.,
2007), whereas pPLAIIa-deficient mutants are more
resistant to Botrytis spp. or avirulent Pseudomonas
syringae infection (La Camera et al., 2005). Among three
isoforms, pPLAIIIa, pPLAIIIb, and pPLAIIId, only
pPLAIIIb is upregulated upon Botrytis spp. and P. syringae infection (La Camera et al., 2005). However,
there is no clear evidence of the involvement of pPLAIII
genes in plant innate immunity. Here we found that
higher expression of pPLAIIIa confers TCV resistance
via the regulation of SA and JA (Fig. 9). Increased levels
of SA with decreased levels of JA may play crucial roles. In Col-0 and the vector control, the ratio of SA/JA was
;0.9 and 1, respectively. In KO mutants, this ratio re-
duced to 0.8 whereas it increased 1.4- to 2-fold in two
pPLAIIIa-OE lines, 8 and 13, respectively (Fig. 9, F and
G), which indicates a 2-fold higher SA level compared
to the JA level in TCV resistance. During TCV infection,
the HR is reportedly mediated by increased expression
of defense genes, such as pathogenesis-related genes,
and the accumulation of SA, phytoalexin, and cama-
lexin (Dempsey et al., 1993; Kachroo et al., 2000). The
2.5-fold increase in PR1 expression and ;50% reduction
in PDF1.2 expression mediated by SA might have in-
creased TCV virus resistance. Plant Materials and Growth Conditions Arabidopsis (Arabidopsis thaliana; Col-0) was used as the wild-type plant. The pplaIIIa knockout mutants (KO 1: SAIL830G12 and KO 2: SALK_040363)
were purchased from the stock center (http://www.arabidopsis.org/). Seeds
were maintained as reported in Jang and Lee (2020a, 2020b). Lipid Extraction and Lipidomic Analysis Elution was achieved through a
gradient of solution B from 27% to 97%, as compared to solvent A within 20 min
at a speed of 0.3 mL min21, and then at 97% for 5 min. Solution B was then
decreased to a 27% enrichment during 7 min for column reequilibration. Rel-
ative quantification of lipid molecular species in samples was achieved with the
software MULTIQUANT (AB Sciex) using intensity values obtained by
extracting masses of the different lipids previously identified, and by normal-
izing based on rosette FW and the internal standard (to control for lipid ex-
traction). In samples, absolute amounts of lipid molecular species (picomole per
milligram FW) in each lipid class were estimated using intensities of the cor-
responding quantification standards of the QC samples (normalized by the
internal standard). For lysoMGDGs and lysoDGDGs, estimates were based on
one of the lysolipid standards (LPE). The estimated absolute amounts of lipid
molecular species were used to calculate total amounts in each lipid class, total
lipid amounts, and lipid class compositions in each line. Role of pPLAIIIa in Cell Elongation and Plant Growth Mostly reduced transcript levels of PLD
genes in pPLAIIIaOE lines (Supplemental Fig. S10)
should support this notion. Although PLDa1 was
slightly upregulated, other PLDs were all down-
regulated. Alternation of lipid species by the reduction
of lysolipids and FFAs (Fig. 4) and hormonal signal-
ing, such as auxin, ethylene, and GAs (Figs. 6 and 7), g
y p
(
)
Trichomes are the outermost epidermal cells, which
develop on almost all aerial structures of Arabidopsis. The pPLAIIIa-induced changes in trichome branching
(Supplemental Fig. S7B) and pollen structure (Fig. 5K)
might also be caused by altered patterns of epidermal
cell elongation. Endogenous JA content is involved in
trichome patterning (Yoshida et al., 2009). Overexpression Plant Physiol. Vol. 184, 2020 1495 1495 Jang et al. could also affect the level of ROS (Corbineau et al.,
2014). Gene Expression Analysis by Reverse Transcription
Quantitative PCR Total RNA extraction, quantification of total RNA, synthesis of comple-
mentary DNA (cDNA), and reverse transcription quantitative PCR (RT-qPCR)
were performed following Jang and Lee (2020a, 2020b). Three independent
experiments were performed for each primer set (Supplemental Table S1). A Higher SA to JA Ratio May Increase the Resistance of
pPLAIIIa-OE to TCV A Higher SA to JA Ratio May Increase the Resistance of
pPLAIIIa-OE to TCV https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. GUS Histochemical Analysis Histochemical
GUS
staining
was
performed
by
incubating
Prop-
PLAIIIa::GUS transformants in staining buffer following Kim et al. (2014). Seedlings were photographed under a microscope (Axio Observer D1; Zeiss). Protein Purification/Extraction, and Immunoblotting The full-length cDNA of pPLAIIIa was cloned into the pET28a vector with
the 63His. The BL21 (DE3) bacteria expressing pPLAIIIa-His fusion protein
were induced with 1 mM of isopropyl 1-thio-b-D-galactopyranoside, and the
fusion protein was purified. The bacterial pellet was resuspended in lysis buffer
(50 mM of NaH2PO4, 300 mM of NaCl, 10 mM of imidazole 1% [v/v] Triton
X-100, adjusted pH to 8.0) containing 1 mg mL21 of lysozyme and 1 mM of
phenylmethanesulfonyl fluoride. The suspension was sonicated. After centrif-
ugation at 10,000g for 20 min, the supernatant was mixed with Ni-NTA agarose
resin (10% [w/v]; Qiagen). The fusion proteins bound to agarose beads were
washed with washing buffer (50 mM of NaH2PO4, 300 mM of NaCl, and 20 mM
of imidazole, adjusted pH to 8.0), and pPLAIIIa proteins were eluted with
elution buffer (50 mM of NaH2PO4, 300 mM of NaCl, and 250 mM of imidazole,
adjusted pH to 8.0). The proteins from 7-d-old seedlings were extracted using
extraction buffer (120 mM of Tris-HCl at pH 7.5, 100 mM of EDTA, 5% [v/v]
glycerol, 2% [w/v] SDS, 1% [v/v] Triton X-100, 300 mM of NaCl, 1 mM of di-
thiothreitol, and 1 mM of phenylmethanesulfonyl fluoride) after sonication and
centrifugation in the same condition as for the above protein purification. The
amount of purified protein was measured with the Bradford method (Bradford,
1976). The immunoblotted membranes were preblotted with 5% (v/v) skim
milk for 30 min and incubated with 63-His tag antibody, horseradish peroxi-
dase (MA1-135; Invitrogen) for 14 h at 4°C. Anti-RFP polyclonal antibody and
goat anti-Rabbit IgG (H1L) horseradish peroxidase were used as primary and
secondary antibody, respectively, for pPLAIIIa-mRFP detection. Bands were
visualized by exposing to the HR-A x-ray film after treatment with enhanced
chemiluminescence substrate (Thermo Fisher Scientific). The following materials are available as supplemental data. Supplemental Figure S1. SDS-PAGE of recombinant pPLAIIIa protein. Supplemental Figure S2. Phylogenetic tree of PLA family proteins in
Arabidopsis. Supplemental Figure S3. Transcript levels of other pPLAIII genes are mod-
ulated by the activity of pPLAIIIa. Supplemental Figure S4. Abundance of minor lipid molecular species in
MGDG, DGDG, SQDG, PG, PC, and PE lipid classes. Supplemental Figure S5. Lipid class composition based on absolute
amounts of lipid molecular species estimated by LC-MS/MS. Supplemental Figure S6. pPLAIIIa expression levels affect petiole length. Supplemental Figure S7. In Vitro Enzymatic Assays Phospholipids (each 16:0-18:1 PA, PC, PE, and PG) were purchased from
Avanti Polar Lipids. Acyl hydrolyzing activities were assayed in a reaction
mixture (25 mM of HEPES at pH 7.5, 10 mM of CaCl2, and 10 mM of MgCl2). Each
0.5 mM of lipids was used as substrate and 0.1 mg of purified protein was added
to the mixture in a final volume of 100 mL. The reaction samples were incubated
at 30°C for 60 min. The released nonesterified free fatty acids (NEFA) products
were measured with a NEFA-HR colorimetric kit (Wako Pure Chemicals,
http://www.wako-chem.co.jp/english/) using an Epoch microplate spectro-
photometer (BioTek) at 546 nm. Phytohormone Analysis Dong Y, Li M, Zhang P, Wang X, Fan C, Zhou Y (2014) Patatin-related
phospholipase pPLAIIId influences auxin-responsive cell morphology
and organ size in Arabidopsis and Brassica napus. BMC Plant Biol 14: 332 Sampleswereextractedtwicewithethylacetate.Theextractswerecombined,
evaporated, and dissolved in 70% methanol. Isotope-labeled standards (2H4-SA
and 2H6-JA) were used as internal standards. UPLC (Waters) coupled with a
quadrupole-time-of-flight instrument (Waters) was used for the analysis. The
chromatographic separation was carried out on a C18 column. The mobile
phases consisted of solvent A (0.1% [v/v] formic acid) and solvent B (acetoni-
trile). MS analysis was conducted in the negative ion mode with electrospray
ionization. Gómez-Lim MA, Valdés-López V, Cruz-Hernandez A, Saucedo-Arias LJ
(1993) Isolation and characterization of a gene involved in ethylene bi-
osynthesis from Arabidopsis thaliana. Gene 134: 217–221 y
Higginson T, Li SF, Parish RW (2003) AtMYB103 regulates tapetum and
trichome development in Arabidopsis thaliana. Plant J 35: 177–192 Holk A, Rietz S, Zahn M, Quader H, Scherer GFE (2002) Molecular
identification of cytosolic, patatin-related phospholipases A from Ara-
bidopsis with potential functions in plant signal transduction. Plant
Physiol 130: 90–101 ACKNOWLEDGMENTS We thank Rae-Dong Jeong (Chonnam National University) for advice on
how to analyze TCV infection. We thank Rae-Dong Jeong (Chonnam National University) for advice on
how to analyze TCV infection. Received May 15, 2020; accepted August 15, 2020; published August 28, 2020. Received May 15, 2020; accepted August 15, 2020; published August 28, 2020. Transgene Constructs and Arabidopsis Transformation The modified pCAMBIA1390 vector containing the CaMV 35S promoter,
YFP, and/or mRFP was used (Lee et al., 2010) to express pPLAIIIa. The full
pPLAIIIa genomic fragment was amplified using primers containing SalI and
AvrII sites (Supplemental Table S1). Enzyme-digested PCR products were
cloned into the vector (Pro-35s: pPLAIIIa-YFP/mRFP). The promoter::GUS fu-
sion construct was generated based on the obtained upstream intergenic region
of pPLAIIIa. The promoter region was amplified using primers as follows: 59-
TC CTG CAG ATC ATC AAT GTA GTC GAA-39 (forward) and 59-TC GTC
GAC TTG CAT CGT AGT TAA CAT-39 (reverse). The amplified PCR product
was subsequently cloned into a pCAM1390 vector containing a gusA reporter
gene. All transgene-confirmed constructs were transformed into Arabidopsis
using Agrobacterium tumefaciens C58C1 (pMP90; Bechtold and Pelletier, 1998). Col-0 and the empty vector line were used as controls for the pplaIIIa mutant
and pPLAIIIa-OE, respectively. Plant Physiol. Vol. 184, 2020 Plant Physiol. Vol. 184, 2020 1496 https://plantphysiol.org
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. pPLAIIIa Affects Germination Rate LITERATURE CITED Bates PD, Fatihi A, Snapp AR, Carlsson AS, Browse J, Lu C (2012) Acyl
editing and headgroup exchange are the major mechanisms that direct
polyunsaturated fatty acid flux into triacylglycerols. Plant Physiol 160:
1530–1539 Protein Purification/Extraction, and Immunoblotting OE of pPLAIIIa affects the direction of cell elon-
gation in filament and style and trichome length. Supplemental Figure S8. Phenotypic differences in KO mutant and OE
lines against TCV infection. Supplemental Figure S9. Lipase activity assay using total protein from
Col-0, KO, and OE plants. Supplemental Figure S10. Relative gene expression patterns of PLDa1,
PLDz1, and PLDz2 in KO mutant and OE lines. Supplemental Table S1. Primers used in confirmation of DNA insertion
and PCR 3,39-Diaminobenzidine Staining For the in situ detection of H2O2, leaves were detached and stained with 3,39-
diaminobenzidine (DAB) solution for 4 to 5 h. DAB solution was generated by
dissolving 1 mg mL21 of DAB in sterile water and adjusting to pH 3.0 with 0.2 M
of HCl. Additionally, 25 mL of TWEEN 20 (0.05% [v/v]) and 2.5 mL of 200 mM of
Na2HPO4 were added. This process generated 10 mM of Na2HPO4 DAB staining
solution and increased the pH. Bechtold N, Pelletier G (1998) In planta Agrobacterium-mediated transfor-
mation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82: 259–266 Bradford MM (1976) A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 72: 248–254 TCV Infection and HR Response Analysis Corbineau F, Xia Q, Bailly C, El-Maarouf-Bouteau H (2014) Ethylene, a
key factor in the regulation of seed dormancy. Front Plant Sci 5: 539 Transcripts synthesized in vitro from a cloned cDNA of the TCV genome
using T7 RNA polymerase were used for viral infections as described in
Dempsey et al. (1993). Resistance and susceptibility were confirmed by RT-
qPCR. Dempsey DA, Wobbe KK, Klessig DF (1993) Resistance and susceptible
responses of Arabidopsis thaliana to turnip crinkle virus. Phytopathology
83: 1021–1029 de Wit M, Galvão VC, Fankhauser C (2016) Light-mediated hormonal reg-
ulation of plant growth and development. Annu Rev Plant Biol 67: 513–537 Supplemental Data Protein Purification/Extraction, and Immunoblotting https://plantphysiol.org
Downloaded on March 11, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. Accession Numbers Hong JK, Yun BW, Kang JG, Raja MU, Kwon E, Sorhagen K, Chu C,
Wang Y, Loake GJ (2008) Nitric oxide function and signalling in plant
disease resistance. J Exp Bot 59: 147–154 Arabidopsis Genome Initiative locus identifiers (http://www.arabidopsis. org) for the genes described in this study are depicted in Supplemental Table S1. Plant Physiol. Vol. 184, 2020 1497 https://plantphysiol.org
Downloaded on March 11, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. https://plantphysiol.org
Downloaded on March 11, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved Jang et al. Huang S, Cerny RE, Bhat DS, Brown SM (2001) Cloning of an Arabidopsis
patatin-like gene, STURDY, by activation T-DNA tagging. Plant Physiol
125: 573–584 Liu G, Zhang K, Ai J, Deng X, Hong Y, Wang X (2015) Patatin-related
phospholipase A, pPLAIIIa, modulates the longitudinal growth of
vegetative tissues and seeds in rice. J Exp Bot 66: 6945–6955 Jang JH, Bae EK, Choi YI, Lee OR (2019) Ginseng-derived patatin-related
phospholipase PgpPLAIIIb alters plant growth and lignification of xylem
in hybrid poplars. Plant Sci 288: 110224 g
p
Lohman KN, Gan S, John MC, Amasino RM (1994) Molecular analysis of
natural leaf senescence in Arabidopsis thaliana. Physiol Plant 92: 322–328 Matos AR, Pham-Thi AT (2009) Lipid deacylating enzymes in plants: Old
activities, new genes. Plant Physiol Biochem 47: 491–503 y
p p
Jang JH, Lee OR (2020a) Overexpression of ginseng patatin-related phos-
pholipase pPLAIIIb alters the polarity of cell growth and decreases lig-
nin content in Arabidopsis. J Ginseng Res 44: 321–331 activities, new genes. Plant Physiol Biochem 47: 491–503 Meijer HJ, Munnik T (2003) Phospholipid-based signaling in plants. Annu
Rev Plant Biol 54: 265–306 g
Jang JH, Lee OR (2020b) Patatin-related phospholipase AtpPLAIIIa affects
lignification of xylem in Arabidopsis and hybrid poplars. Plants 9: 451 Park CH, Roh J, Youn JH, Son SH, Park JH, Kim SY, Kim TW, Kim SK
(2018) Arabidopsis ACC oxidase 1 coordinated by multiple signals
mediates ethylene biosynthesis and is involved in root development. Mol Cells 41: 923–932 Kachroo P, Yoshioka K, Shah J, Dooner HK, Klessig DF (2000) Resistance
to turnip crinkle virus in Arabidopsis is regulated by two host genes and
is salicylic acid dependent but NPR1, ethylene, and jasmonate inde-
pendent. Accession Numbers Plant Cell 22:
1812–1825 Wang C, Zien CA, Afitlhile M, Welti R, Hildebrand DF, Wang X (2000)
Involvement of phospholipase D in wound-induced accumulation of
jasmonic acid in Arabidopsis. Plant Cell 12: 2237–2246 Li M, Bahn SC, Fan C, Li J, Phan T, Ortiz M, Roth MR, Welti R, Jaworski
J, Wang X (2013) Patatin-related phospholipase pPLAIIId increases seed
oil content with long-chain fatty acids in Arabidopsis. Plant Physiol 162:
39–51 Wang X (2001) Plant phospholipases. Annu Rev Plant Physiol Plant Mol
Biol 52: 211–231 Wang X, Devaiah SP, Zhang W, Welti R (2006) Signaling functions of
phosphatidic acid. Prog Lipid Res 45: 250–278 Li M, Bahn SC, Guo L, Musgrave W, Berg H, Welti R, Wang X (2011)
Patatin-related phospholipase pPLAIIIb-induced changes in lipid me-
tabolism alter cellulose content and cell elongation in Arabidopsis. Plant
Cell 23: 1107–1123 Yang C, Li L (2017) Hormonal regulation in shade avoidance. Front Plant
Sci 8: 1527 Yang W, Devaiah SP, Pan X, Isaac G, Welti R, Wang X (2007) AtPLAI is an
acyl hydrolase involved in basal jasmonic acid production and Arabi-
dopsis resistance to Botrytis cinerea. J Biol Chem 282: 18116–18128 Li M, Wei F, Tawfall A, Tang M, Saettele A, Wang X (2015) Over-
expression of patatin-related phospholipase AIIId altered plant growth
and increased seed oil content in camelina. Plant Biotechnol J 13:
766–778 Yao H, Wang G, Guo L, Wang X (2013) Phosphatidic acid interacts with a
MYB transcription factor and regulates its nuclear localization and
function in Arabidopsis. Plant Cell 25: 5030–5042 Li-Beisson Y, Shorrosh B, Beisson F, Andersson MX, Arondel V, Bates
PD, Baud S, Bird D, Debono A, Durrett TP, et al (2013) Acyl-lipid
metabolism. Arabidopsis Book 11: e0161 Yoshida S (2003) Molecular regulation of leaf senescence. Curr Opin Plant
Biol 6: 79–84 Lin CC, Chu CF, Liu PH, Lin HH, Liang SC, Hsu WE, Lin JS, Wang HM,
Chang LL, Chien CT, et al (2011) Expression of an Oncidium gene en-
coding a patatin-like protein delays flowering in Arabidopsis by reducing
gibberellin synthesis. Plant Cell Physiol 52: 421–435 Yoshida Y, Sano R, Wada T, Takabayashi J, Okada K (2009) Jasmonic acid
control of GLABRA3 links inducible defense and trichome patterning in
Arabidopsis. Development 136: 1039–1048 Zheng Y, Li M, Wang X (2014) Proteomic insight into reduced cell elon-
gation resulting from overexpression of patatin-related phospholipase
pPLAIIId in Arabidopsis thaliana. Accession Numbers Plant Cell 12: 677–690 Platre MP, Noack LC, Doumane M, Bayle V, Simon MLA, Maneta-Peyret
L, Fouillen L, Stanislas T, Armengot L, Pejchar P, et al (2018) A com-
binatorial lipid code shapes the electrostatic landscape of plant endo-
membranes. Dev Cell 45: 465–480.e11 Kim YJ, Lee OR, Oh JY, Jang MG, Yang DC (2014) Functional analysis of
3-hydroxy-3-methylglutaryl coenzyme a reductase encoding genes in
triterpene saponin-producing ginseng. Plant Physiol 165: 373–387 Qiao Y, Piao R, Shi J, Lee SI, Jiang W, Kim BK, Lee J, Han L, Ma W, Koh
HJ (2011) Fine mapping and candidate gene analysis of dense and erect
panicle 3, DEP3, which confers high grain yield in rice (Oryza sativa L.). Theor Appl Genet 122: 1439–1449 p
p
p
g g
g
y
Kirik A, Mudgett MB (2009) SOBER1 phospholipase activity suppresses
phosphatidic acid accumulation and plant immunity in response to
bacterial effector AvrBsT. Proc Natl Acad Sci USA 106: 20532–20537 Koornneef M, Bentsink L, Hilhorst H (2002) Seed dormancy and germi-
nation. Curr Opin Plant Biol 5: 33–36 Ryu SB (2004) Phospholipid-derived signaling mediated by phospholipase
A in plants. Trends Plant Sci 9: 229–235 Labusch C, Shishova M, Effendi Y, Li M, Wang X, Scherer GFE (2013)
Patterns and timing in expression of early auxin-induced genes imply
involvement of phospholipases A (pPLAs) in the regulation of auxin
responses. Mol Plant 6: 1473–1486 Sang Y, Cui D, Wang X (2001) Phospholipase D and phosphatidic acid-
mediated generation of superoxide in Arabidopsis. Plant Physiol 126:
1449–1458 Scherer GFE, Ryu SB, Wang X, Matos AR, Heitz T (2010) Patatin-related
phospholipase A: Nomenclature, subfamilies and functions in plants. Trends Plant Sci 15: 693–700 La Camera S, Geoffroy P, Samaha H, Ndiaye A, Rahim G, Legrand M,
Heitz T (2005) A pathogen-inducible patatin-like lipid acyl hydrolase
facilitates fungal and bacterial host colonization in Arabidopsis. Plant J
44: 810–825 Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997) Subcellular
localization of H2O2 in plants. H2O2 accumulation in papillae and hy-
persensitive response during the barley-powdery mildew interaction. Plant J 11: 1187–1194 Lee OR, Kim SJ, Kim HJ, Hong JK, Ryu SB, Lee SH, Ganguly A, Cho HT
(2010) Phospholipase A2 is required for PIN-FORMED protein traffick-
ing to the plasma membrane in the Arabidopsis root. Accession Numbers Plant Signal Behav 9: e28519 Linkies A, Müller K, Morris K, Turecková V, Wenk M, Cadman CSC,
Corbineau F, Strnad M, Lynn JR, Finch-Savage WE, et al (2009) Eth-
ylene interacts with abscisic acid to regulate endosperm rupture during
germination: A comparative approach using Lepidium sativum and Ara-
bidopsis thaliana. Plant Cell 21: 3803–3822 Zhong C, Xu H, Ye S, Zhang S, Wang X (2016) Arabidopsis seed germi-
nation assay with gibberellic acid. Bio Protoc 6: 1–5 1498 Plant Physiol. Vol. 184, 2020 https://plantphysiol.org
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Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Studia Iuridica Lublinensia vol. XXVI, 1, 2017
DOI: 10.17951/sil.2017.26.1.93 Studia Iuridica Lublinensia vol. XXVI, 1, 2017
DOI: 10.17951/sil.2017.26.1.93 Studia Iuridica Lublinensia vol. XXVI, 1, 2017
DOI: 10.17951/sil.2017.26.1.93 Artykuły Konrad Marciniuk
Uniwersytet Warszawski
k.marciniuk@wpia.uw.edu.pl 4 Trybunał Konstytucyjny prowadzi postępowanie pod sygn. K 36/16. Zarówno skarga
Rzecznika Praw Obywatelskich, jak i późniejsze pisma procesowe w tej sprawie są dostępne
w domenie publicznej. Zob. http://ipo.trybunal.gov.pl/ipo/Sprawa?&pokaz=dokumenty&sygnatu
ra=K%2036/16 [dostęp: 10.04.2017]. 1 Nieruchomość rolna (grunt rolny) stanowi najistotniejszy składnik gospodarstwa rolnego
w rozumieniu art. 553 k.c. Bez tego składnika nie może istnieć gospodarstwo rolne, podczas gdy
występowanie pozostałych składników wymienionych w art. 553 k.c. nie jest konieczne dla ist-
nienia gospodarstwa rolnego. Zob. np. postanowienie SN z dnia 7 maja 1997 r., II CKN 137/97,
Legalis. 2 Ustawa z dnia 11 kwietnia 2003 r. o kształtowaniu ustroju rolnego (t.j. Dz.U. z 2012 r., poz.
803 z późn. zm.). STRESZCZENIE
UM Przedmiotem rozważań prezentowanych w artykule jest kształtowanie się normatywnej tre-
ści definicji pojęcia nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego. Analiza prowadzona jest na gruncie szeregu aktów prawnych na przestrzeni lat – przed uchwa-
leniem kodeksu cywilnego, zgodnie z treścią art. 461 Kodeksu cywilnego, na gruncie ustawy
o kształtowaniu ustroju rolnego. Przedmiotem rozważań są także: kryteria uznawania nierucho-
mości za wykorzystywaną na cele produkcji rolnej, ocena prawnego charakteru wpisu klasyfi-
kacji gruntów jako rolnych w ewidencji gruntów i budynków, określania gruntów jako rolnych
w aktach planowania przestrzennego oraz na gruncie samej ustawy o planowaniu i zagospodaro-
waniu przestrzennym. Autor opisuje również historię reglamentacji obrotu ziemią rolną w Polsce,
poświęcając dużo miejsca zmianom wprowadzonym ustawą z dnia 14 kwietnia 2016 r. o wstrzy-
maniu sprzedaży nieruchomości Zasobu Własności Rolnej Skarbu Państwa oraz o zmianie nie-
których ustaw. Opracowanie obejmuje ponadto analizę podstaw materialnoprawnych i postępo-
wania w sprawie wyrażania zgody na zbycie nieruchomości rolnej, udzielanej w drodze decyzji
administracyjnej wydawanej przez Prezesa Agencji Nieruchomości Rolnych, oraz potencjalne
konsekwencje jej wadliwości dla obrotu gruntami rolnymi. Zostały też opisane inne zagadnienia
związane z ograniczeniami obrotem nieruchomościami rolnymi w Polsce. U Słowa kluczowe: nieruchomość rolna; obrót nieruchomościami rolnymi; prawo pierwokupu;
kształtowanie ustroju rolnego 3 Dz.U. z 2016 r., poz. 585. 5 Z. Truszkiewicz dostrzega w tym pewien paradoks: definicję nieruchomości rolnej za-
mieszczono w Kodeksie cywilnym nowelą z 1990 r., która jednocześnie uchyliła przepisy szcze-
gólne dotyczące obrotu własnościowego takimi nieruchomościami. Natomiast w okresie obowią-
zywania kodeksowych przepisów szczególnych, regulujących obrót własnościowy nieruchomoś-
ciami rolnymi (tj. art. 160–168 k.c.), trzeba było sięgać do definicji takiej nieruchomości zamiesz-
czonej poza Kodeksem cywilnym – w rozporządzeniu Rady Ministrów z dnia 28 listopada 1964 r.
Zob. szerzej: Z. Truszkiewicz, Nieruchomość rolna i gospodarstwo rolne w rozumieniu U.K.U.R.,
„Krakowski Przegląd Notarialny” 2016, nr 2, s. 139–140. 6 Doktryna prawa rolnego rozwiązanie to oceniła zdecydowanie negatywnie. Zwrócił na to
uwagę np. Z. Truszkiewicz, wskazując, że nowelizacja Kodeksu cywilnego z dnia 29 lipca 1990 r.
doprowadziła do dekompozycji uregulowań dotyczących obrotu własnościowego gruntami rolny-
mi. Por. Z. Truszkiewicz, [w:] Prawo rolne, Warszawa 2015, s. 155. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 94 Konrad Marciniuk Pojęcie nieruchomości rolnej, będącej podstawowym składnikiem gospodar-
stwa rolnego1, jest jednym z kluczowych pojęć nie tylko z punktu widzenia regu-
lacji zasad obrotu tego rodzaju nieruchomościami, ale także nauki prawa rolnego
w ogóle. Zapewne z tego powodu problematyce nieruchomości rolnych poświęco-
no bardzo dużo miejsca w literaturze przedmiotu. Już na wstępie można jednak
wyrazić żal, że pomimo licznych wypowiedzi wybitnych przedstawicieli doktry-
ny prawa rolnego nie udało się dotąd wypracować, jeśli nawet nie jednolitej, co
być może wcale nie jest potrzebne, to choćby wystarczająco precyzyjnej z punktu
widzenia potrzeb obrotu definicji normatywnej tego pojęcia. Jak wiadomo, współ-
czesny ustawodawca przyjął rozwiązanie polegające na definiowaniu pojęcia „nie-
ruchomość rolna” w sposób odmienny na potrzeby uregulowań różnych obszarów
prawa rolnego. Z punktu widzenia niniejszego opracowania najistotniejszy jest
jednak sposób rozumienia pojęcia nieruchomości rolnej jako przedmiotu obrotu
własnościowego. Kluczowe znaczenie będą więc miały tu definicje legalne nie-
ruchomości rolnych zawarte w Kodeksie cywilnym i w ustawie o kształtowaniu
ustroju rolnego2. MCS Choć obie definicje funkcjonują już w systemie prawa od wielu lat, to – jak
trafnie zaznaczył Rzecznik Praw Obywatelskich w uzasadnieniu skargi wniesio-
nej do Trybunału Konstytucyjnego dotyczącej niezgodności z Konstytucją sze-
regu przepisów ustawy o kształtowaniu ustroju rolnego – w wyniku nowelizacji
tej ustawy, dokonanej na mocy ustawy z dnia 14 kwietnia 2016 r. o wstrzymaniu
sprzedaży nieruchomości Zasobu Własności Rolnej Skarbu Państwa oraz o zmia-
nie niektórych ustaw3, całkowicie zmienił się paradygmat, w jakim pojęcie to
funkcjonuje4. Fakt ten skłania do ponownego spojrzenia na normatywne rozu-
mienie pojęcia „nieruchomość rolna”. UM Ustawodawca, przechodząc na system powszechnego koncesjonowania obro-
tu ziemią rolną, całkowicie zmienił dotychczasowy kontekst normatywny obecnej
definicji. Pamiętać przy tym należy, że nowelizacja Kodeksu cywilnego dokonana
w 1990 r., na podstawie której została sformułowana obecna definicja normatywna
pojęcia „nieruchomość rolna”, była wyrazem daleko idącej liberalizacji obrotu nie-
U 2 Ustawa z dnia 11 kwietnia 2003 r. o kształtowaniu ustroju rolnego (t.j. Dz.U. z 2012 r., poz. 803
óź
) Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego 95 ruchomościami rolnymi5. Uchylono wówczas obowiązujące wcześniej konstrukcje
normatywne, które określały podmiotowe i przedmiotowe przesłanki obrotu nieru-
chomościami rolnymi (art. 160, 161, 163 k.c.)6, a tym samym w zasadzie zniesiono
ograniczenia dotyczące nabywania nieruchomości rolnych w drodze umów. S Tymczasem zmiany wprowadzone z dniem 30 kwietnia 2016 r. 10 Gromada była jednostką podziału administracyjnego. W skład gromad wchodziły wsie
położone na terenie jednej gminy lub sąsiadujących ze sobą gmin, powiązane komunikacyjnie
oraz posiadające wspólne urządzenia gospodarcze, kulturalne lub zdrowotne. Tworzenie, łączenie
i znoszenie gromad, zmiana ich granic oraz ustalanie siedzib gromadzkich rad narodowych regu-
lowała ustawa z dnia 25 września 1954 r. o reformie podziału administracyjnego wsi i powołaniu
gromadzkich rad narodowych (Dz.U. z 1954 r., nr 43, poz. 191). g
y
(
p
)
11 Ustawa z dnia 29 czerwca 1963 r. o ograniczeniu podziału gospodarstw rolnych (Dz.U.
z 1963 r., nr 28, poz. 168); Kodeks cywilny w wersji obowiązującej przed 1 października 1990 r. 7 K. Maj, Zmiany w ustawie o kształtowaniu ustroju rolnego obowiązujące od dnia 30 kwiet-
nia 2016 r., „Krakowski Przegląd Notarialny” 2016, nr 2, s. 60.
8 Rozporządzenie tymczasowe Rady Ministrów z dnia 1 września 1919 r. normujące przeno-
szenie własności nieruchomości ziemskich (Dz.U. z 1919 r., nr 73, poz. 428).
9 Przede wszystkim w dekrecie PKWN z dnia 6 września 1944 r. o przeprowadzeniu reformy
rolnej.
10 Gromada była jednostką podziału administracyjnego W skład gromad wchodziły wsie 7 K. Maj, Zmiany w ustawie o kształtowaniu ustroju rolnego obowiązujące od dnia 30 kwiet-
nia 2016 r., „Krakowski Przegląd Notarialny” 2016, nr 2, s. 60.
8 Rozporządzenie tymczasowe Rady Ministrów z dnia 1 września 1919 r. normujące przeno-
szenie własności nieruchomości ziemskich (Dz.U. z 1919 r., nr 73, poz. 428).
9 Przede wszystkim w dekrecie PKWN z dnia 6 września 1944 r. o przeprowadzeniu reformy
rolnej.
10 Gromada była jednostką podziału administracyjnego. W skład gromad wchodziły wsie
położone na terenie jednej gminy lub sąsiadujących ze sobą gmin, powiązane komunikacyjnie
oraz posiadające wspólne urządzenia gospodarcze, kulturalne lub zdrowotne. Tworzenie, łączenie
i znoszenie gromad, zmiana ich granic oraz ustalanie siedzib gromadzkich rad narodowych regu-
lowała ustawa z dnia 25 września 1954 r. o reformie podziału administracyjnego wsi i powołaniu
gromadzkich rad narodowych (Dz.U. z 1954 r., nr 43, poz. 191).
11 Ustawa z dnia 29 czerwca 1963 r. o ograniczeniu podziału gospodarstw rolnych (Dz.U.
z 1963 r., nr 28, poz. 168); Kodeks cywilny w wersji obowiązującej przed 1 października 1990 r. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 w ustawie
o kształtowaniu ustroju rolnego poszły w zupełnie przeciwnym kierunku, zmie-
rzając do objęcia obrotu nieruchomościami rolnymi szczegółową reglamentacją
CS Tymczasem zmiany wprowadzone z dniem 30 kwietnia 2016 r. w ustawie
o kształtowaniu ustroju rolnego poszły w zupełnie przeciwnym kierunku, zmie-
rzając do objęcia obrotu nieruchomościami rolnymi szczegółową reglamentacją
– przede wszystkim o charakterze administracyjnoprawnym. W tym kontekście
ustalenie, czy dana nieruchomość jest nieruchomością rolną, nabiera zupełnie in-
nego znaczenia, determinuje bowiem reżim prawny właściwy dla dokonywania
czynności zmierzających do przeniesienia własności tej nieruchomości, a także
rzutuje na sytuację prawną (przede wszystkim na zakres obowiązków) jej nabywcy. W szczególności fakt spełniania bądź niespełniania przez konkretną nieruchomość
kryteriów decydujących o jej rolnym charakterze przesądza o tym, czy: na nabycie
takiej nieruchomości przez osobę niebędącą rolnikiem indywidualnym wymagana
jest zgoda administracyjna; Agencji Nieruchomości Rolnych przysługuje wobec ta-
kiej nieruchomości prawo pierwokupu lub prawo nabycia; nabywca takiej nierucho-
mości będzie miał obowiązek osobistego prowadzenia na niej działalności rolniczej
przez okres 10 lat, a w określonych przypadkach nawet obowiązek stałego zamiesz-
kiwania na terenie gminy, na obszarze której położona jest jedna z nieruchomości
rolnych wchodzących w skład tworzonego przez nabywcę gospodarstwa rolnego. Rozstrzygnięcie o rolnym charakterze nieruchomości, przesądzając o poddaniu
czynności obrotu tą nieruchomością rygorom ustawy o kształtowaniu ustroju rol-
nego, decyduje także o objęciu danej czynności sankcją bezwzględnej nieważności
w przypadkach naruszeń wskazanych w art. 9 tej ustawy. Może to nawet spowo-
dować, że nabywca takiej nieruchomości będzie narażony na naruszenie miru do-
mowego w związku z uprawnieniami kontrolnymi Agencji Nieruchomości Rolnych
wynikającymi z art. 8a znowelizowanej ustawy o kształtowaniu ustroju rolnego. UMCS Kluczowa, z punktu widzenia regulacji zasad obrotu nieruchomościami, de-
finicja nieruchomości rolnej została sformułowana w art. 2 pkt 1 ustawy o kształ- 7 K. Maj, Zmiany w ustawie o kształtowaniu ustroju rolnego obowiązujące od dnia 30 kwiet-
nia 2016 r., „Krakowski Przegląd Notarialny” 2016, nr 2, s. 60. (
p
)
9 Przede wszystkim w dekrecie PKWN z dnia 6 września 1944 r. o przeprowadzeniu reformy
rolnej. 12 Dz.U. z 1983 r., nr 19, poz. 86. Rozporządzenie to utraciło moc z dniem 24 grudnia 1990 r.
na podstawie rozporządzenia Rady Ministrów z dnia 12 grudnia 1990 r. w sprawie warunków
dziedziczenia ustawowego gospodarstw rolnych, wydanym po dodaniu z dniem 1 października
1990 r. do Kodeksu cywilnego art. 461. 13 Zob. A. Stelmachowski, [w:] System prawa prywatnego, t. 3: Prawo rzeczowe, red.
T. Dybowski, Warszawa 2007. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Konrad Marciniuk Konrad Marciniuk 96 towaniu ustroju rolnego, zgodnie z którym nieruchomością rolną w rozumieniu tej
ustawy jest nieruchomość rolna w rozumieniu Kodeksu cywilnego, z wyłączeniem
nieruchomości położonych na obszarach przeznaczonych w planach zagospoda-
rowania przestrzennego na cele inne niż rolne. Definicja ta ma zatem w zasadzie
charakter odsyłający – odwołuje się bowiem w zasadniczej części do koncepcji
nieruchomości rolnej określonej w Kodeksie cywilnym, wyłączając jednocześnie
z kategorii nieruchomości rolnych nieruchomości położone na obszarach prze-
znaczonych w planach zagospodarowania przestrzennego na cele inne niż rolne. Taka konstrukcja art. 2 pkt 1 ustawy o kształtowaniu ustroju rolnego powoduje,
że badanie, czy konkretna nieruchomość jest nieruchomością rolną w rozumieniu
tej ustawy, jest wielostopniowe, a pierwszym etapem jest ustalenie, czy jest to nie-
ruchomość rolna w rozumieniu art. 461 k.c. Pozytywna odpowiedź powoduje ko-
nieczność ustalenia, czy jest ona objęta obowiązującym planem zagospodarowania
przestrzennego, a jeśli tak, to jakie jest jej przeznaczenie zgodnie z tym planem7. MCS p
g
j
j
j
j j p
g
y
p
Koncepcje normatywne nieruchomości rolnych kształtowały się w polskim
porządku prawnym stopniowo. Pomijając pojęcie nieruchomości ziemskich, któ-
rym posługiwano się w dwudziestoleciu międzywojennym8 oraz w okresie po-
wojennej przebudowy ustroju rolnego państwa9, do polskiego porządku praw-
nego pojęcie „nieruchomość rolna” zostało wprowadzone w latach 50. XX w. Po raz pierwszy ustawodawca posłużył się nim w ustawie z dnia 13 lipca 1957 r. o obrocie nieruchomościami rolnymi. Zgodnie z brzmieniem art. 1 ust. 2 tej usta-
wy nieruchomość uważano za rolną, jeżeli była użytkowana na cele produkcji
rolnej, nie wyłączając produkcji ogrodniczej, sadowniczej, leśnej i rybnej, albo
jeżeli nieruchomość była przeznaczona do takiej produkcji według planów za-
gospodarowania przestrzennego. Jednocześnie art. 1 ust. 1 tej ustawy ograniczał
zakres pojęcia „nieruchomość rolna” wyłącznie do nieruchomości, które posia-
dały wskazane cechy i dodatkowo były położone na terenie gromad10. Głównym
celem tej ustawy, podobnie jak szeregu późniejszych regulacji, było ograniczenie
podziału gospodarstw rolnych11. Z kolei zgodnie z przepisami rozporządzenia
UMC g
y
(
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11 Ustawa z dnia 29 czerwca 1963 r. o ograniczeniu podziału gospodarstw rolnych (Dz.U. z 1963 r., nr 28, poz. 168); Kodeks cywilny w wersji obowiązującej przed 1 października 1990 r. 14 Por. wyrok SN z dnia 2 czerwca 2000 r., II CKN 1067/98, OSP 2001, nr 2, poz. 27.
15 A. Lichorowicz, Glosa do wyroku SN z 2.6.2000 r., II CKN 1067/98, OSP 2001, nr 2, poz. 27,
s. 87.
16 R. Budzinowski, [w:] Kodeks cywilny, t. 1: Komentarz do art. 461, red. Gutowski, War-
szawa 2016.
17 Por. postanowienie SN z dnia 15 maja 2009 r., II CSK 9/09, Legalis. Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego 97 Rady Ministrów z dnia 28 listopada 1964 r. w sprawie przenoszenia własności
nieruchomości rolnych, znoszenia współwłasności takich nieruchomości oraz
dziedziczenia gospodarstw rolnych12, za nieruchomość rolną uznawano taką nie-
ruchomość, która była lub mogła być użytkowana na cele produkcji rolnej, nie
wyłączając produkcji ogrodniczej, sadowniczej i rybnej, chyba że stosownie do
przepisów o planowaniu przestrzennym decyzją właściwego organu prezydium
rady narodowej została przeznaczona na cele niezwiązane bezpośrednio z pro-
dukcją rolną. Na gruncie wskazanego rozporządzenia nie uważało się jednak za
nieruchomość rolną terenów położonych w obrębie zwartej zabudowy miasta lub
osiedla, a także nieruchomości należących do tej samej osoby lub osób, jeżeli
ich łączny obszar nie przekraczał 0,2 ha, który to pułap następnie podniesiono
do 0,5 ha i w końcu do 1 ha. Obok funkcjonującej wówczas minimalnej normy
obszarowej podstawowym kryterium kwalifikacji danej nieruchomości do nie-
ruchomości rolnych, podobnie jak ma to miejsce dzisiaj, było to, czy „są one lub
mogą być użytkowane na cele produkcji rolnej, nie wyłączając produkcji ogrod-
niczej, sadowniczej i rybnej”. Wskazane rozporządzenie Rady Ministrów z dnia
28 listopada 1964 r. utraciło moc z dniem 24 grudnia 1990 r. na podstawie roz-
porządzenia Rady Ministrów z dnia 12 grudnia 1990 r. w sprawie warunków
dziedziczenia ustawowego gospodarstw rolnych, wydanego po dodaniu, z dniem
1 października 1990 r., do Kodeksu cywilnego art. 461, w którym sformułowa-
no obecną definicję normatywną nieruchomości rolnej. Definicja ta, jak widać,
w dużej mierze opiera się na definicji przyjętej w rozporządzeniu Rady Mini-
strów z dnia 28 listopada 1964 r., choć – co stanowi istotną różnicę – w Kodeksie
cywilnym przy definiowaniu nieruchomości rolnej całkowicie pominięto stoso-
wane wcześniej kryterium obszarowe. UMCS Kodeks cywilny, będący podstawowym aktem prawnym regulującym sto-
sunki cywilnoprawne, definiuje nieruchomość rolną (grunt rolny) jako nierucho-
mości, które są lub mogą być wykorzystywane do prowadzenia działalności wy-
twórczej w rolnictwie w zakresie produkcji roślinnej i zwierzęcej, nie wyłączając
produkcji ogrodniczej, sadowniczej i rybnej. U Należy zwrócić uwagę, że brzmienie art. 461 k.c. na pozór zrównuje pojęcia
nieruchomości rolnej oraz gruntu rolnego, czego konsekwencją byłoby trakto-
wanie obu pojęć jako synonimów. Pogląd taki poddano jednak krytyce. A. Stel-
machowski zwrócił uwagę, że choć konstrukcja art. 461 k.c., który łączy pojęcie
nieruchomości i gruntu rolnego, może sugerować, że nieruchomość rolna i grunt
rolny stanowią tożsame kategorie prawne, to takie podejście jest błędne13. Formu- 18 J. Bieluk, Ustawa o kształtowaniu ustroju rolnego. Komentarz, Warszawa 2016, s. 31.
19 Prawo rolne, red. A. Oleszko, Kraków 2009, s. 61.
20 R. Budzinowski, op. cit.
21 Z. Truszkiewicz, Nieruchomość rolna i gospodarstwo rolne…, s. 143–151. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Konrad Marciniuk Konrad Marciniuk 98 ła nieruchomości rolnej opiera się bowiem zawsze na kryterium własnościowym,
tymczasem istota gruntu rolnego oderwana jest od problematyki własnościowej. Użyte w art. 461 k.c. nawiązanie do gruntów rolnych oznacza jedynie, że nieru-
chomość rolna nie może być nieruchomością budynkową ani tym bardziej loka-
lową. Rozumienie obu pojęć jako synonimów mogłoby doprowadzić do wielu
komplikacji, m.in. gospodarstwo rolne w rozumieniu kodeksowym mogłoby być
prowadzone wyłącznie na gruntach własnych właściciela, a tym samym wyklu-
czyć z gospodarstwa rolnego grunty dzierżawione. CS Wobec zastosowania w konstrukcji wskazanej normy alternatywy zwykłej,
aby dana nieruchomość została uznana za rolną wystarcza, aby spełniała ona tylko
jeden ze wskazanych warunków, tzn. aby „była” lub „mogła być” wykorzystywana
na cele produkcji rolnej. MCS Zarówno w piśmiennictwie, jak i orzecznictwie panuje zgodny pogląd, że
o rolniczym charakterze gruntu przesądza nie tylko aktualny sposób jego wyko-
rzystania, ale także możliwość takiego wykorzystania14. A. Lichorowicz zauwa-
żył, iż dla kwalifikacji nieruchomości jako rolnej decydujące są:
MC […] czysto agronomiczne cechy gruntu, z których wynika, że uzyskiwanie na nim produk-
tów rolnych jest fizycznie możliwe. Co więcej, uzyskiwanie z gruntu płodów rolnych nie musi
stanowić faktu. Artykuł 461 k.c. zadowala się tutaj możliwością, istnieniem pewnej potencjalnej
perspektywy uzyskiwania z gruntu płodów rolnych znajdującej swe uzasadnienie w fizyczno-
-agronomicznych właściwościach gruntu15. UM Podobne stanowisko zajmuje R. Budzinowski, wskazując, że nieruchomość
rolna to taka, na której, ze względu na te właściwości, prowadzi się uprawy, ale są
to też grunty, które z jakichś względów nie są uprawiane (np. odłogi). Te drugie
nie tracą charakteru rolnego, jeśli przy wykorzystaniu obecnej techniki mogą
być włączone do procesu produkcji rolnej16. To stwierdzenie dotyczy także nie-
ruchomości, które przez dłuższy czas były faktycznie wykorzystywane na cele
niezwiązane z produkcją rolną17. U Choć powyższe poglądy na temat wykładni art. 461 k.c. należy podzielić,
to nie sposób nie dostrzec, że stosowanie sformułowanej w tym przepisie de-
finicji pojęcia „nieruchomość rolna” rodzi szereg komplikacji. Trudno bowiem
uznać za precyzyjne kryterium uznania nieruchomości za rolną w oparciu o samą
„techniczną” możliwość wykorzystania jej na cele produkcji rolnej po wykonaniu
bliżej niesprecyzowanych prac agrotechnicznych czy rekultywacyjnych. Czysto 21 Z. Truszkiewicz, Nieruchomość rolna i gospodarstwo rolne…, s. 143–151. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 100 Konrad Marciniuk Konrad Marciniuk cjonalne. Z. Truszkiewicz, dostrzegając potrzebę bardziej racjonalnego kwalifi-
kowania jako rolnych nieruchomości o niewielkiej powierzchni, wyraża pogląd
(który należy podzielić), że pomimo braku w ustawie wyraźnie określonej mini-
malnej normy obszarowej samo kryterium „możliwości wykorzystania nierucho-
mości do prowadzenia działalności wytwórczej w rolnictwie” jest na tyle pojem-
ne, że umożliwia wzięcie pod uwagę nie tylko samych właściwości gruntu, ale też
innych okoliczności wpływających na ocenę przydatności gruntu do prowadzenia
działalności wytwórczej w rolnictwie przy uwzględnieniu zasad prawidłowej go-
spodarki rolnej. Okolicznościami takimi mogą być zatem również: powierzchnia
gruntu (choć oczywiście nie sposób tu wskazać żadnej konkretnej minimalnej
wielkości powierzchni nieruchomości uznawanej za rolną), sposób zagospoda-
rowania, ukształtowanie granic, konfiguracja terenu itp.22 Należy jednocześnie
zaznaczyć, że wobec określonej w art. 9 ustawy o kształtowaniu ustroju rolnego
sankcji nieważności czynności prawnej dokonanej niezgodnie z przepisami usta-
wy, opisane kryteria powinny być stosowane z dużą ostrożnością. MCS wy, opisane kryteria powinny być stosowane z dużą ostrożnością. Drugim zagadnieniem występującym na tle kwalifikowania nieruchomości
jako rolnych w oparciu o postanowienia art. 461 k.c. jest problem nieruchomości
obejmujących zarówno grunty nadające się do produkcji rolniczej, jak i grunty
o innym przeznaczeniu. W szczególności problemem jest właściwe kwalifikowa-
nie nieruchomości w przypadkach, gdy użytkiem rolnym jest jedynie znikoma
część nieruchomości, a jej przeważająca część takiego charakteru nie ma. Jak za-
uważa Z. Truszkiewicz, kwestia ta była przedmiotem rozważań Sądu Najwyższe-
go w sprawie o sygn. IV CSK 93/1223. W uzasadnieniu wyroku, który zapadł w tej
sprawie, sąd zauważył, że w odniesieniu do nieruchomości o niejednorodnym
charakterze możliwe są trzy stanowiska: 1) nie jest ona objęta regulacją ustawy
niezależnie od zakresu przeznaczenia jej na inne cele, 2) podpada pod jej uregu-
lowania, jeśli nie jest w całości wykorzystywana i przeznaczona na inne cele niż
rolne albo 3) wskazane wymagania ustawowe spełnia nieruchomość, której wio-
dącym przeznaczeniem jest prowadzenie działalności wytwórczej w rolnictwie. Zdaniem sądu opowiedzenie się za jednym z tych stanowisk powinno uwzględ-
niać zamierzenia, które legły u podstaw ustawy z dnia 11 kwietnia 2003 r., które
zostały określone w art. 1. Ustawa ma za zadanie kształtowanie ustroju rolnego
przez wprowadzenie do obrotu nieruchomościami rolnymi mechanizmów, któ-
re powinny prowadzić do poprawy struktury obszarowej gospodarstw rolnych,
przeciwdziałać nadmiernej koncentracji nieruchomości rolnych oraz zapewniać
prowadzenie gospodarstw rolnych przez osoby o odpowiednich kwalifikacjach. 23 http://sn.pl/orzecznictwo/SitePages/Baza_orzeczen.aspx [dostęp: 10.04.2017]. 22 Ibidem. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego 99 teoretycznie bowiem można sobie wyobrazić, że praktycznie każda nieruchomość
gruntowa może zostać wykorzystana do produkcji rolnej, przy czym czasem może
to wymagać poniesienia znacznych nakładów, np. związanych z rozbiórką zloka-
lizowanych na nieruchomości budynków i rekultywacją gruntu. Z tego punktu
widzenia wydaje się, że za rolne powinny być uznawane tylko takie nierucho-
mości, których wykorzystanie do prowadzenia działalności wytwórczej będzie
wynikało nie tylko z fizyko-chemicznych cech danej nieruchomości (gruntu), ale
również będzie pozostawało w zgodzie z przepisami prawa – przede wszystkim
z regulacjami dotyczącymi planowania przestrzennego. Innymi słowy, wymogi
zawarte w art. 461 k.c. rozumieć należy nie tylko jako faktyczną, ale i zgodną
z prawem możliwość wykorzystywania nieruchomości do celów rolniczych18. MCS Przyjęcie, że faktyczne lub potencjalne wykorzystanie danej nieruchomości
do produkcji rolnej decyduje o jej rolnym charakterze powoduje, że kodekso-
wa treść pojęcia nieruchomości rolnej została skonstruowana w sposób niepełny,
także w aspekcie posłużenia się przez ustawodawcę pojęciem prowadzenia dzia-
łalności wytwórczej w rolnictwie. Jak zwraca się uwagę w piśmiennictwie, jedno
z podstawowych pojęć dla prawa rolnego, jakim jest „rolnictwo”, jest pojęciem
o obszernym zakresie desygnatów, czego skutkiem jest problem w jasnym i precy-
zyjnym jego zdefiniowaniu19. Ustawodawca dosyć szeroko rozumie „działalność
wytwórczą w rolnictwie”, obejmując nią nie tylko produkcję roślinną i zwierzę-
cą, lecz także produkcję ogrodniczą, sadowniczą i rybną. To sformułowanie ma
zapewne podkreślać, że nie chodzi wyłącznie o tzw. polową uprawę roślin oraz
(związane z nią) chów i hodowlę zwierząt. Nie jest to jednak rozszerzenie pojęcia
rolnictwa, gdyż nie wychodzi ono poza związek działalności z rolniczym wy-
korzystywaniem gruntu. Z zakresu działalności wytwórczej w rolnictwie zosta-
ła wyłączona produkcja leśna, aczkolwiek grunty leśne mogą wchodzić w skład
gospodarstwa rolnego. Należy ponadto podkreślić, że ustawodawca sprowadza
wykorzystywanie gruntów rolnych tylko do działalności wytwórczej, co wyłą-
cza w zasadzie możliwość rolnej kwalifikacji gruntów wykorzystywanych np. do
działalności przetwórczej czy usługowej20. UMC W doktrynie podkreśla się, że współczesna definicja nieruchomości rolnej
jest źródłem dwóch podstawowych problemów21. Po pierwsze, czy – ze wzglę-
du jedynie na funkcjonalne kryterium przydatności do prowadzenia działalności
rolniczej – niewielkie obszarowo nieruchomości winny być kwalifikowane jako
rolne, nawet jeżeli ze względu na ich cechy (takie jak np. położenie, charakter
sąsiednich nieruchomości, konfiguracja granic czy aktualny stan zagospodaro-
wania) ewentualne prowadzenie działalności rolniczej byłoby całkowicie niera- Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego 101 z przedstawionych stanowisk, ponieważ wprowadza ono warunek, który nie zo-
stał przewidziany w art. 2 pkt 1 ustawy o kształtowaniu ustroju rolnego, nadto
rodzi możliwość pozostawania poza ustawowym zasięgiem nieruchomości, które
jedynie w nieznacznym zakresie są wykorzystywane w inny sposób, a miałyby
wpływ na realizację jej założeń. Odnośnie do drugiego stanowiska sąd zauważył,
że z jednej strony zapatrywanie to uwzględnia założenia wprowadzenia ograni-
czeń w obrocie nieruchomościami, zaś z drugiej określa jednoznaczne kryterium
oceny charakteru nieruchomości, wolne od uznaniowości rodzącej niejedno-
krotnie problemy i utrudniające obrót nieruchomościami. Trzecie zapatrywanie
natomiast jest zdaniem sądu zbliżone do drugiego stanowiska. Oparte zostało
bowiem na ocenie charakteru nieruchomości w odniesieniu do wiodącego, czy
też zasadniczego, sposobu jej wykorzystywania oraz przeznaczenia objętego pla-
nem zagospodarowania przestrzennego, uwzględniającego również cel ustawy. Za wiodące, czy też zasadnicze, należałoby uznać przeznaczenie nieruchomości,
gdy jej powierzchnia w przeważającym zakresie stanowi nieruchomość rolną,
a część związana z innego rodzaju działalnością nie jest znaczna, co przesądza
o objęciu całej nieruchomości ustawą z dnia 11 kwietnia 2003 r. UMCS Jednocześnie sąd zauważył, że charakter tych przesłanek może być ocenny,
co z kolei mogłoby wywoływać wątpliwości i trudności w stosowaniu ustawy. W rozpoznawanej sprawie Sąd Najwyższy w konsekwencji za miarodajne uznał
stanowisko drugie jako odpowiadające także zasadzie pewności obrotu. Do sta-
nowiska tego krytycznie odniósł się Z. Truszkiewicz, wskazując jako – jego zda-
niem – bardziej zasadne funkcjonalne podejście do oceny charakteru danej nie-
ruchomości, zwłaszcza że – jak słusznie zauważa ten autor – w treści art. 2 pkt 1
ustawy o kształtowaniu ustroju rolnego nie ma zastrzeżenia, że ustawa dotyczy
tylko nieruchomości obejmujących jedynie użytki rolne ani że jako rolną należy
traktować nieruchomość obejmującą choćby w części użytki rolne bez względu
na ich powierzchnię24. Za trafny wreszcie należy uznać pogląd Z. Truszkiewicza,
że o stosowaniu ustawy o kształtowaniu ustroju rolnego do nieruchomości obej-
mujących użytki o różnym sposobie użytkowania winien przesądzać dominujący
sposób korzystania z nieruchomości czy też jej dominujące przeznaczenie. UM Dla ustalenia rolnego charakteru nieruchomości rolnej pomocne są wpisy
dokonane w ewidencji gruntów. Co do zasady rolne właściwości nieruchomości
rolnych odzwierciedla kategoria użytków rolnych. Zgodnie z § 68 ust. 1 rozporzą-
dzenia Ministra Rozwoju Regionalnego i Budownictwa z dnia 29 marca 2001 r. 24 Z. Truszkiewicz, Nieruchomość rolna i gospodarstwo rolne…, s. 150.
25 Dz.U. z 2016 r., poz. 1034. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Istotnego znaczenia nabiera zatem rozważenie, czy i jaki wpływ na realizację
tych zamierzeń może mieć konkretna nieruchomość, która w pewnym zakresie
obszarowym została przeznaczona na inne cele niż rolne. Sąd odrzucił pierwsze
UM Drugim zagadnieniem występującym na tle kwalifikowania nieruchomości
jako rolnych w oparciu o postanowienia art. 461 k.c. jest problem nieruchomości
obejmujących zarówno grunty nadające się do produkcji rolniczej, jak i grunty
o innym przeznaczeniu. W szczególności problemem jest właściwe kwalifikowa-
nie nieruchomości w przypadkach, gdy użytkiem rolnym jest jedynie znikoma
część nieruchomości, a jej przeważająca część takiego charakteru nie ma. Jak za-
uważa Z. Truszkiewicz, kwestia ta była przedmiotem rozważań Sądu Najwyższe-
go w sprawie o sygn. IV CSK 93/1223. W uzasadnieniu wyroku, który zapadł w tej
sprawie, sąd zauważył, że w odniesieniu do nieruchomości o niejednorodnym
charakterze możliwe są trzy stanowiska: 1) nie jest ona objęta regulacją ustawy
niezależnie od zakresu przeznaczenia jej na inne cele, 2) podpada pod jej uregu-
lowania, jeśli nie jest w całości wykorzystywana i przeznaczona na inne cele niż
rolne albo 3) wskazane wymagania ustawowe spełnia nieruchomość, której wio-
dącym przeznaczeniem jest prowadzenie działalności wytwórczej w rolnictwie. UM Zdaniem sądu opowiedzenie się za jednym z tych stanowisk powinno uwzględ-
niać zamierzenia, które legły u podstaw ustawy z dnia 11 kwietnia 2003 r., które
zostały określone w art. 1. Ustawa ma za zadanie kształtowanie ustroju rolnego
przez wprowadzenie do obrotu nieruchomościami rolnymi mechanizmów, któ-
re powinny prowadzić do poprawy struktury obszarowej gospodarstw rolnych,
przeciwdziałać nadmiernej koncentracji nieruchomości rolnych oraz zapewniać
prowadzenie gospodarstw rolnych przez osoby o odpowiednich kwalifikacjach. Istotnego znaczenia nabiera zatem rozważenie, czy i jaki wpływ na realizację
tych zamierzeń może mieć konkretna nieruchomość, która w pewnym zakresie
obszarowym została przeznaczona na inne cele niż rolne. Sąd odrzucił pierwsze Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 26 Dz.U. z 2015 r., poz. 520 z późn. zm.
27 Tak np. wyrok WSA w Warszawie z dnia 30 lipca 2010 r., VII SA/Wa 950/10.
28 A. Gryszczyńska, [w:] G. Szpor, M. Durzyńska, A. Gryszczyńska, I. Kamińska, K. Mą-
czewski, W. Radzio, Prawo geodezyjne i kartograficzne. Komentarz, Warszawa 2013, s. 178 – cyt.
za uzasadnieniem wniosku RPO do TK z dnia 7 lipca 2016 r. w sprawie K 36/16. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 w sprawie ewidencji gruntów i budynków25 grunty rolne dzielą się na użytki rolne
oraz nieużytki. Do kategorii użytków zalicza się: a) grunty orne, b) sady, c) łąki
trwałe, d) pastwiska trwałe, e) grunty rolne zabudowane, f) grunty pod stawami, g)
grunty pod rowami oraz h) grunty zadrzewione i zakrzewione na użytkach rolnych. 26 Dz.U. z 2015 r., poz. 520 z późn. zm. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego 103 że za nieruchomości rolne należy również uznawać grunty stanowiące nieużytki,
tj. grunty nienadające się bez znacznych nakładów do działalności wytwórczej
w rolnictwie, w szczególności: 1) bagna (błota, topieliska, trzęsawiska, mocza-
ry, rojsty), 2) piaski (piaski ruchome, piaski nadbrzeżne, wydmy), 3) naturalne
utwory fizjograficzne, takie jak: urwiska, strome stoki, uskoki, skały, rumowiska,
zapadliska, nisze osuwiskowe, piargi, 4) grunty pokryte wodami, które nie nada-
ją się do produkcji rybnej (sadzawki, wodopoje, doły potorfowe). Należy zatem
uznać, że oznaczenie nieruchomości w ewidencji gruntów dla ustalenia, czy dana
nieruchomość podlega przepisom ustawy, ma znaczenie pomocnicze i nie zawsze
przesądzające29. W szczególności nie decyduje w razie istnienia miejscowego pla-
nu zagospodarowania przestrzennego określającego przeznaczenie nieruchomości
na cele inne niż rolne. MCS W piśmiennictwie panuje w zasadzie zgoda co do tego, że nieruchomość rol-
na traci swój rolny charakter w wyniku wyłączenia jej z produkcji rolnej. W tym
miejscu warto jednak podkreślić, że niektórzy autorzy, moim zdaniem nietrafnie,
wskazują, że chodzi tu o dokonanie wyłączenia na podstawie ostatecznej decy-
zji administracyjnej o zgodzie na takie wyłączenie. Tymczasem warto zwrócić
uwagę, że w obecnym stanie prawnym wyłączenie gruntu z produkcji rolnej,
rozumiane jako rozpoczęcie innego niż rolnicze użytkowanie gruntów, podlega
reglamentacji prawnej jedynie w ściśle wskazanych w ustawie wypadkach. Zgod-
nie z brzmieniem art. 11 ust. 1 ustawy z dnia 3 lutego 1995 r. o ochronie gruntów
rolnych i leśnych30 wyłączenie z produkcji gruntu rolnego wymaga wcześniej-
szego uzyskania decyzji zezwalających na takie wyłączenie jedynie w odniesie-
niu do użytków rolnych wytworzonych z gleb pochodzenia mineralnego i or-
ganicznego, zaliczonych do klas I, II, III, IIIa, IIIb, oraz użytków rolnych klas
IV, IVa, IVb, V i VI wytworzonych z gleb pochodzenia organicznego, a także
gruntów, o których mowa w art. 2 ust. 1 pkt 2–10. A contrario należy przyjąć, że
np. wyłączenie z produkcji rolnej gruntu rolnego gruntu klasy IV, wytworzonego
z gleb pochodzenia mineralnego, nie będzie podlegało kontroli administracyjnej
(przynajmniej w płaszczyźnie ustawy o ochronie gruntów rolnych i leśnych). Na
marginesie należy także zaznaczyć, że czym innym jest utrata przez nierucho-
mość jej rolnego charakteru w rozumieniu wskazanej ustawy o ochronie gruntów
rolnych i leśnych (i pośrednio w rozumieniu art. 461 k.c.), a czym innym utrata
takiego charakteru w związku z przepisami kreującymi szczególne zasady obrotu
nieruchomościami rolnymi, tj. 29 K. Maj, op. cit., s. 68.
30 Dz.U. z 2015 r., poz. 909. 29 K. Maj, op. cit., s. 68. 30 Dz.U. z 2015 r., poz. 909. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 102 Konrad Marciniuk Szczegółowe zasady zaliczania gruntów do poszczególnych użytków gruntowych
oraz określenie cech gruntów i innych przesłanek, które decydują o zaliczaniu
gruntów do poszczególnych użytków gruntowych, określa załącznik nr 6 do wska-
zanego rozporządzenia. S Szczegółowe zasady zaliczania gruntów do poszczególnych użytków gruntowych
oraz określenie cech gruntów i innych przesłanek, które decydują o zaliczaniu
gruntów do poszczególnych użytków gruntowych, określa załącznik nr 6 do wska-
zanego rozporządzenia. S Zaznaczyć przy tym należy, że określanie charakteru nieruchomości jako
rolnej w rozumieniu art. 461 k.c. w oparciu o wpisy w ewidencji gruntów, pro-
wadzonej na podstawie wskazanego wyżej rozporządzenia oraz ustawy z dnia
17 maja 1989 r. – Prawo geodezyjne i kartograficzne26, prowadziłoby do trafnych
rozstrzygnięć jedynie w zakresie tych nieruchomości, które „są” wykorzysty-
wane do prowadzenia działalności wytwórczej w rolnictwie, natomiast mogło-
by się okazać kryterium zawodnym w stosunku do tych nieruchomości, których
rolny charakter nie wynika z obecnego, lecz z potencjalnego sposobu wykorzy-
stania (zagospodarowania) gruntu, tj. w stosunku do tych nieruchomości, które
wprawdzie nie „są”, lecz „mogą być” wykorzystane do prowadzenia działalności
rolniczej. Ewidencja gruntów ma bowiem charakter „odwzorowawczy”, tj. słu-
ży ujawnianiu informacji dotyczących m.in. aktualnego rodzaju użytków grun-
towych występujących w obrębie poszczególnych nieruchomości, natomiast nie
przesądza w jakikolwiek sposób o możliwościach alternatywnego zagospodaro-
wania nieruchomości, co jest domeną przede wszystkim regulacji i rozstrzygnięć
podejmowanych w zakresie prawa planowania przestrzennego. UMCS Pogląd taki wyraził Naczelny Sąd Administracyjny w uzasadnieniu wyroku
z dnia 15 maja 2014 r. (sygn. I OSK 2566/12), wskazując, że:
UM […] dane zawarte w ewidencji gruntów i budynków mają charakter informacyjny, a sam
rejestr ewidencji gruntów jest wyłącznie odzwierciedleniem aktualnego stanu prawnego dotyczą-
cego danej nieruchomości. Ma on charakter deklaratoryjny, a nie konstytutywny, co oznacza, że
nie tworzy nowego stanu prawnego nieruchomości, a jedynie potwierdza stan faktyczny. U Fakt ewentualnego nieujawnienia zmian sposobu zagospodarowania nieru-
chomości w ewidencji gruntów świadczy jedynie o nieaktualności wpisu, nie
przesądza jednak o prawdziwości zawartych w nim danych27. Należy więc uznać,
że wpisy w ewidencji gruntów i budynków mają charakter czysto informacyjny,
a „przepisy prawa nie wiążą z nią domniemania zgodności z rzeczywistym sta-
nem prawnym ani jakiegokolwiek innego skutku materialnoprawnego”28. Podkreślić należy przy tym, że stosowanie wprost zapisów ewidencji gruntów
do ustalania rolnego charakteru nieruchomości skutkowałoby także przyjęciem, 33 Załącznik nr 1 do Rozporządzenia Ministra Infrastruktury z dnia 26 sierpnia 2003 r.
w sprawie wymaganego zakresu projektu miejscowego planu zagospodarowania przestrzennego
– Podstawowe barwne oznaczenia graficzne i literowe dotyczące przeznaczenia terenów, które
należy stosować na projekcie rysunku planu miejscowego.
34 K M j
i
68 32 Dz.U. z 2003 r., nr 164, poz. 1587. 31 Dz.U. z 2016 r., poz. 778. 34 K. Maj, op. cit., s. 68. 31 Dz.U. z 2016 r., poz. 778.
32 Dz.U. z 2003 r., nr 164, poz. 1587.
33 Załącznik nr 1 do Rozporządzenia Ministra Infrastruktury z dnia 26 sierpnia 2003 r.
w sprawie wymaganego zakresu projektu miejscowego planu zagospodarowania przestrzennego
– Podstawowe barwne oznaczenia graficzne i literowe dotyczące przeznaczenia terenów, które
należy stosować na projekcie rysunku planu miejscowego.
34 K. Maj, op. cit., s. 68. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 ustawy z dnia 11 kwietnia 2003 r. o kształtowaniu
ustroju rolnego. UMC Na potrzeby regulacji zasad obrotu nieruchomościami rolnymi, które zostały
przyjęte w ustawie o kształtowaniu ustroju rolnego, przedstawiony zakres pojęcia
nieruchomości rolnej w rozumieniu art. 461 k.c. uległ istotnemu ograniczeniu. Prze- Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego 105 cele nierolne, mogą być wykorzystywane w sposób dotychczasowy aż do czasu
ich docelowego zagospodarowania, nie przesądza o tym, że nieruchomości poło-
żone na ich obszarze zachowują charakter rolny w rozumieniu ustawy o kształto-
waniu ustroju rolnego. W świetle art. 2 pkt 1 ustawy o kształtowaniu ustroju rol-
nego już sama, wynikająca z miejscowego planu zagospodarowania, możliwość
przeznaczenia nieruchomości na cele nierolne powoduje, że nieruchomość ta
traci swój rolny charakter w rozumieniu tej ustawy. Należy wszakże podkreślić,
że nieruchomość taka zachowa jednocześnie swój rolny charakter w rozumieniu
ustawy o ochronie gruntów rolnych i leśnych i jako taka będzie objęta ochroną na
podstawie tej ustawy do momentu wyłączenia jej z produkcji rolnej oraz będzie
nadal traktowana jako nieruchomość rolna. MCS Na tle przeznaczenia nieruchomości w planach zagospodarowania na cele
nierolne, co przesądzałoby o utracie przez taką nieruchomość charakteru nieru-
chomości rolnej w rozumieniu przepisów dotyczących obrotu nieruchomościami
rolnymi, pojawiają się problemy wynikające z realizowanych w planach miejsco-
wych koncepcji tzw. wielofunkcyjności terenów. Częstym zjawiskiem jest bowiem
ustalanie w miejscowych planach zagospodarowania przestrzennego różnych, naj-
częściej komplementarnych względem siebie funkcji, które mogą być realizowane
na danym terenie. W tym kontekście pojawia się pytanie, jak należy traktować
nieruchomości położone na terenach, które mogą być wykorzystywane jednocześ-
nie na cele nierolne (np. tereny budownictwa mieszkaniowego jednorodzinnego)
i cele rolne (np. utrzymywanie zabudowy zagrodowej). Czy w takim razie nieru-
chomość położoną na takim terenie należy traktować jako rolną w rozumieniu
przepisów dotyczących obrotu nieruchomościami czy też nie? Choć na tak posta-
wione pytanie ustawodawca nie daje jednoznacznej odpowiedzi, to moim zdaniem
w przypadku, gdy miejscowy plan zagospodarowania przestrzennego dopuszcza
nierolne zagospodarowanie terenu, spełniona jest przesłanka określona w art. 2
pkt 1 ustawy o kształtowaniu ustroju rolnego przeznaczenia nieruchomości na cele
„inne niż rolne”, a zatem należy uznać, że nieruchomość taka traci swój rolny
charakter, nawet jeżeli ten sam plan będzie dopuszczał możliwość dalszego wy-
korzystywania danej nieruchomości na cele produkcji rolnej. Przepis art. 2 pkt 1
ustawy o kształtowaniu ustroju rolnego wyłącza z zakresu pojęcia nieruchomości
rolnej nieruchomości położone na obszarach przeznaczonych w planach zagospo-
darowania przestrzennego na cele inne niż rolne. Nie wymaga natomiast, aby na
obszarach tych możliwość prowadzenia działalności była wyłączona. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 104 Konrad Marciniuk de wszystkim wyłączono z tego zakresu nieruchomości, które wprawdzie spełniają
przesłanki uznania ich za rolne w rozumieniu art. 461 k.c., lecz są położone na ob-
szarach przeznaczonych w planach zagospodarowania przestrzennego na cele inne
niż rolne. S Problematykę planów zagospodarowania przestrzennego reguluje ustawa
z dnia 27 marca 2003 r. o planowaniu i zagospodarowaniu przestrzennym31. Zgod-
nie z brzmieniem art. 14 ust. 8 tej ustawy miejscowy plan zagospodarowania prze-
strzennego jest aktem prawa miejscowego, a więc jest źródłem prawa powszech-
nie obowiązującego. Normy zawarte w planie miejscowym ustalają przeznaczenie
terenu, rozmieszczenie inwestycji celu publicznego oraz określają dopuszczalne
sposoby zagospodarowania i warunków zabudowy terenu (art. 4 ust. 1 ustawy
o planowaniu i zagospodarowaniu przestrzennym). Wymagany zakres regulacji
przyjmowanych w miejscowych planach zagospodarowania przestrzennego okre-
ślają przepisy art. 15 ust. 2 i 3 ustawy o planowaniu przestrzennym oraz przepisy
rozporządzenia Ministra Infrastruktury z dnia 26 sierpnia 2003 r. w sprawie wyma-
ganego zakresu projektu miejscowego planu zagospodarowania przestrzennego32. Tereny przeznaczone na cele rolne w miejscowych planach zagospodarowania
przestrzennego oznaczane są jako: 1) tereny rolnicze (oznaczane na rysunku planu
literą R i kolorem żółtym), 2) tereny obsługi produkcji w gospodarstwach rolnych,
hodowlanych, ogrodniczych oraz gospodarstwach leśnych i rybackich (oznaczane
na rysunku planu literą RU i kreskowaniem żółto-czerwonym), a także 3) tereny
zabudowy zagrodowej w gospodarstwach rolnych, hodowlanych i ogrodniczych
(oznaczane na rysunku planu literą RM i kreskowaniem żółto-jasnobrązowym)33. Zaznaczyć przy tym należy, że zgodnie z § 9 ust. 4 wymienionego rozporządzenia
dopuszcza się stosowanie na projekcie rysunku planu miejscowego uzupełniających
i mieszanych oznaczeń barwnych i jednobarwnych oraz literowych i cyfrowych,
w zależności od specyfiki i zakresu ustaleń dotyczących przeznaczenia terenów
oraz granic i linii regulacyjnych. Dotyczy to również nieruchomości, których prze-
znaczenie w planie zostało ustalone jako rolne. W każdym przypadku oznaczenie
na rysunku planu nie wyznacza jednak zakresu przeznaczenia danej nieruchomości
– wynika on z części opisowej (tekstowej) planu34. UMCS Z. Truszkiewicz zauważa, że fakt, iż zgodnie z brzmieniem art. 35 ustawy
o planowaniu i zagospodarowaniu przestrzennym tereny dotychczas rolne, które
zostały następnie przeznaczone w wyniku wejścia w życie planu miejscowego na Z. Truszkiewicz zauważa, że fakt, iż zgodnie z brzmieniem art. 35 ustawy
o planowaniu i zagospodarowaniu przestrzennym tereny dotychczas rolne, które
zostały następnie przeznaczone w wyniku wejścia w życie planu miejscowego na 35 Pkt II załącznika do Zarządzenia Prezesa ANR nr 27/10.
36 Stanowisko Krajowej Rady Notarialnej w sprawie stosowania w praktyce notarialnej
ustawy o kształtowaniu ustroju rolnego, www.anr.gov.pl/web/guest/stanowisko-krajowej-rady-
-notarialnej-i-agencji-nieruchomosci-rolnych-ukur [dostęp: 10.04.2017].
37 Z. Truszkiewicz, Nieruchomość rolna i gospodarstwo rolne…, s. 150. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 UMC Podobny problem powstaje, gdy miejscowy plan zagospodarowania prze-
strzennego na cele nierolne przeznacza jedynie część danej nieruchomości. W tym
wypadku także próżno jest szukać w treści art. 2 pkt 1 ustawy jednoznacznej odpo-
wiedzi na pytanie, czy nieruchomość taką należy traktować jako rolną. Wątpliwo-
ści budzi przede wszystkim kwalifikacja nieruchomości, której jedynie niewielki
fragment będzie miał charakter rolny, podczas gdy pozostała, przeważająca część 38 Zgodnie z art. 4 ustawy o planowaniu i zagospodarowaniu przestrzennym ustalenie prze-
znaczenia terenu, rozmieszczenie inwestycji celu publicznego oraz określenie sposobów zagospo-
darowania i warunków zabudowy terenu następuje w miejscowym planie zagospodarowania prze-
strzennego, natomiast w przypadku braku miejscowego planu zagospodarowania przestrzennego
określenie sposobów zagospodarowania i warunków zabudowy terenu następuje w drodze decyzji
o warunkach zabudowy i zagospodarowania terenu, przy czym lokalizację inwestycji celu publicz-
nego ustala się w drodze decyzji o lokalizacji inwestycji celu publicznego, a sposób zagospodaro-
wania terenu i warunki zabudowy dla innych inwestycji ustala się w drodze decyzji o warunkach
zabudowy. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 2 ustawy o planowaniu i zagospodarowaniu przestrzen-
nym w przypadku braku miejscowego planu zagospodarowania przestrzennego
określenie sposobów zagospodarowania i warunków zabudowy terenu następuje
w drodze decyzji o warunkach zabudowy i zagospodarowania terenu, przy czym Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego 107 lokalizację inwestycji celu publicznego ustala się w drodze decyzji o lokalizacji
inwestycji celu publicznego, a sposób zagospodarowania terenu i warunki zabu-
dowy dla innych inwestycji – w drodze decyzji o warunkach zabudowy38. S y
y
y j
y j
y
W oparciu o literalną wykładnię przepisów obecnej ustawy – w tym zwłasz-
cza art. 11 ust. 2 ustawy z dnia 14 kwietnia 2016 r. o wstrzymaniu sprzedaży nie-
ruchomości Zasobu Własności Rolnej Skarbu Państwa oraz o zmianie niektórych
ustaw, przepisów ustawy o kształtowaniu ustroju rolnego, w brzmieniu nadanym
jej tą ustawą – nie stosuje się do nieruchomości rolnych, które w dniu wejścia
w życie ustawy w ostatecznych decyzjach o warunkach zabudowy i zagospoda-
rowania terenu przeznaczone są na cele inne niż rolne. Pojawia się jednak pyta-
nie, czy uzasadnione jest rozumowanie a contrario, zgodnie z którym fakt prze-
znaczenia nieruchomości rolnej na cele nierolne w drodze decyzji o warunkach
zabudowy, wydanej po wejściu w życie ustawy z dnia 14 kwietnia 2016 r. (albo
decyzji, które zostały wprawdzie wydane wcześniej, ale które stały się ostateczne
dopiero po tej dacie), nie wyłącza w stosunku do tej nieruchomości stosowania
ustawy o kształtowaniu ustroju rolnego. MCS Wydaje się, że takie rozumowanie byłoby zbyt daleko idące. Przede wszyst-
kim należy podzielić poglądy reprezentowane w piśmiennictwie, że to nie tyle
decyzje lokalizacyjne (w tym wypadku decyzje o warunkach zabudowy) stano-
wią podstawę do zmiany przeznaczenia nieruchomości na cele nierolne, ile prze-
pisy ustaw, na podstawie których decyzje te są wydawane. UM Należy zgodzić się z poglądem, że decyzja o warunkach zabudowy i zago-
spodarowania terenu jest aktem deklaratoryjnym, niekonstytuującym żadnych
praw ani obowiązków nieprzewidzianych w ustawie czy akcie wykonawczym. Decyzja ta nie tworzy porządku prawnego, jak ma to miejsce w przypadku pla-
nu miejscowego. Na obszarach pozbawionych planu obowiązuje bowiem „pań-
stwowy” porządek planistyczny wyznaczany przez przepisy ustaw szczególnych. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 106 Konrad Marciniuk Konrad Marciniuk będzie nieruchomością nierolną, przeznaczoną np. na cele budownictwa mieszka-
niowego. Na stanowisku, że wyłączenie stosowania przepisów ustawy o kształto-
waniu ustroju rolnego przewidziane w art. 2 pkt 1 dotyczy jedynie nieruchomości
w całości przeznaczonych w planach zagospodarowania przestrzennego na cele
inne niż rolne, stoi Agencja Nieruchomości Rolnych35. Zdaniem Prezesa Agencji
zasada ta obowiązuje niezależnie od tego, czy na nieruchomość składa się jedna czy
więcej działek. W przypadku rozporządzania prawem własności nieruchomości,
której jedynie część – bez względu na to, czy została geodezyjnie wyodrębniona
jako działka – stanowi nieruchomość rolną, zastosowanie będą miały przepisy usta-
wy o kształtowaniu ustroju rolnego ograniczające swobodę obrotu. Podobny pogląd
wyraziła Krajowa Rada Notarialna, formułując swoje stanowisko w sprawie sto-
sowania w praktyce notarialnej ustawy o kształtowaniu ustroju rolnego36, zgodnie
z którym wyłączenie z zakresu nieruchomości rolnych, spełniających warunki do
uznania ich za rolne w rozumieniu art. 461 k.c., dotyczy wyłącznie nieruchomości
przeznaczonych w całości w planie zagospodarowania przestrzennego na cele inne
niż rolne. Z kolei Z. Truszkiewicz słusznie zauważa, że w treści art. 2 pkt 1 ustawy
o kształtowaniu ustroju rolnego brak jest zastrzeżenia, że ustawa dotyczy tylko
nieruchomości obejmujących jedynie użytki rolne ani że jako rolną należy trakto-
wać nieruchomość obejmującą choćby w części użytki rolne, bez względu na ich
powierzchnię. Należy zgodzić się z poglądem tego autora, że o stosowaniu ustawy
o kształtowaniu ustroju rolnego do nieruchomości o różnym sposobie użytkowania
winna przesądzać funkcja dominująca nieruchomości, tj. dominujący sposób ko-
rzystania z nieruchomości czy też dominujące przeznaczenie37. UMCS Kolejny problem z ustalaniem rolnego charakteru nieruchomości powstaje
w przypadku nieruchomości położonych poza obszarami objętymi miejscowy-
mi planami zagospodarowania przestrzennego. W stosunku do nieruchomości
położonych na takich obszarach, obejmujących zdecydowaną większość, bo ok. 3/4 powierzchni kraju, pojawia się pytanie, czy o nierolniczym charakterze nie-
ruchomości mogą rozstrzygać przepisy szczególne, które mogą podlegać kon-
kretyzacji w odniesieniu do poszczególnych nieruchomości w postaci decyzji
o warunkach zabudowy i zagospodarowania terenu (bądź innego rodzaju decy-
zji lokalizacyjnych). U Zgodnie z art. 4 ust. 39 Zob. szerzej: J. Goździewicz-Biechońska, Decyzja o warunkach zabudowy i zagospodaro-
wania terenu (cechy szczególne), „Państwo i Prawo” 2010, z. 2, s. 95–108.
40 Ibidem. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Decyzje o warunkach zabudowy i zagospodarowania terenu przesądzają o zgod-
ności zamierzonych inwestycji z tym porządkiem (ustawami szczególnymi), tak
jak pod rządami ustawy o zagospodarowaniu przestrzennym na obszarach obję-
tych miejscowym planem zagospodarowania przestrzennego czyniły to decyzje
o warunkach zabudowy i zagospodarowania terenu, przesądzając o zgodności
zamierzonych inwestycji z planem. Ich deklaratoryjny charakter przejawia się też
U 42 Warto zaznaczyć, że w ostatnim zdaniu § 1 wskazanego rozporządzenia Rady Ministrów
z dnia 28 listopada 1964 r. posłużono się kategorią tzw. normy obszarowej, która stanowiła je-
den z ważniejszych instrumentów ingerencji władz w sferę obrotu nieruchomościami rolnymi.
W późniejszym okresie (nowelizując rozporządzenie) podniesiono wysokość minimalnej normy
obszarowej do 0,5 ha. 41 Rozporządzenie Rady Ministrów z dnia 28 listopada 1964 r. w sprawie przenoszenia włas-
ności nieruchomości rolnych, znoszenia współwłasności takich nieruchomości oraz dziedziczenia
gospodarstw rolnych (Dz.U. z 1970 r., nr 24, poz. 199). Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 108 Konrad Marciniuk Konrad Marciniuk w tym, iż z punktu widzenia zagospodarowania terenu decyzje takie stwierdzają
istnienie pewnego stanu faktycznego i prawnego39. W literaturze wskazuje się nadto, iż decyzja o warunkach zabudowy i zago-
spodarowania terenu nie ma charakteru uznaniowego. Organowi nie przysługują
uprawnienia kształtujące ani swoboda inna niż przewidziana w przepisach odręb-
nych stanowiących podstawę decyzji. Decyzja o warunkach zabudowy i zagospo-
darowania terenu jest aktem związanym w tym sensie, że organ jest zobowiązany
wydać decyzję zgodną z żądaniem wnioskodawcy, jeśli nie stwierdzi niezgod-
ności wniosku z przepisami prawa. W przeciwnym przypadku ma obowiązek
wydać decyzję odmowną. Związany charakter decyzji o warunkach zabudowy
i zagospodarowania terenu wynika z art. 56 ustawy o planowaniu i zagospodaro-
waniu przestrzennym. Pogląd ten znajduje też potwierdzenie w orzecznictwie40. MCS W konsekwencji można, moim zdaniem, sformułować pogląd, że nierucho-
mość objęta decyzją o warunkach zabudowy, dopuszczającą jej nierolnicze prze-
znaczenie, w ogóle przestaje być nieruchomością rolną w rozumieniu art. 461 k.c. Decyzja jedynie potwierdza, że zgodnie z przepisami rangi ustawowej (zamiesz-
czonymi zarówno w samej ustawie o planowaniu przestrzennym, jak i w innych
ustawach regulujących zagadnienia zagospodarowania przestrzennego) dana nie-
ruchomość może być zagospodarowana na cele nierolne. UMC Na ten temat wypowiedział się także Z. Truszkiewicz. Opierając się przede
wszystkim na wykładni celowościowej, systemowej i historycznej, stwierdził, że
decyzje lokalizacyjne, w tym decyzje o ustaleniu warunków zabudowy, wyłą-
czają stosowanie ustawy o kształtowaniu ustroju rolnego bez względu na to, czy
zostały wydane i stały się ostateczne przed czy po 30 kwietnia 2016 r. UM Wskazane powyżej wątpliwości interpretacyjne w zakresie wykładni pojęcia
nieruchomości rolnej, będącego przecież pojęciem kluczowym z punktu widzenia
współczesnej regulacji obrotu gruntami rolnymi, przemawiają za koniecznością
wypracowania bardziej precyzyjnego sformułowania definicji normatywnej tego
pojęcia. Przy okazji zasadne byłoby zapewnienie wzajemnej korelacji i spójności
przy określaniu nieruchomości rolnej jako przedmiotu obrotu własnościowego,
ale także jako przedmiotu ochrony w płaszczyźnie ochrony gruntów rolnych. Cele objęcia ochroną gruntów rolnych na podstawie ustawy z dnia 3 lutego 1995 r. oraz objęcia nieruchomości rolnych szczególnymi zasadami obrotu są przecież
zbliżone. W obu przypadkach chodzi niewątpliwie m.in. o zapewnienie warun-
ków do właściwego zagospodarowania ziemi rolnej jako podstawowego i nieza-
stępowalnego środka produkcji rolnej. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Dodatkowo zasadne wydaje się ściślejsze
powiązanie definicji nieruchomości rolnej z regulacjami z zakresu planowania
przestrzennego, które w systemie prawa wydają się najwłaściwszą płaszczyzną
U Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 110 Konrad Marciniuk mość położona w obrębie zwartej zabudowy miasta, chociażby była użytkowana
rolniczo lub nadawała się do takiego użytkowania, nie była zatem uznawana za
nieruchomość rolną. Na marginesie warto zaznaczyć, że obręb zwartej zabudowy
miasta nie był równoznaczny z granicami administracyjnymi miasta. S Wydaje się, że współczesny prawodawca, skoro zdecydował się na bardziej
aktywną i głębszą ingerencję w obrót gruntami rolnymi, wprowadzając do środ-
ków oddziaływania na ten obrót również instrumenty administracyjnoprawne,
powinien rozważyć przynajmniej częściowy powrót do dawnych rozwiązań przez
odniesienie definicji nieruchomości rolnej do rozstrzygnięć dotyczących plano-
wania przestrzennego. To właśnie przepisy z zakresu planowania przestrzennego
są, moim zdaniem, podstawową płaszczyzną regulowania kwestii związanych
z przeznaczaniem terenów na określone cele. Rozstrzygnięcia podejmowane na
podstawie przepisów o planowaniu przestrzennym – z punktu widzenia właści-
ciela nieruchomości są to miejscowy plan zagospodarowania przestrzennego oraz
decyzja o warunkach zabudowy – mają tę zaletę, że na ogół są na tyle precyzyj-
ne, że zarówno uczestnicy obrotu, jak i notariusze, sądy i organy administracji
posiadające kompetencje w zakresie kontroli obrotu nieruchomościami unikaliby
ryzyk związanych z nieprawidłową kwalifikacją przedmiotu czynności prawnej,
co w sposób oczywisty przyczyniłoby się do ochrony bezpieczeństwa obrotu. UMCS p
y
y p
y
y
y
ę
y
p
Biorąc pod uwagę obecny kształt rozwiązań prawnych systemu planowania
przestrzennego, moim zdaniem zasadne byłoby odniesienie definicji nierucho-
mości rolnej nie tylko do postanowień miejscowych planów zagospodarowania
przestrzennego, jak ma to miejsce obecnie, ale także – w zakresie, w jakim doty-
czy to nieruchomości położonych na obszarach nieobjętych takimi planami – do
wyraźnego nawiązania do powszechnego (tzw. państwowego) porządku plani-
stycznego wynikającego bezpośrednio z ustawy o planowaniu i zagospodarowa-
niu przestrzennym oraz z ustaw szczególnych. Porządek ten jest konkretyzowa-
ny w indywidualnych sprawach w postaci decyzji o warunkach zabudowy lub
w innych decyzjach lokalizacyjnych przewidzianych w ustawach szczególnych. Innymi słowy z zakresu pojęcia „nieruchomość rolna” powinny być wyraźnie
wyłączone także nieruchomości przeznaczone na cele nierolne, zgodnie z osta-
teczną decyzją o warunkach zabudowy lub inną decyzją lokalizacyjną. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego 109 do podejmowania rozstrzygnięć dotyczących przeznaczenia nieruchomości (tj. określania sposobów ich zagospodarowania). Jak wspomniano na wstępie, obecna definicja nieruchomości rolnej została
sformułowana w art. 461 k.c. na mocy nowelizacji Kodeksu cywilnego z 1990 r., tj. równolegle ze zniesieniem w zasadzie wszystkich, obowiązujących do tego czasu,
ograniczeń w obrocie własnościowym dotyczących nabywania nieruchomości rol-
nych w drodze umów. Wprawdzie wraz z uchwaleniem w kwietniu 2003 r. ustawy
o kształtowaniu ustroju rolnego ustawodawca powrócił do reglamentacji obrotu
gruntami rolnymi, jednak aż do nowelizacji tej ustawy w kwietniu 2016 r. regla-
mentacja ta miała w zasadzie charakter szczątkowy, przynajmniej w wymiarze
praktycznym. Dopiero wspomniana nowelizacja zasadniczo zmieniła uwarunko-
wania obrotu nieruchomościami rolnymi, poddając dużą część obrotu regulacji
administracyjnoprawnej, co stanowi już bardzo wyraźny powrót do rozwiązań
obowiązujących przed 1990 r. Pomimo zmiany kontekstu i de facto znaczenia de-
finicji nieruchomości rolnych ustawodawca nie zdecydował się na wprowadzenie
jakichkolwiek zmian tej definicji. MCS Znamienne jest, że w czasach poprzedzających liberalizację zasad obrotu nie-
ruchomościami rolnymi definicja pojęcia nieruchomości rolnej w znacznie szer-
szym stopniu nawiązywała do regulacji dotyczących planowania przestrzennego. W rozporządzeniu Rady Ministrów z dnia 28 listopada 1964 r.41 przyjęto, że:
UM Nieruchomość uważa się za rolną, jeżeli jest lub może być użytkowana na cele produkcji
rolnej, nie wyłączając produkcji ogrodniczej, sadowniczej i rybnej, chyba że stosownie do przepi-
sów o planowaniu przestrzennym decyzją właściwego organu prezydium rady narodowej została
przeznaczona na cele niezwiązane bezpośrednio z produkcją rolną. Nie uważa się jednak za nie-
ruchomości rolne terenów położonych w obrębie zwartej zabudowy miasta oraz nieruchomości,
które należą do tej samej osoby (osób) i których łączny obszar nie przekracza 0,2 ha42. U W treści przytoczonej definicji widać wyraźnie, że wolą ówczesnego prawo-
dawcy było ograniczenie zakresu oddziaływania regulacji dotyczących obrotu nie-
ruchomościami rolnymi (a szerzej – oddziaływania na strukturę agrarną) do nieru-
chomości, które na mocy obowiązujących w tamtym czasie przepisów o planowaniu
przestrzennym nie były przeznaczone do prowadzenia produkcji rolnej, a także – co
wydaje się istotne – wszystkich nieruchomości położonych w obrębie zwartej zabu-
dowy miasta. Na gruncie wskazanego rozporządzenia Rady Ministrów nierucho- 44 Projekt Kodeksu urbanistyczno-budowlanego zakłada istotne zwiększenie w stosunku do
obecnie funkcjonujących studiów uwarunkowań i kierunków zagospodarowania przestrzennego,
szczegółowości studium rozwoju. W szczególności zakłada się, że studium rozwoju będzie za-
wierało ustalenia i obligatoryjne wskaźniki zabudowy i zagospodarowania przestrzennego, które
mają stanowić podstawę przy sporządzaniu planów miejscowych, a także mają służyć ustalaniu
warunków brzegowych w przypadku udzielania zgody inwestycyjnej na obszarach pozbawionych
planów miejscowych. Obligatoryjnym elementem treści studium mają być także ustalenia doty-
czące kierunków i zasad kształtowania rolniczej przestrzeni produkcyjnej. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 UM Na marginesie trzeba zaznaczyć, że już dzisiaj, wskutek braku korelacji
między definicjami przedmiotu ochrony na podstawie ustawy o kształtowaniu
ustroju rolnego oraz ustawy o ochronie gruntów rolnych i leśnych, dochodzi do
paradoksalnej sytuacji, w której właściciel nieruchomości rolnej położonej na ob-
szarze nieobjętym miejscowym planem zagospodarowania przestrzennego, ale
w stosunku do której została wydana decyzja o warunkach zabudowy na cele
nierolne, napotyka na przeszkody w rozporządzaniu tą nieruchomością (przed-
stawiony w niniejszej pracy pogląd, że także w obecnym stanie prawnym decy-
zja o warunkach zabudowy przesądza o utracie przez nieruchomość charakteru Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego 111 rolnego nie jest podzielany zarówno przez Agencję Nieruchomości Rolnych, jak
i Krajową Radę Notarialną), ale jednocześnie nie doznaje praktycznie żadnych
przeszkód, aby dokonać zmiany przeznaczenia tej nieruchomości na cele nierolne
we własnym zakresie. S Uwzględniając kierunek spodziewanych zmian w systemie planowania prze-
strzennego43, zakładających wzmocnienie rangi studium (według proponowanej
terminologii będzie to studium rozwoju)44, należałoby postulować, aby w przy-
szłości definicja nieruchomości rolnej opierała się na przeznaczeniu nieruchomo-
ści w miejscowym planie zagospodarowania przestrzennego, jak ma to miejsce
obecnie, a na obszarach takich planów pozbawionych – na postanowieniach stu-
dium rozwoju gminy. MCS Dodatkowo zasadne wydaje się wprowadzenie do treści samej definicji nie-
ruchomości rolnej – w zakresie, w jakim pojęcie to jest definiowane na potrzeby
obrotu – wyraźnego określenia minimalnej powierzchni użytków rolnych ob-
jętych daną nieruchomością, przesądzającej o traktowaniu całej nieruchomości
jako rolnej bez względu na obszar innych użytków objętych tą samą nierucho-
mością. Wydaje się, że zasadne byłoby, aby wielkość tej powierzchni odpowia-
dała wielkości przyjętej już dzisiaj w treści art. 1a pkt 1 lit. b. Nie ma bowiem
powodu, aby wyłączenie stosowania szczególnych zasad obrotu nieruchomościa-
mi obejmowało nieruchomości rolne o powierzchni mniejszej niż 0,3 ha, a nie
obejmowało nieruchomości o większym obszarze, w którym jednak powierzch-
nia użytków rolnych (względnie powierzchnia gruntu przeznaczona na cele rolne
zgodnie z przepisami o planowaniu przestrzennym) także nie przekracza wska-
zanej wielkości. UMC Kwestią wymagającą podjęcia rozstrzygnięcia jest również zasadność utrzy-
mywania ograniczeń w obrocie nieruchomościami rolnymi położonymi w grani-
cach administracyjnych miast. Należy bowiem zaznaczyć, że już dzisiaj, zgod-
nie z treścią art. 10a ustawy z dnia 3 lutego 1995 r. 43 Zob. opracowany przez Ministra Infrastruktury i Budownictwa projekt ustawy – Kodeks
urbanistyczno-budowlany. Jest on w trakcie konsultacji. Zob. www.konsultacje.gov.pl/node/4338
[dostęp: 14.04.2017]. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 112 Konrad Marciniuk Konrad Marciniuk rolnymi położonymi na obszarze miasta wydaje się wynikać głównie z faktu, że
na tego typu obszarach, wskutek naturalnie dominujących procesów urbaniza-
cyjnych, działalność rolnicza ma charakter zanikowy. Utrzymywanie ograniczeń
w obrocie nieruchomościami rolnymi na obszarach miast nie przyczynia się do
realizacji deklarowanych przez ustawodawcę celów regulacji i stanowi jedynie
zbędną barierę inwestycyjną. S BIBLIOGRAFIA
C Bieluk J., Ustawa o kształtowaniu ustroju rolnego. Komentarz, Warszawa 2016. CS Borkowski M., Pojęcia „nieruchomości rolnej” i „nieruchomości leśnej” w rozumieniu ustawy
o nabywaniu nieruchomości przez cudzoziemców, „Rejent” 2007, nr 7–8. MC dzinowski R., [w:] Kodeks cywilny, t. 1: Komentarz do art. 461, red. Gutowski, Warszawa 2016
MC Czerwińska-Koral K., Pojęcie nieruchomości rolnej jako wyznacznik zasad obrotu nieruchomoś-
ciami rolnymi, „Rejent” 2016, nr 6. MC Goździewicz-Biechońska J., Decyzja o warunkach zabudowy i zagospodarowania terenu (cechy
szczególne), „Państwo i Prawo” 2010, z. 2. MC Grabarek W., Określenie nieruchomości podlegającej dziedziczeniu na zasadach dziedzicze
d
l
h
N
P
l d N
i l
” 2005
2
UM Grabarek W., Określenie nieruchomości podlegającej dziedziczeniu na zasadach dziedziczenia
gospodarstw rolnych, „Nowy Przegląd Notarialny” 2005, nr 2. UM Grabarek W., Określenie nieruchomości podlegającej dziedziczeniu
gospodarstw rolnych, „Nowy Przegląd Notarialny” 2005, nr 2. UM gospodarstw rolnych, „Nowy Przegląd Notarialny” 2005, nr 2. UM Gryszczyńska A., [w:] G. Szpor, M. Durzyńska, A. Gryszczyńska, I. Kamińska, K. Mączewski,
W. Radzio, Prawo geodezyjne i kartograficzne. Komentarz, Warszawa 2013. UM http://ipo.trybunal.gov.pl/ipo/Sprawa?&pokaz=dokumenty&sygnatura=K%2036/16-[dostęp:
10.04.2017]. UM http://sn.pl/orzecznictwo/SitePages/Baza_orzeczen.aspx [dostęp: 10.04.2017]. U Klat-Górska E., Przeniesienie własności nieruchomości rolnej – przykłady ograniczeń, Wrocław
2013. U Klat-Górska E., Ustawa o kształtowaniu ustroju rolnego. Komentarz, Warszawa 2014. U Kurowska T., Upowszechnienie prawa własności nieruchomości, Katowice 1994. U Lichorowicz A., Glosa do wyroku SN z 2.6.2000 r., II CKN 1067/98, OSP 2001, nr 2, p Lichorowicz A., Instrumenty oddziaływania na strukturę gruntową Polski w ustawie z dnia 11
kwietnia 2003 r. o kształtowaniu ustroju rolnego, „Kwartalnik Prawa Prywatnego” 2004, nr 2. Lichorowicz A., Instrumenty oddziaływania na strukturę gruntową Polski w ustawie z dnia 11
kwietnia 2003 r. o kształtowaniu ustroju rolnego, „Kwartalnik Prawa Prywatnego” 2004, nr 2. Lichorowicz A., O nowy kształt zasad w obrocie nieruchomościami rolnymi w kodeksie cywilnym,
„Rejent” 1997, nr 6. Lichorowicz A., Regulacja obrotu gruntami rolnymi według ustawy z 11 kwietnia 2003 r. o kształ-
towaniu ustroju rolnego na tle ustawodawstwa agrarnego Europy Zachodniej, „Przegląd
Legislacyjny” 2004, nr 3(43). Maj K., Zmiany w ustawie o kształtowaniu ustroju rolnego obowiązujące od dnia 30 kwietnia 2016 r.,
„Krakowski Przegląd Notarialny” 2016, nr 2. Marciniuk K., [w:] Prawo rolne, red. P. Czechowski, Warszawa 2015. Postanowienie SN z dnia 7 maja 1997 r., II CKN 137/97, Legalis. Postanowienie SN z dnia 15 maja 2009 r., II CSK 9/09, Legalis. Prawo rolne, red. A. Oleszko, Kraków 2009. Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 o ochronie gruntów rolnych
i leśnych, nieruchomości takie są wyłączone spod działania przepisów tej ustawy
w zakresie ograniczania przeznaczania gruntów na cele nierolnicze i nieleśne. Zasadność rezygnacji z aktywnego oddziaływania na obrót nieruchomościami
U Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego o wstrzymaniu sprzedaży nieruchomości Zasobu Własności
R l
j Sk
b P ń t
i
i
i któ
h
t
(D U
2016
585)
U Rolnej Skarbu Państwa oraz o zmianie niektórych ustaw (Dz.U. z 2016 r., poz. 585). Rolnej Skarbu Państwa oraz o zmianie niektórych u
www.konsultacje.gov.pl/node/4338 [dostęp: 14.04.2017]. www.konsultacje.gov.pl/node/4338 [dostęp: 14.04.2017]. www.konsultacje.gov.pl/node/4338 [dostęp: 14.04.2017]. Wyrok SN z dnia 2 czerwca 2000 r., II CKN 1067/98, OSP 2001, nr 2, poz. 27. Wyrok WSA w Warszawie z dnia 30 lipca 2010 r., VII SA/Wa 950/10. Wyrok WSA w Warszawie z dnia 30 lipca 2010 r., VII SA/Wa 950/10. Zarządzenie Prezesa ANR nr 27/10. Zarządzenie Prezesa ANR nr 27/10. Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego 113 Rozporządzenie Ministra Rozwoju Regionalnego i Budownictwa z dnia 29 marca 2001 r. w spra-
wie ewidencji gruntów i budynków (Dz.U. z 2016 r., poz. 1034). Rozporządzenie Rady Ministrów z dnia 28 listopada 1964 r. w sprawie przenoszenia własności
nieruchomości rolnych, znoszenia współwłasności takich nieruchomości oraz dziedziczenia
gospodarstw rolnych (Dz.U. z 1970 r., nr 24, poz. 199; Dz.U. z 1983 r., nr 19, poz. 86). S g
p
y
(
,
, p
;
,
, p
)
Rozporządzenie tymczasowe Rady Ministrów z dnia 1 września 1919 r. normujące przenoszenie
własności nieruchomości ziemskich (Dz.U. z 1919 r., nr 73, poz. 428). S Stanowisko Krajowej Rady Notarialnej w sprawie stosowania w praktyce notarialnej ustawy
o kształtowaniu ustroju rolnego, www.anr.gov.pl/web/guest/stanowisko-krajowej-rady-nota-
rialnej-i-agencji-nieruchomosci-rolnych-ukur [dostęp: 10.04.2017]. CS ańska K., Poszukiwanie indywidualnego modelu gospodarstwa rolnego, „Studia Iuridica
Agraria” 2001. CS g
Stelmachowski A., [w:] System prawa prywatnego, t. 3: Prawo rzeczowe, red. T. Dybowski, War-
szawa 2007. MCS Truszkiewicz Z., [w:] Prawo rolne, Warszawa 2015. MC Truszkiewicz Z., Nieruchomość rolna i gospodarstwo rolne w rozumieniu U.K.U.R., „Krakowski
Przegląd Notarialny” 2016, nr 2. MC Truszkiewicz Z., Wpływ planowania przestrzennego na pojęcie nieruchomości rolnej w rozumie-
niu Kodeksu cywilnego, „Studia Iuridica Agraria” 2007, t. 6. MC Ustawa z dnia 25 września 1954 r. o reformie podziału administracyjnego wsi i powołaniu gro-
madzkich rad narodowych (Dz.U. z 1954 r., nr 43, poz. 191). UM Ustawa z dnia 29 czerwca 1963 r. o ograniczeniu podziału gospodarstw rolnych (Dz.U. z 1963 r.,
nr 28, poz. 168). UM Ustawa z dnia 17 maja 1989 r. – Prawo geodezyjne i kartograficzne (Dz.U. z 2015 r., poz. 520
z późn. zm.). UM Ustawa z dnia 3 lutego 1995 r. o ochronie gruntów rolnych i leśnych (Dz.U. z 2015 r., poz. 909
U Ustawa z dnia 27 marca 2003 r. o planowaniu i zagospodarowaniu przestrzennym (Dz.U. z 2016 r.,
poz. 778). U Ustawa z dnia 11 kwietnia 2003 r. o kształtowaniu ustroju rolnego (t.j. Dz.U. z 2012 r., poz. 803
z późn. zm.). U a z dnia 14 kwietnia 2016 r. o wstrzymaniu sprzedaży nieruchomości Zasobu Własności
U Ustawa z dnia 14 kwietnia 2016 r. o wstrzymaniu sprzedaży nieruchomości Zasobu Własności
Rolnej Skarbu Państwa oraz o zmianie niektórych ustaw (Dz.U. z 2016 r., poz. 585). U Ustawa z dnia 14 kwietnia 2016 r. BIBLIOGRAFIA
C Ptaszyk M., Nowe zasady obrotu nieruchomościami rolnymi inter vivos, „Państwo i Prawo” 1991,
z. 7, poz. 52. Rozporządzenie Ministra Infrastruktury z dnia 26 sierpnia 2003 r. w sprawie wymaganego zakre-
su projektu miejscowego planu zagospodarowania przestrzennego (Dz.U. z 2003 r., nr 164,
poz. 1587). Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 Pojęcie nieruchomości rolnej jako przedmiotu reglamentacji obrotu własnościowego SUMMARY The article introduces a definition of agricultural real property based on various legal acts
– before the adoption of the Civil Code, pursuant to Article 461 of the Civil Code and the Act on
Shaping the Agricultural Regime; it also describes the criteria of establishing the property as be-
ing used for agricultural properties. It features the assessment of the legal character of a property
being recorded in the cadaster as agricultural and establishing properties as agricultural in zoning
acts and the Act on Zoning and Development. The article also tells the story of limitation of trad-
ing agricultural land in Poland, focusing largely on the most recent and very extreme limitation
introduced by the Act on Ceasing the Sale of Property Owned by the State Treasury and amending Pobrane z czasopisma Studia Iuridica Lublinensia http://studiaiuridica.umcs.pl
Data: 24/10/2024 07:43:46 114 114 Konrad Marciniuk certain other acts of 14 April 2016 and reviews the procedure and legal conditioning of obtaining
a permit to sell agricultural property, granted by means of an administrative decision issued by
the President of the Agricultural Property Agency and the potential consequences of its invalidity. The article provides a complex review of a wide range of issues related to the limitations on trans-
actions involving agricultural real property. S certain other acts of 14 April 2016 and reviews the procedure and legal conditioning of obtaining
a permit to sell agricultural property, granted by means of an administrative decision issued by
the President of the Agricultural Property Agency and the potential consequences of its invalidity. The article provides a complex review of a wide range of issues related to the limitations on trans-
actions involving agricultural real property. S Keywords: agricultural property; trading in agricultural properties; right of pre-emption;
shaping of the agricultural regime
CS
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Remain, leave, or return? Mothers’ location continuity after separation in Belgium
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DEMOGRAPHIC RESEARCH
VOLUME 42, ARTICLE 9, PAGES 245-292
PUBLISHED 13 FEBRUARY 2020
https://www.demographic-research.org/Volumes/Vol42/9/
DOI: 10.4054/DemRes.2020.42.9
Research Article
Remain, leave, or return? Mothers’ location
continuity after separation in Belgium
Christine Schnor
Júlia Mikolai
This publication is part of the Special Collection on “Separation, Divorce, and
Residential Mobility in a Comparative Perspective,” organized by Guest
Editors Júlia Mikolai, Hill Kulu, and Clara Mulder.
© 2020 Christine Schnor & Júlia Mikolai.
This open-access work is published under the terms of the Creative Commons
Attribution 3.0 Germany (CC BY 3.0 DE), which permits use, reproduction,
and distribution in any medium, provided the original author(s) and source
are given credit.
See https://creativecommons.org/licenses/by/3.0/de/legalcode.
Contents
1
Introduction
246
2
2.1
2.2
2.3
Background
Moves following separation
Location continuity after separation
The Belgian context
248
248
249
252
3
Hypotheses
255
4
4.1
4.2
4.3
Data and methods
Data
Analytical strategy
Variables
256
256
257
260
5
Descriptive results
261
6
6.1
6.1.1
6.1.2
6.1.3
6.2
6.2.1
6.2.2
6.2.3
Multivariate results
High, moderate, or low levels of location continuity
Woman’s characteristics
Characteristics of the place of residence
Ex-partner characteristics and repartnering
Returning to the pre-separation municipality
Woman’s characteristics
Characteristics of the place of residence
Ex-couple characteristics and repartnering
262
262
264
265
266
266
268
269
269
7
Conclusions
270
8
Acknowledgments
274
References
275
Appendix
283
Demographic Research: Volume 42, Article 9
Research Article
Remain, leave, or return?
Mothers’ location continuity after separation in Belgium
Christine Schnor1
Júlia Mikolai2
Abstract
BACKGROUND
Partnership dissolution can mark an extended period of residential instability for
mothers and their children. Location continuity, i.e., the ability to stay in or return to the
same neighbourhood after separation, is essential to reduce the negative consequences
of separation.
OBJECTIVE
We focus on mothers’ post-separation location continuity in the three years following
separation and study the role of socioeconomic resources and local ties (to a home,
neighbourhood, and region) in remaining in or returning to their pre-separation
neighbourhood.
METHODS
Using linked Belgian Census (2001) and register data (2001–2006), we estimate
multinomial logistic regression models (N = 25,802). Based on the occurrence,
frequency, and destination of moves, we distinguish between high, moderate, and low
degrees of location continuity. We also study the probability of remaining in, leaving,
or returning to the pre-separation neighbourhood.
RESULTS
Mothers who live at their place of birth (a measure of local ties) tend to stay in or return
to their pre-separation neighbourhood or region; if they have more socioeconomic
resources they are more likely to remain in the family home. Mothers from
disadvantaged backgrounds move further and more often.
CONCLUSION
If separated mothers lack socioeconomic resources and local ties, they are less likely to
1
Centre for Demographic Research, Université Catholique de Louvain (UCLouvain), Louvain-La-Neuve,
Belgium. Email: christine.schnor@uclouvain.be.
2
University of St Andrews, St Andrews, UK.
http://www.demographic-research.org
245
Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
maintain location continuity. Policy programmes should target these women in order to
provide better opportunities for separated mothers and their children.
CONTRIBUTION
We introduce the concept of post-separation location continuity and account for
separation-induced as well as post-separation residential changes in the first three years
after separation.
1. Introduction
In recent decades, separation and divorce rates have substantially increased in many
countries, which not only means that individual life courses have become more diverse
and non-linear, but also that people’s residential biographies have become more
complex (Thomas, Mulder and Cooke 2017a). Partnership dynamics and residential
mobility3 are strongly interrelated, as partnership formation or dissolution implies a
move for at least one of the partners (Clark 2013; Dewilde 2008; Mulder 2006; Mulder
and Lauster 2010). Separation-driven moves are usually urgent and rely on a restricted
budget (Feijten 2005; Feijten and van Ham 2007, 2010; Mulder et al. 2012; Mulder and
Wagner 2012). An unfavourable residential situation may imply further moves to readjust to pre-separation housing quality; for example, in terms of neighbourhood or
area (Gram-Hanssen and Bech-Danielsen 2008). Therefore, separation is likely to have
long-term consequences for separated individuals’ residential mobility (Mikolai and
Kulu 2018a, 2018b).
When separating, people may have to leave the joint home, but they might not
necessarily want to leave the place where they live (Mulder 1996; Mikolai and Kulu
2019). Most people feel attached to their environment (Gindes 1998) and a move may
imply a change in housing circumstances, discontinuation of daily routines, and even a
change in employment. Nevertheless, whereas for the separating adult a move may also
appear advantageous, research on residential mobility after parental separation has
unanimously shown that children fare worse if they move away from their earlier home
(Braver, Ellman, and Fabricius 2003). A separation-driven move is psychologically
disruptive and disorienting to children, because the loss of friends and familiar
surroundings that are sources of support can hamper their adjustment to the family
crisis (Tucker, Marx, and Long 1998; Austin 2008; Amato 2000). Residential mobility
may have a negative effect on children, translating into, e.g., school behavioural
3
Throughout this paper we use the term ‘residential mobility’ to describe a change in individuals’ place of
residence.
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Demographic Research: Volume 42, Article 9
problems, less academic success, higher dropout rates, and lower well-being
(McLanahan and Sandefur 1994). In fact, a large part of the detrimental effect of
divorce on child outcomes can be explained by children’s higher residential mobility
(McLanahan and Sørensen 1984; Astone and McLanahan 1994; Simpson and Fowler
1994). Researchers and mental health professionals agree that after separation, location
continuity, such as staying in the family home, in the same school, or at least in the
same community, is advantageous for children (Gindes 1998; Hagan, MacMillan, and
Wheaton 1996). Although joint custody is becoming increasingly common across some
Western and Northern European countries, most children still stay with their mother
after parental separation (for Europe see, for example, Bernardi and Mortelmans 2018;
for Belgium see Sodermans, Matthijs, and Swicegood 2013). Therefore, it is especially
relevant to study the post-separation residential situation of mothers and the
determinants of their moving behaviour.
In this study we focus on mothers’ location continuity; that is, whether a mother
remains in the pre-separation neighbourhood4 (by not moving or by moving within the
same or to a nearby neighbourhood) or returns to it later. We are interested in
determining how characteristics of the separated mother, the ex-couple, and the place
where the family lived at the time of separation relate to a mother’s probability of
remaining at or returning to the pre-separation location. We assume that location
continuity is the preferred option for most mothers, unless they have ties to another
location (Mulder 1993; Mulder and Malmberg 2011; Feijten and van Ham 2007) or
their current location is suboptimal (Cooke, Mulder, and Thomas 2016). However,
whether location continuity is feasible may depend on the mother’s socioeconomic
resources, her relative bargaining position in the (ex-)couple, and her local ties. The
social and economic ties that bind people to a place can be fixed to a home, a
neighbourhood, or the surrounding region. We introduce a qualitative concept to
understand separated mothers’ degree of location continuity, which is highest if a
woman remains at her pre-separation address, moves only within the neighbourhood
(e.g., municipality) where she lived at the time of separation, or returns to her preseparation neighbourhood sometime after separation. If a woman moves to a different
neighbourhood in the same region (e.g., province), her degree of location continuity is
moderate, and it is low if she moves to a different region. By introducing this new
perspective, this study contributes to existing research on the post-separation residential
mobility of mothers.
The second novelty is that we follow mothers from the time of separation up to
three years after separation, which allows us to account for both separation-induced and
4
Throughout the paper we use the term ‘neighbourhood’ in a general sense to refer to the area and
environment where individuals live, rather than as a spatial unit. It is explained later on in the paper that the
smallest spatial unit that we can measure are municipalities, followed by provinces.
http://www.demographic-research.org
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Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
post-separation residential changes. This is important because the effect of separation
on residential mobility can be lagged (Oakil 2013), meaning that some individuals may
not move out of their pre-separation home at the same time that separation occurs.
Furthermore, repartnering is common after separation and may lead to additional
residential moves with consequences for mothers’ and children’s location continuity
(Feijten and van Ham 2010). Additionally, several moves may be necessary to re-adjust
to pre-separation residential standards and moving away from the pre-separation
location can be temporary (Gram-Hanssen and Bech-Danielsen 2008). Therefore, we
also explicitly study separated mothers’ propensity to return to their pre-separation
neighbourhood.
We study post-separation residential mobility in the context of Belgium, a country
with high rates of union dissolution and an increasing share of lone parent families, a
key driver of increasing housing demand. Belgium is rooted in a residential tradition of
homeownership and single-family houses; there is limited supply of good quality and
affordable rental housing (Dewilde and Lancee 2013; Winters and Heylen 2014) and
the rental market is inflexible. These conditions are likely to accentuate the negative
consequences of separation for separated mothers’ housing careers and well-being. We
use unique, linked population data from the Belgian Census (2001) and the Population
Register (2001–2006). The individual-level data covers the complete Belgian
population and includes the exact date of all residential moves between 1 October 2001
and 1 January 2006. We focus on mothers whose marriage or cohabitation ended in
2002 (N = 25,802); this design allows us to follow them for a period of three years after
separation.
2. Background
2.1 Moves following separation
Separation may mark the beginning of a period of increased residential mobility
(Feijten and Mulder 2005; Feijten and van Ham 2010). Sub-optimal and transitory
residential situations can further increase already high levels of stress associated with
separation (Gram-Hanssen and Bech-Danielssen 2008). The effect of separation on
residential mobility can be lagged, as one partner might initially stay in the family
home and only move after the house is sold or rented (Oakil 2013). Separation-driven
moves are specific as they frequently are the result of a negotiation process between the
ex-partners and do not necessarily follow from individual decision-making. The
(monetary and non-monetary) costs of moving for both ex-partners determine who
moves out and who stays in the joint home upon separation (Mulder and Wagner 2010).
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Demographic Research: Volume 42, Article 9
Those who leave the home more often declare that the housing outcome of the
separation was to their disadvantage than those who stay (Mulder and Wagner 2012).
In general, women are more likely to move out of the joint home after separation
than men (Dewilde 2008; Feijten 2005; Mulder 2013). If the ex-couple has children,
men are somewhat more likely to leave the family home than women (Thomas, Mulder,
and Cooke 2017a; Fiori 2019). Whether moving or not, the negative financial
consequences of separation are more severe for women than for men, which may
translate to an unfavourable residential situation (Andreß et al. 2006; Feijten 2005;
Mulder 2013). Women’s relatively higher vulnerability at separation has commonly
been attributed to their weaker absolute and relative socioeconomic position in
partnerships. In practice, it is often easier for the man to keep the joint home because of
his better income prospects (Mulder 2013; Feijten and Mulder 2010). Women are
especially likely to move out of the joint home if they have low levels of education or
are less educated than their partner (Theunis, Willaert, and Van Bavel 2016).
The impact of relative resources is not gender-neutral, as women need
comparatively more resources than men to negotiate staying in the home (Mulder 2013;
Andreß and Hummelsheim 2009; McCarthy and Simpson 1991). Living in social
housing, however, which is more prevalent among the less educated, reduces women’s
likelihood of moving out in Denmark and the United Kingdom (Gram-Hanssen and
Bech-Danielssen 2008; Thomas, Mulder, and Cooke 2017a). Educational differences in
moving propensities following separation persist among both parents and the childless
(Theunis, Eeckhaut, and van Bavel 2018). Women also move out of the joint home
more often if they live in a rural area (Feijten and van Ham 2007). If homeowners,
separated women are also more likely both to move and to move longer distances than
men (Feijten and van Ham 2007). Additionally, already having a new partner at the
time of separation lowers the costs of moving and increases the probability of a move
(Mulder and Wagner 2010). Starting a new partnership is one reason for a move among
separated people and a potential way for women to recover their housing quality
(Feijten and van Ham 2010; Jalovaara and Kulu 2019).
2.2 Location continuity after separation
Location-specific capital, defined as the ties that bind people to a specific place, plays
an important role in defining the costs of a move and can serve as a resource that
enables people to stay at a given location (Mulder and Wagner 2012). Social, human, or
economic capital that cannot be taken to a new location can create local ties (DaVanzo
1981). These ties can be fixed to the home (e.g., being a homeowner or living in social
housing), or they can be fixed to its location (e.g., the community or region) through
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social and family networks and familiarity with the location (Mulder and Wagner
2012). The concept of neighbourhood encompasses the idea of a familiar environment
in which people interact for utility, support, and socialization. A neighbourhood can
contribute to one’s identity and become a reflection of oneself and one’s values,
aspirations, and socioeconomic conditions (Lebel, Pampalon, and Villeneuve 2007).
Next to the neighbourhood, the surrounding environment, such as the district or region,
is also a source of social and economic ties (Lebel, Pampalon, and Villeneuve 2007).
Hence, location-specific capital can increase the benefits of remaining in a specific
location and make a longer-distance move costlier. Ties may also increase opportunities
to remain at the same location following separation: the local social network can
provide help in finding a nearby place (Mulder and Wagner 2012).
Next to ties, the attractiveness of the location also affects the decision to move and
the destination of moves following separation. The attractiveness of a location is
determined by its proximity to daily amenities such as schools and cultural and sports
facilities (Reginster and Goffette-Nagot 2005). Ideally, facilities should be within
walking or cycling distance even in suburban or rural areas (Boussauw, van Meeteren,
and Witlox 2014). Proximity to childcare and schools becomes more important when
both parents are engaged in paid work and is essential for maintaining employment
activity after separation (Karsten 2007). People often move within the same
neighbourhood and within the constraints formed by access to these daily amenities
(Clark and Rivers 2012).
Remaining in close proximity to the pre-separation family home may bring
benefits for the mother and her child(ren), as it leads to continuity in their social
networks, daily routines, and emotional attachments (Thomas, Willaert, and van Bavel
2017a). However, this may also be a costly decision as it constrains housing choice: it
might be more difficult to find affordable accommodation when the search radius is
smaller. If separated mothers wish to remain in the pre-separation environment, tight
markets may restrict their opportunity to find appropriate housing (Thomas, Willaert,
and van Bavel 2017a; Mulder and Hooimeijer 1999).
There may be situations in which changing environments after separation is
attractive to women. For example, geographic proximity to family is crucial to
receiving support (Mulder and Van der Meer 2009) and may be a key factor in the
decision of where to move after separation for those living further from family
(Petterson and Malmberg 2009). Other reasons to change location might be moving in
with a new partner, escaping the ex-partner (Bowstead 2015), or simply the desire for a
new start in a new location (Schier 2015). Moreover, the pre-separation location may
reflect the ex-partner’s residential preferences rather than that of the mother, in which
case mothers are likely to move to a place which better suits their needs (Cooke,
Mulder, and Thomas 2016). It has also been shown that separated persons are
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especially likely to move to cities (Feijten and van Ham 2007). However, Thomas,
Adam, and Verhetsel (2017) show that the actual post-separation mobility behaviour of
parents often contradicts pre-separation locational preferences. It is likely that separated
parents are still tied to their pre-separation place of residence because of their children.
A handful of studies have analysed the distance of a move after separation: these
studies typically analyse the likelihood of moving followed by the distance moved
among the movers using the Euclidian distance (in logged kilometres) between the
origin and destination location. They show that when children are present, people tend
to remain in their neighbourhood, as the social networks of the children are considered
crucial (Gram-Hanssen and Bech-Danielssen 2008; Mulder and Malmberg 2011;
Feijten and van Ham 2007, 2013; Ferrari, Bonnet, and Solaz 2019). Most studies find
that mothers stay in closer proximity to the pre-separation family home than fathers
(Clark and Rivers 2012; Feijten and van Ham 2007), whereas Mulder and Malmberg
(2011) find no difference.
Mulder and Malmberg (2011) study moves in the first year following separation
among people with and without children using Swedish register data. They show that
separation-driven moves involve shorter distances from the former home than moves
for other reasons, especially for parents. Local social ties, measured as grandparents
living less than 2 km away, decrease the likelihood of moving and the distance moved.
They do not find an effect of education on moving distance, but as their analysis
includes childless persons as well as parents we do not know whether education
influences the distance moved by separated parents. Similarly, using longitudinal Dutch
data, Feijten and van Ham (2007) study whether separation has an effect on the
likelihood, distance, and destination of moves. They exclude ‘event moves’ from the
analysis, i.e., moves related to a change in partnership status (e.g., separation or
repartnering), and only analyse ‘state moves,’ i.e., moves whilst already in a given
partnership status (e.g., among separated or repartnered individuals). They show that
even when excluding event moves, separated people move more often and over shorter
distances than those who are in a first relationship. However, separated mothers and
mothers in relationships have similar moving distances.
Additionally, Thomas, Mulder, and Cooke (2017a) study a small (N = 354) sample
of British parents and investigate which parent moves out upon separation and the
distance between the centroids of separated parents’ neighbourhoods in the first year
after separation. The educational composition of the ex-couple and whether the mother
repartnered are not significant predictors of the distance between parents. Using the
same sample, Thomas, Mulder, and Cooke (2017b) study the distance between parents,
following them for several years after separation. They show that the distance is
determined by moves in the first year, but tends to further increase over time since
separation. Net of other characteristics, highly educated parents live further away from
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each other than parents without a degree. Unfortunately, these analyses do not allow us
to draw firm conclusions regarding the location continuity of separated mothers.
Some other studies have focused on categorical measures of distance to capture the
degree of location continuity after separation. A study on Danish separated persons
(Gram-Hanssen and Bech-Danielssen 2008) shows that the large majority of moves
after separation are within the same municipality, especially if children are involved
(less than 15% moved to another region). According to studies conducted in the United
States, about 17% of custodial parents move to another city within two years after
separation (Braver and O’Connell 1998; Kelly and Lamb 2003).
Separation may force people to leave their neighbourhood if they do not find
appropriate housing nearby. However, if pre-separation locational ties remain
important, separated people may later return to their pre-separation neighbourhood.
Existing evidence shows that a considerable proportion of separated people moves to
temporary housing and thus faces several residential transitions. For example, GramHanssen and Bech-Danielssen (2008) show that about half of separated individuals who
moved from the matrimonial home to a new address moved again one year later to yet
another new address. The lack of empirical evidence to date makes it difficult, if not
impossible, to quantify the probability of returning to the pre-separation location after a
temporary period of absence.
2.3 The Belgian context
Belgium is characterized by high union dissolution rates and an inflexible housing
market, which can make it difficult for mothers to find a new place of residence after
separation. We describe the Belgian context based on previous studies and official
statistics from 2014, the latest year for which data is available.
In 2014 there were 6 divorces to every 10 newly formed marriages in Belgium.
Apart from marriage, since 2000 couples have also been able to register cohabitations,
and official statistics provide information on these unions. Informal cohabitations are
not registered and thus are not covered in official statistics. In 2014 around 50% of
newly formed registered cohabitations ended in separation. More registered
cohabitations than marriages were formed (45,000 registered cohabitations vs. 40,000
marriages). Since the 1970s social policies have provided practical support for families
through widely available childcare that allows couples to combine work and family
(Esping-Andersen 1999; Neels and de Wachter 2010). Most children live with their
mother after separation, but time spent with the father has increased over the last
decades (Sodermans, Matthijs, and Swicegood 2013).
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Belgium is a small country (30,528 km2) comprising 589 municipalities with an
average area of 52 km2.5 Population density in the municipalities is on average 773
inhabitants per km2 (Statistics Belgium 2015).6 From the north to the south and the west
to the east the maximum distance is around 200 km. The division of the country into a
more densely populated Dutch-speaking North (Flanders) and a less densely populated
French-speaking South (Wallonia) contributes to small moving distances, as people
tend to remain in their language communities. Furthermore, moving and commuting
behaviours7 reveal the pre-eminence of the borders of the 10 provinces (Thomas,
Adam, and Verhetsel 2017). Moving distances are generally short and take place within
provinces, because Belgians want to stay close to relatives, social relationships,
neighbours, and sports and collective activities situated in the same province (Thomas,
Adam, and Verhetsel 2017).
Provincial borders have a long-standing history, rooted in the 18th century. Some
institutional competences are exerted at the provincial level (such as education, leisure
activities, social assistance, healthcare, and maintenance of provincial roads and
waterways)8, which may strengthen the bonds between provinces and their inhabitants.
Childcare and primary school education are organized by language communities and
municipalities. Municipalities are thus another relevant spatial structure in people’s
everyday lives as they provide local infrastructure (such as schools and leisure
organizations), a sense of identity, and location-specific capital (e.g., friends and
relatives living nearby). Research has shown that Belgians identify foremost with their
local environment (town or village) (Hooghe 2004). Furthermore, rental allowance is
organized at the municipality level. Eligibility requirements for social housing can be
based on existing local ties (already living or working in the municipality) and thus
motivate within-municipality moves (CGKR 2014).
Residential mobility is hampered by housing market rigidity, e.g., the high
prevalence of homeownership and the limited supply of good quality and affordable
rental housing (OECD Economic Survey 2015, 2017). About 72% of the Belgian
population are homeowners (the European Union (EU-28) average is 70%; Eurostat
2018). Rising rents and the weakening income profile of tenants have contributed to the
5
Median: 40 km²; Inter-quartile range: [27 km², 67 km²]
Median: 308; Inter-quartile range: [166, 606]
7
Belgians have longer home–workplace commuting distances than moving distances (Thomas, Adam, and
Verhetsel 2017). In Flanders the average one-way commuting distance is 19 km, whereas the commuting
distance to educational institutions (including primary and secondary schools as well as higher education) is
around 9.5 km (Janssens et al. 2011). The mean distance to elementary schools is much shorter (1.8 km),
indicating that most children live within walking or cycling distance of their school (Boussauw, van
Meeteren, and Witlox 2014).
8
The provinces are autonomous institutions but under the supervision of the regions (Flanders and BrusselsWallonia) or the communities (Dutch-, French-, German-speaking). For example, a provincial school is
managed by the community, whereas an initiative on land use is monitored by the region.
6
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Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
‘residual character’ of the private rental market (OECD Economic Survey 2015: 85).
The social housing sector is very small (about 6% of the housing stock); thus, many
people who would qualify for social housing based on their financial resources have to
resort to the private rental market (Andrews, Sanchez, and Johansson 2011). Belgium’s
rental market is rather inflexible as tenants can only decide between a three-year (shortterm) and a nine-year (long-term) contract (De Decker 2001). A long-term contract can
only be terminated prematurely with a payment of compensation to the landlord. A
short-term contract cannot be easily terminated by the tenant before its end. The
landlord has the right to choose between enforcing the rental agreement and dissolving
the contract with additional compensation charges to be paid by the tenant. When
contracts are negotiated landlords are in a more powerful position and they favour
short-term contracts, because these allow them to review the rental price every three
years (De Decker 2001). Altogether, these renting conditions result in unfavourable
situations for tenants in case of partnership separation.
Housing prices have been on the rise for decades and are higher than in other
European countries (OECD 2017; Caldera Sánchez and Andrews 2011; Warisse 2017).
One reason for this is that transaction costs are particularly large in Belgium (Caldera
Sánchez and Andrews 2011). The proportion of tenants whose housing costs exceeded
40% of their equivalised disposable income is 34%, higher than the European average
of 27%; whereas the percentage spent on housing by owner-occupiers in Belgium is
below the European mean (2.4% vs. 6.7%; Eurostat 2018). The ownership of a
detached house is traditionally viewed as the ideal housing tenure, providing the highest
degree of housing security (Halleux and Strée 2013). This view has contributed to
Belgium’s housing structure, which is dominated by its suburban style of one-family
houses. Around a quarter (22%) of the Belgian population lives in apartments,
compared to 42% in Europe. The share of apartments is low by international standards,
even in cities. In 2014 most Belgians (53%) lived in suburban areas in comparison to
31% in the European Union.
As the supply of rental housing is low and concentrated in disadvantaged
communities, people from lower socioeconomic backgrounds or with financial
constraints have limited locational choice. In addition, discrimination contributes to
limit the choice of location; for example, single mothers tend to be discriminated
against in the private rental market (CGKR 2014). As the number of single-parent
families is increasing, the demand for housing in Belgium is also projected to increase
(OECD 2015).
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3. Hypotheses
We assume that location continuity in the years following separation is the preferred
outcome for most separated mothers, unless they have ties to another location or their
current location is suboptimal (Mulder and Wagner 2010; Mulder and Malmberg 2011;
Feijten and van Ham 2007). Whether location continuity is feasible is expected to
depend on the mother’s socioeconomic resources, her local ties, and her relative
bargaining position in the ex-couple. First, we formulate three sets of hypotheses
relating to the propensity to experience a high (1.1), moderate (1.2), or low (1.3) degree
of location continuity. We define location continuity as high if the mother either stays
in the same municipality or returns to it later, moderate if she changes municipality but
stays in the same province, and low if she leaves the pre-separation province. Then, we
formulate hypotheses about the determinants of return moves to the pre-separation
location (2.1 and 2.2).
We expect mothers to be more likely to remain in the pre-separation municipality
(and thus the degree of location continuity to be highest) if they have socioeconomic
resources, location-specific capital, and are in an advantageous bargaining position
relative to their ex-partner (Hypothesis 1.1). The latter should especially increase a
mother’s likelihood of staying in the family home. Mothers in a disadvantaged position
in the housing market are expected to have difficulties finding new housing nearby
immediately; these women will be more likely to leave their pre-separation location
than those who have better circumstances. However, they might move to another
municipality within the province if they have ties to that municipality, thereby keeping
at least some (i.e., moderate) degree of location continuity (Hypothesis 1.2). We expect
that a lack of socioeconomic resources and local ties is associated with an increased
likelihood of moving to a different province following separation and thus leads to a
low degree of location continuity (Hypothesis 1.3).
Women who have to leave their neighbourhood may only move away temporarily
until they can move back again. We hypothesize that women with a disadvantaged
profile on the housing market follow this strategy, as they may face more difficulties to
find housing nearby immediately following separation than their more advantaged
counterparts (Hypothesis 2.1). Beyond that, local ties should increase the likelihood of
returning to the pre-separation location. Women who lack these ties will likely not
return to their pre-separation municipality (Hypothesis 2.2).
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4. Data and methods
4.1 Data
We use a dataset provided by Statistics Belgium, which links information from the
National Population Registers (2001–2006) and the 2001 Belgian Census. The 2001
Belgian Census provides information on the demographic and socioeconomic
characteristics of the entire Belgian population on 1 October 2001. National Population
Registers include information on all residential moves between and within
municipalities from 1 October 2001 to 1 January 2006. The large sample size enables
us to study the mobility patterns of separated mothers in more detail than prior studies.
We have information on the date of residential moves and the destination municipality.
Although information on the actual distance moved is not available in the dataset, we
can calculate Euclidian distances between municipality halls to give a rough estimate of
the distance moved. Our geographical information is limited to the municipality level:
we know whether a person moved within a municipality, but we have no information
on the distance of these moves, as we do not have information on the exact address.
The census allows us to identify married and cohabiting couples. The
identification of married couples (as well as multiple married couples in the same
household) is based on the relationship between household members. Consensual
unions are identified based on the LIPRO typology (van Imhoff and Keilman 1991).
We focus on the physical separation of both previously married and previously nonmarried (i.e., cohabiting) women. Separation is defined as household dissolution; the
time of separation is defined as the date when one of the partners (or both) leaves the
joint household. If only one partner moved between 2001 and 2006, the date of this
move is considered to be the date of separation (in the case of several moves, we take
the earliest move). If both partners moved but on a different date, the earliest move is
considered the date of separation. We know that a couple separated through information
on their union status in 2006. If a couple is known to be separated in 2006, but the only
move is on the same date and to the same municipality, then this move is considered as
the separation move.
Our sample is restricted to women with coresident children, who separated in
2002, were residing in Belgium between October 2001 and January 2006, and were
between 18 and 49 years old at the time of separation (N = 25,802).9 We chose to study
women who separated in 2002 because this allows us to follow the residential
9
The sampling design is similar to that in the study by Gram-Hanssen and Bech-Danielssen (2008), who used
Danish register data on all couples who moved apart in the year 2002, including those who were not legally
married, and focused on the housing situation in 2002 and 2003. In contrast to this Danish study, we focus on
mothers only and extend the observation window to three years after separation.
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biographies of these women for three years following separation. All women were
observed throughout the entire observation period. Our estimate of union dissolution is
close to official divorce statistics. Official Belgian divorce statistics report 30,628
divorces for 2002 (Eurostat 2018). We have identified 48,902 separations for 2002, of
which 28,814 were separations from marriage. The difference between our estimate and
the official statistics can be explained by the fact that we focus (a) on household
dissolution, which in most cases precedes divorce, (b) on couples that were still living
together in 2001, and (c) on a specific age range.
4.2 Analytical strategy
The analyses are divided into two parts. In the first we analyse the degree of location
continuity after separation and in the second we evaluate the likelihood of ‘return
moves’. In the first part we distinguish between high, moderate, and low degree of
location continuity (Figure 1). Separated women may experience several moves, each
of which may have different levels of location continuity. To this end we apply a spatial
hierarchy to the moving patterns and group women according to the move that
disrupted their location continuity the most in the first 36 months following
separation.10 This results in six moving patterns.
Figure 1:
Moving patterns of mothers following separation
Location continuity is considered high if the woman remains in her pre-separation
municipality throughout the observation period. This can be achieved by either staying
in the pre-separation family home throughout the three years following separation
(pattern 1) or by moving to a new dwelling in the same municipality (pattern 2).
Distinguishing between these outcomes is important, as previous literature has shown
that staying in the family home is associated with certain factors that may not apply to
10
For example, mothers who moved across municipalities may have had within-municipality moves as well,
or those who moved across provinces might have also moved within provinces.
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Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
within-municipality moves. The degree of location continuity is moderate if women
remain in the same province by moving once (pattern 3) or several times across
municipalities (pattern 4) and it is low if they change provinces either once (pattern 5)
or multiple times (pattern 6).
Distinguishing single from multiple moves is analytically important, because it
takes into account that some women changed their environment several times within a
few years after separation. Women who moved once to another municipality or
province could by definition not return to their pre-separation location. Multiple moves
could mean that women move from one place to another, implying that their location
continuity is somewhat lower compared to women who only had one move of that kind.
However, it is possible that additional moves are related to moving back to the preseparation neighbourhood. In that case the degree of location continuity would be
higher than for mothers who left their pre-separation location and did not return to it.
To understand whether these patterns relate to the characteristics of the mother, we
distinguish between women who moved multiple times and those who only moved
once. Then, to understand whether repeated moves are related to a higher or lower
degree of location continuity, in the second part of the analysis we explicitly address
separated mothers’ propensity to return to their pre-separation municipality.
Furthermore, we compare the characteristics of mothers who returned to their preseparation neighbourhood to those of women who remained in their municipality or
who left and did not return.
The six mobility patterns are (Figure 1 and Table 1): (1) no move (32%); (2)
move(s) within the same municipality only (26%); (3) one move to a different
municipality in the same province, i.e., a within province move (21%); (4) several
within province moves (10%); (5) one move to a different province, i.e., a crossprovince move (9%); and (6) several cross-province moves (3%). In line with previous
studies (Gram-Hanssen and Bech-Danielssen 2008), these proportions demonstrate that
most mothers remain in their pre-separation municipality and only a minority leaves the
province. About 7% (N = 1,595) of separated mothers who moved away returned to
their pre-separation municipality during the three-year follow-up period.
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Table 1:
Descriptive information on mobility patterns of separated mothers in
the first three years following separation, N = 25,802, column
percentages (for total sample and by mobility pattern)
Total
N = 25,802
(100%)
Mobility pattern
High degree of location
continuity:
Remaining in same
municipality
(1) No
(2) Withinmove
municipality
move(s)
Moderate degree of location
continuity:
Remaining in same province, but
moving to different municipality
(3) One cross- (4) Several crossmunicipality
municipality
move
moves
Low degree of location continuity:
Moving to different province
(5) One crossprovince move
(6) Several crossprovince moves
8,127
(32%)
5,339
(21%)
2,656
(10%)
2,298
(9%)
765
(3%)
6,617
(26%)
Number of moves
0 (32%)
100%
0%
0%
0%
0%
1 (30%)
0%
59%
56%
0%
42%
0%
0%
2 (25%)
0%
32%
34%
54%
39%
40%
3+ (13%)
0%
9%
10%
46%
19%
60%
Number of moves within municipality
0 (56%)
100%
0%
56%
69%
64%
69%
1 (28%)
0%
59%
34%
23%
28%
23%
2 (11%)
0%
32%
7%
6%
6%
6%
3+ (4%)
0%
9%
3%
2%
2%
2%
Number of moves across municipalities - within the same province
0 (63%)
100%
100%
0%
0%
71%
73%
1 (25%)
0%
0%
100%
0%
23%
20%
2 (10%)
0%
0%
0%
78%
5%
5%
3+ (3%)
0%
0%
0%
22%
1%
2%
0%
Number of moves across provinces
0 (86%)
100%
100%
100%
100%
0%
1 (10%)
0%
0%
0%
0%
100%
0%
2 (3%)
0%
0%
0%
0%
0%
82%
3+ (1%)
0%
0%
0%
0%
0%
18%
0%
68%
70%
85%
73%
82%
0%
46%
2%
42%
% moved at separation
(53%)
% moved back to pre-separation municipality
0%
0%
Total distance moved (km) *
Mean (SD)
0
0
22.20 (27.03)
55.69 (64.35)
58.60 (47.22)
123.57 (98.58)
Median [IQR]
0
0
13 [7; 26]
33 [18; 66]
49 [21; 85]
106 [48; 174]
100%
100%
78%
45%
35%
15%
16.45 (26.92)
51.62 (40.49)
26.76 (35.19)
43 [17; 77]
12 [0; 40]
39%
69%
% less than 30km
Distance between first and last address (km) *
Mean (SD)
0
0
22.20 (27.03)
Median [IQR]
0
0
13 [7; 26]
100%
100%
% Less than 30km
78%
8 [0; 20]
83%
Note: IQR = Interquartile range. * Calculated as distance between municipality halls.
Source: Authors’ own calculations using linked Belgian Census (2001) and Population Register data (2001–2006).
In both empirical parts of the analysis we apply multinomial logistic regression
models. The multinomial logistic regression estimates coefficients for five outcomes
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relative to the sixth outcome (reference outcome). Relative risk ratios indicate how the
risk of an outcome compared to the risk of the reference outcome changes with the
variable in question. We present the results as average marginal effects (with 95%
confidence intervals), which show the percentage point difference in the predicted
probabilities of one category compared to the predicted probabilities of a reference
category for all six outcomes (predicted probabilities and relative risk ratios are shown
in the Appendix). The use of marginal effects enables us to compare the probability of
belonging to one of the six mobility patterns by independent variable category, as well
as to compare this probability between the three degrees of location continuity. As we
use population data, p-values, confidence intervals, and standard errors can be
interpreted as illustrations of the effect strengths.
4.3 Variables
The characteristics of the woman, the dissolved couple, and the area where the woman
lived at the time of separation matter for women’s post-separation residential mobility.
Characteristics of the woman include woman’s education, measured as the highest
level of education achieved by the time of the Census (2001). Following the ISCED97
classification we distinguish between three levels of education: low (ISCED97 0−2; no
qualification or degrees up to lower secondary school), medium, (ISCED97 3−4; upper
secondary education), and high (ISCED97 5−6; tertiary education). Other
characteristics are her age at separation (measured in years), her employment status
during the last 12 months before separation (full-time, part-time, unemployed, missing),
the number of children at the time of separation (one, two, three, and four or more
children), and the age of the youngest coresident child at separation (younger than 7
years old, 7–11 years old, 12–17 years old, and at least 18 years old). Unfortunately, we
cannot distinguish children from a previous relationship from those born within the
dissolved union, as we do not have information on the start date of the union if it was
non-marital. Education and employment status are proxies for women’s socioeconomic
resources. Women are considered disadvantaged on the housing market if their
educational level is low or if they have more than two children (i.e., more than the
average number of children).
We do not have information on the place of residence of the separated mothers’
parents or siblings or her social networks. However, a proxy for these local ties is
whether at the time of separation the woman lived in the municipality or province
where she was born. If she was living where she was born, this was most likely where
she was socialized, and where her network of family and friends were.
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To measure characteristics of the place where mothers lived at separation, we
include whether the woman lived in the municipality or in the province where she was
born, the degree of urbanization (urban, suburban, rural) based on the classification of
Belgian municipalities into city regions (Luyten and van Hecke 2007), and the region
(Flanders vs. Brussels/Wallonia). A further measure of location-specific capital is
whether separated mothers have access to social housing, as this is organized at the
municipality level. Further, we consider information on the housing tenure at the time
of separation (owner, private renting, social renting, or missing information/living rentfree).
Several characteristics of the ex-couple are available in the 2001 Census, such as
their educational composition (woman equally educated as her ex-partner, woman
more/less educated than ex-partner), marital status (married vs. cohabiting), and the age
difference between the ex-partners (same age, she is older than him, he is 2–4 years
older than her, he is 5 or more years older than her). Women are in an advantageous
bargaining position when deciding who stays in the family home after separation if they
are more educated or older than their ex-partner. Recent research shows that marital
status affects the probability to leave the family home (Murinko 2019). We also control
for whether the woman started a new coresidential partnership at the time of union
dissolution or thereafter. This information is reconstructed based on movements in the
register data and the household situation in 2006. The condition for identifying
repartnering is that the newly formed couple lived together in 2006, which means that
we are not able to capture living-apart-together relationships or short coresidential
unions that were formed and dissolved between 2002 and 2006.
5. Descriptive results
Separated mothers can move several times within the observation window. As
explained above, we applied a spatial hierarchy to classify the moving patterns and
gave priority to the move that likely had the most impact on separated mothers’
location. Table 1 describes the moving patterns of separated mothers for the overall
sample as well as for the six moving patterns. A considerable proportion (38%) of
separated mothers moved several times in the three-year period following separation.
Among the mobile mothers, within-municipality moves were common: between 31%
and 44% of the mothers who moved across municipalities (patterns 3–6) also moved at
least once within municipalities. Among mothers who moved across provinces (patterns
5–6), about one in three had also moved across municipalities within the same
province.
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Overall, about half of the mothers did not move at the time of separation, which
confirms prior studies (e.g., Thomas, Mulder, and Cooke 2017a). Looking at mobile
mothers only, the percentage of women who did not move at the time of separation is
much lower, especially for women in the very mobile patterns 4 and 6. However, even
in these patterns, 15% and 18% respectively first remained at their address when
separating and only moved later. This stresses the importance of taking an extended
time window when studying residential mobility after separation.
Apart from the number and destination of moves, Table 1 also displays how often
mothers returned to their pre-separation location. Surprisingly, 46% of women who had
several moves within the same province and 42% of those who had several crossprovince moves eventually moved back to their pre-separation municipality. This also
becomes apparent when comparing the total distance moved and the distance between
the pre-separation address and the last observed place of residence. Mothers who
moved several times moved more kilometres in total than other mothers, but more often
found themselves close to their pre-separation municipality towards the end of the
observation window.
Table A-1 describes the moving patterns of separated mothers (for the overall
sample as well as for the six moving patterns) by the characteristics of the woman, the
characteristics of the place of residence at the time of separation, the characteristics of
the ex-couple, and by whether separated mothers have formed a new coresidential
relationship (either at the time of separation or thereafter). In the next section we
compare women with different mobility patterns to each other, using multinomial
logistic regression.
6. Multivariate results
6.1 High, moderate, or low levels of location continuity
Figure 2 shows the results of the first multinomial logistic regression model, where we
estimate the likelihood of separated mothers belonging to one of the six mobility
patterns: staying at the same address, moving only within the same municipality,
moving to another municipality in the same province (once or several times), or moving
to a different province (once or several times). The results are displayed as average
marginal effects (with 95% confidence intervals; see Notes below the Figure for more
information on how to interpret these figures). The results are shown as predicted
probabilities in Table A-2 and as relative risk ratios in Table A-4 in the Appendix.
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Figure 2:
Average marginal effects of six mobility patterns
Women's characteristics
Education
(ref=Medium)
Low
High
Employment status
(ref=Full-time)
Part-time
Unemployed
Number of children
(ref=1 child)
2 children
3 children
4+ children
Age youngest child
(ref=7–11years)
<7 years
12–17years
18+ years
Age at separation
Age in years
-.2
-.15
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-.1
-.05
0
.05
Average marginal effect
.1
.15
.2
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Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
Figure 2:
(Continued)
Ex-couple characteristics & repartnering
Age difference
(ref=Same age)
Woman older
Man 2–4 years older
Man 4+ years older
Educational difference
(ref=Same level)
Man>woman
Woman>man
Missing
Type of dissolved union
(ref=Cohabitation)
Marriage
-.2
-.15
-.1
-.05
0
.05
Average marginal effect
.1
.15
.2
Repartnering
New partner
(ref=No partner)
Repartnered at
separation
Repartnered after
separation
-.25
-.2
-.15
HIGH: no move
MODERATE: within-prov (1)
LOW: cross-prov (1)
-.1
-.05
0
Average marginal effect
.05
.1
.15
.2
HIGH: within munic move(1+)
MODERATE: within-prov (2+)
LOW: cross-prov (2+)
Source: Authors’ own calculations using linked Belgian Census (2001) and Population Register data (2001–2006).
Notes: The Figure illustrates average marginal effects. Average marginal effect of a variable is the average change in the predicted
probability of a category compared to the reference category. Taking the example of woman’s education, we predict that 31% of
women with medium educational level, 28% of women with low educational level and 37% of women with high educational level do
not move (see predicted probabilities in Table A-2). In terms of marginal effects, we thus observe that compared to medium
educated women, the predicted probability of not moving is 3 percentage points lower for low educated women and 6 percentage
points higher for high educated women (black squared boxes in Figure 2). The maximum range of change in predicted probabilities
goes from -26 percentage points to +28 percentage points. The strength of the effect size varies across variables and is strongest for
birth place, housing and repartnering. Beyond comparing variables, the Figure allows to capture characteristic-specific moving
patterns. When comparing marginal effects across outcomes, it is however important to consider that the rarer the outcome the
closer the marginal effects are.
6.1.1 Woman’s characteristics
We expected the degree of location continuity to be high if mothers have
socioeconomic resources (measured using level of education and employment status)
(Hypothesis 1.1). We find that net of other characteristics, education is positively
related to the propensity to stay in the family home and negatively related to the
propensity to move within the municipality. By contrast, women’s education and
employment status are not strong predictors of the probability to move once or several
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times within or across provinces (except that highly educated women have a somewhat
lower probability than those who are less educated of moving once to a different
municipality within the same province, and moves within the same municipality are
more likely when not working full-time). We also speculated that mothers with a higher
than average number of children would find it more difficult to remain in the preseparation municipality. Mothers with four or more children are indeed more likely
than mothers with one child to change municipalities multiple times or even to move to
a different province, and they are less likely to only move within their pre-separation
municipality. Women with two or three children have a higher probability of staying in
the family home (but having three children decreases the probability of moving only
within the municipality). Beyond that, we find that the older the youngest child is, the
lower separated mothers’ propensity to stay in the family home and the higher the
likelihood that they will move to another municipality in the same or another province
(even several times). Mother’s age at separation is positively related to the probability
of staying in the family home, but it is not a significant predictor of other mobility
patterns. In sum, the results suggest that children’s characteristics are related to
separated mothers’ degree of location continuity, whereas mothers’ characteristics
predict the likelihood of staying in the family home.
6.1.2 Characteristics of the place of residence
We expected that mothers who have location-specific capital in the pre-separation
location (measured using information on their birthplace and access to social housing)
would have a high degree of location continuity following separation (Hypothesis 1.1).
Figure 2 shows that location-specific capital is indeed an important predictor of
women’s post-separation moving patterns. In line with Hypothesis 1.2, ties to another
municipality within the pre-separation province (i.e., being born in the province)
increase women’s likelihood of moving within the province, thereby keeping a
moderate degree of location continuity. If women lack these ties they are more likely to
leave the province and hence experience a low degree of location continuity
(confirming Hypothesis 1.3). Living in the birth municipality or birth province
especially decreases the probability of a single move to another province. Furthermore,
social renting seems to be a strong indicator of structural local ties: the propensity of
mothers who lived in social housing at the time of separation to stay in the family home
is very large. Compared to homeowners, mothers living in a rental dwelling have a
higher probability of moving within the pre-separation municipality or of moving once
to another municipality in the same province. In addition, women who lived in
suburban areas at the time of separation have the highest probability of moving within
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Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
and across provinces. Women who lived in an urban area or in Flanders at the time of
separation are the most likely to remain in their municipality.
6.1.3 Ex-partner characteristics and repartnering
Finally, we speculated that mothers who are in an advantageous bargaining position
relative to their ex-partner (i.e., older or more educated than their ex-partner) would be
especially likely to remain in the family home. Indeed, we observe that women who
were older than their ex-partner at the time of separation have the highest probability of
not moving at all. The (relatively) older the ex-partner, the less likely it is that the
woman stays in the family home. Women who are better educated than their ex-partner
have a higher probability of remaining at the same address, but the influence of
educational differences between ex-partners is less pronounced than that of age
differences. Furthermore, we find that mothers who were married are somewhat more
likely to remain in the family home than those who cohabited prior to separation, but
we find no differences between their probability of belonging to any of the other
moving patterns. Repartnering is an important predictor of mothers’ moving behaviour.
It decreases the probability of remaining in the family home and increases the
probability of moving to another municipality or province. The timing of repartnering is
also important for moving: women who had a new partner at the time of separation
have a higher probability of only changing the municipality or province once, whereas
women who repartnered after separation are more likely to move several times both
within and across provinces.
6.2 Returning to the pre-separation municipality
In this part we analyse how separated mothers’ characteristics, the characteristics of
their place of residence at the time of separation, the characteristics of the ex-couple,
and whether they formed a new relationship influence separated women’s likelihood of
returning to their pre-separation municipality. We estimate a multinomial logistic
regression model and present average marginal effects (with 95% confidence intervals
see Notes below the figure for more information on how to interpret these figures) for
each outcome (Figure 3). The results are shown as predicted probabilities in Table A-3
and as relative risk ratios in Table A-5 in the Appendix.
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Figure 3:
Average marginal effects of leaving, remaining in, or returning to the
pre-separation municipality
Women's characteristics
Education
(ref=Medium)
Low
High
Employment status
(ref=Full-time)
Part-time
Unemployed
Number of children
(ref=1 child)
2 children
3 children
4+ children
Age ygst child
(ref=7–11years)
<7 years
12–17years
18+ years
Age at separation
Age in years
-.25
-.2
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-.15
-.1
-.05
0
.05
Average marginal effect
.1
.15
.2
.25
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Figure 3:
(Continued)
Source: Authors’ own calculations using linked Belgian Census (2001) and Population Register data (2001–2006).
Notes: The Figure illustrates average marginal effects. Average marginal effect of a variable is the average change in the predicted
probability of a category compared to the reference category. Taking the example of woman’s education, we predict that 36% of
women with medium educational level, 35% of women with low educational level and 35% of women with high educational level
leave their municipality (see predicted probabilities in Table A-3). In terms of marginal effects, we thus observe that compared to
medium educated women, the predicted probability of leaving is 1 percentage point lower for low educated and high educated
women (grey squared boxes in Figure 3). The maximum range of change in predicted probabilities goes from -25 percentage points
to +26 percentage points. The strength of the effect size varies across variables and is strongest for birth place, housing and
repartnering. Beyond comparing variables, the Figure allows to capture characteristic-specific moving patterns. When comparing
marginal effects across outcomes, it is however important to consider that the rarer the outcome the closer the marginal effects are.
6.2.1 Woman’s characteristics
Women who leave their neighbourhood following separation may temporarily move
away until they can move back to their pre-separation neighbourhood. We hypothesized
that especially women who are in a disadvantaged position in the housing market
(measured using level of education, employment status, and number of children) follow
this strategy, whereas advantaged women will remain in the same municipality
(Hypothesis 2.1). Women with low education have a somewhat higher probability of
returning to their pre-separation municipality, whereas the highly educated are more
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likely to stay there in the first place. We find no educational differences in the
propensity to leave the pre-separation municipality and not return to it.
Women who work part-time are less likely to leave their municipality than women
who work full-time, but we find no significant differences in the probability of
returning to the pre-separation municipality. The number of children is not a significant
predictor of the probability of leaving or remaining in the pre-separation municipality,
but having four or more children increases the propensity to return to the pre-separation
neighbourhood. Women who have adult children (18+) have the lowest probability of
remaining in their pre-separation municipality and the highest probability of leaving or
returning to it. Overall, it is the characteristics of the children rather than those of the
mother that determine the separated mother’s propensity to return to the pre-separation
place.
6.2.2 Characteristics of the place of residence
In sum, we find that the characteristics of the place where the woman lived at the time
of separation are associated with the probability of remaining or leaving, but they do
not seem to influence the probability of returning to that place. We expected local ties
to increase women’s probability of either remaining in their neighbourhood or returning
to it. Consequently, the absence of local ties should be linked to an increased likelihood
of leaving the pre-separation municipality and not returning to it (Hypothesis 2.2).
Women who at the time of separation lived in the same municipality they were born in
(our proxy for local ties) have the highest probability of staying in the pre-separation
municipality and the lowest probability of leaving it. Living in the birth province is also
linked to a higher probability of staying in and a lower probability of leaving the preseparation municipality. However, local ties do not seem to matter for the probability of
returning to the pre-separation municipality. Women who lived in social renting were
most likely to remain in the pre-separation municipality, the least likely to leave it, and
the least likely to return to it. Living in a non-urban area increases women’s probability
of leaving the municipality, but the probability of returning does not vary by area type.
6.2.3 Ex-couple characteristics and repartnering
The ex-couple’s characteristics are related to the separated mother’s probability of
staying in and leaving the pre-separation municipality. Repartnering is linked to a
higher probability of leaving the pre-separation municipality and a reduced probability
of staying. Additionally, those who repartnered sometime after separation have a higher
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probability of returning to the pre-separation municipality than those who either did not
repartner or already had a new partner at the time of separation.
7. Conclusion
Partnership dissolution can mark the beginning of a period of residential instability for
mothers and their children. Location continuity, i.e., the ability to stay in the same
neighbourhood, is essential to reduce the negative consequences of parental separation
for mothers and their children. However, not all mothers and their children can stay in
the neighbourhood they lived in prior to separation: some of them leave at the time of
separation, others leave some time thereafter. Previous studies have focused on the
distance of moves among those who move following separation (e.g., Mulder and
Malmberg 2011; Thomas, Mulder, and Cooke 2017b; Ferrari, Bonnet, and Solaz 2019),
but the aspect of location continuity following separation has not been explicitly
addressed.
Using large-scale population data and an extended observation period of three
years following separation, we studied separated mothers’ mobility patterns in more
detail than before. We also analysed separated mothers who returned to their preseparation neighbourhood after a temporary absence, an aspect that has not previously
been addressed empirically. Our study provides a new perspective by introducing the
concept of post-separation location continuity. Location continuity is defined based on
whether separated mothers remain in the pre-separation neighbourhood (by not moving
or by moving within the same neighbourhood) or return to it in the first three years after
separation. We assumed that location continuity is the preferred option for separated
mothers, unless they have ties to another location. Our research questions asked to what
extent separated mothers experience location continuity and whether and how their
degree of location continuity depends on certain factors. Whether staying in or
returning to the pre-separation neighbourhood is feasible should depend on the
mother’s socioeconomic resources (e.g., educational level and employment status), her
relative bargaining position in the ex-couple, her position in the housing market, and
her local ties.
Within the first three years following separation, about half (58%) of the mothers
remained in the pre-separation municipality: 26% moved within this municipality and
about one-third (32%) even stayed in the family home. In addition, 7% of the separated
mothers returned to their pre-separation municipality after a temporary absence. These
mothers and their children are assumed to experience a high degree of location
continuity. Among those who moved to another municipality, two-thirds remained in
the same province, thereby maintaining a moderate degree of location continuity.
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Although only a minority of mothers experienced a low degree of location continuity
because they left the province, a considerable proportion (38%) of separated mothers
moved several times during the observation window. This finding shows that many
families experience an extended period of residential instability after separation, ,
confirming prior findings for the United Kingdom (Mikolai and Kulu 2018a, 2018b,
2019) and other industrialised countries (Kulu et al. 2017, Mikolai et al. 2019).
Even when the move is within the same municipality, changing homes may mean
the loss of a familiar environment (e.g., a change in the route to school, friends, leisure
activities), which can negatively influence the well-being of children. Experts believe
that a change in context extenuates the negative effects of parental separation in
children (Austin 2008; Gindes 1998). Overall, mothers who moved several times across
municipalities or across provinces move more kilometres than other mothers, but more
often find themselves close to or even at their pre-separation municipality towards the
end of the observation window. This suggests that mothers strive to achieve some
degree of location continuity. However, if they do not find appropriate housing nearby
they may be forced to leave their neighbourhood temporarily.
We found that mothers’ socioeconomic situation and local ties play a role in their
degree of post-separation location continuity and their probability of moving several
times. In line with our hypothesis, local ties are linked to a higher probability of
remaining in the pre-separation municipality. Socioeconomic resources also predict
high location continuity: mothers with more resources are more likely to remain in the
family home. A higher relative age of the woman has a similar effect, suggesting that it
is associated with a higher bargaining power in the discussion of which ex-partner
remains in the family home. In addition, structural local ties, such as living in social
housing, increase the likelihood of staying in the family home. Contrary to what we
expected, separated mothers’ lack of resources is not associated with an increased
probability of leaving the pre-separation municipality. However, women with a more
than average number of children are more likely to move several times both within and
across provinces. Moreover, women with local ties are more likely to move within the
province than those who do not have local ties. We found that women with a
disadvantaged profile in the housing market are more likely to leave their preseparation municipality and return to it later: low-educated women and those with four
or more children have a higher probability of returning to their pre-separation
neighbourhood than other women. Local ties are a significant predictor of separated
mothers’ probability of staying in their municipality but – contrary to our expectations
– they are not linked to the probability of return moves.
To summarise, five profiles emerged based on separated mothers’ characteristics.
The first group consists of socioeconomically advantaged women and those in an
advantageous bargaining position in the ex-couple who stayed in their pre-separation
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home during the entire observation period. Next, we identified three groups of socially
disadvantaged women who have high location continuity but differ with respect to their
local ties and their mobility patterns: women who remain in the family home because
they live in social housing; those who move within their pre-separation municipality as
they were born there; and those who return to their pre-separation municipality after a
temporary absence. The latter group tends to have more children. This finding suggests
that these mothers had difficulties finding accommodation near the family home
immediately after separation. The last group consists of women who lack local ties,
have a weak bargaining position in the ex-couple, and have adult children: these women
leave their (often suburban) neighbourhood following separation and settle down
elsewhere. It may be that other locations are more suitable or offer better opportunities
for these women. Socioeconomic resources did not play a role for this group of women.
The lack of location continuity is likely to lead to adverse consequences for
children. Mothers who are likely to move several times shortly after separation should
be targeted in policy programmes. Future research should further investigate this group
and gather more information on the reasons for their discontinuous and interrupted
residential careers. We especially encourage studying sequences of moves depending
on the women’s characteristics. Studying the timing, order, and combination of moves
can reveal under which conditions a woman first left and then returned to her preseparation location and whether the return is temporary or followed by other moves.
This study provides a first insight into the complexity of Belgian mothers’ postseparation residential biographies.
Belgian post-separation families may be more exposed to residential instability
than families in other countries. Recent cross-national research shows that in contrast to
other European countries, the risk of a residential move after separation remains high in
Belgium (Kulu et al. 2017). We argue that Belgium’s inflexible rental market is likely
to accentuate the negative consequences of separation for separated mothers’ housing
careers. It remains for future research to compare the post-separation residential
mobility of mothers and their children across countries.
Women’s moving behaviour after separation reflects a complex combination of
changes in their preferences, opportunities, and housing needs as well as their social
and economic constraints. Moving back to the pre-separation municipality may be a
response to the preference for being close to kin in emotionally difficult times, to the
opportunity to use living space offered by the family, or to the need for cheap
accommodation. Previous research has showed that well-off men are often the winners
and socially marginalised men are the losers when it comes to deciding who remains in
the family home after separation (Gram-Hanssen and Bech-Danielsen 2007). Focusing
on women and distinguishing between different degrees of location continuity, this
study has shown that disadvantaged mothers (and their children) also have less
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favourable moving outcomes than those who are well off. Future research should
investigate whether and how children are affected by residential mobility and housing
outcomes related to parental separation.
It is important to note that separated individuals are a specific group and their
residential mobility behaviour seems to be driven by different processes and underlying
mechanisms than the residential mobility decisions of the general population. For
example, we argue that those with fewer socioeconomic resources will be more likely
to move following separation than those who are in a more advantageous position. Our
analysis supports this claim, at least regarding who remains in the family home.
However, for the general population a lack of resources was found to be related to
reduced opportunities for individuals to move or to migrate over longer distances
(Feijten and van Ham 2007). Taking the two arguments together, this might explain the
lack of educational gradient in location continuity in this study.
This research has some limitations. First, we were restricted in the measurement of
local ties, as we were not able to identify the place of residence of mothers’ family of
origin. Although in the Belgian context the place of birth is a proxy for family and
social relations to a location, future research with more information on family ties is
needed. Second, we did not analyse fathers’ moving behaviour but focused only on
mothers because they are the main custodian of children in Belgium. As information on
custody arrangements is not available in administrative data, future research may
benefit from using survey data to investigate custodial fathers. Third, temporary
housing solutions, such as staying with friends or relatives, are likely to be
underreported in register data (Gram-Hanssen and Bech-Danielsen 2007). Nevertheless,
we observe a substantial proportion of (multiple) moves.
Taken together, this study is the first to highlight that location continuity is an
important aspect of separated mothers’ residential mobility outcomes. We have shown
that the degree of location continuity following separation is mainly related to separated
mothers’ local ties. However, a lack of socioeconomic resources is linked to
experiencing (repeated) moves. This should concern policymakers, because residential
instability has implications for the well-being of separated mothers and their children.
To ensure that children have equal opportunities, policymakers have the responsibility
to develop measures that can offset the negative effects of separation for mothers and
their children (Mooney, Oliver, and Smith 2009). Our results suggest that social
housing helps separated mothers with fewer resources to avoid post-separation moves.
However, only a small share (7%) of separated mothers lived in social housing at the
time of separation. Inequalities in separated mothers’ moving opportunities highlight
the need for policies that support separated mothers with fewer resources to find
accommodation close to their pre-separation family home; for example, by increasing
the availability of social housing.
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8. Acknowledgements
We thank Didier Willaert for the data preparation during his time at the Interface
Demography, Free University in Brussels (VUB), as well as Lindsay Theunis and Clara
H. Mulder for their comments on earlier drafts of this paper. Christine Schnor
acknowledges support from the European Research Council (ERC) under the European
Union’s Horizon 2020 research and innovation programme (FAMILYTIES project:
Grant Agreement No. 740113 (2017‒2022), PI: Clara H. Mulder, Rijksuniversiteit
Groningen; GENDERBALL project: Grant No. 312290 (2013‒2017), PI: Jan Van
Bavel, KU Leuven). Júlia Mikolai acknowledges support from the Economic and Social
Research Council (PartnerLife project; Grant No.: ES/L01663X/1 (2014‒2017), PIs:
Clara H. Mulder (University of Groningen), Michael Wagner (University of Cologne),
and Hill Kulu (University of St Andrews) under the Open Research Area (ORA) Plus
scheme. Part of the research was conducted at the Center for Sociological Research,
KU Leuven and at the Interface Demography research group, VUB. The research data
was distributed by FOD Economie, AD Statistiek – Statistics Belgium.
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References
Amato, P.R. (2000). The consequences of divorce for adults and children. Journal of
Marriage and Family 62(4): 1269–1287. doi:10.1111/j.1741-3737.2000.012
69.x.
Andreß, H. and Hummelsheim, D. (2009). When marriage ends: Economic and social
consequences of partnership dissolution. Northampton: Edward Elgar.
doi:10.4337/9781848447202.
Andreß, H., Borgloh, B., Bröckel, M., Giesselmann, M., and Hummelsheim, D. (2006).
The economic consequences of partnership dissolution: A comparative analysis
of panel studies from Belgium, Germany, Great Britain, Italy, and Sweden.
European Sociological Review 22(5): 533–560. doi:10.1093/esr/jcl012.
Andrews, D., Sanchez, A.C., and Johansson, Å. (2011). Housing markets and structural
policies in OECD countries. Paris: OECD (OECD Economics Department
Working Papers 836). doi:10.1787/5kgk8t2k9vf3-en.
Astone, N.M. and McLanahan, S.S. (1994). Family structure, residential mobility, and
school dropout: A research note. Demography 31(4): 575–584. doi:10.2307/
2061791.
Austin, W.G. (2008). Relocation, research, and forensic evaluation: Part I: Effects of
residential mobility on children of divorce. Family Court Review 46(1): 137–
150. doi:10.1111/j.1744-1617.2007.00188.x.
Bernardi, L. and Mortelmans, D. (2018). Lone parenthood in the life course. Cham:
Springer. doi:10.1007/978-3-319-63295-7.
Boussauw, K., van Meeteren, M., and Witlox, F. (2014). Short trips and central places:
The home-school distances in the Flemish primary education system (Belgium).
Applied Geography 53: 311–322. doi:10.1016/j.apgeog.2014.06.025.
Bowstead, J.C. (2015). Forced migration in the United Kingdom: Women’s journeys to
escape domestic violence. Transactions of the Institute of British Geographers
40(3): 307–320. doi:10.1111/tran.12085.
Braver, S. and O’Connell, D. (1998). Divorced dads: Shattering the myths. New York:
Putnam.
Braver, S.L., Ellman, I.M., and Fabricius, W.V. (2003). Relocation of children after
divorce and children’s best interests: New evidence and legal considerations.
Journal of Family Psychology 17(2): 206–219. doi:10.1037/0893-3200.17.2.206.
http://www.demographic-research.org
275
Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
Caldera Sánchez, A. and Andrews, D. (2011). Residential mobility and public policy in
OECD countries. OECD Journal: Economic Studies Vol. 2011/1.
doi:10.1787/eco_studies-2011-5kg0vswqt240.
Centrum voor Gelijkheid van Kansen en voor Racismebestrijding [Centre for equal
chances and the fight against racism, CGKR] (2014). Diversiteitsbarometer
huisvesting [Diversity barometer housing]. Brussels: CGKR. https://www.unia.
be/files/Z_ARCHIEF/de_diversiteitsbarometer_werk_0.pdf.
Clark, W.A.V. (2013). Life course events and residential change: Unpacking age effects
on the probability of moving. Journal of Population Research 30(4): 319–334.
doi:10.1007/s12546-013-9116-y.
Clark, W.A.V. and Rivers, N. (2012). Community choice in large cities: Selectivity and
ethnic sorting across neighbourhoods. In: van Ham, M., Manley, D., Bailey, N.,
Simpson, L., and Maclennan, D. (eds.). Understanding neighbourhood
dynamics. Dordrecht: Springer: 255–279. doi:10.1007/978-94-007-4854-5_12.
Cooke, T.J., Mulder, C.H., and Thomas, M.J. (2016). Union dissolution and migration.
Demographic Research 34(26): 741–760. doi:10.4054/DemRes.2016.34.26.
DaVanzo, J. (1981). Repeat migration, information costs, and location-specific capital.
Population and Environment 4(1): 45–73. doi:10.1007/BF01362575.
De Decker, P.D. (2001). Jammed between housing and property rights: Belgian private
renting in perspective. European Journal of Housing Policy 1(1): 17‒39.
Dewilde, C. (2008). Divorce and the housing movements of owner-occupiers: A
European comparison. Housing Studies 23(6): 809–832. doi:10.1080/02673030
802423151.
Dewilde, C. and Lancee, B. (2013). Income inequality and access to housing in Europe.
European Sociological Review 29(6): 1189–1200.
Esping-Andersen, G. (1999). Social foundations of postindustrial economies. Oxford:
Oxford University Press. doi:10.1093/0198742002.001.0001.
Eurostat (2018). Housing Statistics. https://ec.europa.eu/eurostat/statistics-explained/
index.php/Housing_statistics.
Feijten, P. (2005). Union dissolution, unemployment and moving out of homeownership. European Sociological Review 21(1): 59–71. doi:10.1093/esr/jci004.
Feijten, P. and Mulder, C.H. (2005). Life-course experience and housing quality.
Housing Studies 20(4): 571–587. doi:10.1080/02673030500114474.
276
http://www.demographic-research.org
Demographic Research: Volume 42, Article 9
Feijten, P. and Mulder, C.H. (2010). Gender, divorce and housing: A life course
perspective. In: Reuschke, D. (ed.). Wohnen und Gender: Theoretische,
politische, soziale und räumliche Aspekte [Living and gender: Theoretical,
political, social and spatial aspects]. Wiesbaden: VS Verlag für
Sozialwissenschaften: 175–193. doi:10.1007/978-3-531-92110-5_10.
Feijten, P. and van Ham, M. (2007). Residential mobility and migration of the divorced
and separated. Demographic Research 17(21): 623–654. doi:10.4054/DemRes.
2007.17.21.
Feijten, P. and van Ham, M. (2010). The impact of splitting up and divorce on housing
careers in the UK. Housing Studies 25(4): 483–507. doi:10.1080/02673031003
711477.
Feijten, P. and van Ham, M. (2013). The consequences of divorce and splitting up for
spatial mobility in the UK. Comparative Population Studies 38(2): 405–432.
doi:10.4232/10.CPoS-2013-10en.
Ferrari, G., Bonnet, C., and Solaz, A. (2019). ‘Will the one who keeps the children keep
the house?’ Residential mobility after divorce by parenthood status and custody
arrangements in France. Demographic Research 40(14): 359–394.
doi:10.4054/DemRes.2019.40.14.
Fiori, F. (2019). Who leaves, who stays? Gendered routes out of the family home
following union dissolution in Italy. Demographic Research 40(20): 533–560
doi:10.4054/DemRes.2019.40.20.
Gindes, M. (1998). The psychological effects of relocation for children of divorce.
Journal of the American Academy of Matrimonial Lawyers 15: 119–148.
doi:10.1037/0893-3200.17.2.206.
Gram-Hanssen, K. and Bech-Danielsen, C. (2008). Home dissolution: What happens
after separation? Housing Studies 23(3): 507–522. doi:10.1080/0267303080
2020635.
Hagan, J., MacMillan, R., and Wheaton, B. (1996). New kid in town: Social capital and
the life course effects of family migration on children. American Sociological
Review 61(3): 368–385. doi:10.2307/2096354.
Halleux, J.M. and Strée, J. (2012). Production de l’habitat et enjeux territoriaux
[Habitat production and territorial issues]. Liège: Conférence Permanente du
Développement Territorial. https://orbi.uliege.be//bitstream/2268/144127/3/RI5_
Annexe.pdf.
http://www.demographic-research.org
277
Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
Hooghe, L. (2004). Belgium: Hollowing the Center. In: Amoretti, U.M. and Bermeo,
N.G.(eds.). Federalism and territorial cleavages. Baltimore: Johns Hopkins
Press.
Jalovaara, M. and Kulu, H. (2019). Homeownership after separation: A longitudinal
analysis of Finnish register data. Demographic Research 41(29): 847–872.
doi:10.4054/DemRes.2019.41.29.
Janssens, D., Cools, M., Miermans, W., Declercq, K., and Wets, G. (2011). Onderzoek
verplaatsingsgedrag Vlaanderen 4.2 (2009–2010): Analyserapport [Research on
Flemish moving behaviour 4.2 (2009–2010): A report of analyses]. Diepenbeek:
Transportation Research Institute (IMOB). https://orbi.uliege.be/bitstream/2268/
143241/1/ovg42-analyse-globaal.pdf.
Karsten, L. (2007). Housing as a way of life: Towards an understanding of middle-class
families’ preference for an urban residential location. Housing Studies 22(1):
83‒98.
Kelly, J.B. and Lamb, M.E. (2003). Developmental issues in relocation cases involving
young children: When, whether, and how? Journal of Family Psychology 17(2):
193–205. doi:10.1037/0893-3200.17.2.193.
Kulu, H., Mikolai, J., Thomas, M.J., Vidal, S., Schnor, C., Willaert, D., Visser, F.H.L.,
and Mulder, C.H. (2017). Separation and spatial mobility: A cross-national
comparison. Paper presented at the Annual Meeting of the Population
Association of America, April 27–29, 2017, Chicago, USA.
Lebel, A., Pampalon, R., and Villeneuve, P.Y. (2007). A multi-perspective approach for
defining neighbourhood units in the context of a study on health inequalities in
the Quebec City region. International Journal of Health Geographics 6(27): 1–
15. doi:10.1186/1476-072X-6-27.
Luyten, S. and Van Hecke, E. (2007). De Belgische stadsgewesten 2001 [The Belgian
regions]. Brussels: Algemene Directie Statistiek en Economische Informatie
(Statistics Belgium Working Paper 14).
McCarthy, P. and Simpson, B. (1991). Issues in post-divorce housing. Aldershot:
Avebury Academic Publishing Group.
McLanahan, S. and Sandefur, G. (1994). Growing up with a single parent: What hurts,
what helps. Cambridge: Harvard University Press.
278
http://www.demographic-research.org
Demographic Research: Volume 42, Article 9
McLanahan, S.S. and Sørensen, A.B. (1984). Life events and psychological well-being:
A reexamination of theoretical and methodological issues. Social Science
Research 13(2): 111–128. doi:10.1016/0049-089X(84)90017-6.
Mikolai, J. and Kulu, H. (2018a). Divorce, separation, and housing changes: A
multiprocess analysis of longitudinal data from England and Wales.
Demography 55(1): 83–106. doi:10.1007/s13524-017-0640-9.
Mikolai, J. and Kulu, H. (2018b). Short- and long-term effects of divorce and
separation on housing tenure in England and Wales. Population Studies 72(1):
17–39. doi:10.1080/00324728.2017.1391955.
Mikolai, J. and Kulu, H. (2019). Union dissolution and housing trajectories in Britain.
Demographic Research 41(7): 161–196. doi:10.4054/DemRes.2019.41.7.
Mikolai, J., Kulu, H., Vidal, S., van der Wiel, R., and Mulder, C.H. (2019). Separation,
divorce, and housing tenure: A cross-country comparison. Demographic
Research 41(39): 1131–1146. doi:10.4054/DemRes.2019.41.39.
Mooney, A., Oliver, C., and Smith, M. (2009). Impact of family breakdown on
children’s well-being: Evidence review. London: Thomas Coram Research Unit
Institute of Education, University of London (Research Report DCSF-RR113).
Mulder, C.H. (1993). Migration dynamics: A life course approach. Amsterdam: Thesis.
Mulder, C.H. (1996). Housing choice: Assumptions and approaches. Netherlands
Journal of Housing and the Built Environment 11(3): 209–232. doi:10.1007/
BF02496589.
Mulder, C.H. (2006). Population and housing: A two-sided relationship. Demographic
Research 15(13): 401–412. doi:10.4054/DemRes.2006.15.13.
Mulder, C.H. (2013). Family dynamics and housing: Conceptual issues and empirical
findings. Demographic Research 29(14): 355–378. doi:10.4054/DemRes.2013.
29.14.
Mulder, C.H. and Hooimeijer, P. (1999). Residential relocations in the life course. In:
van Wissen, L.J.G. and Dykstra, P.A. (eds.). Population issues: An
interdisciplinary focus. Dordrecht: Springer: 159–186. doi:10.1007/978-94-0114389-9_6.
Mulder, C.H. and Lauster, N.T. (2010). Housing and family: An introduction. Housing
Studies 25(4): 433–440. doi:10.1080/02673031003771109.
http://www.demographic-research.org
279
Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
Mulder, C.H. and Malmberg, G. (2011). Moving related to separation: Who moves and
to what distance. Environment and Planning A: Economy and Space 43(11):
2589–2607. doi:10.1068/a43609.
Mulder, C.H. and van der Meer, M.J. (2009). Geographical distances and support from
family members. Population, Space and Place 15(4): 381–399. doi:10.1002/
psp.557.
Mulder, C.H. and Wagner, M. (2010). Union dissolution and mobility: Who moves
from the family home after separation? Journal of Marriage and Family 72(5):
1263‒1273.
Mulder, C.H. and Wagner, M. (2012). Moving after separation: The role of locationspecific capital. Housing Studies 27(6): 839–852. doi:10.1080/02673037.2012.
651109.
Mulder, C.H., Ten Hengel, B., Latten, J., and Das, M. (2012). Relative resources and
moving from the joint home around divorce. Journal of Housing and the Built
Environment 27(2): 153–168. doi:10.1007/s10901-011-9250-9.
Murinkó, L. (2019). Housing consequences of divorce and separation in a ‘super home
ownership’ regime. Demographic Research 40(34): 975‒1014. doi:10.4054/
DemRes.2019.40.34.
Neels, K., and De Wachter, D. (2010). Postponement and recuperation of Belgian
fertility: How are they related to rising female educational attainment. Vienna
Yearbook of Population Research 8: 77‒106
Oakil, A.T. (2013). Temporal dependence in life trajectories and mobility decisions
[PhD thesis]. Utrecht: Utrecht University.
OECD (2015). Economic Surveys: Belgium. Paris: OECD. https://doi.org/10.1787/
eco_surveys-bel-2015-en.
OECD (2017). Economic Surveys: Belgium. Paris: OECD. https://doi.org/10.1787/
eco_surveys-bel-2017-en.
Pettersson, A. and Malmberg, G. (2009). Adult children and elderly parents as mobility
attractions in Sweden. Population, Space and Place 15(4): 343‒357.
Reginster, I. and Goffette-Nagot, F. (2005). Urban environmental quality in two
Belgian cities, evaluated on the basis of residential choices and GIS data.
Environment and Planning A: Economy and Space 37(6): 1067–1090.
doi:10.1068/a3735a.
280
http://www.demographic-research.org
Demographic Research: Volume 42, Article 9
Schier, M. (2015). Post-separation families: Spatial mobilities and the need to manage
multi-local everyday life. In: Aybek, C.M., Huinink, J., and Muttarak, R. (eds.).
Spatial mobility, migration, and living arrangements. Cham: Springer: 205–224.
doi:10.1007/978-3-319-10021-0_10.
Simpson, G.A. and Fowler, M.G. (1994). Geographic mobility and children’s
emotional/behavioral adjustment and school functioning. Pediatrics 93(2): 303–
309.
Sodermans, A.K., Matthijs, K., and Swicegood, G. (2013). Characteristics of joint
physical custody families in Flanders. Demographic Research 28(29): 821–848.
doi:10.4054/DemRes.2013.28.29.
Statistics Belgium (2015): Chiffres de population au 1ier janvier 2015.
https://www.ibz.rrn.fgov.be/fileadmin/user_upload/fr/pop/statistiques/population
-bevolking-20150101.pdf.
Theunis, L., Eeckhaut, M., and Van Bavel, J. (2018). Who leaves the joint home after
separation? The role of partners’ absolute and relative education in Belgium.
European Sociological Review 34(6): 659–674. doi:10.1093/esr/jcy035.
Theunis, L., Willaert, D., and Van Bavel, J. (2016). Wie verlaat de gezinswoning bij
een echtscheiding? Het belang van de socio-economische positie van partners
[Who leaves the family home upon separation? The importance of partners’
socio-economic position]. Sociologos 38(3): 221–247.
Thomas, I., Adam, A., and Verhetsel, A. (2017). Migration and commuting interactions
fields: A new geography with community detection algorithm? Belgeo: Revue
Belge de Géographie 4: 1–17. doi:10.4000/belgeo.20507.
Thomas, M.J., Mulder, C.H., and Cooke, T.J. (2017a). Linked lives and constrained
spatial mobility: The case of moves related to separation among families with
children. Transactions of the Institute of British Geographers 42(4): 597–611.
doi:10.1111/tran.12191.
Thomas, M.J., Mulder, C.H., and Cooke, T.J. (2017b). Geographical distances between
separated parents: A longitudinal analysis. European Journal of Population
34(4): 463–489. doi:10.1007/s10680-017-9437-1.
Tucker, C.J., Marx, J., and Long, L. (1998). ‘Moving on’: Residential mobility and
children’s school lives. Sociology of Education 71(2): 111–129. doi:10.2307/
2673244.
http://www.demographic-research.org
281
Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
van Imhoff, E. and Keilman, N. (1991). LIPRO 2.0: An application of a dynamic
demographic projection model to household structure in the Netherlands. The
Hague: Netherlands Interdisciplinary Demographic Institute. https://www.nidi.
knaw.nl/shared/content/output/lipro/nidicbgs-publ-23.pdf.
Warisse, C. (2017). Analysis of the developments in residential property prices: Is the
Belgian market overvalued? Economic Review (i): 61‒77.
Winters, S. and Heylen, K. (2014). How housing outcomes vary between the Belgian
regions. Journal of Housing and the Built Environment 29(3): 541–556.
doi:10.1007/s10901-013-9364-3.
282
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Appendix
Table A-1:
Six mobility patterns by separated mother’s characteristics, the
characteristics of the woman’s place of residence at the time of
separation, the characteristics of the ex-couple, and by whether the
woman formed a new coresidential relationship, N = 25,802, column
percentages (for total sample and by mobility patterns)
Total
Mobility pattern
High degree of location
Moderate degree of location
continuity:
continuity:
Remaining in same municipality Remaining in same province, but
moving to different municipality
(1) No move
(2) Within(3) One cross- (4) Several
municipality
municipality crossmove(s)
move
municipality
moves
Low degree of location
continuity:
Moving to different province
(5) One cross- (6) Several
province move cross-province
moves
Woman’s characteristics
Education
Low (34%)
32%
38%
34%
36%
34%
38%
Medium (39%)
37%
40%
42%
43%
38%
39%
High (23%)
29%
19%
22%
18%
26%
19%
2%
3%
3%
3%
3%
4%
Missing (3%)
Employment status 12 months prior to separation
Full-time (44%)
44%
41%
46%
44%
47%
42%
Part-time (27%)
27%
28%
27%
27%
24%
21%
Unemployed (19%)
19%
20%
19%
18%
20%
24%
Missing (10%)
10%
11%
9%
11%
10%
12%
39
36
36
34
36
35
1 child (36%)
31%
38%
38%
42%
40%
40%
2 children (41%)
43%
41%
41%
38%
39%
35%
3 children (16%)
18%
14%
15%
15%
15%
15%
4+ children (7%)
8%
7%
6%
6%
6%
10%
Mean age at separation (years)
36
Number of children at separation
Age of the youngest child at separation
< 7 (46%)
41%
46%
47%
53%
51%
56%
7–11 (25%)
27%
25%
25%
22%
23%
19%
12–17 (20%)
22%
21%
19%
17%
18%
16%
18+ (9%)
10%
9%
10%
8%
9%
9%
56%
Characteristics of the place of residence at the time of separation
Living at the place of birth
Elsewhere (31%)
30%
27%
27%
23%
62%
Birth municipality (19%)
21%
29%
12%
14%
9%
9%
Birth province (50%)
49%
45%
61%
64%
29%
35%
Owner (65%)
68%
62%
65%
65%
62%
57%
Private renting (23%)
16%
27%
26%
25%
28%
30%
Social renting (7%)
12%
6%
5%
5%
5%
7%
5%
5%
5%
5%
5%
6%
Housing
Missing/rent free (5%)
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Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
Table A-1:
(Continued)
Mobility pattern
High degree of location
Moderate degree of location
continuity:
continuity:
Remaining in same municipality Remaining in same province, but
moving to different municipality
Total
(1) No move
(2) Within(3) One cross- (4) Several
municipality
municipality crossmove(s)
move
municipality
moves
Characteristics of the place of residence at the time of separation
Low degree of location
continuity:
Moving to different province
(5) One cross- (6) Several
province move cross-province
moves
Area type
Urban (43%)
44%
45%
35%
37%
34%
37%
Suburban (34%)
34%
30%
40%
37%
40%
42%
Rural (22%)
22%
26%
25%
26%
27%
21%
Flanders (55%)
52%
58%
56%
56%
52%
50%
Brussels/Wallonia (45%)
48%
42%
44%
44%
48%
50%
Cohabitation (28%)
24%
28%
29%
31%
31%
31%
Marriage (72%)
76%
72%
71%
69%
69%
69%
Same age (28%)
32%
28%
28%
25%
27%
24%
Woman older (14%)
19%
13%
12%
11%
12%
13%
Man 2–4 years older (31%)
29%
31%
32%
31%
32%
30%
Man 4+ years older (26%)
20%
28%
28%
33%
29%
33%
Same level (49%)
49%
50%
50%
49%
50%
51%
Man > Woman (18%)
16%
19%
18%
19%
18%
18%
Woman > Man (25%)
28%
24%
25%
25%
24%
22%
8%
8%
7%
7%
7%
9%
1%
5%
8%
4%
7%
2%
10%
17%
23%
33%
29%
29%
88%
78%
69%
63%
64%
69%
Region
Ex-couple characteristics
Type of dissolved union
Age difference
Educational difference
Missing (8%)
New Partner
Repartnered at time of
separation (4%)
Repartnered after separation
(19%)
Not repartnered (76%)
2
Note: all differences were tested using chi test (categorical variables) or F-test (continuous variable) and found to be significant at
p < 0.001 level.
Source: Authors’ own calculations using linked Belgian Census (2001) and Population Register data (2001–2006).
284
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Demographic Research: Volume 42, Article 9
Table A-2:
Predicted probability (with 95% confidence intervals) of belonging to
different mobility patterns among separated mothers, N = 25,802
Mobility patter
High degree of location
continuity: Remaining in the
same municipality
(1) No move
(2) Withinmunicipality
move(s)
Moderate degree of location
continuity:
Remaining in same province, but
moving to a different municipality
(3) One cross- (4) Several
municipality
crossmove
municipality
moves
Low degree of location
continuity:
Moving to a different province
(5) One cross- (6) Several
province move cross-province
moves
Woman’s characteristics
Education
0.28
0.30
0.21
[0.27; 0.29]
[0.29; 0.31]
[0.20; 0.22]
0.31
0.27
0.22
Medium
[0.30; 0.32]
[0.26; 0.28]
[0.22; 0.23]
0.37
0.24
0.21
High
[0.36; 0.38]
[0.23, 0.25]
[0.19; 0.22]
0.18
0.31
0.25
Missing
[0.15; 0.22]
[0.27; 0.36]
[0.20; 0.29]
Employment status 12 months prior to separation
0.31
0.26
0.23
Full-time
[0.30; 0.32]
[0.25; 0.27]
[0.22; 0.23]
0.30
0.29
0.22
Part-time
[0.29; 0.31]
[0.28; 0.30]
[0.21; 0.23]
0.30
0.28
0.22
Unemployed
[0.29; 0.31]
[0.27; 0.29]
[0.20; 0.23]
0.32
0.29
0.20
Missing
[0.30; 0.34]
[0.27; 0.31]
[0.18; 0.21]
Number of children at separation
0.29
0.28
0.22
1 child
[0.28; 0.30]
[0.27; 0.29]
[0.21; 0.23]
0.31
0.28
0.22
2 children
[0.30; 0.32]
[0.27; 0.29]
[0.21; 0.23]
0.32
0.25
0.22
3 children
[0.31; 0.34]
[0.23; 0.26]
[0.20; 0.23]
0.30
0.26
0.22
4+ children
0.28; 0.32]
[0.24; 0.28]
[0.19; 0.24]
Age of the youngest child at separation
0.32
0.27
0.22
<7
[0.31; 0.34]
[0.26; 0.28]
[0.21; 0.23]
0.32
0.28
0.22
7–11
[0.30; 0.33]
[0.27; 0.29]
[0.21; 0.23]
0.28
0.29
0.21
12–17
[0.27; 0.30]
[0.28; 0.31]
[0.20; 0.23]
0.25
0.25
0.24
18+
[0.23; 0.27]
[0.22; 0.27]
[0.21; 0.26]
0.26
0.29
0.18
Missing
[0.11; 0.41]
[0.13; 0.44]
[0.05; 0.31]
Characteristics of the place of residence at the time of separation
Low
0.11
[0.10; 0.11]
0.10
[0.09, 0.11]
0.08
[0.07; 0.09]
0.16
[0.12; 0.20]
0.07
[0.07; 0.08]
0.08
[0.07; 0.08]
0.08
[0.07; 0.09]
0.07
[0.04; 0.09]
0.03
[0.02; 0.03]
0.03
[0.02; 0.03]
0.02
[0.02; 0.03]
0.03
[0.02; 0.05]
0.10
[0.09; 0.11]
0.10
[0.09; 0.11]
0.09
[0.09; 0.10]
0.10
[0.08; 0.11]
0.08
[0.07; 0.08]
0.07
[0.06; 0.08]
0.08
[0.07; 0.09]
0.07
[0.06; 0.08]
0.03
[0.02; 0.03]
0.02
[0.02; 0.03]
0.03
[0.03; 0.04]
0.02
[0.02; 0.03]
0.10
[0.09; 0.10]
0.09
[0.09; 0.10]
0.11
[0.10; 0.12]
0.11
[0.10; 0.13]
0.08
[0.07; 0.09]
0.07
[0.07; 0.08]
0.08
[0.07; 0.08]
0.07
[0.06; 0.08]
0.02
[0.02; 0.03]
0.02
[0.02; 0.03]
0.03
[0.02; 0.03]
0.04
[0.03; 0.05]
0.09
[0.08; 0.10
0.09
[0.08; 0.10]
0.11
[0.10; 0.12]
0.14
[0.12; 0.16]
0.16
[0.03; 0.29]
0.08
[0.07; 0.08]
0.07
[0.07; 0.08]
0.07
[0.06; 0.08]
0.08
[0.07; 0.10]
0.09
[–0.01;0.18]
0.02
[0.02; 0.03]
0.02
[0.02; 0.03]
0.03
[0.02; 0.03]
0.05
[0.03; 0.06]
0.03
[–0.03; 0.09]
0.07
[0.06; 0.08]
0.12
[0.12; 0.13]
0.08
[0.07; 0.08]
0.04
[0.04; 0.05]
0.05
[0.05; 0.05]
0.18
[0.17; 0.19]
0.01
[0.01; 0.02]
0.02
[0.02; 0.02]
0.05
[0.05; 0.06]
Living at the place of birth
Birth municipality
Birth province
Elsewhere
0.34
[0.33; 0.36]
0.30
[0.29; 0.31]
0.27
[0.25; 0.28]
http://www.demographic-research.org
0.39
[0.38; 0.40]
0.25
[0.24; 0.25]
0.23
[0.22; 0.24]
0.14
[0.13; 0.15]
0.26
[0.25; 0.27]
0.19
[0.18; 0.20]
285
Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
Table A-2:
(Continued)
Mobility pattern
High degree of location
continuity: Remaining in the
same municipality
(1) No move
(2) Withinmunicipality
move(s)
Moderate degree of location
continuity:
Remaining in same province, but
moving to a different municipality
(3) One cross- (4) Several
municipality
crossmove
municipality
moves
Low degree of location
continuity:
Moving to a different province
(5) One cross- (6) Several
province move cross-province
moves
Housing
Owner
Private renting
Social renting
Missing/rent free
0.32
[0.31; 0.32]
0.23
[0.21; 0.24]
0.54
[0.51; 0.56]
0.31
[0.29; 0.34]
0.27
[0.26; 0.27]
0.31
[0.30; 0.32]
0.20
[0.18; 0.22]
0.27
[0.25; 0.30]
0.22
[0.21; 0.22]
0.25
[0.24; 0.26]
0.14
[0.12; 0.15]
0.21
[0.19; 0.24]
0.10
[0.10; 0.11]
0.10
[0.09; 0.11]
0.06
[0.05; 0.07]
0.10
[0.08; 0.12]
0.07
[0.07; 0.08]
0.09
[0.08; 0.10]
0.05
[0.04; 0.06]
0.08
[0.06; 0.09]
0.02
[0.02; 0.03]
0.03
[0.02; 0.03]
0.02
[0.01; 0.02]
0.03
[0.02; 0.04]
0.32
[0.31; 0.33]
0.30
[0.29; 0.31]
0.29
[0.28; 0.30]
0.30
[0.29; 0.30]
0.24
[0.23; 0.25]
0.29
[0.27; 0.30]
0.20
[0.19; 0.21]
0.23
[0.22; 0.24]
0.22
[0.21; 0.23]
0.09
[0.08; 0.09]
0.11
[0.10; 0.11]
0.10
[0.10; 0.11]
0.07
[0.06; 0.07]
0.09
[0.08; 0.09]
0.07
[0.07; 0.08]
0.02
[0.02; 0.03]
0.03
[0.03; 0.03]
0.02
[0.02; 0.03]
0.32
[0.31; 0.33]
0.30
[0.29; 0.31]
0.25
[0.25; 0.26]
0.29
[0.28; 0.30]
0.22
[0.21; 0.23]
0.21
[0.21; 0.22]
0.10
[0.10; 0.11]
0.10
[0.09; 0.10]
0.08
[0.07; 0.08]
0.07
[0.07; 0.08]
0.03
[0.02; 0.03]
0.02
[0.02; 0.03]
0.33
[0.32; 0.35]
0.38
[0.37; 0.40]
0.30
[0.29; 0.31]
0.25
[0.24; 0.26]
0.27
[0.26; 0.28]
0.24
[0.23; 0.26]
0.28
[0.27; 0.29]
0.29
[0.28; 0.30]
0.21
[0.20; 0.22]
0.19
[0.18; 0.21]
0.22
[0.21; 0.23]
0.24
[0.23; 0.25]
0.09
[0.08; 0.10]
0.09
[0.08; 0.10]
0.10
[0.09; 0.10]
0.12
[0.11; 0.13]
0.08
[0.07; 0.08]
0.06
[0.05; 0.07]
0.08
[0.07; 0.09]
0.08
[0.07; 0.08]
0.02
[0.02; 0.03]
0.03
[0.02; 0.03]
0.02
[0.02; 0.03]
0.03
[0.02; 0.03]
0.30
[0.29; 0.31]
0.29
[0.27; 0.30]
0.32
[0.31; 0.34]
0.35
[0.32; 0.38]
0.28
[0.27; 0.28]
0.27
[0.26; 0.29]
0.27
[0.26; 0.29]
0.26
[0.24; 0.29]
0.22
[0.21; 0.23]
0.23
[0.21; 0.24]
0.21
[0.20; 0.22]
0.21
[0.18; 0.23]
0.10
[0.09; 0.10]
0.10
[0.09; 0.11
0.10
[0.09; 0.11]
0.08
[0.06; 0.10]
0.08
[0.07; 0.08]
0.08
[0.07; 0.09]
0.07
[0.06; 0.08]
0.07
[0.06; 0.09]
0.03
[0.02; 0.03]
0.03
[0.02; 0.03]
0.02
[0.02; 0.03]
0.03
[0.02; 0.03]
0.29
[0.28; 0.30]
0.32
[0.31; 0.32]
0.27
[0.26; 0.28]
0.27
[0.27; 0.28]
0.23
[0.22; 0.24]
0.22
[0.21; 0.22]
0.10
[0.10; 0.11]
0.10
[0.09; 0.10]
0.08
[0.08; 0.09]
0.07
[0.07; 0.08]
0.02
[0.02; 0.03]
0.03
[0.02; 0.03]
0.36
[0.36; 0.37]
0.10
[0.08; 0.12]
0.18
[0.16; 0.19]
0.27
[0.27; 0.28]
0.29
[0.27; 0.32]
0.23
[0.22; 0.24]
0.19
[0.19; 0.20]
0.37
[0.34; 0.40]
0.26
[0.25; 0.27]
0.08
[0.08; 0.09]
0.09
[0.07; 0.10]
0.17
[0.16; 0.18]
0.06
[0.06; 0.07]
0.14
[0.12; 0.16]
0.12
[0.11; 0.13]
0.02
[0.02; 0.02]
0.01
[0.01; 0.02]
0.04
[0.04; 0.05]
Area type
Urban
Suburban
Rural
Region
Flanders
Brussels/Wallonia
Ex-couple characteristics
Age difference
Same age
Woman older
Man 2–4 years older
Man 4+ years older
Educational difference
Same level
Man > Woman
Woman > Man
Missing
Type of dissolved union
Cohabitation
Marriage
New partner
Not repartnered
Repartnered at time of
separation
Repartnered after
separation
Source: Authors’ own calculations using linked Belgian Census (2001) and Population Register data (2001–2006).
286
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Demographic Research: Volume 42, Article 9
Table A-3:
Predicted probability (with 95% confidence intervals) of leaving,
remaining in, or returning to the pre-separation municipality,
N = 25,802
LEAVE
(N = 14,744)
REMAIN
(N = 9,463)
RETURN
(N = 1,595)
0.58
[0.57; 0.59]
0.57
[0.56; 0.58]
0.61
[0.59; 0.62]
0.49
[0.44; 0.54]
0.07
[0.06; 0.07]
0.06
[0.06; 0.07]
0.04
[0.04; 0.05]
0.10
[0.07; 0.13]
0.57
[0.56; 0.58]
0.59
[0.58; 0.60]
0.58
[0.56; 0.59]
0.62
[0.59; 0.64]
0.06
[0.05; 0.06]
0.07
[0.06; 0.07]
0.06
[0.05; 0.06]
0.06
[0.05; 0.07]
0.58
[0.57; 0.59]
0.59
[0.58; 0.60]
0.57
[0.56; 0.59]
0.56
[0.53; 0.59]
0.06
[0.05; 0.06]
0.06
[0.05; 0.06]
0.06
[0.05; 0.07]
0.08
[0.06; 0.09]
0.59
[0.58; 0.60]
0.60
[0.59; 0.61]
0.58
[0.56; 0.59]
0.50
[0.47; 0.53]
0.55
[0.37; 0.72]
0.05
[0.05; 0.06]
0.06
[0.05; 0.06]
0.07
[0.06; 0.08]
0.08
[0.06; 0.10]
0.06
[–0.02; 0.15]
0.20
[0.19; 0.21]
0.38
[0.37; 0.39]
0.44
[0.42; 0.45]
0.75
[0.73; 0.76]
0.56
[0.55; 0.57]
0.55
[0.50; 0.52]
0.05
[0.05; 0.06]
0.06
[0.06; 0.07]
0.06
[0.05; 0.06]
0.36
[0.35; 0.36]
0.41
[0.39; 0.42]
0.24
[0.22; 0.26]
0.36
[0.33; 0.39]
0.58
[0.57; 0.59]
0.54
[0.52; 0.55]
0.72
[0.70; 0.75]
0.58
[0.55; 0.61]
0.06
[0.06; 0.07]
0.06
[0.05; 0.06]
0.04
[0.03; 0.05]
0.06
[0.04; 0.07]
Woman’s characteristics
Education
0.35
[0.34; 0.36]
0.36
Medium
[0.35; 0.37]
0.35
High
[0.33; 0.36]
0.41
Missing
[0.36; 0.46]
Employment status 12 months prior to separation
0.37
Full-time
[0.36; 0.38]
0.34
Part-time
[0.33; 0.35]
0.36
Unemployed
[0.35; 0.38]
0.33
Missing
[0.31; 0.35]
Number of children at separation
0.36
1 child
[0.35; 0.38]
0.35
2 children
[0.34; 0.36]
0.37
3 children
[0.35; 0.38]
0.36
4+ children
[0.34; 0.39]
Age of the youngest child at separation
0.36
<7
[0.34; 0.37]
0.34
7–11
[0.33; 0.36]
0.36
12–17
[0.34; 0.37]
0.42
18+
[0.39; 0.45]
0.39
Missing
[0.22; 0.56]
Characteristics of the place of residence at the time of separation
Low
Living at the place of birth
Birth municipality
Birth province
Elsewhere
Housing
Owner
Private renting
Social renting
Missing/rent free
http://www.demographic-research.org
287
Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
Table A-3:
(Continued)
LEAVE
(N = 14,744)
REMAIN
(N = 9,463)
RETURN
(N = 1,595)
0.33
[0.32; 0.34]
0.40
[0.39; 0.41]
0.36
[0.34; 0.37]
0.62
[0.61; 0.63]
0.54
[0.53; 0.55]
0.58
[0.56; 0.59]
0.05
[0.05; 0.06]
0.06
[0.06; 0.07]
0.07
[0.06; 0.07]
0.37
[0.36; 0.38]
0.35
[0.34; 0.36]
0.57
[0.56; 0.58]
0.59
[0.58; 0.60]
0.06
[0.06; 0.06]
0.06
[0.05; 0.06]
0.34
[0.33; 0.35]
0.31
[0.30; 0.33]
0.37
[0.36; 0.38]
0.39
[0.38; 0.40]
0.60
[0.59; 0.61]
0.63
[0.61; 0.65]
0.58
[0.57; 0.59]
0.54
[0.53; 0.55]
0.06
[0.05; 0.06]
0.06
[0.05; 0.07]
0.05
[0.05; 0.06]
0.07
[0.06; 0.07]
0.36
[0.35; 0.37]
0.37
[0.36; 0.39]
0.35
[0.33; 0.36]
0.34
[0.31; 0.37]
0.58
[0.57; 0.59]
0.56
[0.55; 0.58]
0.60
[0.58; 0.61]
0.62
[0.59; 0.64]
0.06
[0.06; 0.07]
0.06
[0.06; 0.07]
0.06
[0.05; 0.06]
0.05
[0.04; 0.06]
0.38
[0.37; 0.40]
0.35
[0.34; 0.36]
0.56
[0.55; 0.57]
0.59
[0.58; 0.60]
0.06
[0.05; 0.06]
0.06
[0.06; 0.06]
0.31
[0.30; 0.31]
0.57
[0.54; 0.60]
0.52
[0.51; 0.54]
0.64
[0.63; 0.64]
0.39
[0.36; 0.42]
0.40
[0.39; 0.42]
0.06
[0.05; 0.06]
0.04
[0.03; 0.05]
0.07
[0.07; 0.08]
Area type
Urban
Suburban
Rural
Region
Flanders
Brussels/Wallonia
Ex-couple characteristics
Age difference
Same age
Woman older
Man 2–4 years older
Man 4+ years older
Educational difference
Same level
Man > Woman
Woman > Man
Missing
Type of dissolved union
Cohabitation
Marriage
New partner
Not repartnered
Repartnered at time of separation
Repartnered after separation
Source: Authors’ own calculations using linked Belgian Census (2001) and Population Register data (2001–2006).
288
http://www.demographic-research.org
Demographic Research: Volume 42, Article 9
Table A-4:
Relative risk ratio (standard error) of belonging to different mobility
patterns among separated mothers. Base outcome: moving within the
same municipality (pattern 2), N = 25,802
Base outcome: Moving within the same municipality (2)
Remaining in
Remaining in province, but moving to
municipality
different municipality
No move (1)
One move (3)
Several moves (4)
Moving to different province
One move (5)
Several moves
(6)
Woman’s characteristics
Education (ref = Medium)
Low
0.83***
(0.04)
High
1.38***
(0.07)
Missing
0.87***
(0.04)
0.88*
0.94
(0.06)
(0.10)
1.05
0.94
(0.06)
(0.07)
0.52***
(0.07)
0.98
(0.06)
1.20***
(0.08)
1.02
(0.12)
0.96
1.39*
0.76
1.07
(0.14)
(0.25)
(0.15)
(0.29)
Employment status 12 months prior to separation (ref = Full-time)
Part-time
0.88***
(0.04)
Unemployed
0.90**
(0.04)
Missing
0.77***
(0.06)
1.07**
(0.03)
Age at separation2
0.89**
(0.05)
0.92
(0.06)
Age at separation
0.86***
(0.04)
0.97
(0.03)
0.90*
(0.05)
0.86**
(0.06)
0.83**
(0.07)
0.93**
(0.03)
0.79***
(0.05)
0.73***
(0.08)
0.94
1.08
(0.07)
(0.11)
0.79**
(0.08)
0.94*
(0.04)
0.76**
(0.11)
0.88**
(0.05)
1.00
1.00
1.00
1.00
1.00
(0.00)
(0.00)
(0.00)
(0.00)
(0.00)
Number of children at separation (ref = One child)
2 children
1.09**
(0.04)
3 children
1.26***
(0.07)
4 or more children
1.02
0.98
0.92
0.97
(0.05)
(0.05)
(0.06)
(0.09)
1.14**
(0.07)
1.12
1.08
(0.09)
(0.09)
1.25***
(0.10)
1.28**
(0.14)
1.09
(0.09)
0.99
(0.12)
1.32**
(0.17)
1.81***
(0.29)
Age of youngest child at separation (ref = < 7 years)
7–11
12–17
1.08
1.06
1.07
1.13
1.11
(0.05)
(0.06)
(0.07)
(0.08)
(0.13)
0.87***
(0.05)
18+
0.91
(0.07)
Missing
0.95
(0.06)
1.24**
(0.11)
1.17**
(0.09)
1.72***
(0.21)
0.98
1.26*
(0.08)
(0.18)
1.30**
(0.16)
2.46***
(0.47)
0.81
0.80
1.71
1.18
1.45
(0.38)
(0.43)
(0.97)
(0.82)
(1.56)
Characteristics of the place of residence at the time of separation
Living at the place of birth (ref = Elsewhere)
Birth municipality
0.77***
(0.04)
Birth province
1.07
(0.04)
http://www.demographic-research.org
0.44***
(0.03)
1.29***
(0.06)
0.53***
(0.04)
1.51***
(0.09)
0.13***
(0.01)
0.26***
(0.01)
0.15***
(0.02)
0.35***
(0.03)
289
Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
Table A-4:
(Continued)
Base outcome: Moving within the same municipality (2)
Remaining in
Remaining in province, but moving to
municipality
different municipality
No move (1)
One move (3)
Several moves (4)
Moving to different province
One move (5)
Several moves
(6)
Housing tenure (ref = Owner)
Private renting
0.61***
(0.03)
Social renting
2.30***
(0.16)
Missing
0.99
(0.05)
0.86*
(0.08)
0.82***
(0.05)
0.77**
(0.09)
1.02
0.99
(0.06)
(0.10)
0.88
1.07
(0.11)
(0.18)
0.98
0.97
0.96
1.01
1.12
(0.08)
(0.09)
(0.11)
(0.12)
(0.20)
Area type at separation (ref = Urban)
Suburban
1.13***
(0.05)
Rural
0.92*
(0.04)
Living in Flanders
0.82***
(0.03)
1.42***
(0.06)
1.14***
(0.06)
0.85***
(0.03)
1.49***
(0.08)
1.22***
(0.07)
0.84***
(0.04)
1.52***
(0.09)
1.58***
(0.14)
1.07
1.05
(0.07)
(0.11)
0.84***
(0.04)
0.82**
(0.07)
Ex-couple characteristics
Marriage
1.08*
0.93
0.92
(0.05)
(0.04)
(0.05)
0.84***
(0.05)
1.01
(0.09)
Age difference (ref = Same age)
Woman older
1.25***
(0.07)
Man 2–4 years older
0.85***
(0.04)
Man > 4 years older
0.70***
(0.03)
0.99
1.09
0.89
1.24
(0.07)
(0.09)
(0.08)
(0.17)
1.00
1.02
1.00
1.03
(0.05)
(0.06)
(0.07)
(0.11)
1.03
(0.05)
1.20***
(0.08)
0.95
1.16
(0.06)
(0.12)
Educational difference (ref = Same level)
Man > Woman
Woman > Man
Missing
0.96
1.03
1.06
1.07
0.96
(0.05)
(0.05)
(0.07)
(0.08)
(0.11)
1.09*
0.96
1.02
0.93
0.89
(0.05)
(0.05)
(0.06)
(0.06)
(0.09)
1.23**
(0.10)
New partner (ref = Not repartnered)
Repartnered at time of
0.25***
separation
(0.03)
Repartnered after separation
0.58***
(0.03)
Constant
0.23***
(0.12)
0.97
0.84
0.98
0.99
(0.09)
(0.10)
(0.12)
(0.18)
1.77***
(0.14)
1.60***
(0.08)
0.96
(0.11)
2.37***
(0.13)
2.15***
(0.23)
2.40***
(0.15)
0.53**
(0.14)
2.29***
(0.21)
1.35
2.21
2.66
4.03
(0.71)
(1.38)
(1.82)
(3.97)
Note: *** p < 0.01, ** p < 0.05, * p < 0.1
Source: Authors’ own calculations using linked Belgian Census (2001) and Population Register data (2001–2006).
290
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Demographic Research: Volume 42, Article 9
Table A-5:
Results of multinomial logistic regression, relative risk ratio
(standard errors) of remaining in or returning to the pre-separation
municipality. Base outcome: leaving the pre-separation municipality
and not returning to it, N = 25,802
Base outcome: LEAVE (N = 9,463)
REMAIN
RETURN
N = 14,744
N = 1,595
Woman’s characteristics
Woman’s education (ref = Medium)
Low
High
Missing
Age at separation
Age at separation squared
Number of children (ref = One child)
2 children
3 children
4 or more children
Age of youngest child (ref = < 7 years)
7–11
12–17
18+
Missing
Employment status at separation (ref = Full-time)
Part-time
Unemployed
Missing
1.04
(0.04)
1.11***
(0.04)
0.76**
(0.08)
1.06**
(0.02)
1.00
(0.00)
1.15**
(0.08)
0.73***
(0.06)
1.44*
(0.31)
0.91**
(0.04)
1.00
(0.00)
1.07**
(0.04)
0.99
(0.04)
0.98
(0.06)
1.06
(0.07)
1.09
(0.10)
1.40***
(0.17)
0.95
(0.04)
0.93*
(0.04)
0.68***
(0.04)
0.80
(0.30)
0.91
(0.07)
1.17
(0.11)
1.16
(0.16)
0.97
(0.74)
1.13***
(0.04)
1.04
(0.04)
1.23***
(0.07)
1.23***
(0.08)
1.01
(0.08)
1.14
(0.12)
Characteristics of the place of residence at the time of separation
Housing tenure (ref = Owner)
Private renting
Social renting
Missing
http://www.demographic-research.org
0.80***
(0.03)
1.87***
(0.12)
0.98
(0.06)
0.77***
(0.05)
0.89
(0.11)
0.87
(0.11)
291
Schnor & Mikolai: Remain, leave, or return? Mothers’ location continuity after separation in Belgium
Table A-5:
(Continued)
Base outcome: LEAVE (N = 9,463)
REMAIN
RETURN
N = 14,744
N = 1,595
Living at the place of birth (ref = Elsewhere)
Living at birth municipality
3.19***
(0.14)
Living at birth province
1.25***
(0.04)
2.03***
(0.18)
1.28***
(0.08)
Area (ref = Urban)
Suburban
0.72***
(0.02)
Rural
0.85***
(0.03)
Living in Flanders (ref = Brussels/Wallonia)
1.08**
(0.03)
0.93
(0.06)
1.11
(0.08)
1.02
(0.06)
Ex-couple characteristics
Married (ref = Cohabiting)
1.16***
(0.04)
1.16**
(0.08)
Age difference (ref = Same age)
Woman older
1.16***
(0.05)
Man 2–4 years older
0.90***
(0.03)
Man 5 years or more older
0.79***
(0.03)
1.09
(0.11)
0.89*
(0.06)
1.05
(0.08)
Educational difference (ref = Same level of education)
Man > woman
Woman > man
0.95
1.02
(0.04)
(0.08)
1.09**
(0.04)
Missing
1.14**
(0.08)
0.99
(0.07)
0.82
(0.12)
New partner (ref = Not repartnered)
Repartnered at separation
0.33***
(0.02)
Repartnered after separation
0.37***
(0.01)
Constant
0.34***
(0.13)
0.41***
(0.06)
0.77***
(0.05)
1.55
(1.08)
Note: *** p < 0.01, ** p < 0.05, * p < 0.1.
Source: Authors’ own calculations using linked Belgian Census (2001) and Population Register data (2001–2006).
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English
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Emotional Reactivity to Binge Food and Erotic Cues in Women With Bulimia Nervosa Symptoms
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Research Square (Research Square)
| 2,021
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cc-by
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Abstract Background: Studies on food cue reactivity have documented that altered responses to high-calorie food are associ‑
ated with bulimic symptomatology, however, alterations in sexual motivations and behaviors are also associated
clinical features in this population, which justify their inclusion as a research target. Here, we study responses to erotic
cues—alongside food, neutral and aversive cues—to gain an understanding of specificity to food versus a general‑
ized sensitivity to primary reinforcers. Methods: We recorded peripheral psychophysiological indices –the startle reflex, zygomaticus, and corrugator
responses—and self-reported emotional responses (valence, arousal, and dominance) in 75 women completing the
Bulimia Test-Revised (BULIT-R). Multiple regression analysis tested whether BULIT-R symptoms were predicted by self-
reported and psychophysiological responses to food versus neutral and erotic versus neutral images. Results: The results showed that individuals with higher bulimic symptoms were characterized by potentiated eye
blink startle response during binge food (vs. neutral images) and more positive valence ratings during erotic (vs. neutral) cues. Conclusions: The results highlight the negative emotional reactivity of individuals with elevated bulimic symptoms
toward food cues, which could be related to the risk of progression to full bulimia nervosa and thereby addressed in
prevention efforts. Results also point to the potential role of reactivity to erotic content, at least on a subjective level. Theoretical models of eating disorders should widen their conceptual scope to consider reactivity to a broader spec‑
trum of primary reinforcers, which would have implications for cue exposure-based treatments. Plain English summary: We examined appetitive and aversive cue responses in college women to investigate how
bulimic symptoms relate to primary reinforcers such as food and erotic images. We recorded peripheral psychophysi‑
ological indices (the startle reflex, zygomaticus, and corrugator responses) and self-reported emotional responses
(valence, arousal, and dominance) in 75 college women that were presented with the Spanish version of the Bulimia
Test-Revised. The results showed that bulimic symptoms increase both psychophysiological defensiveness toward
food cues and subjective pleasure toward erotic cues. The findings suggest a generalized sensitivity to primary rein‑
forcers in the presence of bulimic symptoms, and emphasize the relevance of adopting a wider framework in research
and treatment on bulimia nervosa. Keywords: Bulimia nervosa, Binge food cues, Erotic cues, Psychophysiology, Valence, Startle reflex Emotional reactivity to binge food and erotic
cues in women with bulimia nervosa symptoms Isabel Hernández‑Rivero1, Jens Blechert2, Laura Miccoli1, Katharina Naomi Eichin2,
M. Carmen Fernández‑Santaella1 and Rafael Delgado‑Rodríguez3* Isabel Hernández‑Rivero1, Jens Blechert2, Laura Miccoli1, Katharina Naomi Eichin2,
M Carmen Fernández‑Santaella1 and Rafael Delgado‑Rodríguez3* Isabel Hernández‑Rivero1, Jens Blechert2, Laura Miccoli1, Katharina Nao
M. Carmen Fernández‑Santaella1 and Rafael Delgado‑Rodríguez3* M. Carmen Fernández‑Santaella1 and Rafael Delgado‑Rodríguez3* Hernández‑Rivero et al. J Eat Disord (2021) 9:120
https://doi.org/10.1186/s40337-021-00475-9 © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Eating and reproductive behaviors are among the most
essential goal-directed behaviors due to their high rele-
vance for human survival. Thus, food-related and erotic *Correspondence: rfdelgad@ujaen.es
3 Department of Psychology, University of Jaén, Campus Las Lagunillas,
23009 Jaén, España
Full list of author information is available at the end of the article However, the LPP is primarily sensitive to
emotional arousal; it increases with the motivational sali-
ence of the stimuli, regardless their positive or negative
valence [31], whereas other psychophysiological meas-
ures are sensitive to the appetitive or aversive nature of
the stimuli. cues are generally experienced as motivating and pleas-
ing, reliably engaging the appetitive motivational system
[1, 2]. Nevertheless, some psychiatric syndromes, such
as bulimia nervosa (BN), which especially affects women
[3], tend to deviate from this general pattern, at least for
food cues. Patients with this debilitating disorder are
characterized by binge eating, inappropriate compensa-
tory behaviors, undue influence of body weight and/or
shape on self-esteem [4]. These symptoms appear to be
related to altered food cue responses, although it recently
emerged that erotic stimuli also tend to be processed dif-
ferently in patients with BN, at least in terms of subjec-
tive emotional reactivity [5, 6].hi The scientific literature has documented the presence
of altered food cue reactivity in women with BN [7–10]. According to studies examining peripheral and subjec-
tive reactivity to food cues, high-calorie food pictures
are processed as moderately pleasant and activating by
healthy women [1, 2], however, the same pictures evoke
affective ambivalence in women with BN and individuals
with binge eating, since their urge to eat is accompanied
by anxiety and fear of weight gain [7]. This ambivalence
takes the form of a discrepancy between psychophysi-
ological responses—that show enhancement of the cor-
rugator supercilii muscle activity and the eye-blink startle
reflex, both indicating aversive responses—, and subjec-
tive responses—that tend to indicate appetitive food pro-
cessing [11, 12]. Previous studies that analyzed peripheral psychophysi-
ological responses to food stimuli in BN patients and
women with subclinical bulimic symptoms have used
standardized food cues, including high-calorie foods
[11–13]. However, not all foods have the same emotional
meaning for people with food-related problems [14, 15],
since binge-food preferences are highly idiosyncratic [16,
17]. Thus, the present study individually selected binge
food images for each participant (see also [6, 16]. © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hernández‑Rivero et al. J Eat Disord (2021) 9:120 Hernández‑Rivero et al. J Eat Disord (2021) 9:120 Page 2 of 11 Page 2 of 11 show greater sexual activity [23, 25] and interest in erotic
cues compared to healthy women [26], although, at the
same time, they experience greater sexual dissatisfaction
and lower sexual self-esteem [23, 27]. Sexual dysfunc-
tions are associated with different forms of compensa-
tory behaviors [28], which, according to the authors,
might be used by bulimic women to achieve their body
ideal and feel more comfortable during sexual intimacy. Despite the relationship between BN and intimacy and
sexuality issues, research on this topic has been limited to
clinical observations and self-reports [23, 29, 30]. To the
best of our knowledge, only one study specifically used
erotic pictures to explore BN subjective reactions [5] and
found that women with BN subjectively report less pleas-
ure and greater dyscontrol than healthy participants. Recently, another study provided objective evidence that
women with BN process food and erotic cues differently:
Delgado-Rodríguez and colleagues [6] investigated emo-
tional brain responses in BN patients and showed that
pictures of personal binge foods and erotic couples were
accompanied by a larger late positive potential (LPP) in
full-diagnosis BN patients, whereas patients did not dif-
fer from controls in their reactivity to unpleasant and
neutral cues. Explor-
ing subjective and psychophysiological reactivity to spe-
cific binge food cues has implications for clinical practice,
since binge foods are the cue target of exposure therapy
[18–20], through which BN patients can develop a more
adaptive emotional pattern and prevent binge-purge
cycles [21].i The present study follows up on Delgado-Rodríguez
and colleagues’ findings by examining the motivational
significance of food and erotic cues in a nonclinical sam-
ple of women with bulimic symptoms. To characterize
the valence dimension, we examined peripheral indica-
tors, such as corrugator and zygomatic muscle activ-
ity together with the eye-blink startle, that are sensitive
to the activity of both the aversive and the appetitive
motivational systems [1]. In addition, affective ratings
of valence, arousal, and dominance were also evalu-
ated using the Self-Assessment Manikin (SAM; [32]. We
examined a sample of college women with bulimic symp-
toms (a) to evaluate whether altered cue reactivity to
food and erotic images are visible also at early subclini-
cal stages of the disorder and (b) to explore the dimen-
sional characteristics of such cue reactivity. Disordered
eating patterns tend to emerge early, during adolescence
[33], with the result that they are very common among
university women [34, 35]. It is therefore relevant to
investigate eating disorders within nonclinical samples
of college women because they may still benefit from
broad preventive programs aimed at the early stages of
food-related disorders [34, 36]. From a more theoretical
perspective, confirming that subclinical BN symptoms Although, based on the fifth edition of the Diagnostic
and Statistical Manual of Mental Disorders (DSM-5; [4],
sexual dysfunction is not a core symptom of eating disor-
ders, a body of clinical and empirical literature has linked
BN with sexuality and intimacy dysfunctions [22, 23]. Some authors have suggested similarities between eating
and sexual behaviors in women with BN, stressing that
eating more food than the normal population, but feeling
more discomfort associated with that intake, is paralleled
by their sexual behavior [24]: women with BN tend to Hernández‑Rivero et al. J Eat Disord (2021) 9:120 Page 3 of 11 are also accompanied by altered reactivity to food and
body-related cues would further support the necessity of
such prevention programs and help refining their target. Previous findings of altered response to high-calorie food
cues in adolescents with risky dieting practices [37] and
in women with binge-purge eating patterns [11] suggest
as a whole that food processing disturbances are present
also at subclinical levels of the disorder. However, erotic
cue processing has not been previously investigated in
nonclinical women with bulimic symptoms. Thus, to
sum up, the present study aims to examine, in college
women, how bulimic symptoms relate to appetitive and
aversive cue responses across a wide spectrum of stimuli,
both related and unrelated to binge food and erotica. Our
ultimate goal is to understand BN cue reactivity within a
broader frame of reference. sources in order to avoid selecting more explicit sexual
content (e.g., opposite-sex erotica) which in women,
although subjectively perceived as pleasant and activat-
ing, also prompt psychophysiological reactions that indi-
cate the activation of the aversive system, i.e., increase in
corrugator muscle activity and startle reflex [2]. There-
fore, all selected erotic pictures depicted erotic couples:
romantic couples during physical intimacy, either par-
tially unclothed or naked (i.e., non-explicit erotic cues),
in order to avoid women’s mixed motivation toward more
sexually explicit pictures. Indeed, pictures of erotic cou-
ples prompt women’s greatest startle probe inhibition,
indicative of highly appetitive motivation [2]. g
y pp
Food pictures were individually selected for each par-
ticipant based on foods they “found personally irresistible
and that they could keep eating until binging”. It has been
shown that personally significant stimuli, such as faces
of loved ones or cues of personal phobias, are associated
with high levels of physiological reactivity [40, 41]. In our
previous study [6], the presentation of personal binge
food cues successfully prompted subjective feelings of
arousal that were higher than those observed using non-
personal high-calorie food cues. Thus, during a telephone
interview, participants reported their six personal binge
foods, describing them meticulously and giving at least
two descriptors of each food (e.g., lasagna with meat,
cheese, and bechamel). Foods had to be high in calories,
and could be sweet as well as salty. Based on each partici-
pant’s description, the experimenter searched the Inter-
net for four different images of each personal binge food
(n = 24), therefore, participants did not see them before
the experiment. 1 IAPS codes for unpleasant: 1525, 2683, 2688, 2800, 2981, 3110, 3530, 6200,
6313, 6315, 6350, 6415, 6510, 6540, 6550, 6555, 6570, 8485, 9075, 9163, 9410,
9630, 9635, 9810; IAPS codes for neutral: 5531, 7000, 7002, 7004, 7009, 7010,
7012, 7020, 7025, 7035, 7040, 7045, 7055, 7059, 7080, 7090, 7150, 7175, 7185,
7224, 7233, 7235, 7705, 7950); IAPS codes for erotic: 4604, 4607, 4608, 4623,
4643, 4651, 4652, 4653, 4658, 4659, 4668, 4669, 4670, 4672, 4676, 4680, 4687,
4695, 4697). 2 NAPS codes for erotic: Opposite-sex_couple_011_h, Opposite-sex_couple_
015_h, Opposite-sex_couple_016_v, Opposite-sex_couple_017_v, and Oppo-
site- sex_couple_020_h Participants A total of 82 healthy women from the University of Gra-
nada participated in this study between November 2018
and April 2019. We excluded women who reported preg-
nancy; past and present drug abuse; a psychotic disorder;
visual, auditory, cardiovascular, or neurological diseases;
or current or past treatment for eating or weight-related
disorders. Female university students have high rates of
eating disorders problems [34], therefore the sample was
considered to be adequate and representative for study-
ing the association between BN symptoms and emotional
reactivity to binge food and erotic cues. Some partici-
pants were excluded due to technical problems with the
startle probe (n = 2), corrugator electromyographic
(EMG) (n = 1), and body mass index (BMI) measure
(n = 2), for suffering from blood phobia (n = 1), or for
being in a fasting state at the experiment onset (n = 1). Thus, complete data were available for 75 participants. Picture selection followed the same
rationale as in previous studies [42, 43]: selected food
pictures had a high digital resolution and displayed food
on a naturalistic, nonuniform background that corre-
sponded with the perceptual properties of nonfood IAPS
pictures. Even though our selection procedure could lead
to select food images differing in perceptual features
from the IAPS images, it should not affect results since
previous emotional literature indicates that perceptual
features as color, contrast, spatial frequency, and com-
plexity does not influence peripheral reactivity [1, 44]. Stimulih The participants viewed a total of 96 pictures belonging
to 4 different categories: unpleasant (n = 24) and neutral
(n = 24) pictures were taken from the International Affec-
tive Picture System (IAPS)[38],1 and erotic pictures were
taken from both the IAPS (n = 19) and the Erotic sub-
set of the Nencki Affective Picture System (NAPS ERO;
[39]2 (n = 5). The pictures were taken from two different Self‑report measuresh The Bulimia Test-Revised (BULIT-R, 47) is a self-report
measure of BN symptoms that consists of 36 items that,
in the Spanish version, covers four main factors –body
concerns, binge, diuretics, and laxatives—[48]. This test
was designed to detect the risk of suffering from BN in
the general population. In Thelen et al.’s [47] study, scores
of ≥ 104 were associated with a BN diagnosis, though
they proposed a score of ≥ 85 as an alternative cut-off
to reduce the number of false negatives. In the present
study, internal consistency was high (α = .90), similar to
the results reported by Thelen et al. [47] (α = .92–.98;
1991). The Spanish version of the BULIT-R has reliable
psychometric properties in college students [48]. The second session was devoted to the psychophysi-
ological data recording during the affective picture
viewing and took place one week after the first session,
either in the morning (8:30 AM–12:30 PM) or in the
afternoon (3:00–5:00 PM). Upon arrival at the labora-
tory, a glucose test assessed whether participants had
adhered to the instructions of having a small snack. After sensor and headphone placement, their hunger
level was assessed. A 19″ flat screen, located 60 cm
from the participants, was used to present the pictures. Picture delivery was controlled by a computer running
Presentation (v.16.3, Neurobehavioral Systems, San
Francisco, CA). Participants were asked to view each
picture the entire time that it was on the screen. Four
pseudorandomized picture orders were used across
the participants. Each order included 96 picture trials,
avoiding more than two repetitions of the same picture
category. After a five-minute baseline, the 96 picture
trials were displayed, each comprising 4 s of baseline
and 6 s of picture viewing, followed by a variable inter-
trial interval (ITI) of 10–22 s. The startle probes con-
sisted of 50 ms white noise bursts (105 dB) presented
through headphones during 75% of the trials after 4,
4.5, 5, or 5.5 s from picture onset. To decrease their
predictability, the probes were also randomly delivered
during 1/8 of the ITIs. y
g
The Self-Assessment Manikin (SAM; [32]) is a nonver-
bal pictorial rating scale often used along with the IAPS
[38] to rate the valence (pleasant vs. unpleasant), arousal
(relaxed vs. activated), and dominance (feeling in control
vs. feeling controlled) elicited by images. Self‑report measuresh Nine inten-
sity levels are represented by 5 humanoid figures and
the spaces between these figures. The SAM has been
widely validated and is extensively used in cue reactivity
research [49, 50]. It has demonstrated a strong consist-
ency in the covariation between physiological responses
and self-reports of valence and arousal [32]. Apparatus and physiological measures Signal acquisition and stimulus presentation were car-
ried out with a PC running VPM software, v.12.6 [45]. Startle reflex and EMG activity of the zygomaticus major
and corrugator supercilii muscles were recorded through
a Coulbourn polygraph, LabLink V model (Coulbourn
Instruments, Lehigh Valley, PA), using three V75-04 Page 4 of 11 Page 4 of 11 Hernández‑Rivero et al. J Eat Disord (2021) 9:120 Hernández‑Rivero et al. J Eat Disord (2021) 9:120 In the first session, they signed an informed con-
sent form and completed the BULIT-R questionnaire
[48]. To obtain a broader understanding of the sam-
ple population, other ED characteristics were also
assessed using the Eating Disorder Examination Ques-
tionnaire (EDE‐Q) ([51]; Spanish version by [52]), the
Yale Food Addiction Scale (YFAS 2.0) ([53]; Spanish
version by [54]), the Snaith-Hamilton Pleasure Scale
(SHAPS) ([55]; Spanish version by [56]), and the Body
Shape Questionnaire (BSQ) [57, 58]. The participants’
BMI was estimated using a Leicester height measure
stadiometer recorded to the nearest millimeter and an
electronic body composition analyzer (Tanita Model
300MA, Chicago, IL). Participants were then invited
to a second session for the psychophysiological record-
ing and were made aware of the testing requirements
for this session: not having their period, not taking any
medication, not consuming drugs or alcoholic bever-
ages, and avoiding excessive exercise 24 h before the
session. To avoid food deprivation and to match the
hunger level between participants, they were instructed
to eat a piece of toasted bread with olive oil or butter
(between 400 and 415 kcal) without eating or drink-
ing anything but water, 2.5 h before the physiological
recording. bioamplifiers with miniature electrodes filled with con-
ductive gel. For startle responses, following Blumenthal
and colls.’ guidelines [46], startle probes were delivered
through headphones (model Telephonics TDH49P) by a
Coulbourn S81‐02 white noise generator. A Brüel & Kjaer
sonometer (model 2235) and an artificial ear (model
4153) were used to calibrate the noise intensity. The raw
EMG signals were bandpass-filtered (28–500 Hz) and
transformed into integrated EMG in μV with a V76-24
integrator. The time constant for the zygomaticus and
corrugator was 500 ms, with a sampling rate of 100 Hz,
whereas the time constant for the orbicularis was 20 ms
with a sampling rate of 1000 Hz. Skin conductance and
electroencephalography were also recorded; however,
these measures were not included in the present paper. Data reduction and statistical analysisil To define the startle reflex amplitude, we used the differ-
ence in microvolts between the peak and the onset of the
response in a time window between 20 and 120 ms after
stimulus onset, using the algorithm described by Balaban
and colls [59]. Corrugator and zygomatic EMGs were
scored as the mean activity during the first 4 s of picture
presentation, before startle probe delivery. To remove
between-subject variability, startle amplitudes and cor-
rugator and zygomatic EMG responses to each picture
category were t-score standardized (mean = 50; SD = 10)
using the individual mean across all picture categories. We performed a hierarchical linear regression to exam-
ine whether psychophysiological and subjective reac-
tions to food and erotic pictures predicted the presence
of bulimic symptoms. Following previous literature [60–
63], for startle reflex, corrugator and zygomatic muscle
reactivity, and for valence, arousal, and dominance rat-
ings, the difference in reactivity to food minus neutral
cues, and to erotic minus neutral cues were introduced
as predictors. Thus, we assessed whether the baseline-
corrected amplitude of each woman’s reaction to food
and erotic cues predicted the amount of bulimic symp-
toms she reported. Predictor scores greater or less than 3
SD were winsorized to ensure that distributions were less
sensitive to outliers while maintaining statistical power. A small number of self-reported and psychophysiological
responses were winsorized (corrugator: 4; zygomatic: 1;
valence: 1). in Table 1. To get an overview of the degree of clinical
eating disorder symptomatology, we examined the par-
ticipants according to BULIT-R and EDE-Q established
cutoffs. In the sample, 7.7% (n = 6) passed the BULIT-R
cut-off for BN diagnosis (i.e., 85; [47]), and 21% (n = 16)
had an EDE-Q Global score above 2.8, which falls into
the 85th percentile of Spanish normative EDE-Q data
[52]. Breaking down behavioral symptoms of BN, 19%
of women (n = 15) endorsed at least one objective binge
eating episode in the past month and reported a mean
of 2.4 binge eating episodes during the last month; 40%
(n = 31) engaged in at least one compensatory behavior
to control weight and informed of 4.8 inappropriate eat-
ing behaviors in the last month. Approximately one third
of the sample (31%, n = 24) reported high body dissatis-
faction (scores above 105; [58]), the most potent predic-
tor for eating disorders [65]. Procedure
Th The participants were contacted at their lecture class-
room where the experiment was introduced. Women
who decided to participate were later interviewed by
phone. In this interview, the experimenter checked that
participants did not meet the exclusion criteria and gath-
ered information about the six personal binge foods that
they could “eat and eat until binging” (see Stimuli). Eli-
gible participants were invited to two experimental ses-
sions at the laboratory. After the picture viewing, once the sensors and head-
phones were removed, the women reported their hunger Hernández‑Rivero et al. J Eat Disord (2021) 9:120 Page 5 of 11 Table 1 Participants’ characteristics level and rated all pictures on the SAM scales of valence,
arousal, and dominance [32]. After an explanation about
the purpose of the study, the participants were thanked
and compensated for their time with course credits. Table 1 Participants’ characteristics Values are presented as the means and standard deviations
BULIT-R = Bulimia Test Revised; SHAPS = Snaith-Hamilton Pleasure Scale; EDE-Q:
Eating Disorder Questionnaire; BSQ = Body Shape Questionnaire; YFAS = Yale
Food Addiction Scale
1 Questions on hunger level were measured on a 1–9 Likert scale
Demographics
Age
20.06(2.78)
Body mass index (kg/m2)
22.54(3.54)
Blood glucose (mg/dL)
81.04(27.90)
Questionnaires
BULIT-R
56.03(16.083)
SHAPS
20.79(4.54)
EDE-Q
1.51(1.22)
Bingeing
0.46(1.10)
Compensatory behaviours
1.90(4.76)
BSQ
82.31(33.04)
YFAS
1.35(2.42)
Hunger:
Level of hunger pre psychophysiological registration1
4.30(1.77)
Level of hunger post psychophysiological registration1
6.26(1.88)
Are you hungry? (yes/no)
35/43
How strong is your desire to eat?1
4.13(2.09)
How full do you feel?1
3.94(1.91)
How much food do you think you could eat?1
5.18(1.77)
How long since your last meal? (in min)
214(24.8) Data reduction and statistical analysisil Regarding food addiction, a
food problem that seems to overlap with bulimic symp-
toms even in individuals with no clinical diagnoses of
disordered eating [66, 67], 10.3% of women (n = 8) could
be classified as food addicted. Notwithstanding the mean
BMI of the whole sample (20.06) indicating normal-
weight, five women were underweight, 16 overweight,
and 2 obese. In order to select the predictors to be included into the
multiple linear regression analysis, we ran, in a first step,
Pearson’s bivariate correlations between each of the psy-
chophysiological and self-reported measures (food vs. neutral and erotic vs. neutral) on the one hand and the
BULIT-R score on the other. Only measures with signifi-
cant correlations with the BULIT-R score were included
into the following two-step hierarchical linear regres-
sion analysis predicting BULIT-R scores. Based on its
relevance in food-related disorders [64], BMI was also
entered into the regression analysis (at the first step),
along with psychophysiological and self-report predic-
tors (at the second step). Significance was set at 0.05. Results Descriptive statistics
Descriptive statistics for the BULIT-R, EDE-Q Global
and subscale scores, BSQ, YFAS, and BMI are presented Descriptive statistics Descriptive statistics for the BULIT-R, EDE-Q Global
and subscale scores, BSQ, YFAS, and BMI are presented Hernández‑Rivero et al. J Eat Disord (2021) 9:120 Page 6 of 11 Table 2 Pearson’s bivariate correlations between psychophysiological and self-reported measures and BULIT-R total score
BULIT-R, Bulimia Test Revised; BMI, Body Mass Index. Hunger refers to level of hunger pre psychophysiological registration
* p < .05
** p < .01
Hunger
BMI
Binge food-neutral
Erotic-neutral
Valence
Arousal
Zygomatic
Corrugator
Startle
Valence
Arousal
Zygomatic
Corrugator
Startle
BULIT-R
− .14
.30*
.11
− .02
.20
.05
.31**
.25*
.05
.12
.11
.16
Fig. 1 Graphical representation of the Pearson’s bivariate correlations. a Represents Pearson’s bivariate correlations between BULIT-R scores and
Startle binge food versus neutral and b represents Pearson’s bivariate correlations between BULIT-R scores and Valence erotic versus neutral Table 2 Pearson’s bivariate correlations between psychophysiological and self-reported measures and BULIT-R total score
BULIT-R, Bulimia Test Revised; BMI, Body Mass Index. Hunger refers to level of hunger pre psychophysiological registration
* p < .05
** p < .01
Hunger
BMI
Binge food-neutral
Erotic-neutral
Valence
Arousal
Zygomatic
Corrugator
Startle
Valence
Arousal
Zygomatic
Corrugator
Startle
BULIT-R
− .14
.30*
.11
− .02
.20
.05
.31**
.25*
.05
.12
.11
.16 Fig. 1 Graphical representation of the Pearson’s bivariate correlations. a Represents Pearson’s bivariate correlations be
Startle binge food versus neutral and b represents Pearson’s bivariate correlations between BULIT-R scores and Valence Fig. 1 Graphical representation of the Pearson’s bivariate correlations. a Represents Pearson’s bivariate correlations between BULIT-R scores and
Startle binge food versus neutral and b represents Pearson’s bivariate correlations between BULIT-R scores and Valence erotic versus neutral Table 3 Hierarchical linear regression predicting BULIT-R scores
BULIT-R, Bulimia Test Revised; BMI, body mass index; Adj. R2, adjusted R2. ΔR2 for
model 2 = .19
* p < .05
** p < .01
*** p < .001
Predictors
Standardized B
Adj. R2
Model 1
.07*
BMI
.29*
Model 2
.239***
BMI
.30**
Startle binge food-neutral
.35**
Valence erotic-neutral
.29** the difference between food and neutral cues; and, for
valence ratings, the difference between erotic and neutral
cues. The second model resulting from the hierarchical
regression analyses predicted 23.9% of the variance in
BULIT-R scores (Table 3). Descriptive statistics In line with linear correla-
tions, BULIT-R scores were positively associated with
BMI, Startle food-neutral, and Valence erotic-neutral:
Greater startle reflex amplitudes when viewing food
compared to neutral images—indicative of defen-
sive processing during food cues—were associated
with more bulimic symptoms. Moreover, more posi-
tive valence responses when viewing erotic vs. neutral
images–indicative of appetitive processing during
erotic cues—were also associated with more bulimic
symptoms. Finally, consistent with literature [64], BMI
was a significant predictor of BULIT-R scores, so that
having a higher BMI was related to more bulimic symp-
toms. Although based on Pearson’ bivariate correla-
tions hunger was not related to BULIT-R scores, we
run an additional hierarchical regression that included
all predictors from Table 3 and hunger level (reported
right before the psychophysiological task) to control
for the possible confounding impact of hunger on the
results. The data did not change. Discussionh The current study examined the peripheral psychophysi-
ological and subjective emotional cue reactivity to two
classes of primary reinforcers with relevance to eating
disorders—i.e., binge food and erotic cues—, together
with neutral and unpleasant stimuli, in a nonclinical
sample of college women varying on bulimic symptoms. In line with literature, BMI significantly correlated with
bulimic symptoms. Moreover, the findings also revealed
that bulimic symptoms were associated with a defensive
emotional reactivity to binge food cues—potentiation of
the startle reflex—and an exaggerated hedonic processing
of erotic cues—high valence responses. Taken together,
the data point out that the presence of bulimic symptoms
at subclinical stage of BN are accompanied by both aver-
sive psychophysiological reactivity to food cues and high
subjective pleasure towards erotic cues, overall suggest-
ing the necessity to approach a wider framework for BN
research and treatment. Food and erotic cues reactivity as predictors of Bulimic
Symptoms Table 2 displays the results of Pearson’s bivariate cor-
relations between all psychophysiological and self-
reported measures and the BULIT-R scores; only the
three measures that significantly correlated with bulimic
symptoms were included into the multiple linear regres-
sion model (see Fig. 1): BMI; for startle blink amplitude, Page 7 of 11 Hernández‑Rivero et al. J Eat Disord (2021) 9:120 Hernández‑Rivero et al. J Eat Disord (2021) 9:120 A post hoc power analysis was conducted using the
software G*Power 3.1 [68]. A sample size of 75 was
used for the statistical power analyses with three pre-
dictor variables, an alpha value of 0.05, and an observed
R2 of 0.27 (f2 = 0.37) for the whole regression model. The post hoc analyses showed an observed statistical
power of 0.99 for this multiple regression study. enhanced startle potentiation; however, subjective data
do not indicate affective ambivalence, as valence does not
suggest exaggerated appetitive processing of binge food
cues. Discrepant findings may be due to differences in sam-
ple characteristics: while BN-like participants from
Drobes et al.’s study [11] were selected according to
scores on binge-purge and dietary restraint items (“sub-
jects who scored in the upper 10% on binge-purge items
and in the bottom two-thirds on dietary restraint items”,
p. 167), for participants’ selection we used the BULIT-
R scores that not only include binge and inappropriate
compensatory behaviors, but also body-concerns items
[48]. In this regard, Racine et al. [60], likewise examin-
ing the peripheral psychophysiology during food cues in
a nonclinical sample, observed that binge eating episodes
and eating disorder cognitions (especially weight and
shape concerns) were associated with different emotional
patterns: while binge eating was associated with moti-
vational ambivalence, eating disorder cognitions were
associated with aversive psychophysiological reactivity to
food cues. We hypothesize therefore that in the present
research the low number of binge eating episodes (87%
of women who binge reported less than 4 episodes dur-
ing the last month), together with the fact that BULIT-
R scores reflect also body-concerns, might have biased
the results toward an aversive processing of food cues. Accordingly, we expect that a greater presence of binge
eating symptoms might have led to an ambivalent emo-
tional response to food cues. Elevated subjective reactivity to erotic pictures
as a predictor of bulimic symptoms f
Another relevant aspect related to erotic cues is that
naked or semi-naked female bodies appear in these
images. Body cues (i.e., pictures depicting other’s female
body or body parts) are differently processed by women
with eating-related problems, specifically, women with
BN subjectively assess ‘idealized, thin’ female bodies
as highly anxious [13]. Although women appearing in
erotic cues (as used in the current study) mostly have
idealized bodies, female bodies are not determining the
association between bulimic scores and valence ratings
(reported in Table 3). Erotic cues reflect a sexual/intimate
environment depicting a woman sexually interacting
with a naked or semi-naked man; conversely, body cues
only depict female body or body parts (typically without
head or face; e.g., [13, 76]). Moreover, female bodies or
body parts depicted in body cues are clearly presented to
facilitate the participants’ body comparison (e.g., wearing
bikinis or fitted sports clothing to highlight body shape)
(e.g., [76–78], however, erotic cues not always fulfill this
assumption (e.g., some women are dressed with clothes
that do not highlight their shape). Despite the above-
mentioned differences between erotic and body cues, we
performed extra analyses to examine the possible impact
of female bodies on the current valence result (i.e., the
association between valence reactions to erotic cues and
bulimic symptoms). Additional analyses (see Additional
file 1) showed that valence ratings did not depend on
how female bodies appeared in the pictures, but only on
BULIT-R scores. Although the influence of female bod-
ies does not seem to be critical for the current results,
we consider that future studies might benefit from con-
trolling if women with BN symptoms are influenced
by female bodies while viewing erotic cues. Recording
objective measures to identify the time and gaze pat-
tern (e.g., using an eye tracker) while viewing erotic cues
might allow to have a deeper understanding of erotic cue
processing in BN.h Sexual dysfunction is not a diagnostic criterion for BN;
however, previous literature links BN symptoms with
sexuality and intimacy difficulties, and points to these
problems as influencing factors in the development and
maintenance of the disorder [23, 72, 73]. Clinical obser-
vations and self-report studies have hypothesized that,
in women with BN, sexual behaviors may resemble their
food cue reactivity in terms of a marked ambivalence. Aversive reactivity towards binge food pictures
as a predictor of bulimic symptoms Another possible explanation is that in women with
more bulimic symptoms personalized binge food pic-
tures might have potentiated the threat to control over
food cues, again biasing the results toward aversive pro-
cessing. Participants in the current study were presented
with the “specific foods that they found irresistible and
that they could keep eating until binging”, thus, in women
with bulimic traits, these foods are capable of prompt-
ing the irresistible cravings associated with binge onset
[71]. That is to say, in some individuals psychophysiologi-
cally aversive reactions might be generated by idiosyn-
cratic binge food cues: aversion and defensiveness might
emerge as a result of the need to avoid cues that are per-
ceived as a threat. y
Previous research examining standardized food stimulus
processing in women with subthreshold [11] and full syn-
drome BN [12] showed aversive reactions to standard-
ized food cues: a significant potentiation of startle blink
magnitudes. Moreover, in contrast to the startle reflex
findings, both Drobes and Mauler found that in women
with disordered eating self-reported valence was consist-
ent with an appetitive reaction to nonpersonal food cues. This divergent pattern between psychophysiological and
subjective responses led to the hypothesis that in this
population food cues might prompt a state of motiva-
tional ambivalence [11, 12, 69]. Interestingly, the opposite
pattern of motivational conflict has also been observed
[13]: BN females had attenuated (i.e., appetitive) startle
responsiveness along with aversive subjective responses
to food cues. However, in Friederich and colls.’ [13] study
there was no control of food deprivation, a key variable
that modulates emotional responses to food cues [11, 70]. Our findings on nonclinical women with BN symptoms
extend past research [11]: based on psychophysiological
data, the greater presence of bulimic symptoms is asso-
ciated with negative processing of binge foods, visible in During the food pictures selection procedure (in which
participants were asked to describe their six high-calorie
irresistible foods), we did not instruct participants to bal-
ance individual foods on regard to food content (sweet
vs. salty), thus, the number of both food contents dif-
fer between participants. Extra analyses (see Additional
file 1) indicated that food contents did not influence
emotional reactivity. We highlight the relevance of this
procedure because it allows to select the most irresistible Hernández‑Rivero et al. Aversive reactivity towards binge food pictures
as a predictor of bulimic symptoms J Eat Disord (2021) 9:120 Page 8 of 11 Page 8 of 11 food (i.e., asking participants to select sweet and fat foods
[e.g., 3 sweet and 3 fat foods] might result in not selecting
the most irresistible foods). Then, this procedure allows
to explore the emotional reactivity toward emotionally
charged foods, which has implications for clinical prac-
tice since binge foods are the cue targets of exposure
treatments [18–20] that aim to generate more adaptive
emotional patterns to prevent binge-purge cycles [21]. explicit sexual contents (i.e., opposite-sex erotica) tend
to prompt more aversive reactions, visible in women’s
increased corrugator and startle reflex [2]. Future stud-
ies might further investigate how different erotic con-
tents are processed in subclinical and full-diagnosis BN
in order to elucidate whether explicit and non-explicit
erotic contents provoke different emotional reactions or,
rather, whether the worsening of the disorder disrupts
the affective processing of erotic cues. Elevated subjective reactivity to erotic pictures
as a predictor of bulimic symptoms Individuals suffering from BN, compared to healthy
women, tend to have more sexual activity and sexual
partners, greater sexual desire and fantasies [23, 73, 74],
and more impulsive sexual profiles [22, 75]; however,
they also experience increased performance pressure
[25, 27], less sense of control [5], and high levels of over-
all sexual dissatisfaction [23, 73]. Being more sexually
active but feeling more dissatisfied might be comparable
to their eating behavior; women with BN consume more
food during binges than healthy women, but feel greater
discomfort associated with their food intake [23]. Findings from the current study did not show nega-
tive feelings during sexual pictures: bulimic symp-
tomatology was associated with heightened subjective
pleasure responses toward erotic couple pictures. Con-
versely, previous research on self-reported emotions to
more heterogeneous erotic contents (i.e., erotic couples
and opposite-sex erotica) found that women with full-
diagnosis BN felt less pleasure and greater dyscontrol
than healthy women, which, according to the authors,
supported BN coactivation of aversive and appetitive
mechanisms when processing erotic pictures [5]. Two
hypotheses might be suggested to explain such discrep-
ancy. The first one proposes that although erotic cues
might prompt increased pleasure at an early, subclinical
level, the worsening of such symptoms into full BN may
lead to affective ambivalence, similar to the mixed moti-
vation observed with food stimuli [12, 13]. Alternatively,
divergence in valence ratings might be due to stimuli
selection: in healthy women, erotic couples (i.e., nonex-
plicit erotic content) tend to provoke appetitive subjec-
tive and psychophysiological processing, whereas more The implications of the current results should be
evaluated while considering some methodological
limitations. The primary limitations are related to our
sample selection. Recruiting an unselected sample of
undergraduate women resulted in only six participants Hernández‑Rivero et al. J Eat Disord (2021) 9:120 Page 9 of 11 a wider framework in research and treatment on BN,
encompassing a broader spectrum of primary reinforcers. exceeding BULIT-R cut-off for BN; increasing the num-
ber of women exceeding this cut-off might uncover dif-
ferences in psychophysiological reactivity to erotic cues
between healthy and subclinical BN women. Moreover,
the use of a diagnostic interview (e.g., EDE; [79]) should
be used instead of questionnaires to select a subclinical
or clinical sample of BN. Abbreviations APA: American Psychiatric Association; BMI: Body mass index; BN: Bulimia
nervosa; BSQ: Body shape questionnaire; BULIT-R: Bulimia test-revised; EDE‐Q:
Eating disorder examination questionnaire; EMG: Electromyographic; IAPS:
International affective picture system; LPP: Late positive potential; NAPS:
Nencki affective picture system; SAM: Self-assessment Manikin; SHAPS: Snaith-
Hamilton pleasure scale; YFAS: Yale food addiction scale. Authors’ contributions IHR: Investigation, Formal analysis, Writing- Original draft, Writing—Review &
Editing. JB: Formal analysis, Writing—Review and Editing. LM: Conceptualiza‑
tion, Methodology, Writing—Review and Editing, Visualization. KNE: Formal
analysis, Writing—Review and Editing. MCFS: Project administration, Writing—
Review and Editing. RDR: Conceptualization, Methodology, Software, Formal
analysis, Data curation, Writing- Original Draft, Supervision, Writing—Review
and Editing. All authors read and approved the final manuscript. Acknowledgements The authors would like to thank all participants of the current study for their
vital contribution to this study. Elevated subjective reactivity to erotic pictures
as a predictor of bulimic symptoms Another limitation, which is
common in eating disorder studies, is that the sample is
limited to women, which might raise concerns that these
findings can be generalized to men with BN. Lastly, it
should be considered that most of the previous research
used to discuss results from food and erotic cue reactivity
referred to women with a full diagnosis of BN, while our
results are based on a nonclinical sample of women with
bulimic symptoms.hi Supplementary Information The online version contains supplementary material available at https://doi.
org/10.1186/s40337-021-00475-9. Additional file 1. Extra analyses to examine the influence of food
contents on emotional reactivity and the influence of female bodies on
valence results. The current study is the first that examines peripheral
psychophysiological responses in a wider field of rele-
vant stimuli for women with bulimic symptoms. This is
remarkable because the use of cues other than food helps
to understand the complexity of binge-purge symptoma-
tology. In this sense, although scarce, previous research
that used non-eating disorder-related stimuli (e.g., facial
expression images or tones in a tone discrimination
task) has found that individuals with BN may exhibit
impaired cognitive processing across different stimulus
classes [9, 80–82]. Another strength is that food cues are
highly individualized; using personalized—emotionally
charged—binge foods to measure emotional responses
to cues directly related to altered behavioral responses in
BN. Funding This work was supported by the MECD [grant numbers FPU16/01200], ‘Doc‑
toral College "Imaging the Mind" (FWF; W1233-B)’, a grant from Junta de Anda‑
lucía, Spain [Grant Code P12.SEJ.391], and a grant from the Spanish Ministry of
Economy and Competitiveness/MINECO (Grant Code: PSI2013- 43777-P). Consent for publication
Not applicable. Not applicable. Availability of data and materials The current findings highlight that in a nonclinical sam-
ple of women, bulimic symptoms are accompanied by
altered peripheral psychophysiology—increased startle
reflex—during idiosyncratic binge food cues, indicat-
ing the early appearance of defensive reactions to food
cues in correspondence with bulimic symptomatology. Moreover, rating data point out that the presence of BN
symptoms is accompanied by the early appearance of
alterations in the processing—increased subjective pleas-
ure—during cues depicting sexual intimacy. Therefore,
early BN symptoms increase both psychophysiological
defensiveness toward food cues and subjective pleasure
during erotic cues. Interestingly, as mentioned above, it
has been observed that full-diagnosis BN is accompanied
by larger brain potentials (LPP, indicative of motivational
relevance) during both binge food and erotic cues [6],
overall suggesting the late emergence of BN psychophysi-
ological sensitivity to both food and erotic cues. Taken
as a whole, the data emphasize the relevance of adopting The datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request. Ethics approval pp
The Ethics Committee of the University of Granada (Spain) approved this
study (IRB# 699). This research was conducted according to the Declaration of
Helsinki. All participants provided written informed consent and were offered
course credits for their participation. Competing interests The authors declare that they have no competing interests. References 1. Bradley MM, Codispoti M, Cuthbert BN, Lang PJ. Emotion and motiva‑
tion I: defensive and appetitive reactions in picture processing. Emo‑
tion. 2001;1(3):300–19. 24. Moreno Dominguez S, Ortega-Roldán B, Rodríguez-Ruiz S. Impulsivi‑
dad en mujeres con bulimia nerviosa. eduPsykhé. 2009;8(1):63–77. 2. Bradley MM, Codispoti M, Sabatinelli D, Lang PJ. Emotion and motiva‑
tion II: sex differences in picture processing. Emotion. 2001;1(3):300–19 25. Katzman MA, Wolchik SA. Bulimia and binge eating in college women:
a comparison of personality and behavioral characteristics. J Consult
Clin Psychol. 1984;52(3):423–8. 3. Keski-Rahkonen A, Mustelin L. Epidemiology of eating disorders in
Europe: prevalence, incidence, comorbidity, course, consequences,
and risk factors. Curr Opin Psychiatr. 2016;29(6):340–5. y
26. Casper RC. Bulimia: its incidence and clinical importance in patients
with anorexia nervosa. Arch Gen Psychiatry. 1980;37(9):1030. 4. American Psychiatric Association. Diagnostic and statistical manual of
mental disorders. 5th ed. Washington: American Psychiatric Associa‑
tion; 2013. 27. Raciti M, Hendrick SS. Relationships between eating disorder charac‑
teristics and love and sex attitudes. Sex Roles. 1992;27(9–10):553–64. 28. Tobin DL, Griffing AS. Coping, sexual abuse, and compensatory beha
ior. Int J Eat Disord. 1996;20(2):143–8. 5. Rodríguez S, Mata JL, Lameiras M, Fernández MC, Vila J. Dyscontrol
evoked by erotic and food images in women with bulimia nervosa. Eur
Eat Disord Rev. 2007;15(3):231–9. 29. Castellini G, Lelli L, Lo Sauro C, Fioravanti G, Vignozzi L, Maggi M, et al. Anorectic and bulimic patients suffer from relevant sexual dysfunc‑
tions. J Sex Med. 2012;9(10):2590–9. 6. Delgado-Rodríguez R, Hernández-Rivero I, Fernández-Santaella MC,
Vila J, Guerra P, Miccoli L. Neural processing of food and erotic cues in
bulimia nervosa. Psychosom Med. 2019;81(6):527–35. 30. Pinheiro AP, Raney TJ, Thornton LM, Fichter MM, Berrettini WH, Goldman
D, et al. Sexual functioning in women with eating disorders. Int J Eat
Disord. 2010;123–9. 7. Giel KE, Teufel M, Friederich H, Hautzinger M, Enck P. Processing of pic‑
torial food stimuli in patients with eating disorder-A systematic review. Int J Eat Disord. 2011;44:105–17. 31. Schupp HT, Schmälzle R, Flaisch T, Weike AI, Hamm AO. Affective picture
processing as a function of preceding picture valence: an ERP analysis. Biol Psychol. 2012;91(1):81–7. 8. Chami R, Cardi V, Lautarescu A, Mallorquí-Bagué N, McLoughlin G. Neural responses to food stimuli among individuals with eating and
weight disorders: a systematic review of event-related potentials. Int
Rev Psychiatry. 2019;31(4):318–31. 32. Bradley MM, Lang PJ. Measuring emotion: the self-assessment
manikin and the semantic differential. Author details 1 Mind, Brain, and Behavior Research Center (CIMCYC), University of Granada,
Campus de Cartuja s/n, 18071 Granada, España. 2 Department of Psychol‑
ogy, Centre for Cognitive Neuroscience, Paris-Lodron-University of Salzburg,
Hellbrunnerstraße 34, 5020 Salzburg, Austria. 3 Department of Psychology,
University of Jaén, Campus Las Lagunillas, 23009 Jaén, España. Received: 11 July 2021 Accepted: 10 September 2021 Page 10 of 11 Page 10 of 11 Hernández‑Rivero et al. J Eat Disord (2021) 9:120 Hernández‑Rivero et al. J Eat Disord (2021) 9:120 23. Wiederman MW, Pryor T, Morgan CD. The sexual experience of women
diagnosed with anorexia nervosa or bulimia nervosa. Int J Eat Disord. 1996;19(2):109–18. References J Behav Ther Exp Psychiatry. 1994;25(1):49–59. 9. Hiluy JC, David IA, Daquer AF, Duchesne M, Volchan E, Appolinario
JC. A systematic review of electrophysiological findings in binge-
purge eating disorders: a window into brain dynamics. Front Psychol. 2021;12:619780. 33. Rohde P, Auslander BA, Shaw H, Raineri KM, Gau JM, Stice E. Dissonance-
based prevention of eating disorder risk factors in middle school girls:
results from two pilot trials: middle school prevention pilots. Int J Eat
Disord. 2014;47(5):483–94. 10. Wolz I, Fagundo AB, Treasure J, Fernández-Aranda F. The processing of
food stimuli in abnormal eating: a systematic review of electrophysiol‑
ogy. Eur Eat Disord Rev. 2015;23(4):251–61. 34. Eisenberg D, Nicklett EJ, Roeder K, Kirz NE. Eating disorder symptoms
among college students: prevalence, persistence, correlates, and
treatment-seeking. J Am Col Health. 2011;59(8):700–7. 11. Drobes DJ, Miller EJ, Hillman CH, Bradley MM, Cuthbert BN, Lang PJ. Food deprivation and emotional reactions to food cues: implications
for eating disorders. Biol Psychol. 2001;57(1–3):153–77. 35. Tavolacci MP, Grigioni S, Richard L, Meyrignac G, Déchelotte P, Ladner J. Eating disorders and associated health risks among university students. J
Nutr Educ Behav. 2015;47(5):412-420.e1. 12. Mauler BI, Hamm AO, Weike AI, Tuschen-Caffier B. Affect regulation
and food intake in bulimia nervosa: emotional responding to food
cues after deprivation and subsequent eating. J Abnorm Psychol. 2006;115(3):567–79. 36. Taylor CB, Bryson S, Luce KH, Cunning D, Doyle AC, Abascal LB, et al. Prevention of eating disorders in at-risk college-age women. Arch Gen
Psychiatry. 2006;63(8):881. 37. Miccoli L, Martínez-Fiestas M, Delgado-Rodríguez R, Díaz-Ferrer S,
Rodríguez-Ruiz S, Fernández-Santaella MC. Adolescent emotions toward
sweet food cues as a function of obesity and risky dieting practices. Food
Qual Prefer. 2018;68:205–14. 13. Friederich HC, Kumari V, Uher R, Riga M, Schmidt U, Campbell IC, et al. Differential motivational responses to food and pleasurable cues in
anorexia and bulimia nervosa: a startle reflex paradigm. Psychol Med. 2006;36(9):1327–35. 38. Lang P, Bradley M, Cuthbert B. International affective picture system
(IAPS): Affective ratings of pictures and instruction manual. Technical
report A-8. University of Florida, Gainesville; 2008. 14. Schulte EM, Avena NM, Gearhardt AN. Which foods may be addictive? The roles of processing, fat content, and glycemic load. PLoS ONE. 2015;10(2):e0117959. 39. Wierzba M, Riegel M, Pucz A, Leśniewska Z, Dragan WŁ, Gola M, Jednoróg
K, Marchewka A. Erotic subset for the Nencki affective picture system
(NAPS ERO): cross-sexual comparison study. Front Psychol. 2015;6:1336. https://doi.org/10.3389/fpsyg.2015.01336. 15. Gearhardt AN. References J Eat Disord (2021) 9:120 46. Blumenthal TD, Cuthbert BN, Filion DL, Hackley S, Lipp OV, Van Boxtel A. Committee report: guidelines for human startle eyeblink electromyo‑
graphic studies. Psychophysiology. 2005;42(1):1–15. 65. Stice E, Marti CN, Durant S. Risk factors for onset of eating disorders: evi‑
dence of multiple risk pathways from an 8-year prospective study. Behav
Res Ther. 2011;49(10):622–7. 47. Thelen MH, Farmer J, Wonderlich S, Smith M. A revision of the bulimia
test: the BULIT-R. Psychol Assess. 1991;3(1):119–24. 66. Meule A, von Rezori V, Blechert J. Food addiction and Bulimia Nervosa. Eur Eat Disord Rev. 2014;22(5):331–7. 48. Berrios-Hernandez MN, Rodríguez-Ruiz S, Perez M, Gleaves DH, Maysonet
M, Cepeda-Benito A. Cross-cultural assessment of eating disorders:
psychometric properties of a Spanish version of the Bulimia test-revised. Eur Eat Disord Rev. 2007;15(6):418–24. 67. Pursey K, Stanwell P, Gearhardt A, Collins C, Burrows T. The Prevalence of
food addiction as assessed by the yale food addiction scale: a systematic
review. Nutrients. 2014;6(10):4552–90. 68. Faul F, Erdfelder E, Buchner A, Lang AG. Statistical power analyses using
G* Power 3.1: tests for correlation and regression analyses. Behav Res
Methods. 2009;41(4):1149–60. 49. Bradley MM, Lang PJ. Emotion and motivation. In: Cacioppo JT, Tassinary
LG, Berntson G, editors. Handbook of psychophysiology. 3th. ed. New
York: Cambridge University Press; 2007, p. 581–607. https://doi.org/10.
1017/CBO9780511546396.025 69. Suissa-Rocheleau L, Benning SD, Racine SE. Associations between self-
report and physiological measures of emotional reactions to food among
women with disordered eating. Int J Psychophysiol. 2019;144:40–6. 50. Coffey SF, Saladin ME, Drobes DJ, Brady KT, Dansky BS, Kilpatrick DG. Trauma and substance cue reactivity in individuals with comorbid
posttraumatic stress disorder and cocaine or alcohol dependence. Drug
Dlcohol Depend. 2002;65(2):115–27. 70. Stockburger J, Schmälzle R, Flaisch T, Bublatzky F, Schupp HT. The impact
of hunger on food cue processing: an event-related brain potential study. Neuroimage. 2009;47(4):1819–29. https://doi.org/10.1016/j.neuroimage.
2009.04.071. 51. Fairburn CG, Beglin SJ. Assessment of eating disorders: interview or self-
report questionnaire? Int J Eat Disord. 1994;16(4):363–70. 71. Ster Wallin G, Norring C, Holmgren S. Binge eating versus nonpurged
eating in bulimics: is there a carbohydrate craving after all? Acta Psychiatr
Scand. 1994;89(6):376–81. 52. Villarroel AM, Penelo E, Portell M, Raich RM. Screening for eating disorders
in undergraduate women: norms and validity of the spanish version of
the eating disorder examination questionnaire (EDE-Q). J Psychopathol
Behav Assess. 2011;33(1):121–8. 72. Castellini G, Lelli L, Ricca V, Maggi M. References Sexuality in eating disorders patients:
etiological factors, sexual dysfunction and identity issues. A systematic
review. Horm Mol Biol Clin Investig. 2016;25(2):71–90. 53. Gearhardt AN, Corbin WR, Brownell KD. Preliminary validation of the yale
food addiction scale. Appetite. 2009;52(2):430–6. 73. Don Morgan C, Wederman MW, Pryor TL. Sexual functioning and atti‑
tudes of eating-disordered women: a follow-up study. J Sex Marital Ther. 1995;21(2):67–77. 54. Granero R, Jiménez-Murcia S, Gearhardt AN, Agüera Z, Aymamí N,
Gómez-Peña M, et al. Validation of the Spanish version of the yale food
addiction scale 2.0 (YFAS 2.0) and clinical correlates in a sample of eating
disorder, gambling disorder, and healthy control participants. Front
Psychiatry. 2018;9:208. 74. Rothschild BS, Fagan PJ, Woodall C, Andersen AE. Sexual functioning of
female eating-disordered patients. Int J Eat Disord. 1991;10(4):389–94.i 75. Westen D, Harnden-Fischer J. Personality profiles in eating dis‑
orders: Rethinking the distinction between axis I and axis II. AJP. 2001;158(4):547–62. 55. Snaith RP, Hamilton M, Morley S, Humayan A, Hargreaves D, Trigwell P. A
Scale for the assessment of hedonic tone the snaith–hamilton pleasure
scale. Br J Psychiatry. 1995;167(1):99–103. 76. Blechert J, Ansorge U, Tuschen-Caffier B. A body-related dot-probe task
reveals distinct attentional patterns for bulimia nervosa and anorexia
nervosa. J Abnorm Psychol. 2010;119(3):575–85. y
y
56. Fresán A, Berlanga C. Traducción al español y validación de la Escala
de Placer Snaith-Hamilton para Anhedonia (shaps). Actas Esp Psiquiatr. 2013;41(4):226–31. 77. Friederich HC, Uher R, Brooks S, Giampietro V, Brammer M, Williams SCR,
Herzog W, Treasure J, Campbell IC. I’m not as slim as that girl: Neural
bases of body shape self-comparison to media images. Neuroimage. 2007;37(2):674–81. 57. Cooper PJ, Taylor MJ, Cooper Z, Fairbum CG. The development and valida‑
tion of the body shape questionnaire. Int J Eat Disord. 1987;6(4):485–94. Á 58. Raich RM, Mora M, Soler A, Ávila C, Clos I, Zapater L. Adaptación de un
instrumento de evaluación de la insatisfacción corporal. Clín Salud. 1996;7(1):51–66. 78. Friederich HC, Brooks S, Uher R, Campbell IC, Giampietro V, Brammer M,
Williams SCR, Herzog W, Treasure J. Neural correlates of body dissatisfac‑
tion in anorexia nervosa. Neuropsychologia. 2010;48(10):2878–85. 59. Balaban MT, Losito B, Simons RF, Graham FK. Off-line latency and ampli‑
tude scoring of the human reflex eye blink with Fortran IV. Psychophysiol‑
ogy. 1986;23(5):612. 79. Fairburn C, Cooper Z, O’Connor M. Eating disorder examination (Edition
16.0D). In: Fairburn C, editor. Cognitive behavior therapy and eating
disorders. References New York: Guilford Press; 2008:265–308. 60. Racine SE, Hebert KR, Benning SD. Emotional reactivity and appraisal of
food in relation to eating disorder cognitions and behaviours: evidence
to support the motivational conflict hypothesis. Eur Eat Disord Rev. 2018;26(1):3–10. 80. Kühnpast N, Gramann K, Pollatos O. Electrophysiologic evidence for
multilevel deficits in emotional face processing in patients with bulimia
nervosa. Psychosom Med. 2012;74(7):736–44. 61. Racine SE, Suissa-Rocheleau L, Martin SJ, Benning SD. Implicit and explicit
motivational responses to high- and low-calorie food in women with
disordered eating. Int J Psychophysiol. 2021;159:37–46. 81. Merlotti E, Mucci A, Volpe U, Montefusco V, Monteleone P, Bucci P,
Galderisi S. Impulsiveness in patients with bulimia nervosa: electrophysi‑
ological evidence of reduced inhibitory control. Neuropsychobiology. 2013;68(2):116–23. 62. Schnepper R, Georgii C, Eichin K, Arend A-K, Wilhelm FH, Vögele C, et al. Fight, flight, – or grab a bite! Trait emotional and restrained eating style
predicts food cue responding under negative emotions. Front Behav
Neurosci. 2020;14:91. 82. Otagaki Y, Tohoda Y, Osada M, Horiguchi J, Yamawaki S. Prolonged P300
latency in eating disorders. Neuropsychobiology. 1998;37(1):5–9. 63. Weygandt M, Schaefer A, Schienle A, Haynes J. Diagnosing different
binge-eating disorders based on reward-related brain activation patterns. Hum Brain Mapp. 2012;33(9):2135–46. References Neural correlates of food addiction. Arch Gen Psychiatry. 2011;68(8):808. 16. Bulik CM, Lawson RH, Carter FA. Salivary reactivity in restrained and
unrestrained eaters and women with bulimia nervosa. Appetite. 1996;27(1):15–24. 40. Guerra P, Campagnoli RR, Vico C, Volchan E, Anllo-Vento L, Vila J. Filial
versus romantic love: contributions from peripheral and central electro‑
physiology. Biol Psychol. 2011;88(2–3):196–203. 17. Fedoroff I, Polivy J, Peter HC. The specificity of restrained versus unre‑
strained eaters’ responses to food cues: general desire to eat, or craving
for the cued food? Appetite. 2003;41(1):7–13. 41. McTeague LM, Lang PJ, Laplante M-C, Cuthbert BN, Shumen JR,
Bradley MM. Aversive imagery in posttraumatic stress disorder: trauma
recurrence, comorbidity, and physiological reactivity. Biol Psychiatry. 2010;67(4):346–56. 18. Ferrer-García M, Gutiérrez-Maldonado J, Pla-Sanjuanelo J, Vilalta-
Abella F, Riva G, Clerici M, et al. A randomised controlled comparison
of second-level treatment approaches for treatment-resistant adults
with bulimia nervosa and binge eating disorder: assessing the benefits
of virtual reality cue exposure therapy: benefits of virtual reality cue
exposure therapy. Eur Eat Disorders Rev. 2017;25(6):479–90. 42. Miccoli L, Delgado R, Rodríguez-Ruiz S, Guerra P, García-Mármol E, Fernán‑
dez-Santaella MC. Meet OLAF, a good friend of the IAPS! The open library
of affective foods: a tool to investigate the emotional impact of food in
adolescents. PLoS ONE. 2014;9(12):e114515. 19. Jansen A, Schyns G, Bongers P, van den Akker K. From lab to clinic:
extinction of cued cravings to reduce overeating. Physiol Behav. 2016;162:174–80. 43. Miccoli L, Delgado R, Guerra P, Versace F, Rodríguez-Ruiz S, Fernández-
Santaella MC. Affective pictures and the open library of affective foods
(OLAF): tools to investigate emotions toward food in adults. PLoS ONE. 2016;11(8):1–13. 20. Pla-Sanjuanelo J, Ferrer-García M, Gutiérrez-Maldonado J, Riva G,
Andreu-Gracia A, Dakanalis A, et al. Identifying specific cues and
contexts related to bingeing behavior for the development of effective
virtual environments. Appetite. 2015;87:81–9. 44. Bradley MM. Brain potentials in perception: PICTURE complexity and
emotional arousal. Psychophysiology. 2007;44(3):364–73. 21. Jansen A. A learning model of binge eating: cue reactivity and cue
exposure. Behav Res Ther. 1998;36(3):257–72. 45. Cook EW. VPM reference manual. Birmingham: University of Alabama;
2000. 22. Eddy KT, Novotny CM, Westen D. Sexuality, personality, and eating
disorders. Eat Disord. 2004;12(3):191–208. 22. Eddy KT, Novotny CM, Westen D. Sexuality, personality, and eating
disorders. Eat Disord. 2004;12(3):191–208. Page 11 of 11 Page 11 of 11 Hernández‑Rivero et al. J Eat Disord (2021) 9:120 Hernández‑Rivero et al. 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. 64. Yilmaz Z, Gottfredson NC, Zerwas SC, Bulik CM, Micali N. Developmental
premorbid body mass index trajectories of adolescents with eating
disorders in a longitudinal population cohort. J Am Acad Child Adolesc
Psychiatr. 2019;58(2):191–9. 64. Yilmaz Z, Gottfredson NC, Zerwas SC, Bulik CM, Micali N. Developmental
premorbid body mass index trajectories of adolescents with eating
disorders in a longitudinal population cohort. J Am Acad Child Adolesc
Psychiatr. 2019;58(2):191–9.
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Dr hab. Aleksandra Kustra-Rogatka – prof. Uniwersytetu Mikołaja Kopernika w Toruniu, Polska;
e-mail: a_kustra@umk.pl; ORCID: 0000-0002-1153-8717. Badania nie są finansowane przez żadną
instytucję. Autorka dziękuje za cenne uwagi krytyczne dr. Wojciechowi Włochowi. „Krytyka Prawa”, tom 12, nr 3/2020, s. 50–69, ISSN 2080-1084, e-ISSN 2450-7938, © 2020 Author.
This is an open access article distributed under the Creative Commons BY 4.0 license:
http://creativecommons.org/licenses/by/4.0 „Krytyka Prawa”, tom 12, nr 3/2020, s. 50–69, ISSN 2080-1084, e-ISSN 2450-7938, © 2020 Author. This is an open access article distributed under the Creative Commons BY 4.0 license:
http://creativecommons.org/licenses/by/4.0 „Krytyka Prawa”, tom 12, nr 3/2020, s. 50–69, ISSN 2080-1084, e-ISSN 2450-7938, © 2020 Author. This is an open access article distributed under the Creative Commons BY 4.0 license:
http://creativecommons.org/licenses/by/4.0 ALEKSANDRA KUSTRA-ROGATKA1 Złożony do redakcji: 23.05.2020. Zaakceptowany do druku: 8.09.2020 Słowa kluczowe: konstytucjonalizm, polityczność, Carl Schmitt,
Chantal Mouffe, John Rawls. ALEKSANDRA KUSTRA-ROGATKA Submitted: 23.05.2020. Accepted: 8.09.2020 Keywords: constitutionalism, the political, Carl Schmitt, Chantal Mouffe,
John Rawls. Streszczenie Charakterystyczny dla współczesnego dyskursu o konstytucjonalizmie zabieg
etykietowania (labelling) doprowadził do powstania gęstej dżungli „przymiotni-
kowych konstytucjonalizmów”, wykraczających poza klasyczny liberalny para-
dygmat. Tworzone koncepcje zmierzają do sprecyzowania zmian zachodzących
w prawie publicznym oraz jego otoczeniu politycznym. Dyskurs dotyczący poj-
mowania konstytucjonalizmu wpisuje się w szerszy problem polityczności (the
political). Celem niniejszego artykułu jest skonfrontowanie ze sobą dwóch tytuło-
wych pojęć: konstytucjonalizmu i polityczności. W pierwszej części tekstu został
poruszony problem metodologicznych niespójności we współczesnej analizie
koncepcji konstytucjonalizmu. Druga część porusza kontrowersje wokół istoty
konstytucjonalizmu i ich fundamentalne znaczenie dla teoretycznej legitymizacji
koncepcji alternatywnych wobec konstytucjonalizmu prawnego i liberalnego
(koncepcje istoty konstytucjonalizmu jako limitacji władzy, samo(u)stanowienia
rozumianego statycznie – mit fundacyjny – oraz samostanowienia rozumianego
dynamicznie). Trzecia część została poświęcona pojęciu polityczności jako kryte-
rium podziału wymienionych koncepcji konstytucjonalizmu. Słowa kluczowe: konstytucjonalizm, polityczność, Carl Schmitt,
Chantal Mouffe, John Rawls. 1
Dr hab. Aleksandra Kustra-Rogatka – prof. Uniwersytetu Mikołaja Kopernika w Toruniu, Polska;
e-mail: a_kustra@umk.pl; ORCID: 0000-0002-1153-8717. Badania nie są finansowane przez żadną
instytucję. Autorka dziękuje za cenne uwagi krytyczne dr. Wojciechowi Włochowi. DOI: 10.7206/kp.2080-1084.395 Tom 12, nr 3/2020 Tom 12, nr 3/2020 „Krytyka Prawa”, tom 12, nr 3/2020, s. 50–69, ISSN 2080-1084, e-ISSN 2450-7938, © 2020 Author. This is an open access article distributed under the Creative Commons BY 4.0 license:
http://creativecommons.org/licenses/by/4.0 „Krytyka Prawa”, tom 12, nr 3/2020, s. 50–69, ISSN 2080-1084, e-ISSN 2450-7938, © 2020 Author. This is an open access article distributed under the Creative Commons BY 4.0 license:
http://creativecommons.org/licenses/by/4.0 Abstract The act of labelling, typical of the contemporary discourse on constitutionalism,
has given rise to a thick jungle of “adjectival constitutionalisms”, reaching beyond
the classic liberal paradigm. The formulated concepts aim at specifying the changes
taking place in public law and in its political surrounding. The discourse on the notion
of constitutionalism is a piece of a greater puzzle – the problem of the political. The
purpose of this paper is to compare the two titular notions: constitutionalism and
the political. The first part of the paper addresses the problem of methodological
inconsistencies in the modern-day analysis of the concept of constitutionalism. The second part raises the controversies around the essence of constitutionalism
and their fundamental significance to the theoretical legitimisation of alternatives
to the concept of legal and liberal constitutionalism (the concepts of the essence
of constitutionalism as limitation of power, statically understood self-establishment
– a founding myth, and dynamically understood self-establishment). Part three dis-
cusses the notion of the political as a criterion of division of the said concepts of con-
stitutionalism. The act of labelling, typical of the contemporary discourse on constitutionalism,
has given rise to a thick jungle of “adjectival constitutionalisms”, reaching beyond
the classic liberal paradigm. The formulated concepts aim at specifying the changes
taking place in public law and in its political surrounding. The discourse on the notion
of constitutionalism is a piece of a greater puzzle – the problem of the political. The
purpose of this paper is to compare the two titular notions: constitutionalism and
the political. The first part of the paper addresses the problem of methodological
inconsistencies in the modern-day analysis of the concept of constitutionalism. The second part raises the controversies around the essence of constitutionalism
and their fundamental significance to the theoretical legitimisation of alternatives
to the concept of legal and liberal constitutionalism (the concepts of the essence
of constitutionalism as limitation of power, statically understood self-establishment
– a founding myth, and dynamically understood self-establishment). Part three dis-
cusses the notion of the political as a criterion of division of the said concepts of con-
stitutionalism. Keywords: constitutionalism, the political, Carl Schmitt, Chantal Mouffe,
John Rawls. Tom 12, nr 3/2020 Tom 12, nr 3/2020 DOI: 10.7206/kp.2080-1084.395 52 Aleksandra Kustra-Rogatka 52 Aleksandra Kustra-Rogatka 52 3
Por. T. Drinóczi, A. Bień-Kacała, Illiberal Constitutionalism: The Case of Hungary and Poland, „German
Law Journal” 2019, 20(8), s. 1140–1166. 2
Por. G. Stopler, Semi-liberal constitutionalism, „Global Constitutionalism” 2019, 8(1), s. 94–122. 5
W polskiej literaturze krytycznie wobec konstytucjonalizmu prawnego/prawniczego: por. M. Stam-
bulski, Nowa gra w mieście. Polityczny konstytucjonalizm jako krytyka dogmatyki konstytucyjne, [w:] Jaki
konstytucjonalizm? Refleksje nad „New Democracies in Cirsis” Paula Blokkera, „Politicon” I, http://www.
bibliotekacyfrowa.pl/Content/92238/politicon_I_Jaki_konstytucjonalizm_Refleksje_nad_New_
Democracies_in_Crisis_Paula_Blokkera.pdf (dostęp: 21.02.2020), s. 7. Por. M. Tushnet, Authoritarian Constitutionalism, „Cornell Law Review” 2015, 100(2), s. 3 15
Problem przekładalności koncepcji konstytucjonalizmu – skrojonej pierwotnie na potrzeby teorii
i praktyki prawa konstytucyjnego państw narodowych – na poziom ponadnarodowy wymaga
odrębnej analizy i wykracza poza zakres treściowy niniejszego artykułu. Z bogatej literatury do-
tyczącej tej problematyki warto w tym miejscu wskazać na monografię N. Krischa, Beyond Consti-
tutionalism: The Pluralist Structure of Postnational Law, Oxford 2011. 20
Por. M. Baranowski, W stronę konfliktowego modelu polityczności, [w:] idem (red.) Demokracja i rola oby-
watela. O napięciu pomiędzy państwem, społeczeństwem i procesami globalizacyjnymi, Poznań 2014, s. 81. Wprowadzenie Współczesna debata na temat zmian w prawie publicznym toczona zarówno na
poziomie dyskursu teoretycznego, jak i krytycznej oceny rozwiązań normatywnych
oraz praktyki ustrojowej nie pomija w swej treści pojęcia konstytucjonalizmu. Coraz powszechniejsze staje się zjawisko dodawania do terminu „konstytucjona-
lizm” rozmaitych przymiotników: konstytucjonalizm liberalny, semi-liberalny2,
illiberalny3, autorytarny4, prawniczy/prawny5, polityczny6, obywatelski (civic)7,
społeczny (societal)8, ludowy (popular)9, populistyczny10 czy abuzywny (abusive consti-
tutionalism)11 tłumaczony w polskiej literaturze również na znieważający (G. Skąp-
ska)12 albo obraźliwy (A. Czarnota)13. Bez dokładniejszej prezentacji poszczególnych
odmian konstytucjonalizmu przymiotnikowego14, za jego podstawową (paradyg- 5
W polskiej literaturze krytycznie wobec konstytucjonalizmu prawnego/prawniczego: por. M. Stam-
bulski, Nowa gra w mieście. Polityczny konstytucjonalizm jako krytyka dogmatyki konstytucyjne, [w:] Jaki
konstytucjonalizm? Refleksje nad „New Democracies in Cirsis” Paula Blokkera, „Politicon” I, http://www. bibliotekacyfrowa.pl/Content/92238/politicon_I_Jaki_konstytucjonalizm_Refleksje_nad_New_
Democracies_in_Crisis_Paula_Blokkera.pdf (dostęp: 21.02.2020), s. 7. 6
Por. m.in. R. Bellamy, Political Constitutionalism. A Republican Defence of the Constitutionality of Demo
cracy, Cambridge 2007. 7
Por. P. Blokker, New Democracies in Crisis? A Comparative Constitutional Study of the Czech Republic,
Hungary, Poland, Romania and Slovakia, Routledge 2015, s. 9. 8
Por. G. Teubner, Constitutional Fragments: Societal Constitutionalism and Globalization, Oxford 2012. 9
Por. M. Tushnet, Popular Constitutionalism as Political Law, „Chicago–Kent Law Review” 2006, 81,
s. 991–1006. 10
Por. A. Czarnota, Populistyczny konstytucjonalizm czy nowy konstytucjonalizm?, „Krytyka Prawa” 2019,
1, s. 27–42. 1
Por. D. Landau, Abusive Constitutionalism, „UC Davis Law Review” 2013, 47, s. 189–260. 12
Por. G. Skąpska, Znieważający konstytucjonalizm i konstytucjonalizm znieważony. Refleksja socjologiczna
na temat kryzysu liberalno-demokratycznego konstytucjonalizmu w Europie pokomunistycznej, „Filozofia
Publiczna i Edukacja Demokratyczna” 2018, 1, s. 276–301. 13
Por. A. Czarnota, Populistyczny konstytucjonalizm…, s. 34 i n. 14
Określenie: D. Kapiszewski (adjectival constitutionalism). Por. M. Tushnet, Varieties of constitutionalism
(editorial), „International Journal of Constitutional Law” 2016, 14(1), s. 1–5. DOI: 10.7206/kp.2080-1084.395 Tom 12, nr 3/2020 16
J.-W. Müller, What is Populism?, Philadelphia, PA 2016.
17
M.A. Graber, S. Levinson, M. Tushnet (red.), Constitutional Democracy in Crisis?, Oxford 2018.
18
M. Król, Filozofia polityczna, Kraków 2008, 129. 21
Ibidem, s. 83. 24
Por. Ł. Dulęba, Michael Freeden: liberalizm i ideologia, „Przegląd Polityczny” 2015, 12, s. 68. Aczkolwiek,
jak wskazuje ten autor, istnieją wątpliwości co do używania kategorii polityczności względem
koncepcji J. Rawlsa. Por. Ł. Dulęba, Liberalizm jako forma ucieczki od polityczności, „Refleksje” 2012,
6, s. 68, przypis nr 50 z przywołaniem następujących pozycji: G. Newey, After Politics. The Rejection
of Politics in Contemporary Liberal Philosophy, Basingstoke 2001, s. 161–166; Por. R. Alejandro, What
Is Political about Rawls’s Political Liberalism?, „The Journal of Politics” 1996, 58(1), s. 1–25. 22
Zoran Oklopcic pisze o trwającej fascynacji (ongoing fascination) koncepcją polityczności C. Schmitta,
por. Z. Oklopic, Imagined ideologies: Populist figures, liberalist projections, and the horizons of constitu-
tionalism, „German Law Journal” 2019, 20, s. 203. 23
Termin agonizm pochodzi od starogreckiego słowa agon, oznaczającego współzawodnictwo,
zarówno sportowe, jak i innego rodzaju. 25
Por. W. Włoch, Pomiędzy czystym prawem a ideą polityczną, Toruń 2018, s. 129–149. Konstytucjonalizm a polityczność Konstytucjonalizm a polityczność 53 matyczną) odsłonę możemy uznać, tę, którą najczęściej w literaturze przedmiotu
określa się jako konstytucjonalizm liberalny oraz prawny/prawniczy (niekiedy sto-
sując te określenia jako krytyczne). Ta w pewnym sensie modelowa koncepcja kon-
stytucjonalizmu jest zazwyczaj przeciwstawiana pozostałym typom: przede wszyst-
kim konstytucjonalizmowi politycznemu, ale też społecznemu, populistycznemu,
illiberalnemu (również semiliberalnemu oraz autorytarnemu) czy abuzywnemu. Obserwowane zmiany w prawie publicznym związane z jednej strony z proce-
sem globalizacji (czego dowodem są takie koncepcje teoretyczne jak global governance,
global constitutionalism, European constitutionalism15), a z drugiej strony przeciwstawną
tendencją podkreślania tradycyjnej roli państwa narodowego (będące nierzadko
podatnym teoretycznym gruntem m.in. dla zjawiska tzw. nowego populizmu16 oraz
postępującej erozji demokracji liberalnej17) wskazują na mocne osadzenie toczącej
się debaty w kontekście sporów politycznych. Dyskurs dotyczący pojmowania konstytucjonalizmu wpisuje się więc w szerszy
problem polityczności (the political) oraz polityczności prawa. Polityczność jest
pojęciem, którym posługują się zarówno prawnicy (przede wszystkim teoretycy
i filozofowie prawa), jak i politolodzy oraz socjologowie, często w dość różnych
kontekstach i znaczeniach. Jak stwierdza Marcin Król, „każdy pogląd z zakresu filo-
zofii politycznej prezentuje jakąś koncepcję polityczności”18, zaś „każda filozofia
polityczna to jakaś filozofia polityczności”19. W opracowaniu przyjmuję maksymal-
nie szeroką i „neutralną” definicję polityczności, przyjmując, że jest ona zbiorem
aprobowanych sposobów przedstawiania propozycji organizowania rzeczywistości
społecznej w perspektywie relacji międzyludzkich. Polityczność poprzedza przed-
miotowe ujęcie rozwiązań instytucjonalnych i stanowi pierwotny obszar politycz-
nej organizacji danej wspólnoty. Jest warunkiem konceptualizacji i legitymizacji
rozwiązań dotyczących sprawowania szeroko rozumianej władzy20. Tak ujęta po-
lityczność ma metateoretyczny charakter i jest predefinicją każdej polityki21. 15
Problem przekładalności koncepcji konstytucjonalizmu – skrojonej pierwotnie na potrzeby teorii
i praktyki prawa konstytucyjnego państw narodowych – na poziom ponadnarodowy wymaga
odrębnej analizy i wykracza poza zakres treściowy niniejszego artykułu. Z bogatej literatury do-
tyczącej tej problematyki warto w tym miejscu wskazać na monografię N. Krischa, Beyond Consti-
tutionalism: The Pluralist Structure of Postnational Law, Oxford 2011. J. W. Müller, What is Populism?, Philadelphia, PA 2016. 17
M.A. Graber, S. Levinson, M. Tushnet (red.), Constitutional Democracy in Crisis?, Oxford 2018. 18
M. Król, Filozofia polityczna, Kraków 2008, 129. 20
Por. M. Baranowski, W stronę konfliktowego modelu polityczności, [w:] idem (red.) Demokracja i rola oby-
watela. O napięciu pomiędzy państwem, społeczeństwem i procesami globalizacyjnymi, Poznań 2014, s. 81. 21
Ibidem, s. 83. DOI: 10.7206/kp.2080-1084.395 Tom 12, nr 3/2020 Aleksandra Kustra-Rogatka Konstytucjonalizm a polityczność 55 nozą Z. Oklopicia, który wskazuje (powołując się na tezy Neila Walkera26), że głów-
nym powodem obecnego kryzysu idei konstytucjonalizmu jest niechęć współczes
nych teoretyków do wyjścia poza dychotomie definiujące konceptualną architekturę
konstytucjonalizmu: indywidualne–zbiorowe, pojedyncze–mnogie, szczególne–
–uniwersalne. Nawet w swojej najbardziej autorefleksyjnej i samokrytycznej współ-
czesnej wyobraźni konstytucyjnej (constitutional imaginary) konceptualna architek-
tura konstytucjonalizmu pozostaje kompulsywnie grupowa, sytuacyjnie obojętna
i polemicznie zdemoralizowana27. 27
Z. Oklopic, Imagined ideologies: Populist figures, liberalist projections, and the horizons of constitutionalism,
„German Law Journal” 2019, 20, s. 203. 26
N. Walker, Populism and Constitutional Tension, „International Journal of Constitutional Law” 2019,
17(2), s. 515–535. 28
Por. A. Sulikowski, Kryzys nowoczesnego konstytucjonalizmu. Między liberalną sędziokracją a postlibe-
ralnym populizmem, „Filozofia Publiczna i Edukacja Demokratyczna” 2018, 7(1), s. 328–340. 29
Por. W.-C. Chang, Back into the political? Rethinking judicial, legal, and transnational constitutionalism,
„International Journal of Constitutional Law” 2019, 17(2), s. 453–460. W polskiej literaturze A. Czar-
nota i M. Stambulski, próbując zdefiniować konstytucjonalizm prawny, wskazują, że określają
go trzy cechy (przyjmując poziom deskrypcji), określane przez wymienionych autorów jako tezy
(na poziomie normatywnym): tezę tekstualną, tezę o determinacji oraz tezę o separacji. Zgodnie
z tezą tekstualną tekst konstytucji „(…) jest bytem niezależnym od interpretatora. Jest to tekst
wewnętrznie uporządkowany. Ponadto, tekst ten jest wynikiem procesu politycznego, jednak po
jego zakończeniu staje się niezależny od bieżącej polityki – teza o separacji. Konstytucja jest wy-
nikiem szerokiego konsensusu politycznego i społecznego. Stanowi minimalną wizję wspólnoty,
która jest wspólna dla wszystkich racjonalnych aktorów politycznych. W procesie stosowania
przepisów konstytucyjnych dyskrecjonalność prawnika jest bardzo ograniczona – teza o deter-
minacji. Orzeczenie sądu konstytucyjnego pozostaje w prostym, dedukcyjnym związku z prze-
pisami konstytucji. Nie ma w nim miejsca na realne konflikty wartości czy kreatywność, dlatego
że konstytucja zawiera w sobie wszelkie odpowiedzi na przyszłe polityczne pytania. Wystarczy Aleksandra Kustra-Rogatka 54 Tekst ma kilkuczęściową strukturę. W pierwszej części poruszam problem
metodologicznych niespójności we współczesnej analizie koncepcji konstytucjo-
nalizmu. Wskazuję w niej na uwikłanie toczącej się debaty w spory o charakterze
ideologicznym i teoretyczno-prawnym, co utrudnia ustalenie, jaki jest de facto
przedmiot dyskusji (konstytucjonalizm jako koncepcja teoretyczna czy konstytucjo-
nalizm jako praktyka ustrojowa). Zwracam też uwagę, że krytycy konstytucjona-
lizmu prawniczego/liberalnego niekiedy w swych negatywnych ocenach praktyki
ustrojowej łączą ze sobą zbyt odległe sfery koncepcji teoretycznej i praktyki ustro-
jowej i gospodarczej. W drugiej części przedstawiam kontrowersje wokół istoty konstytucjonalizmu
i ich fundamentalne znaczenie dla teoretycznej legitymizacji koncepcji alternatyw-
nych wobec konstytucjonalizmu prawnego i liberalnego (koncepcje istoty konsty-
tucjonalizmu jako limitacji władzy, samo(u)stanowienia rozumianego statycznie
– jako mit fundacyjny – oraz samostanowienia rozumianego dynamicznie). Trzecia część artykułu została poświęcona pojęciu polityczności jako kryterium
podziału wymienionych koncepcji konstytucjonalizmu. Wskazuję w niej na dwa
przeciwstawne teoretyczne ujęcia polityczności. Pierwsze (obecnie preferowane22,
szczególnie przez krytyków konstytucjonalizmu prawnego i liberalnego nawiązu-
jące do koncepcji C. Schmitta oraz autorów inspirujących się jego pracami, w tym
przede wszystkim Ch. Mouffe) jako istotę polityczności określa konflikt, antagonizm
społeczny (w terminologii Ch. Mouffe agonizm23). Drugie (którego początek łą-
czony jest niekiedy z pierwszym wydaniem Teorii sprawiedliwości J. Rawlsa w 197124)
istotę polityczności widzi z kolei w próbie złagodzenia tego konfliktu poprzez za-
proponowanie częściowego konsensu opartego na wizji rozumnego pluralizmu25. Twierdzę, że te dwie biegunowo odmienne koncepcje polityczności mogą być
przydatne uporządkowania dyskursu wokół współczesnego konstytucjonalizmu. Jednocześnie jednak uznaję tę dychotomię za niewystarczającą. Zgadzam się diag Tom 12, nr 3/2020 DOI: 10.7206/kp.2080-1084.395 Konstytucjonalizm a polityczność Metodologiczne problemy w dyskursie
wokół koncepcji konstytucjonalizmu Tocząca się debata akademicka wokół pojęcia konstytucjonalizmu, przynajmniej
do pewnego stopnia, jest wynikiem krytycznej oceny praktyki ustrojowej poszcze-
gólnych państw, często uzasadnianej odwołaniem do konstytucjonalizmu okreś
lanego (niekiedy pejoratywnie) jako konstytucjonalizm liberalny lub prawniczy. Krytycy tego ostatniego wskazują przy tym na oświeceniowy, modernistyczny trzon
tak rozumianej koncepcji konstytucjonalizmu28. Za jego główne komponenty uznaje
się na płaszczyźnie dogmatyczno-normatywnej: konstytucję pisaną (formalną),
zasadę nadrzędności konstytucji, niekiedy również sądową kontrolę konstytucyj-
ności prawa29. Krytycy konstytucjonalizmu prawnego twierdzą, że przyznawanie y
p pi
j
y
29
Por. W.-C. Chang, Back into the political? Rethinking judicial, legal, and transnational constitutionalism,
„International Journal of Constitutional Law” 2019, 17(2), s. 453–460. W polskiej literaturze A. Czar-
nota i M. Stambulski, próbując zdefiniować konstytucjonalizm prawny, wskazują, że określają
go trzy cechy (przyjmując poziom deskrypcji), określane przez wymienionych autorów jako tezy
(na poziomie normatywnym): tezę tekstualną, tezę o determinacji oraz tezę o separacji. Zgodnie
z tezą tekstualną tekst konstytucji „(…) jest bytem niezależnym od interpretatora. Jest to tekst
wewnętrznie uporządkowany. Ponadto, tekst ten jest wynikiem procesu politycznego, jednak po
jego zakończeniu staje się niezależny od bieżącej polityki – teza o separacji. Konstytucja jest wy-
nikiem szerokiego konsensusu politycznego i społecznego. Stanowi minimalną wizję wspólnoty,
która jest wspólna dla wszystkich racjonalnych aktorów politycznych. W procesie stosowania
przepisów konstytucyjnych dyskrecjonalność prawnika jest bardzo ograniczona – teza o deter-
minacji. Orzeczenie sądu konstytucyjnego pozostaje w prostym, dedukcyjnym związku z prze-
pisami konstytucji. Nie ma w nim miejsca na realne konflikty wartości czy kreatywność, dlatego
że konstytucja zawiera w sobie wszelkie odpowiedzi na przyszłe polityczne pytania. Wystarczy Tom 12, nr 3/2020 DOI: 10.7206/kp.2080-1084.395 Aleksandra Kustra-Rogatka 56 tym komponentom nadmiernej wagi w dogmatyce prawa konstytucyjnego i prak-
tyce ustrojowej doprowadziło do utożsamienia konstytucji z tekstem konstytu-
cyjnym, przesadnego formalizmu w interpretacji tekstu konstytucyjnego, sztucz-
nego oddzielenia prawa (i konstytucji) od sfery politycznej oraz nadmiernej roli
sądów, w tym przede wszystkim sądów konstytucyjnych w ustalaniu treści norm
konstytucyjnych30. Michał Stambulski w recenzji książki Paula Blokkera New Democracies in Crisis?31
– zwolennika konstytucjonalizmu obywatelskiego (civic constitutionalism) i krytyka
praktyki ustrojowej post-socjalistycznych państw Europy Środkowej – stwierdza,
że konstytucjonalizm prawny/prawniczy (…) „charakteryzuje się: uznaniem wyż-
szości prawa nad polityką, nienaruszalnością praw podstawowych oraz formalną
równością wobec prawa, a także bezwzględnym prymatem procedur w zakresie
zmian instytucjonalnych. Prawniczy konstytucjonalizm argumentuje, że: 1) moż-
liwe jest ustalenie apolitycznych reguł dla tworzenia prawa; 2) reguły te powinni
określać prawnicy. mieć odpowiednią wiedzę techniczną, aby być w stanie to dostrzec”. A. Czarnota, M. Stambulski,
Janusowe oblicze konstytucjonalizmu, „Krytyka Prawa” 2019, 11(1), s. 11. Jako tezy charakteryzujące
konstytucjonalizm polityczny (i konstytucji w znaczeniu politycznym) A. Czarnota i M. Stambul-
ski wskazują (przeciwstawne wobec wskazanej wyżej triady): tezę społeczną, tezę o polityczności
oraz tezę o niedookreśloności, por. ibidem, s. 15. 33
W tym duchu A. Czarnota opisuje konstytucjonalizm liberalny określając go także jako liberalno-
-demokratyczny, por. idem, Populistyczny konstytucjonalizm…, s. 29–30. 31
P. Blokker, New Democracies in Crisis? A Comparative Constitutional Study of the Czech Republic, Hun-
gary, Poland, Romania and Slovakia, Routledge 2015. mieć odpowiednią wiedzę techniczną, aby być w stanie to dostrzec”. A. Czarnota, M. Stambulski,
Janusowe oblicze konstytucjonalizmu, „Krytyka Prawa” 2019, 11(1), s. 11. Jako tezy charakteryzujące
konstytucjonalizm polityczny (i konstytucji w znaczeniu politycznym) A. Czarnota i M. Stambul-
ski wskazują (przeciwstawne wobec wskazanej wyżej triady): tezę społeczną, tezę o polityczności
oraz tezę o niedookreśloności, por. ibidem, s. 15. 30
W.-C. Chang, op. cit., s. 453. 0
W.-C. Chang, op. cit., s. 453. 34
Jako tezy charakteryzujące konstytucjonalizm polityczny (i konstytucji w znaczeniu politycznym)
A. Czarnota i M. Stambulski wskazują (przeciwstawne wobec wskazanej wyżej triady): tezę
społeczną, tezę o polityczności oraz tezę o niedookreśloności, definiując je w następujący sposób:
„Konstytucja jest tutaj postrzegana jako żyjący porządek instytucjonalny – teza społeczna. Jest
to system instytucji dokonujących klasyfikacji konfliktów społecznych. Konstytucjonalizm do-
konuje podziałów na żądania racjonalne/nieracjonalne oraz zgodne/niezgodne z tradycją czy
tożsamością konstytucyjną. Konstruuje także hierarchię wartości. Procesy polityczne wpływają
na konstytucjonalizm – teza o polityczności. Dostęp do konstytucji nie jest egalitarny, lecz zależny
od dostępu do mass mediów, kapitału symbolicznego i kulturowego. Dlatego stosowanie kon-
stytucji poprzez sprawowanie władzy, powinno uwzględniać interesy »innych«, którzy są z niej
wykluczeni. Jest to działanie polityczne w tym sensie, że dochodzi w nim do uzgodnienia wielu
sprzecznych interesów, a stawką tego uzgodnienia jest stabilność i ład wspólnoty. Jednocześnie
tekst konstytucji nie determinuje jej stosowania – teza o nieokreśloności. Znaczenie konstytucji
jest i powinno być otwarte na renegocjacje. Zmiany społeczne oraz artykulacja nowych konfliktów
powoduje konieczność dopasowania znaczenia konstytucji do nowych warunków. Takie dopa-
sowanie ma w sobie moment twórczy”. A. Czarnota, M. Stambulski, op. cit., s. 15. 36
W. Włoch, op. cit., s. 31–32. 35
Np. M. Korycka-Zirk przyjmuje, że „konstytucjonalizm to ujawnione w dyskursie społecznym,
a następnie w dyskursie prawnym pewne koncepcje teoretyczno- i filozoficznoprawne porządku
prawnego co do fundamentalnych wartości politycznych”. M. Korycka-Zirk, Filozoficznoprawny
wymiar kontroli konstytucyjności, Toruń 2017, s. 13. Stwierdza jednocześnie, że dwoma głównymi
implikacjami konstytucjonalizmu są rządy prawa oraz liberalizm, co zawęża przyjętą definicję do
paradygmatu konstytucjonalizmu liberalnego (por. ibidem, s. 178). 37
Badania konstytucjonalizmu dokonywane z poziomu deskrypcji mogą, a nawet powinny wy-
korzystywać różne metody. Oprócz metody dogmatyczno-prawnej oraz komparatystycznej dla
pełnego obrazu konstytucjonalizmu w płaszczyźnie opisowej niezbędne jest również wykorzy-
stanie metod empirycznych, charakterystycznych dla nauk społecznych. Metodologiczne problemy w dyskursie
wokół koncepcji konstytucjonalizmu Blokker wskazuje, że ujęcie takie prowadzi do sytuacji, w któ-
rej prawo staje się zamkniętym systemem, posługującym się swoim językiem
i wykluczającym jakąkolwiek interakcję obywatelską. Innymi słowy – taki system
angażuje prawników, ale nie szerokie rzesze obywateli, którym nie oferuje żadnej
tożsamości”32. Jednocześnie łączy się niekiedy konstytucjonalizm prawny/prawniczy wyłącz-
nie z konstytucjonalizmem liberalnym, najczęściej utożsamiając obie koncepcje
z demokracją liberalną33. Społeczna dezaprobata wobec formalizmu prawnego,
rządów technokratycznych i neoliberalnego kapitalizmu uznawane są za główne
przyczyny zmian społecznych i politycznych, których celem jest urzeczywistnie-
nie lub przynajmniej wzmocnienie idei konstytucjonalizmu politycznego. Kon-
stytucjonalizm polityczny (jak również społeczny, obywatelski i populistyczny)
ustawiony w kontrze do konstytucjonalizmu prawnego przyjmuje przede wszyst-
kim odmienną, szerszą definicję konstytucji, wskazując na jej polityczny charak- Tom 12, nr 3/2020 DOI: 10.7206/kp.2080-1084.395 Konstytucjonalizm a polityczność 57 ter, otwartość znaczeniową oraz wpisany w takie rozumienie konstytucji brak
finalności34. Takie dychotomiczne ujęcie konstytucjonalizmu prawniczego oraz politycznego
jako pewnych kategorii skrajnych wskazuje na pewien brak precyzji w ustalaniu
przedmiotu badań w aktualnej debacie akademickiej. Po pierwsze, nie do końca
jasne jest, czy przedmiotem badań, analizy oraz ocen akademików jest konstytu-
cjonalizm jako normatywna idea, czy konstytucjonalizm jako opis pewnej ustro-
jowej praktyki. W szczególności nie zawsze jest klarowne, czy krytyce poddawana
jest teoria czy praktyka, a także czy niepożądane aspekty praktyki stanowią argu-
ment za całkiem odmienną teorią. Oczywiście możliwa jest również analiza kon-
stytucjonalizmu łącząca aspekt opisowy oraz normatywny, jak również oba te
aspekty, niemniej wymaga ona jasnego wyartykułowania metodologicznych za-
łożeń i przyjętej definicji35. Po drugie, nie wiadomo, czy dychotomiczność konsty-
tucjonalizmu prawnego i politycznego jest konsekwencją teorii czy też zdiagnozo-
wanej praktyki i czy w ogóle – na poziomie opisowym – możliwa jest dychotomia? Jak wskazuje W. Włoch, nawet na poziomie normatywnym idea konstytucjonaliz-
mu jest naznaczona wewnętrznym napięciem między demokracją a rządami
prawa. Pierwszy element powiązać można z czynnikiem politycznym, drugi nato-
miast z prawnym. Teoretycznym ideałem natomiast byłaby koncepcja równoważąca
te elementy36. 35 35
Np. M. Korycka-Zirk przyjmuje, że „konstytucjonalizm to ujawnione w dyskursie społecznym,
a następnie w dyskursie prawnym pewne koncepcje teoretyczno- i filozoficznoprawne porządku
prawnego co do fundamentalnych wartości politycznych”. M. Korycka-Zirk, Filozoficznoprawny
wymiar kontroli konstytucyjności, Toruń 2017, s. 13. Stwierdza jednocześnie, że dwoma głównymi
implikacjami konstytucjonalizmu są rządy prawa oraz liberalizm, co zawęża przyjętą definicję do
paradygmatu konstytucjonalizmu liberalnego (por. ibidem, s. 178). DOI: 10.7206/kp.2080-1084.395 Tom 12, nr 3/2020 Aleksandra Kustra-Rogatka 58 38
Por. M. Atienza, Konstytucja, globalizacja i prawo, https://www.rpo.gov.pl/pliki/12421353110.pdf
(dostęp: 10.01.2020), s. 1. 39
Por. ibidem. Konceptualne problemy z istotą konstytucjonalizmu Usystematyzowanie dyskursu wokół pojęcia konstytucjonalizmu ułatwi posta-
wienie kilku pytań: Czy pojęcie to odnosimy do poziomu deskrypcji czy preskryp-
cji? Jakie są jego granice znaczeniowe? Jaka jest istota konstytucjonalizmu? Nawet
bez pogłębionej analizy spornych koncepcji konstytucjonalizmu łatwo można do-
strzec, że odpowiedzi na te pytania będą różne w zależności od przyjętej koncepcji
konstytucjonalizmu. Po pierwsze, konstytucjonalizm jest pojęciem, którym operuje
się na obu wspomnianych poziomach: deskryptywnym i preskryptywnym. W pierw-
szym przypadku konstytucjonalizm utożsamia się z opisem, ale też i krytyczną
oceną praktyki ustrojowej danego podmiotu politycznego, najczęściej państwa,
aczkolwiek standardem staje się odnoszenie terminu „konstytucjonalizm” również
do opisu funkcjonowania podmiotów o charakterze ponadnarodowym (np. Unii
Europejskiej)37. W ujęciu preskryptywnym konstytucjonalizm skupia się z kolei na
budowaniu normatywnej wizji znaczenia, roli oraz treści szeroko rozumianej kon-
stytucji, czyli struktury wspólnoty politycznej38. Te dwa poziomy należy jednak
bardziej postrzegać jako komponenty całościowej koncepcji konstytucjonalizmu. Chociaż w dogmatyce prawa konstytucyjnego oraz socjologicznych badaniach
nad konstytucjonalizmem przeważa poziom deskrypcji, to krytyczna analiza in-
stytucji ustrojowych wymaga przecież odniesienia do płaszczyzny normatywnej. Z kolei preskryptywne, teoretyczne koncepcje konstytucjonalizmu nie są tworzone
w społeczno-politycznej próżni. Historyczny rozwój ustroju poszczególnych
państw często stanowi dla nich źródło inspiracji. Dwie fundamentalne teoretyczne
tradycje konstytucjonalizmu: prawna oraz polityczna mają swoje źródło w kry-
tycznej obserwacji funkcjonowania w praktyce określonych rozwiązań ustrojowych
będących efektem szerszym zjawisk normatywnych39. Niemniej tytułowy problem
relacji konstytucjonalizmu i polityczności wymaga bardzo jasnego rozgraniczania
odniesień do poziomu opisowego i normatywnego, a tego w obecnym dyskursie
wokół konstytucjonalizmu niekiedy brakuje. Odpowiedź na pytania o granice znaczeniowe pojęcia konstytucjonalizmu
zależy z kolei do pewnego stopnia od ustalenia, który ze wspomnianych poziomów
(opisu lub preskrypcji) jest dominujący. Można przy tym wyróżnić dwa stanowiska 38
Por. M. Atienza, Konstytucja, globalizacja i prawo, https://www.rpo.gov.pl/pliki/12421353110.pdf
(dostęp: 10.01.2020), s. 1. 39
Por ibidem DOI: 10.7206/kp.2080-1084.395 Tom 12, nr 3/2020 Tom 12, nr 3/2020 Konstytucjonalizm a polityczność 59 dostrzegalne w literaturze. Pierwsze przyjmuje, że konstytucjonalizm ma jasno
określone granice znaczeniowe. To stanowisko jest zazwyczaj przyjmowane przez
badaczy poruszających się w ramach paradygmatu konstytucjonalizmu liberalnego
czy, szerzej, w ramach modelu demokracji konstytucyjnej (obejmując tym samym
zarówno konstytucjonalizm prawny nawiązujący do koncepcji liberalizmu, jak i kon-
stytucjonalizm polityczny czerpiący z tradycji republikańskich40). Drugie stanowis-
ko odrzuca paradygmat konstytucjonalizmu liberalnego jako zbyt wąski i skupia
się na poziomie krytycznego opisu praktyki ustrojowej często wykraczającej poza
ramy pojęciowe demokracji liberalnej. Takie otwarcie znaczeniowe pozwala na sfor-
mułowanie koncepcji konstytucjonalizmu autorytarnego, illiberalnego, abuzyw-
nego i ich pochodnych. 40
Szerzej na temat relacji między konstytucjonalizmem a liberalizmem i republikanizmem por.
T. Bekrycht, Republikanizm i liberalizm w świetle idei konstytucjonalizmu. Uwagi o legitymizacji prawa,
„Filozofia Publiczna i Edukacja Demokratyczna” 2018, 7(1), s. 26–39. 42
Por. Z. Elkins, T. Ginsburg, J. Melton, The content of authoritarian constitutions, [w:] T. Ginsburg,
A. Simpser (red.), Constitutions in Authoritarian Regimes, Cambridge 2013, s. 141–164. 41
M. Tushnet, Authoritarian constitutionalism, [w:] T. Ginsburg, A. Simpser (red.), Constitutions in Autho
ritarian Regimes, Cambridge 2013, s. 36–49. 40
Szerzej na temat relacji między konstytucjonalizmem a liberalizmem i republikanizmem por.
T. Bekrycht, Republikanizm i liberalizm w świetle idei konstytucjonalizmu. Uwagi o legitymizacji prawa,
„Filozofia Publiczna i Edukacja Demokratyczna” 2018, 7(1), s. 26–39.
41
M. Tushnet, Authoritarian constitutionalism, [w:] T. Ginsburg, A. Simpser (red.), Constitutions in Autho
ritarian Regimes, Cambridge 2013, s. 36–49.
42
Por. Z. Elkins, T. Ginsburg, J. Melton, The content of authoritarian constitutions, [w:] T. Ginsburg,
A. Simpser (red.), Constitutions in Authoritarian Regimes, Cambridge 2013, s. 141–164. „Filozofia Publiczna i Edukacja Demokratyczna 2018, 7(1), s. 26 39.
41
M. Tushnet, Authoritarian constitutionalism, [w:] T. Ginsburg, A. Simpser (red.), Constitutions in Autho
ritarian Regimes, Cambridge 2013, s. 36–49.
42
Por. Z. Elkins, T. Ginsburg, J. Melton, The content of authoritarian constitutions, [w:] T. Ginsburg, ritarian Regimes, Cambridge 2013, s. 36–49.
42
Por. Z. Elkins, T. Ginsburg, J. Melton, The content of authoritarian constitutions, [w:] T. Ginsburg,
A. Simpser (red.), Constitutions in Authoritarian Regimes, Cambridge 2013, s. 141–164. Konceptualne problemy z istotą konstytucjonalizmu Za
ustrojowy pierwowzór na poziomie deskrypcji i zarazem punkt odniesienia dla
koncepcji normatywnych konstytucjonalizmu politycznego uznaje się najczęściej
ustrój panujący na Wyspach Brytyjskich, z charakterystycznym dla niego brakiem
konstytucji w znaczeniu formalnym oraz obowiązującą zasadą suwerenności
parlamentu. Jednak przeniesienie tej koncepcji na grunt innych porządków ustro-
jowych, w tym przede wszystkim USA, wymusza pewne modyfikacje, przede
wszystkim ze względu na istnienie bardzo mocnego trójpodziału władzy oraz
ugruntowaną pozycję Sądu Najwyższego jako organu dokonującego kontroli kon-
stytucyjności prawa. ta jest utożsamiana z ideą samoograniczenia się suwerena na przyszłość oraz
zapewnieniem kontroli przestrzegania konstytucji pojmowanej kontraktualistycz-
nie i ujętej w formę aktu normatywnego o najwyższej mocy prawnej. Ważną rolę
w tej koncepcji odgrywają w związku z tym: rozróżnienie władzy ustrojodawczej
i ustawodawczej, instytucja sądowej kontroli konstytucyjności oraz komplemen-
tarnie ujmowana idea demokracji konstytucyjnej. Z kolei konstytucjonalizm po-
lityczny bazuje na koncepcji demokracji większościowej, zazwyczaj łączy w sobie
element krytyki judicial review, a jako istotę konstytucjonalizmu uznaje prawo do
samodecydowania o kształcie ustrojowym wspólnoty politycznej. Centralnymi
ideami tej koncepcji stają się w związku z tym zagwarantowanie równości party-
cypacji w podejmowaniu decyzji politycznych przez poszczególnych członków wspól-
noty oraz założenie luźniejszych ograniczeń w zakresie wprowadzania zmian
ustrojowych w przyszłości. Warto jednak zaznaczyć, że o ile konstytucjonalizm
prawny jako pewną szeroko ujętą koncepcję normatywną cechuje dość spójny,
jednolity charakter, o tyle konstytucjonalizm polityczny ma kilka odmian. Za
ustrojowy pierwowzór na poziomie deskrypcji i zarazem punkt odniesienia dla
koncepcji normatywnych konstytucjonalizmu politycznego uznaje się najczęściej
ustrój panujący na Wyspach Brytyjskich, z charakterystycznym dla niego brakiem
konstytucji w znaczeniu formalnym oraz obowiązującą zasadą suwerenności
parlamentu. Jednak przeniesienie tej koncepcji na grunt innych porządków ustro-
jowych, w tym przede wszystkim USA, wymusza pewne modyfikacje, przede
wszystkim ze względu na istnienie bardzo mocnego trójpodziału władzy oraz
ugruntowaną pozycję Sądu Najwyższego jako organu dokonującego kontroli kon-
stytucyjności prawa. Uczestnicy dyskursu o współczesnym konstytucjonalizmie powinni bardzo
precyzyjnie określać, w jakim znaczeniu używają tego terminu, na jakim poziomie
się nim posługują (deskrypcja/preskrypcja), jakie są granice znaczeniowe pojęcia
konstytucjonalizmu w rozumieniu pewnej koncepcji normatywnej (czy jest nim
konstytucjonalizm liberalny, czy też dana koncepcja wykracza poza paradygmat
konstytucjonalizmu liberalnego, jak np. konstytucjonalizm populistyczny), i wresz-
cie jaka jest istota pojęcia konstytucjonalizmu na poziomie preskrypcyjnym (idea
samoograniczenia suwerena bądź też idea demokratycznego samostanowienia). Konceptualne problemy z istotą konstytucjonalizmu Cechą wspólną tych koncepcji jest bazowanie na założeniu,
że konstytucja może pełnić istotną rolę prawną, społeczną i polityczną również
w ustrojach nie mieszczących się w definicji demokracji liberalnej. Badacze przyj-
mujący tę perspektywę zwracają uwagę, że chociaż kuszące jest traktowanie kon-
stytucji w takich ustrojach jako jedynie „aktów pozornych”, to w praktyce mogą
one pełnić w nich istotną rolę41. Badania prawno-porównawcze dowodzą, iż treść
konstytucji w państwach autorytarnych nie odbiega znacząco od tych obowiązu-
jących we współczesnych ustrojach demokratycznych, ale mimo tej pozornie toż-
samej warstwy treściowej koncepcje konstytucjonalizmu wykraczającego poza
paradygmat liberalny zakładają, że konstytucje niedemokratyczne przestają być
narzędziami ograniczania arbitralności władzy, a stają się jednym z elementów roz-
budowanego instrumentarium zwiększania efektywności władzy politycznej42. g
ę
y
y p
y
j
Pytanie o istotę konstytucjonalizmu do pewnego stopnia nawiązuje do podej-
mowanych wyżej problemów. Ma ono zdecydowanie istotniejsze znaczenie na
poziomie preskrypcji, gdy dążymy do uzyskania odpowiedzi na pytanie o właściwą
rolę i znaczenie konstytucji. W ujęciu deskryptywnym kwestia istoty konstytucjo-
nalizmu schodzi na dalszy plan – badacz skupia się na opisie funkcjonowania kon-
stytucji w ramach danego podmiotu politycznego. Na poziomie preskryptywnym
problem ten jest centralną częścią każdej koncepcji konstytucjonalizmu. Trady-
cyjnie wskazuje się na dwie przeciwstawne koncepcje: konstytucjonalizm prawny
oraz konstytucjonalizm polityczny, zaznaczając, że diametralnie odmiennie ujmują
one istotę konstytucjonalizmu. W koncepcji konstytucjonalizmu prawnego istota Tom 12, nr 3/2020 DOI: 10.7206/kp.2080-1084.395 Aleksandra Kustra-Rogatka 60 ta jest utożsamiana z ideą samoograniczenia się suwerena na przyszłość oraz
zapewnieniem kontroli przestrzegania konstytucji pojmowanej kontraktualistycz-
nie i ujętej w formę aktu normatywnego o najwyższej mocy prawnej. Ważną rolę
w tej koncepcji odgrywają w związku z tym: rozróżnienie władzy ustrojodawczej
i ustawodawczej, instytucja sądowej kontroli konstytucyjności oraz komplemen-
tarnie ujmowana idea demokracji konstytucyjnej. Z kolei konstytucjonalizm po-
lityczny bazuje na koncepcji demokracji większościowej, zazwyczaj łączy w sobie
element krytyki judicial review, a jako istotę konstytucjonalizmu uznaje prawo do
samodecydowania o kształcie ustrojowym wspólnoty politycznej. Centralnymi
ideami tej koncepcji stają się w związku z tym zagwarantowanie równości party-
cypacji w podejmowaniu decyzji politycznych przez poszczególnych członków wspól-
noty oraz założenie luźniejszych ograniczeń w zakresie wprowadzania zmian
ustrojowych w przyszłości. Warto jednak zaznaczyć, że o ile konstytucjonalizm
prawny jako pewną szeroko ujętą koncepcję normatywną cechuje dość spójny,
jednolity charakter, o tyle konstytucjonalizm polityczny ma kilka odmian. 50
C. Schmitt oceniał parlamentaryzm jako rozwiązanie wypaczające zasadniczy sens demokracji,
definiowanej za J.J. Rousseau jako tożsamość rządzących z rządzonymi. Por. C. Schmitt, Teologia
polityczna i inne pisma, Kraków 2000, s. 117. 49
Por. ibidem. Szerzej na ten temat por. Ch. Mouffe, E. Laclau, Hegemonia i socjalistyczna strategia.
Przyczynek do projektu radykalnej polityki demokratycznej, Wrocław 2007. Filozoficzne problemy związane z istotą polityczności Przyjęta na wstępie definicja polityczności otwiera to pojęcie zarówno na konflik-
towe, jak i konsensualne ujęcia konceptualizacji i legitymizacji rozwiązań doty-
czących sprawowania szeroko rozumianej władzy. Jak stwierdziła H. Arendt, Tom 12, nr 3/2020 DOI: 10.7206/kp.2080-1084.395 . Arendt, Wprowadzenie w politykę, [w:] eadem, Polityka jako obietnica, Warszawa 2007, s. 124. 43
H. Arendt, Wprowadzenie w politykę, [w:] eadem, Polityka jako obietnica, Warszawa 2007, s. 124.
44
Ł. Dulęba, Liberalizm…, s. 65 z powołaniem na: C. Schmitt, Pojęcie polityczności, [w:] idem, Teologia
polityczna i inne pisma, Warszawa 2012, s. 254.
45
Por. R. Mańko, Orzekanie w polu polityczności, „Filozofia Publiczna i Edukacja Demokratyczna”
2008, 1, s. 67. 47
Ł. Dulęba, Liberalizm…, s. 67–68. 43
H. Arendt, Wprowadzenie w politykę, [w:] eadem, Polityka jako obietnica, Warszawa 2007, s. 124.
44
Ł. Dulęba, Liberalizm…, s. 65 z powołaniem na: C. Schmitt, Pojęcie polityczności, [w:] idem, Teologia
polityczna i inne pisma, Warszawa 2012, s. 254.
45
Por. R. Mańko, Orzekanie w polu polityczności, „Filozofia Publiczna i Edukacja Demokratyczna”
2008, 1, s. 67.
46
Por. ibidem, s. 67–68.
47
Ł. Dulęba, Liberalizm…, s. 67–68.
48
Por. R. Mańko, op. cit.
49
Por. ibidem. Szerzej na ten temat por. Ch. Mouffe, E. Laclau, Hegemonia i socjalistyczna strategia.
Przyczynek do projektu radykalnej polityki demokratycznej, Wrocław 2007.
50
C. Schmitt oceniał parlamentaryzm jako rozwiązanie wypaczające zasadniczy sens demokracji,
definiowanej za J.J. Rousseau jako tożsamość rządzących z rządzonymi. Por. C. Schmitt, Teologia
polityczna i inne pisma, Kraków 2000, s. 117. Ł. Dulęba, Liberalizm…, s. 65 z powołaniem na: C. Schmitt, Pojęcie polityczności, [w:] idem, T
polityczna i inne pisma, Warszawa 2012, s. 254. 46
Por. ibidem, s. 67–68. 51
Por. R. Mańko, op. cit., s. 68. Modelowym rozwiązaniem instytucjonalnym dla demokracji ago-
nistycznej w ujęciu Ch. Mouffe jest parlamentaryzm, pojmowany za Eliasem Canettim jako forum
starcia, gdzie pokonani w głosowaniu (porównanym do bitwy) uznają wynik, co wcale jednak
nie oznacza, że przyjmują pogląd przeciwnika. Ch. Mouffe, Polityczność. Przewodnik Krytyki Poli-
tycznej, przeł. J. Erbel, Warszawa 2008, s. 37 z powołaniem na: E. Canetti, Masa i władza, Warszawa
1996, s. 216. Por też F. Biały, Konflikt jako wartość? Demokracja agonistyczna a populizm europejski w ujęciu
Chantal Mouffe, „Refleksje” 2010, 1, s. 229–230. 52
Jak stwierdza Ch. Mouffe, w koncepcji demokracji agonistycznej chodzi o „takie rozumienie plu-
ralizmu, które znacznie różni się od jego wersji liberalnej. Chodzi o pluralizm, który podobnie jak
ten Nietzschego czy Maxa Webera pociąga za sobą niemożliwość ostatecznego pojednania wszyst-
kich punktów widzenia”. Ch. Mouffe, Demokratyczna polityka musi być stronnicza, https://krytykapo-
lityczna.pl/kraj/mouffe-demokratyczna-polityka-musi-byc-stronnicza/, 21.02.2015 (dostęp: 17.03.2020). 54
Por. Ch. Mouffe, Agonistyka. Polityczne myślenie o świecie, przeł. B. Szelewa, Warszawa 2015 oraz
eadem, Paradoks demokracji, przeł. W. Jach, M. Kamińska, A. Orzechowski, Wrocław 2005. 53
Ch. Mouffe, Polityczność…, s. 24. Por. też F. Biały, op. cit., s. 228–229. 56
J. Rawls, Liberalizm polityczny, przeł. A. Romaniuk, Warszawa 1998, s. 33. 55
Por. Zob. P. Kelly, Liberalizm, Warszawa 2007, s. 140–149. Konstytucjonalizm a polityczność Konstytucjonalizm a polityczność 61 „polityka za swą podstawę ma fakt ludzkiej wielości (…) jest domeną współistnienia
i stowarzyszania się różnych ludzi”43. Fakt niezgody społecznej w dwóch przeciw-
stawnych wizjach polityczności ujmowany jest albo jako trwający konflikt społeczny,
albo jako punkt wyjścia dla pluralizmu opartego na racjonalnym konsensie. l Najczęściej przywoływanym konfliktowym ujęciem polityczności jest to zapro-
ponowane przez C. Schmitta, który polityczność (das Politische) utożsamił ze szcze-
gólną intensywnością antagonizmu – wymiarem: wróg–przyjaciel. Polityczność
u Schmitta jest kategorią samoistną, którą możemy jedynie określić poprzez „jasne
kryterium”, a nie poprzez definicję czy treść44. W schmittowskiej wizji polityczności
chodzi jednak o wrogość, która osiągnęła najwyższe stadium – dążenie do fizyczne-
go zniszczenia wroga45. Cechą dystynktywną schmittowskiej wizji polityczności
jest więc intensywność konfliktu, a nie jego przedmiot. Polityczność dotyczy tylko
konfliktu o krańcowej intensywności i, co istotne, nie ma charakteru indywidual-
nego, lecz kolektywny/zbiorowy46. Polityczność dotyczy więc relacji z innymi pod-
miotami stosunków międzynarodowych i oznacza potencjalność wojny, a więc
możliwość ustalenia wroga i sposobność do jego unicestwienia47. Radykalizm schmittowskiej wizji polityczności został złagodzony przez Ch. Mo-
uffe. Belgijska filozofka w miejsce polityczności antagonistycznej (przyjaciel/wróg)
zaproponowała polityczność agonistyczną, opartą na relacji „przeciwników”, a nie
„wrogów”, a tym samym możliwą do utrzymania w ramach jednej wspólnoty po-
litycznej48. Celem przestaje być zniszczenie wroga, a staje się nim pokonanie prze-
ciwnika i narzucenie własnej hegemonii49. O ile schmittowski decyzjonizm i po-
lityczność antagonistyczna oznaczały negację liberalizmu i parlamentaryzmu50,
o tyle ustrój demokracji agonistycznej w ujęciu Ch. Mouffe aprobuje czy wręcz
preferuje partyjną demokrację parlamentarną, przyjmując, że w obrębie wspólnoty
politycznej konieczne jest stworzenie forum dla artykulacji konfliktów politycz- 48
Por. R. Mańko, op. cit. 50
C. Schmitt oceniał parlamentaryzm jako rozwiązanie wypaczające zasadniczy sens demokracji,
definiowanej za J.J. Rousseau jako tożsamość rządzących z rządzonymi. Por. C. Schmitt, Teologia
polityczna i inne pisma, Kraków 2000, s. 117. DOI: 10.7206/kp.2080-1084.395 DOI: 10.7206/kp.2080-1084.395 Tom 12, nr 3/2020 Aleksandra Kustra-Rogatka 62 nych51. Agonistyczny wymiar stosunków społecznych jest fundamentem politycz-
ności („tego, co polityczne”)52. Ch. Mouffe odróżnia jednak opartą na konflikcie
„polityczność” od „polityki” definiowanej jako „zestaw praktyk i instytucji, które
w obliczu wprowadzanego przez polityczność konfliktu tworzą porządek umożli-
wiający ludzkie współistnienie”53. O ile więc polityczność ze swej istoty opiera się
na konflikcie, o tyle polityka ma na celu działania pozwalające na funkcjonowanie
wspólnoty politycznej pomimo agonizmu leżącego u podstaw każdego społeczeństwa. Z tych względów Ch. 54
Por. Ch. Mouffe, Agonistyka. Polityczne myślenie o świecie, przeł. B. Szelewa, Warszawa 2015 oraz
eadem, Paradoks demokracji, przeł. W. Jach, M. Kamińska, A. Orzechowski, Wrocław 2005.
55
Por Zob P Kelly Liberalizm Warszawa 2007 s 140–149 55
Por. Zob. P. Kelly, Liberalizm, Warszawa 2007, s. 140–149. g
y
y
y
eadem, Paradoks demokracji, przeł. W. Jach, M. Kamińska, A. Orzechowski, Wrocław 2005.
55
Por. Zob. P. Kelly, Liberalizm, Warszawa 2007, s. 140–149.
56
J. Rawls, Liberalizm polityczny, przeł. A. Romaniuk, Warszawa 1998, s. 33. Konstytucjonalizm a polityczność Mouffe można odczytać jako filozofkę biorącą na poważnie
ideę pluralizmu i możliwość jej urzeczywistnienia w ramach konstytucyjnej demo-
kracji i w tym sensie radykalizuje ona demokrację, podkreślając rolę sporu i prawa
do udziału w nim oraz krytykuje apele o konsens i opanowanie konfliktów54. nych51. Agonistyczny wymiar stosunków społecznych jest fundamentem politycz-
ności („tego, co polityczne”)52. Ch. Mouffe odróżnia jednak opartą na konflikcie
„polityczność” od „polityki” definiowanej jako „zestaw praktyk i instytucji, które
w obliczu wprowadzanego przez polityczność konfliktu tworzą porządek umożli-
wiający ludzkie współistnienie”53. O ile więc polityczność ze swej istoty opiera się
na konflikcie, o tyle polityka ma na celu działania pozwalające na funkcjonowanie
wspólnoty politycznej pomimo agonizmu leżącego u podstaw każdego społeczeństwa. Z tych względów Ch. Mouffe można odczytać jako filozofkę biorącą na poważnie
ideę pluralizmu i możliwość jej urzeczywistnienia w ramach konstytucyjnej demo-
kracji i w tym sensie radykalizuje ona demokrację, podkreślając rolę sporu i prawa
do udziału w nim oraz krytykuje apele o konsens i opanowanie konfliktów54. Przeciwstawna wizja polityczności wyłania się z teorii liberalizmu politycznego. Fakt niezgody (pluralizm) jest punktem wyjścia dla częściowego konsensu i akcep-
tacji podstawowych idei demokracji konstytucyjnej. Liberalizm polityczny zakłada,
że cały czas będziemy się różnić politycznie, jednakże będziemy afirmować zasady
konstytucji jako sprawiedliwe. Warto przy tym podkreślić, że nie powoduje to w kon-
sekwencji apolityczności liberalnych koncepcji, a jedynie uwypukla odmienne
podejście do rzeczywistości politycznej55. Problem pogodzenia faktu pluralizmu z ideą stworzenia stabilnego społeczeń-
stwa J. Rawls formułuje w następujący sposób: „jak jest możliwe, by istniało przez
czas dłuższy sprawiedliwe i stabilne społeczeństwo wolnych i równych obywateli,
których wciąż głęboko dzielą rozumne doktryny religijne, filozoficzne i moralne?”56. Odpowiedź Rawlsa na to pytanie wymaga wprowadzenia pojęcia rozumnego 55
Por. Zob. P. Kelly, Liberalizm, Warszawa 2007, s. 140–149. 56
J. Rawls, Liberalizm polityczny, przeł. A. Romaniuk, Warszawa 1998, s. 33 Tom 12, nr 3/2020 DOI: 10.7206/kp.2080-1084.395 Konstytucjonalizm a polityczność 63 pluralizmu (reasonable pluralism)57, czyli wyniku funkcjonowania „wolnego prak-
tycznego rozumu działającego w ramach wolnych instytucji”58. Rozumny pluralizm
(w przeciwieństwie do pluralizmu jako takiego) nawiązuje tylko do tych poglądów,
które spełniają postulat rozumności59. Zdaniem amerykańskiego filozofa „w ro-
zumnym społeczeństwie – najprościej rzecz ilustrując, w społeczeństwie osób,
które w podstawowych sprawach są równe – wszyscy mają własne racjonalne cele,
które mają nadzieję urzeczywistnić, i wszyscy gotowi są zaproponować sprawie-
dliwe warunki, co do których można rozumnie się spodziewać, że inni je przyjmą,
tak by wszyscy mogli czerpać korzyść i zwiększać możliwości własnego działania”60. 57
Termin reasonable pluralism jest też niekiedy tłumaczony w polskiej literaturze na „rozsądny plu-
ralizm”. Por. A. Szahaj, Jednostka czy wspólnota? Spór liberałów z komunitarystami a „sprawa polska”,
Warszawa 2000. Szerzej na temat rozumnego pluralizmu por. C. Błaszczyk, Rozumny pluralizm a ra-
cjonalistyczna tolerancja. Myśl Johna Rawlsa na tle filozofii Johna Locke’a, „Eryda” 2015, 1, s. 15–38. 58
Por. Ł. Dulęba, Liberalizm… s. 73 z powołaniem na J. Rawlsa, op. cit., s. 75.
59
Por. ibidem. 62
Należy przy tym podkreślić, że konstytucjonalizm populistyczny jest dość różnorodnym tego
zjawiskiem, głęboko uwikłanym w określone wymiary historyczne i kontekstowe (takie jak, na
przykład, transformacja postkomunistyczna oraz reaktywny stosunek do liberalnego konstytucjo-
nalizmu). Jak wskazują P. Blokker, B. Bugaric oraz G. Halmai, centralne wymiary zjawiska popu-
listycznego odnoszą się w szczególności do: (a) odmian populistycznego zaangażowania w kon-
stytucjonalizm, które, upraszczając, można sklasyfikować jako populizm lewicowy i prawicowy;
(b) głębszego rozumienia populistycznego sposobu myślenia w swoim podejściu do liberalizmu, 61
Por. F. Biały, op. cit., s. 228. 60
J. Rawls, op. cit., s. 97. 57
Termin reasonable pluralism jest też niekiedy tłumaczony w polskiej literaturze na „rozsądny plu-
ralizm”. Por. A. Szahaj, Jednostka czy wspólnota? Spór liberałów z komunitarystami a „sprawa polska”,
Warszawa 2000. Szerzej na temat rozumnego pluralizmu por. C. Błaszczyk, Rozumny pluralizm a ra-
cjonalistyczna tolerancja. Myśl Johna Rawlsa na tle filozofii Johna Locke’a, „Eryda” 2015, 1, s. 15–38.
58
Por. Ł. Dulęba, Liberalizm… s. 73 z powołaniem na J. Rawlsa, op. cit., s. 75.
59
Por. ibidem.
60
J. Rawls, op. cit., s. 97.
61
Por. F. Biały, op. cit., s. 228.
62
Należy przy tym podkreślić, że konstytucjonalizm populistyczny jest dość różnorodnym tego
zjawiskiem, głęboko uwikłanym w określone wymiary historyczne i kontekstowe (takie jak, na
przykład, transformacja postkomunistyczna oraz reaktywny stosunek do liberalnego konstytucjo-
nalizmu). Jak wskazują P. Blokker, B. Bugaric oraz G. Halmai, centralne wymiary zjawiska popu-
listycznego odnoszą się w szczególności do: (a) odmian populistycznego zaangażowania w kon-
stytucjonalizm, które, upraszczając, można sklasyfikować jako populizm lewicowy i prawicowy;
(b) głębszego rozumienia populistycznego sposobu myślenia w swoim podejściu do liberalizmu, 63
Warto podkreślić, że Ch. Mouffe mocno odróżnia populizm prawicowy i lewicowy. Jej zdaniem
prawicowy populizm jest odpowiedzią na konflikty, których polityczny wyraz został zablokowany
przez liberalną hegemonię. Prawicowy populizm jest w istocie wrogi demokratycznej emancypa-
cji, więc jeżeli chcemy zrealizować czy też uratować demokrację, odwołać się musimy do populizmu
lewicowego. Ta teza ma znaczenie dla odróżnienia stanowiska samej Ch. Mouffe od stanowiska
tych, którzy się na nią powołują. Por. Ch. Mouffe, W obronie lewicowego populizmu, Warszawa 2000. Konstytucjonalizm a polityczność Według Rawlsa, funkcjonowanie społeczeństwa w oparciu o zasady wspólnie sfor-
mułowanej koncepcji sprawiedliwości, zapewni akceptację instytucji przez obywa-
teli61. Współczesny liberalizm, stanowiąc normatywne ujęcie teorii polityki, wiąże
kategorię polityczności z kwestiami zasad legitymizacji ustroju politycznego, które
niewątpliwie posiadają swój moralny wymiar. Jaka jest relacja między przedstawionymi powyżej przeciwstawnymi ujęciami
polityczności a konstytucjonalizmem? Bez wątpienia kategoria polityczności może
pomóc w klasyfikacji poszczególnych koncepcji konstytucjonalizmu, zarówno
w płaszczyźnie normatywnej, jak i opisowej. Oczywisty jest związek między kon-
stytucjonalizmem liberalnym a liberalną formułą polityczności, w której pluralizm
wartości uznawany jest za impuls dla stworzenia stabilnego społeczeństwa równych
i wolnych obywateli. Wyjście poza liberalny paradygmat konstytucjonalizmu po-
stulowane przez jego krytyków oznacza więc również konieczność sformułowania
alternatywnej koncepcji polityczności. Na poziomie normatywnym jedną z takich
alternatyw jest agonistyczna polityczność Ch. Mouffe, najczęściej utożsamiana
z koncepcją konstytucjonalizmu populistycznego62. Pewne problemy wywołuje 62
Należy przy tym podkreślić, że konstytucjonalizm populistyczny jest dość różnorodnym tego
zjawiskiem, głęboko uwikłanym w określone wymiary historyczne i kontekstowe (takie jak, na
przykład, transformacja postkomunistyczna oraz reaktywny stosunek do liberalnego konstytucjo-
nalizmu). Jak wskazują P. Blokker, B. Bugaric oraz G. Halmai, centralne wymiary zjawiska popu-
listycznego odnoszą się w szczególności do: (a) odmian populistycznego zaangażowania w kon-
stytucjonalizm, które, upraszczając, można sklasyfikować jako populizm lewicowy i prawicowy;
(b) głębszego rozumienia populistycznego sposobu myślenia w swoim podejściu do liberalizmu, DOI: 10.7206/kp.2080-1084.395 Tom 12, nr 3/2020 Aleksandra Kustra-Rogatka 64 podział na konstytucjonalizm prawny i polityczny. Obie tradycje mieszczą się
w szeroko ujętym paradygmacie demokracji konstytucyjnej. Jednak analiza modelu
demokracji agonistycznej w ujęciu Ch. Mouffe i nawiązujących do niego głosów
aprobujących i konceptualizujących przemiany polityczne dokonujące się pod
wpływem współczesnych ruchów populistycznych63 prowadzi do wniosku, że kon-
stytucjonalizm populistyczny (na poziomie deskryptywnym) często nawiązuje do
normatywnego modelu konstytucjonalizmu politycznego, szczególnie w zakresie
określania istoty konstytucjonalizmu64. Charakterystyczne dla konstytucjonalizmu
politycznego utożsamienie istoty konstytucjonalizmu z ideą dynamicznego samo-
decydowania wspólnoty politycznej nie powinno być jednak mylone z dopusz-
czalnością dokonywania tzw. korekt ustrojowych poza dotychczasowymi ramami
konstytucyjnymi. Konstytucjonalizm polityczny zakłada konsensualną wizję poli-
tyczności, a konstytucję pojmuje jako zbiór zasad i instytucji, które pozwalają na
zarządzanie powstającymi konfliktami65. Tezy o politycznym charakterze konsty-
tucji oraz nieuchronności korekt ustrojowych poza ramami konstytucyjnymi (tzw. kontrkonstytucjonalizm)66 zdecydowanie bardziej wpisują się w schmittowską
teorię polityczną i charakterystyczne dla niej konfliktowe ujęcie polityczności. Poli-
tyczna teoria konstytucji C. Schmitta uznaje permanentny potencjał konfliktu za
niezbędny dla politycznego charakteru konstytucji. Potencjalne konflikty okazjo-
nalnie aktualizują się jako wyjątkowe (nadzwyczajne) naruszenia konstytucyjne. praworządności i konstytucjonalizmu; (c) stosunku konstytucjonalistów do konstytucjonalizmu
populistycznego; (d) złożonej relacji i nakładania się populizmu z illiberalizmem oraz autoryta-
ryzmem; (e) centralnego charakteru władzy konstytutywnej w projektach populistycznych. Por.
P. Blokker, B. Bugaric, G. Halmai, Introduction: Populist constitutionalism: Varieties, complexities, and
contradictions, „German Law Journal” 2019, 20(3, Special Issue), s. 298. 65
Por. P. Minkkinen, Political constitutionalism versus political constitutional theory: Law, power, and
politics, „International Journal of Constitutional Law” 2013, 11(3), s. 585. 64
Dostrzegalne jest to chociażby w charakterystycznym dla retoryki PiS podkreślaniu znaczenia
parlamentu jako gwaranta suwerenności narodu. praworządności i konstytucjonalizmu; (c) stosunku konstytucjonalistów do konstytucjonalizmu
populistycznego; (d) złożonej relacji i nakładania się populizmu z illiberalizmem oraz autoryta-
ryzmem; (e) centralnego charakteru władzy konstytutywnej w projektach populistycznych. Por.
P. Blokker, B. Bugaric, G. Halmai, Introduction: Populist constitutionalism: Varieties, complexities, and
contradictions, „German Law Journal” 2019, 20(3, Special Issue), s. 298. 66
Por. A. Czarnota, Populistyczny konstytucjonalizm czy nowy konstytucjonalizm?, „Krytyka Prawa” 2019,
11(1), s. 39–40. 67
Por. P. Minkkinen, op. cit., s. 585. 68
Por. ibidem, s. 608. Trzeba jednak dodać, że autor przywołanego artykułu krytykuje koncepcję
konstytucjonalizmu politycznego jako zbyt sztywną. Stwierdza, że zobowiązując się normatyw-
nie do instytucji, zasad i wartości liberalnej demokracji, konstytucjonalizm polityczny sytuuje
je poza zasięgiem konstytutywnej władzy, której konstytucja zawdzięcza swoje istnienie. Zdaniem
P. Minkkinena to polityczna teoria konstytucyjna w ujęciu C. Schmitta pozwala na przyjęcie tezy,
że konstytucja jest faktycznym wyrazem nieograniczonego politycznego samookreślenia. 69
N. Walker, op. cit., s. 515. Konstytucjonalizm a polityczność Naruszenia te w ujęciu Schmitta paradoksalnie potwierdzają suwerenną władzę
konstytuującą stanowiącą podstawę radykalnie demokratycznych fundamentów
wszystkich ustanowionych instytucji politycznych i prawnych67. W tym aspekcie praworządności i konstytucjonalizmu; (c) stosunku konstytucjonalistów do konstytucjonalizmu
populistycznego; (d) złożonej relacji i nakładania się populizmu z illiberalizmem oraz autoryta-
ryzmem; (e) centralnego charakteru władzy konstytutywnej w projektach populistycznych. Por. P. Blokker, B. Bugaric, G. Halmai, Introduction: Populist constitutionalism: Varieties, complexities, and
contradictions, „German Law Journal” 2019, 20(3, Special Issue), s. 298. 63 64
Dostrzegalne jest to chociażby w charakterystycznym dla retoryki PiS podkreślaniu znaczenia
parlamentu jako gwaranta suwerenności narodu. 64
Dostrzegalne jest to chociażby w charakterystycznym dla retoryki PiS podkreślaniu znaczenia
parlamentu jako gwaranta suwerenności narodu. 65
Por. P. Minkkinen, Political constitutionalism versus political constitutional theory: Law, power, and
politics, „International Journal of Constitutional Law” 2013, 11(3), s. 585. 67
Por. P. Minkkinen, op. cit., s. 585. Tom 12, nr 3/2020 DOI: 10.7206/kp.2080-1084.395 Konstytucjonalizm a polityczność Konstytucjonalizm a polityczność 65 konstytucjonalizm polityczny nie jest wcale tak różny od konstytucjonalizmu
prawnego. Bazując na liberalnym ujęciu polityczności, nie legitymizuje „kontrkon-
stytucyjnych korekt ustrojowych”. W ramach paradygmatu konstytucjonalizmu
liberalnego są to zwykłe naruszenia konstytucji68. Warto przy tym podkreślić, że na
poziomie deskrypcji ustroje państw, które najczęściej są punktem odniesienia dla
zwolenników konstytucjonalizmu politycznego, okazują się być wyjątkowo sztywne
i odporne na zmiany konstytucje. Charakterystyczny dla współczesnego dyskursu o konstytucjonalizmie zabieg
etykietowania (labelling) doprowadził do powstania gęstej dżungli „przymiotniko-
wych konstytucjonalizmów”, wykraczających poza klasyczny liberalny paradygmat. Tworzone koncepcje zmierzają do sprecyzowania zmian zachodzących w prawie
publicznym oraz jego otoczeniu politycznym. Jednym z dostrzegalnych przeobrażeń
jest niedawne odrodzenie się populizmu, które stanowi dziś poważne wyzwanie
dla prawa konstytucyjnego i głębszej tradycji współczesnego konstytucjonalizmu69. Jak stwierdza N. Walker, odrębność orientacji konstytucyjnej populizmu opiera się
na zaanektowaniu przestrzeni między konstytucjonalizmem autorytarnym a ludo-
wym (czerpiącym de facto z fundamentalnych założeń konstytucjonalizmu poli-
tycznego). Usytuowany tam populizm jest reakcją na niedoskonałości paradygmatu
konstytucjonalizmu liberalnego. A wspomniana na wstępie niestabilna równowaga
między różnymi dobrami konstytucyjnymi (pluralizm–jedność, indywidualizm– i wych konstytucjonalizmów”, wykraczających poza klasyczny liberalny paradygmat. Tworzone koncepcje zmierzają do sprecyzowania zmian zachodzących w prawie
publicznym oraz jego otoczeniu politycznym. Jednym z dostrzegalnych przeobrażeń
jest niedawne odrodzenie się populizmu, które stanowi dziś poważne wyzwanie
dla prawa konstytucyjnego i głębszej tradycji współczesnego konstytucjonalizmu69. Jak stwierdza N. Walker, odrębność orientacji konstytucyjnej populizmu opiera się
na zaanektowaniu przestrzeni między konstytucjonalizmem autorytarnym a ludo-
wym (czerpiącym de facto z fundamentalnych założeń konstytucjonalizmu poli-
tycznego). Usytuowany tam populizm jest reakcją na niedoskonałości paradygmatu
konstytucjonalizmu liberalnego. A wspomniana na wstępie niestabilna równowaga
między różnymi dobrami konstytucyjnymi (pluralizm–jedność, indywidualizm–
–kolektywizm, uniwersalizm–partykularyzm) pogłębia definiujące napięcie współ-
czesnego konstytucjonalizmu liberalnego i stanowi wyzwanie dla wszystkich,
którzy go popierają70. Niemniej zgadzam się z P. Blokkerem, który twierdzi, że
chociaż populistyczna krytyka liberalnego konstytucjonalizmu zapewnia istotny
wgląd w problemy strukturalne liberalnej demokracji, populistyczny konstytucjo-
nalizm ostatecznie nie spełnia swojej demokratycznej obietnicy. Populizm odwo-
łuje się do zasad demokracji, jednak wyciągając z nich ekstremalne, jednostronne
wnioski, narusza kluczowe wymiary demokratycznego konstytucjonalizmu, takie –kolektywizm, uniwersalizm–partykularyzm) pogłębia definiujące napięcie współ-
czesnego konstytucjonalizmu liberalnego i stanowi wyzwanie dla wszystkich,
którzy go popierają70. Niemniej zgadzam się z P. Blokkerem, który twierdzi, że
chociaż populistyczna krytyka liberalnego konstytucjonalizmu zapewnia istotny
wgląd w problemy strukturalne liberalnej demokracji, populistyczny konstytucjo-
nalizm ostatecznie nie spełnia swojej demokratycznej obietnicy. 70
Ibidem. 69
N. Walker, op. cit., s. 515.
70
Ibidem. 71
P. Blokker, Populism as Constitutional Project, „International Journal of Constitutional Law” 2019,
17(2), s. 535–536. Konstytucjonalizm a polityczność Populizm odwo-
łuje się do zasad demokracji, jednak wyciągając z nich ekstremalne, jednostronne
wnioski, narusza kluczowe wymiary demokratycznego konstytucjonalizmu, takie –kolektywizm, uniwersalizm–partykularyzm) pogłębia definiujące napięcie współ-
czesnego konstytucjonalizmu liberalnego i stanowi wyzwanie dla wszystkich,
którzy go popierają70. Niemniej zgadzam się z P. Blokkerem, który twierdzi, że
chociaż populistyczna krytyka liberalnego konstytucjonalizmu zapewnia istotny
wgląd w problemy strukturalne liberalnej demokracji, populistyczny konstytucjo-
nalizm ostatecznie nie spełnia swojej demokratycznej obietnicy. Populizm odwo-
łuje się do zasad demokracji, jednak wyciągając z nich ekstremalne, jednostronne
wnioski, narusza kluczowe wymiary demokratycznego konstytucjonalizmu, takie DOI: 10.7206/kp.2080-1084.395 Tom 12, nr 3/2020 Aleksandra Kustra-Rogatka 66 jak pluralizm, inkluzja społeczna oraz faktyczne obywatelskie zaangażowanie
w konstytucjonalizm71. y
j
Liczne odmiany „przymiotnikowych konstytucjonalizmów” dowodzą również,
jak złożona jest relacja między konstytucjonalizmem (zarówno na poziomie opiso-
wym, jak i normatywnym) a politycznością. Z jednej strony kategoria polityczności
pomaga uporządkować pojęciowy i metodologiczny chaos, wynikający z mnożenia
się kolejnych typów konstytucjonalizmu. Z drugiej strony rozróżnienie konflik-
towych oraz konsensualnych ujęć polityczności nie zawsze w pełni oddaje różnice
między paradygmatycznym konstytucjonalizmem liberalnym a jego „przymiot-
nikowymi odmianami”. Dwie podstawowe tradycje konstytucjonalizmu – prawna
i polityczna – pomimo położenia nacisku na odmienne idee (samoograniczenie
suwerena versus demokratyczne samostanowienie) należy łączyć z konsensualną
wizją polityczności. W przypadku konstytucjonalizmu prawnego konsens ten ma
wymiar formalny, spisany i tym samym, przynajmniej w założeniu, bardziej stabilny. Z kolei konstytucjonalizm polityczny konceptualnie dopuszcza większą dynamikę
w ramach szeroko ujętego konstytucyjnego konsensusu. Być może jedną z przyczyn
współczesnego kryzysu konstytucjonalizmu liberalnego jest pojmowana zbyt
statycznie idea częściowego konsensu. Koncepcje konstytucjonalizmu podkreśla-
jące konfliktowy charakter polityczności zmieniają ten element konstytucyjnego
krajobrazu współczesności, niemniej na poziomie opisowym przynajmniej konsty-
tucjonalizm populistyczny de facto bardziej współgra z radykalną wizją politycz-
ności C. Schmitta niż adwersaryjną koncepcją Ch. Mouffe. Kategoria polityczności
pokazuje, że konstytucjonalizm liberalny, aby przetrwać coraz widoczniejszy kryzys,
wymaga pewnej samokorekty i wyjścia poza, wspomniane na wstępie, polemicz-
nie zdemoralizowanie oraz sytuacyjną obojętność. Z kolei – analizowane z poziomu
deskrypcji – zjawisko konstytucjonalizmu populistycznego jest, przynajmniej
obecnie, raczej symptomem choroby niż antidotum na niedostatki liberalnego
paradygmatu. Alejandro R., What Is Political about Rawls’s Political Liberalism?, „The Journal of Politics”
1996, 58(1), s. 1–25. Atienza M., Konstytucja, globalizacja i prawo, https://www.rpo.gov.pl/pliki/12421353110.pdf
(dostęp: 10.01.2020). Arendt H., Wprowadzenie w politykę, [w:] H. Arendt, Polityka jako obietnica, Warszawa 2007. Konstytucjonalizm a polityczność Konstytucjonalizm a polityczność 67 Baranowski M., W stronę konfliktowego modelu polityczności, [w:] M. Baranowski (red.)
Demokracja i rola obywatela. O napięciu pomiędzy państwem, społeczeństwem i procesami
globalizacyjnymi, Poznań 2014. Bekrycht T., Republikanizm i liberalizm w świetle idei konstytucjonalizmu. Uwagi o legitymi-
zacji prawa, „Filozofia Publiczna i Edukacja Demokratyczna” 2018, 7(1), s. 26–39. Bellamy R., Political Constitutionalism. A Republican Defence of the Constitutionality of Demo
cracy, Cambridge 2007.l Biały F., Konflikt jako wartość? Demokracja agonistyczna a populizm europejski w ujęciu Chantal
Mouffe, „Refleksje” 2010, 1. Blokker P., Populism as Constitutional Project, „International Journal of Constitutional Law”
2019, 17(2). Blokker P., New Democracies in Crisis? A Comparative Constitutional Study of the Czech
Republic, Hungary, Poland, Romania and Slovakia, Routledge 2015. Blokker P., New Democracies in Crisis? A Comparative Constitutional Study of th Blokker P., New Democracies in Crisis? A Comparative Constitutional Study of the Czech
Republic, Hungary, Poland, Romania and Slovakia, Routledge 2015. Republic, Hungary, Poland, Romania and Slovakia, Routledge 2015. Blokker P., Bugaric B., Halmai G., Introduction: Populist constitutionalism: Varieties, complexi
ties, and contradictions, „German Law Journal” 2019, 20(3) Special Issue. Błaszczyk C., Rozumny pluralizm a racjonalistyczna tolerancja. Myśl Johna Rawlsa na tle
filozofii Johna Locke’a, „Eryda” 2015, 1, s. 15–38. Canetti E., Masa i władza, Warszawa 1996. Chang W.-C., Back into the political? Rethinking judicial, legal, and transnational constitutio
nalism, „International Journal of Constitutional Law” 2019, 17(2), s. 453–460. Czarnota A., Populistyczny konstytucjonalizm czy nowy konstytucjonalizm?, „Krytyka Prawa”
2019, 1, s. 27–42. Czarnota A., Stambulski M., Janusowe oblicze konstytucjonalizmu, „Krytyka Prawa” 2019,
11(1). Drinóczi T., Bień-Kacała A., Illiberal Constitutionalism: The Case of Hungary and Poland,
„German Law Journal” 2019, 20(8), s. 1140–1166. Dulęba Ł., Michael Freeden: liberalizm i ideologia, „Przegląd Polityczny” 2015, 12.l Dulęba Ł., Liberalizm jako forma ucieczki od polityczności, „Refleksje” 2012, 6. Elkins Z., Ginsburg T., Melton J., The content of authoritarian constitutions, [w:] T. Gins-
burg, Simpser A. (red.), Constitutions in Authoritarian Regimes, Cambridge 2013,
s. 141–164. Graber M.A., Levinson S., Tushnet M. (red.), Constitutional Democracy in Crisis?, Oxford
2018. Kelly P., Liberalizm, Warszawa 2007. Korycka-Zirk M., Filozoficznoprawny wymiar kontroli konstytucyjności, Toruń 2017. Krischa N., Beyond Constitutionalism: The Pluralist Structure of Postnational Law, Oxfo Krischa N., Beyond Constitutionalism: The Pluralist Structure of Postnational Law, Oxford 2011. Król M., Filozofia polityczna, Kraków 2008. Król M., Filozofia polityczna, Kraków 2008. Król M., Filozofia polityczna, Kraków 2008. Landau D., Abusive Constitutionalism, „UC Davis Law Review” 2013, 47, s. Bibliografia Alejandro R., What Is Political about Rawls’s Political Liberalism?, „The Journal of Politics”
1996, 58(1), s. 1–25. Arendt H., Wprowadzenie w politykę, [w:] H. Arendt, Polityka jako obietnica, Warszawa 2007. Arendt H., Wprowadzenie w politykę, [w:] H. Arendt, Polityka jako obietnica, Warszawa 2007. Atienza M., Konstytucja, globalizacja i prawo, https://www.rpo.gov.pl/pliki/12421353110.pdf
(dostęp: 10.01.2020). Atienza M., Konstytucja, globalizacja i prawo, https://www.rpo.gov.pl/pliki/12421353110.pdf
(dostęp: 10.01.2020). 71
P. Blokker, Populism as Constitutional Project, „International Journal of Constitutional Law” 2019,
17(2), s. 535–536. DOI: 10.7206/kp.2080-1084.395 Tom 12, nr 3/2020 Konstytucjonalizm a polityczność 189–260.i Mańko R., Orzekanie w polu polityczności, „Filozofia Publiczna i Edukacja Demokratyczna”
2008, 1. DOI: 10.7206/kp.2080-1084.395 Tom 12, nr 3/2020 Aleksandra Kustra-Rogatka 68 Minkkinen P., Political constitutionalism versus political constitutional theory: Law, power,
and politics, „International Journal of Constitutional Law” 2013, 11(3). Minkkinen P., Political constitutionalism versus political constitutional theory: Law, power,
and politics, „International Journal of Constitutional Law” 2013, 11(3). Mouffe Ch., Demokratyczna polityka musi być stronnicza, https://krytykapolityczna.pl/
kraj/mouffe-demokratyczna-polityka-musi-byc-stronnicza/, 21.02.2015 (dostęp:
17.03.2020). Mouffe Ch., Agonistyka. Polityczne myślenie o świecie, przeł. B. Szelewa, Warszawa 2015. Mouffe Ch., Polityczność. Przewodnik Krytyki Politycznej, przeł. J. Erbel, Warszaw Mouffe Ch., Paradoks demokracji, przeł. W. Jach, M. Kamińska, A. Orzechowski, Wrocław
2005. Mouffe Ch., W obronie lewicowego populizmu, Warszawa 2000. Mouffe Ch., Laclau E., Hegemonia i socjalistyczna strategia. Przyczynek do projektu radykalnej
polityki demokratycznej, Wrocław 2007. Müller J.-W., What is Populism?, Philadelphia, PA 2016. Newey G., After Politics. The Rejection of Politics in Contemporary Liberal Philosophy, Basing
stoke 2001. Oklopic Z., Imagined ideologies: Populist figures, liberalist projections, and the horizons of con-
stitutionalism, „German Law Journal” 2019, 20. Rawls J., Liberalizm polityczny, przeł. A. Romaniuk, Warszawa 1998. Schmitt C., Pojęcie polityczności, [w:] C. Schmitt, Teologia polityczna i inne pisma, Warszawa
2012. Schmitt C., Teologia polityczna i inne pisma, Kraków 2000. Skąpska G., Znieważający konstytucjonalizm i konstytucjonalizm znieważony. Refleksja socjo-
logiczna na temat kryzysu liberalno-demokratycznego konstytucjonalizmu w Europie poko-
munistycznej, „Filozofia Publiczna i Edukacja Demokratyczna” 2018, 1, s. 276–301. Stambulski M., Nowa gra w mieście. Polityczny konstytucjonalizm jako krytyka dogmatyki
konstytucyjne, [w:] Jaki konstytucjonalizm? Refleksje nad „New Democracies in Cirsis”
Paula Blokkera, „Politicon” I, http://www.bibliotekacyfrowa.pl/Content/92238/politi-
con_I_Jaki_konstytucjonalizm_Refleksje_nad_New_Democracies_in_Crisis_Paula_
Blokkera.pdf (dostęp: 21.02.2020). Stopler G., Semi-liberal constitutionalism, „Global Constitutionalism” 2019, 8(1), s. 94–122. Sulikowski A., Kryzys nowoczesnego konstytucjonalizmu. Między liberalną sędziokracją
a postliberalnym populizmem, „Filozofia Publiczna i Edukacja Demokratyczna” 2018,
7(1), s. 328–340. Szahaj A., Jednostka czy wspólnota? Spór liberałów z komunitarystami a „sprawa polska”,
Warszawa 2000. Teubner G., Constitutional Fragments: Societal Constitutionalism and Globalization, Oxford
2012. Tushnet M., Varieties of constitutionalism (editorial), „International Journal of Constitutional
Law” 2016, 14(1). Tushnet M., Authoritarian Constitutionalism, „Cornell Law Review” 2015, 100(2),
s. 393–461. Tom 12, nr 3/2020 DOI: 10.7206/kp.2080-1084.395 Konstytucjonalizm a polityczność Konstytucjonalizm a polityczność 69 Tushnet M., Authoritarian constitutionalism, [w:] T. Ginsburg, A. Simpser (red.), Constitu
tions in Authoritarian Regimes, Cambridge 2013, s. 36–49. Tushnet M., Authoritarian constitutionalism, [w:] T. Ginsburg, A. Simpser (red.), Constitu
tions in Authoritarian Regimes, Cambridge 2013, s. 36–49. Tushnet M., Popular Constitutionalism as Political Law, „Chicago–Kent Law Review” 2006,
81, s. 991–1006. Walker N., Populism and Constitutional Tension, „International Journal of Constitutional
Law” 2019, 17(2), s. 515–535. ,
( ),
Włoch W., Pomiędzy czystym prawem a ideą polityczną, Toruń 2018. Włoch W., Pomiędzy czystym prawem a ideą polityczną, Toruń 2018. Tom 12, nr 3/2020 DOI: 10.7206/kp.2080-1084.395 Tom 12, nr 3/2020
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Restoring the Soils of Nauru: Plants as Tools for Ecological Recovery
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Restoring the soils of Nauru
Plants as tools for Ecological Recovery
by
Alexander Feary A thesis in partial fulfilment for a degree in Ecological
Restoration at Victoria University of Wellington, New
Zealand. A thesis in partial fulfilment for a degree in Ecological
Restoration at Victoria University of Wellington, New
Zealand. Abstract The restoration of Nauru’s mined areas is fundamental to the future
wellbeing of the people and ecosystems of Nauru. Extensive open cast
phosphate mining on Nauru over the last 100 years has led to soil losses and
landscape degradation to the extent that over 70% of this South-Western
Pacific island state is now uninhabitable and almost all productive land has
been lost. Significant landscape degradation has occurred and as a
consequence the soils that remain are insufficient in volume and quality to
achieve the Government’s restoration goals which support the long-term
development of Nauru and the well-being of its people. The aim of this
research is to evaluate aspects of cover-crop use as a means for soil
restoration in Nauru. This research evaluates biomass production,
phytoremediation potential, and germination success for a range of species
in Nauruan soils. Field trials exploring biomass production and cadmium
phytoextraction were performed, as was an experiment assessing the effects
of cadmium on germination success. It was found that, in the circumstances
assessed, biomass productivity was significantly determined by species,
mulch use, soil type, and to a small degree – cadmium. Phytoextraction was
significantly determined by tissue type. Germination success was not
determined by soil cadmium, but soil type was a significant factor. i ii ii Acknowledgements I have been extremely fortunate to have had the support of a large
number of generous and brilliant people while working on this
report. My supervisors Dr Murray Williams and Dr Sean Weaver
have provided invaluable support. Xavier Barker and Alamanda Lauti of the Nauru branch of the
University of the South Pacific provided sponsorship, resources,
and accommodated me in their homes. For this I am deeply
indebted to them, and to their families. George Fraser, Australian Consul-General and Special
Representative to Nauru gave me time and personal introductions. The following Nauru government officials met with me at various
times and granted me the benefit of their insight: Minister of
Education Rus Kun; Minister of Foreign Affairs and Trade Dr
Kieran Keke; Minister of Commerce, Industry and Enterprise (CIE),
Minister for Rehabilitation, and Minister for Mining Frederick
Pitcher; and Director for Agriculture, CIE, Frankie Ribauw. The following NRC staff members provided information and
direction - John Gray – Project Engineer, Lahn Debao – NRC
Surveyor, Creedance Halstead – NRC Assistant Project Engineer,
Yanjol Issacs – Planning Engineer. The Freemasons and NZAid provided financial support, without
which I would have been unable to visit Nauru. Credit must go to the lovely support staff of the School of Biological
Sciences at Victoria University, who keep our department together. iii Dr Joel Baker and Dr Marc-Alban Millet provided ICP-MS facilities
and technical support for sample analysis. Warmest thanks to those who reviewed and proofed this report-
Connie May Nisbett, Elise Ruth Broadbent, and Thomas Brydone
Taptiklis. Thom’s parsing was brutal. Your meticulous attention to
detail has significantly improved the clarity of my writing. I was extremely fortunate to have worked with the Nursery Staff of
the Nauru Rehabilitation Corporation. I would particularly like to
thank Harry Duncan Stroggoffadam, Lio Eauwe Agadio, Frangipani
Detogia, Timex Dabwido, Jeriel Jeremiah, Gideon Teabuge,
Maxwell Kosam, Louisiana Grundler, the Nursery Foreman Noah
Telani and Assistant Nursery Foreman Justin Fine Olsson. The time
that we spent together has been for me the most memorable part of
this thesis. I was extremely fortunate to have worked with the Nursery Staff of
the Nauru Rehabilitation Corporation. I would particularly like to
thank Harry Duncan Stroggoffadam, Lio Eauwe Agadio, Frangipani
Detogia, Timex Dabwido, Jeriel Jeremiah, Gideon Teabuge, To my friends and family who have listened to me ramble on about
Nauru ad absurdum, thank you for your tolerance and kind support. Acknowledgements iv iv Table of Contents
Abstract ............................................................................................... i
Acknowledgements ......................................................................... iii
Table of Contents .............................................................................. v
Terms and Abbreviations .................................................................xi
Chapter One - Introduction and background to the
rehabilitation of the soils of Nauru ...................................................... 1
Introduction ........................................................................................................... 1
Discovery of Phosphate Deposits ..................................................................... 2
Nauru Land Rehabilitation ................................................................................. 4
Experimental planning and design .......................................................... 6
Soil ........................................................................................................................... 7
Climate .................................................................................................................. 9
Freshwater ........................................................................................................... 10
Mandate to rehabilitate ............................................................................... 11
Ecological restoration ................................................................................... 14
Soil restoration ................................................................................................... 17
Ecological relevance ........................................................................................ 17
Nauru soil ecology ............................................................................................. 17
Increasing soil quantity and quality ............................................................... 18
Biomass production ........................................................................................... 22
Phytoremediation of soils ................................................................................. 26
Germination ........................................................................................................ 32
Adaptive management ............................................................................... 32
Research approach and thesis content .............................................. 34
Biomass production in Nauru ........................................................................... 35
Cadmium phytoremediation .......................................................................... 36 v Effects of cadmium on germination success ............................................... 36 . 36 vi
Effects of cadmium on germination success ............................................... 36
References .......................................................................................................... 36
Chapter Two - Biomass production in Nauru: A preliminary
field trial ................................................................................................................. 53
Abstract ................................................................................................................ 53
Introduction ........................................................................................................ 53
Aim .......................................................................................................................... 58
Hypotheses ......................................................................................................... 58
Method .................................................................................................................. 58
Variables ............................................................................................................. 59
Complications .................................................................................................... 61
Field plots ............................................................................................................ 62
Sample Preparation and Analysis .................................................................. 66
Results .................................................................................................................... 70
Soil cadmium ...................................................................................................... 70
Biomass productivity ......................................................................................... 72
Biomass productivity of specific soil types .................................................... 75
Discussion ............................................................................................................ 78
Research outcomes .......................................................................................... 78
Research limitations .......................................................................................... 80
Relevance to academic literature ................................................................ 80
Conclusion .......................................................................................................... 81
References .......................................................................................................... 82
Chapter Three - Cadmium phytoaccumulation in Nauruan
plants: A preliminary field trial .......................................................... 87
Abstract ................................................................................................................ 87 vi Introduction ......................................................................................................... 87
Aim .......................................................................................................................... 93
Hypotheses .......................................................................................................... 93
Method .................................................................................................................. 93
Variables .............................................................................................................. 93
Complications .................................................................................................... 96
Field trials ............................................................................................................. 97
Sample Preparation and Analysis ................................................................ 101
Results ................................................................................................................. 106
Soil cadmium ................................................................................................... 106
Cadmium phytoaccumulation .................................................................... 107
Discussion .......................................................................................................... 111
Research outcomes ....................................................................................... 111
Research limitations ........................................................................................ 112
Relevance to academic literature ............................................................. 113
Conclusion ....................................................................................................... 114
References ........................................................................................................ 116
Chapter Four - Germination success in Nauruan soils: The
effects of different cadmium bearing soils on selected species
.................................................................................................................. 123
Abstract .............................................................................................................. 123
Introduction ...................................................................................................... 123
Aim ....................................................................................................................... 125
Hypotheses ....................................................................................................... 125
Method ............................................................................................................... 125
Variables ........................................................................................................... 125
Experimental process ..................................................................................... 128 vii Complications .................................................................................................. 129
Sample Analysis ............................................................................................... 129
Results ................................................................................................................. 133
Soil cadmium .................................................................................................... 133
Analysis of germination success ................................................................... 135
Discussion ......................................................................................................... 139
Research outcomes ........................................................................................ 139
Research limitations ........................................................................................ 139
Relevance to other studies ............................................................................ 141
Conclusion ........................................................................................................ Acknowledgements 142
References ....................................................................................................... 144
Chapter Five - Discussion. ................................................................ 149
Introduction ..................................................................................................... 149
Results ................................................................................................................. 149
Seedbank .......................................................................................................... 149
Biomass .............................................................................................................. 149
Cadmium .......................................................................................................... 150
Germination ..................................................................................................... 150
Significance to rehabilitation ................................................................. 151
Seedbank .......................................................................................................... 151
Biomass .............................................................................................................. 152
Cadmium .......................................................................................................... 153
Germination ..................................................................................................... 155
Soil stockpiles .................................................................................................... 156
Cadmium waste .............................................................................................. 158
Ecological restoration ..................................................................................... 159
Adaptive management ................................................................................. 162
Concluding discussion
165 Freshwater ........................................................................................................ 166
Community restoration .................................................................................. 166
Tension between mining and rehabilitation .............................................. 167
Buada lagoon ................................................................................................. 167
Biochar .............................................................................................................. 168
Composting toilets .......................................................................................... 169
Human resources ............................................................................................ 170
Recommendations ....................................................................................... 170
References ........................................................................................................ 172 ix x x Terms and Abbreviations Black soil - Soil removed from the surface of mining areas which
has been stockpiled for land rehabilitation. Bottomside - The coastal fringe of land surrounding Topside. This
is where most of the islands occupants reside. Cover crop – A crop which is planted to cover and protect the soil
beneath Ecological restoration – The process of modifying landscapes from
a degraded ecological state into a more desired state; usually a
historic state. Ecosystems functions - Exchanges of energy and nutrients within
ecosystems, involving decomposition and production of biomass. Ecosystem services - Services to humanity provided by ecosystems
functions. Green manure – A crop which is grown to provide biomass,
smother weeds, improve soil tilth, and possibly for nitrogen
fixation. Green mulch – A crop which is grown to suppress weeds, reduce
soil erosion, enhance soil fertility, improve water infiltration and
maintain soil moisture. Hyperaccumulator – A plant species that accumulates high
concentrations of soil toxins in its tissues. For cadmium the critical xi xi value above which a species is regarded as a hyperaccumulator is
100ppm. ICP-MS - Inductively coupled mass spectrometry. ICP-MS provides
accurate assessment of rare elements. Karrenfield - An undulating landscape of vertical pillars of
fossilised coral. Karrenfield - An undulating landscape of vertical pillars of
fossilised coral. Land rehabilitation - The process of modifying landscapes so that
they are suitable for human habitation. Mollisol – A soil type containing high concentrations of organic
matter. Considered to be the most productive soil for agriculture NRC - Nauru Rehabilitation Corporation. NPC - Nauru Phosphate Corporation. Phytoaccumulation – The accumulation of contaminants in plant
tissues. Phytoaccumulation – The accumulation of contaminants in plant
tissues. Phytoextraction - The uptake by plants of contaminants from soil. Phytoremediation – The use of plants to reduce the toxic potential
of contaminants. Species - The term is used in two ways within this document. The
first refers to the biological definition of the word, to describe a
genetically distinct grouping of organisms. The other is used in the
chemical sense, to describe the diversity of chemical forms in which
elements (in this case cadmium) can exist. xii SOM – Soil Organic Matter - The total biologically derived organic
material found in or on the soil, excluding the aboveground portion
of live plants. Terms and Abbreviations Topsoil - The raised plateau in central Nauru which is occupied by
the phosphate mine Topsoil - The raised plateau in central Nauru which is occupied by
the phosphate mine USP – University of the South Pacific. USP – University of the South Pacific. xiii xiv Introduction & Background INTRODUCTION Open-cast mining of phosphate rock on Nauru has directly affected
over 70 per cent of the land surface area of the island, in particular
the raised central plateau known as Topside (Ferguson 1999). Topside has incurred the greatest impacts due to open-cast mining
of the majority of the area. Topside comprises a raised plateau
which is physically distinct from the coastal fringe area known as
‘Bottomside’ where the majority of the population resides (Manner,
Thaman et al. 1984; Gowdy and McDaniel 1999). Open-cast mining of phosphate rock on Nauru has directly affected
over 70 per cent of the land surface area of the island, in particular
the raised central plateau known as Topside (Ferguson 1999). Before mining began, Topside was thickly covered with tropical
rainforest dominated in parts by the Pacific mahogany (Ijo or
Tomano) tree (Callophyllum inophyllum), or pandanus (Pandanus
tectorius) (Manner 1984; Thaman 1992; Thaman, Fosberg et al. 1994;
Thaman, Hassall et al. 2008). Throughout most of the early 20th
century Topside remained relatively undeveloped, despite a clear
delineation of land ownership through inheritance, conquest, and
customary use (Barker 2009). The land was managed by an
agroforestry system, whereby productive tree species were planted,
tended and cultivated within an environment that was otherwise
largely unmodified (Weeramantry 1992). Nauru is a 21km2 raised limestone island 41km south of the equator
at longitude 166° 56’ E in the Central Pacific (Fig. 1.1). It is
surrounded by a fringing coral reef that sharply drops away 4,300m
to the Pacific floor. ‚Bottomside‛ is the local name for the thin 1 1 Introduction & Background coastal plain surrounding the interior of the island, known as
‚Topside‛. Topside is a 1,620ha raised plateau that ranges from 20-
60m in elevation, and is largely divided from Bottomside by a steep
escarpment. A brackish lagoon, called ‚Buada‛ is in a large interior
depression, and is hydraulically connected with the Pacific Ocean
(Hunter, Harris et al. 1994; Kingston, P.A. 2004) (See Appendices I & II). Figure 1.1 – Map of Nauru’s position in the Pacific Ocean Figure 1.1 – Map of Nauru’s position in the Pacific Ocean Discovery of Phosphate Deposits In 1886, Germany annexed the island. In 1900, Nauru was
discovered to have phosphate of a quality surpassing all other
sources then known (Gowdy and McDaniel 1999). By this time
phosphate rock had come to be a valuable commodity due the
following factors: its value as an essential macronutrient for plant
based agriculture; the development of superphosphate
(monocalcium phosphate), by treating rock phosphate with
sulphuric acid thereby significantly increasing its availability to
plants; and (laterly) aerial topdressing, which made widespread
application of fertilisers economically feasible. With the phosphate 2 2 Introduction & Background discovery of 1900, Nauru suddenly gained international
prominence. discovery of 1900, Nauru suddenly gained international
prominence. From 1901 Germany proceeded to exploit the phosphate deposits
(Weeramantry 1992). Meagre compensation was given to the
Nauruan people in exchange for the extraction of what was a very
valuable commodity (Weeramantry 1992; Fagence 1996). German rule
in Nauru came to an end during World War I. After World War I,
governance of Nauru was passed to the League of Nations. In 1919,
at the Paris Peace Conference the island was entrusted to joint
administration by the United Kingdom, Australia and New
Zealand. Later that year, the Nauru Island Agreement was signed
entitling the three administrative powers to the phosphate of Nauru
at the cost of extraction rather than the international market price. After WWII governance of Nauru was passed to the UN, who re-
established the UK, NZ and Australian administration (Drummond
1921; Macdonald 1988; Weeramantry 1992; CIA 2007). On the 31st of
January 1968, Nauru gained independence and became a sovereign
state (Weeramantry 1992). From this point, phosphate extraction was
nationalised and the profits from the continued exports provided
the Nauruan government with the vast majority of its revenue
(Fagence 1996; Gowdy & McDaniel 1999). By 2005, 100 million tonnes
of phosphate had been removed from Topside, Nauru (Morrison &
Manner 2005). Unlike other Small Island Developing States (SIDS) that have been
reliant on international aid to a greater or lesser extent, Nauru was
able to be economically self sustaining. Exploitation of Nauruan
phosphate rock might have allowed for the state to achieve a form 3 Introduction & Background of economic sustainability were the mining profits effectively
managed, however the capital has been substantially eroded (Gowdy
& McDaniel 1999). Discovery of Phosphate Deposits Open-cast mining of phosphate has resulted in substantially
compromised ecosystems services, radically transforming the
majority of the land of Nauru into an inhospitable landscape no
longer suitable for human use (Manner et al. 1984). Bottomside and
Buada are the only currently inhabitable and arable areas on the
island. They are densely populated and cannot provide sufficient
ecosystem services or resources for subsistence. Unsustainable land use, notably mining, has greatly reduced soil
quantity and quality on Nauru (Manner et al. 1984). In preparation
for mining topsoil was cleared from mining sites and stored for
later use in post-mining land rehabilitation. 650,000m3 of soil has
been stockpiled (Hunter, Harris et al. 1994). However, this volume of
soil alone is insufficient to adequately rehabilitate the island
(Carstairs 1994). Soil quality is also an issue. Low levels of organic
matter and soil organisms mean that the remnant soils are infertile
and have low water retaining qualities (Bhogal, Nicholson et al. 2009). Management practices which increase organic matter and soil
organisms are likely to improve the quality of these soils. Nauru Land Rehabilitation In 1994, the Nauru Australia Cooperation Rehabilitation and
Development Feasibility Study (RDFS) initiated the long term
process of determining how to rehabilitate the mined areas of the
island. The document provides an outline of how the landscape can 4 Introduction & Background be modified to achieve land rehabilitation goals. Further
assessments by contractors and NRC staff have resulted in the
development of the Pit 6 rehabilitation project (Burns 2007; Masters
2007; Debao 2009). The current plans for Pit 6 rehabilitation describe
the engineering necessary to prepare the landscape for planting
(levelling pinnacles, layering gravel and soil on top), but lacks
guidance for the establishment of vegetative communities that will
achieve rehabilitation goals. It will be necessary to re-establish
ecosystems services essential for human habitation in Topside; for
this to occur, ecosystem functions will need to be reinstated. be modified to achieve land rehabilitation goals. Further
assessments by contractors and NRC staff have resulted in the
development of the Pit 6 rehabilitation project (Burns 2007; Masters
2007; Debao 2009). The current plans for Pit 6 rehabilitation describe
the engineering necessary to prepare the landscape for planting
(levelling pinnacles, layering gravel and soil on top), but lacks
guidance for the establishment of vegetative communities that will
achieve rehabilitation goals. It will be necessary to re-establish
ecosystems services essential for human habitation in Topside; for
this to occur, ecosystem functions will need to be reinstated. There are specific abiotic barriers in Topside which will need to be
resolved in order to accelerate biotic succession for the re-
establishment of ecosystem functions. Specifically these are lack of
soil and water, and high levels of solar irradiance on the island. There are specific abiotic barriers in Topside which will need to be
resolved in order to accelerate biotic succession for the re-
establishment of ecosystem functions. Specifically these are lack of
soil and water, and high levels of solar irradiance on the island. Desertification is an issue for the island, especially for Topside due
to infrequent rainfall, lack of groundcover and ground porosity. An
additional factor may be the presence of high levels of
environmental cadmium which may affect community composition
due to cadmium’s toxic effects. Biotic factors include greatly
reduced sources of propagules and invasive weed species. soil and water, and high levels of solar irradiance on the island. Nauru Land Rehabilitation Desertification is an issue for the island, especially for Topside due
to infrequent rainfall, lack of groundcover and ground porosity. An
additional factor may be the presence of high levels of
environmental cadmium which may affect community composition
due to cadmium’s toxic effects. Biotic factors include greatly
reduced sources of propagules and invasive weed species. Desertification is an issue for the island, especially for Topside due
to infrequent rainfall, lack of groundcover and ground porosity. An
additional factor may be the presence of high levels of The high level of solar irradiance is potentially an asset in landscape
restoration if the incident energy can be effectively captured
through photosynthesis. As the landscape is currently devegetated
this quality is not being utilised for the benefit of the restoration
process and instead, exposed soils are subject to dramatic
temperature fluctuations and ultraviolet radiation. This leads to
abiotic degradation of soil organic matter, desiccation, and 5 5 Introduction & Background ultimately, soils which are denuded of soil organisms. Accordingly,
the degraded soils support little biomass and low soil biodiversity
leading to poor fertility and plant growth. Soil removed from the landscape prior to mining has been
stockpiled, but is of reduced quality and limited in quantity. The
author proposes that management of this soil resource is of
fundamental concern to the success of Nauruan rehabilitation. This
report provides a preliminary assessment of key components of the
management of this resource: determining the potential for biomass
production on different soils, the potential for selected species to
contribute to cadmium phytoremediation and the effects of soil
cadmium on germination success. EXPERIMENTAL PLANNING AND DESIGN The research involved three trips to Nauru. The first trip was from
the 4th until the 12th of September 2008. The first trip involved
meeting with a number of Nauruan officials and discussing
prospects for research. The second trip from 31st March until the 1st
of May 2009 was to establish field trials and conduct other
experiments and site assessments. During the third trip from the
14th of November until the 7th of December 2009 samples required
for analysis were collected. A key part of the planning was making connections with relevant
individuals and organisations to consult and collaborate, to benefit
from their knowledge and ensure that the project design was
relevant to the environment and community found in Nauru. 6 Introduction & Background Introduction & Background After consultation and a literature review of soil restoration
techniques a field trial exploring the utility of plants for biomass
accumulation and soil cadmium phytoremediation, and an
experiment determining the effects of soil cadmium on germination
success were planned. Unexpected results early on forced a
significant re-evaluation of the direction of the research. The
species trialled within the field plots were extirpated by the
combination of severe environmental stresses and overcrowding by
adventitious species that germinated from the soil seed bank. However, the success of the adventitious species provided a source
of data relevant to the issues of biomass production and cadmium
phytoremediation, as well as a valuable lesson in the utility of
adaptive management principles in rehabilitation projects. Soil For over 30 years, when the mined areas of the island were cleared
of vegetation and soil to access the phosphate rock beneath them,
the covering vegetation and soil were lost. This continued until the
1930s, when a policy to stockpile soil cleared for later use in
landscape restoration was implemented. 1,236ha requires soil of a
total rehabilitation area of 1,405ha. Topsoil stockpiles Topsoil stockpiles
Topsoil was deposited in two large stockpiles on Topside
(Stockpiles A & B). The stockpiles are situated in exposed locations
in Topside. Although only a small fraction of the soil that existed
prior to mining, these stockpiles are a considerable asset to the
rehabilitation process. However, long term stockpiling has reduced 7 Introduction & Background soil fertility, volume and quality. Stockpiling in large mounds has
increased drainage and exposed the soil to dry winds, reducing
levels of soil moisture, and leading to the loss of a significant
proportion of the organic matter in the soil. Stockpiles and more recently removed soils account for around
1,000,000m3 of soil. Prior to mining, Nauru had an overall topsoil
depth of 15-300mm (Carstairs 1994). Of the available soil left, if a soil
depth of 150mm was to be used to rehabilitate the landscape, 700ha
may be rehabilitable. This equates to less than half of the area
(1404ha) that requires soil. Soil organisms
Soil organisms thrive in well aerated soils with organic material and
protection from fluctuations in temperature, moisture, pH, and
gases. Stockpiling of soils has resulted in the formation of large
anoxic areas leading to extensive loss of soil organisms. Desiccation
due to exposure has further reduced soil organism populations. Cadmium Previous reports have identified that the topsoil deposits contain
high levels of cadmium (Blake 1992; Hunter, Harris et al. 1994). Cadmium is toxic to a wide range of organisms and thus may affect
restoration processes due to toxicity (Maksymiec and Krupa 2006; Lin,
Wang et al. 2007; Dell'Amico, Cavalca et al. 2008; Sun, Zhou et al. 2008;
Ci, Jiang et al. 2009). Additionally, soils used for agricultural
purposes may pose risks to human health. Minimising the
ecological effects of cadmium and the risk of cadmium exposure via
contaminated soils is an important aspect of soil restoration. Weeds The Black soil stockpiles are colonised by weedy non-native species. Stockpile A is almost entirely dominated by Leucaena leucocephala
while Stockpile B has a wider range of species. Weed species seed
in the soil may pose ongoing issues if the soil is used in areas where
non-natives are not desired. In Nauru, while weedy non-natives
have been successful in colonising mined areas, native species tend
to succeed the non-native species. Accordingly, these colonisers
may promote the re-establishment of native vegetation, providing 8 8 Introduction & Background the soil with cover and leaf litter, initiating processes which
establish soil structure. Climate change g
Projected climate change impacts in Nauru may reduce rainfall due
to increased frequency and intensity of El Nino events. This would
result in more frequent and intense drought events for Nauru
further threatening water security. Any increase in vegetation
cover may help to mitigate the worst effects of future drought
patterns. An effective and comprehensive programme to
rehabilitate soils on this island will enable the reestablishment of
forest cover and the related climate change mitigation effects. Research, careful monitoring, and adaptive management
approaches to ecological restoration are current best practice and
provide mechanisms by which future climate change can be
integrated into existing management models (Harrington 1999; Lasch,
Lindner et al. 2002; Cummings, Reid et al. 2005). Climate The climate of Nauru is hot and humid due to its proximity to the
equator. Temperatures are very stable, with monthly lows ranging
from 24-26°C up to monthly highs of 29-31°C. Easterly winds
predominate for most of the year, but stronger and gustier westerly
winds are common toward the end of the year. The wet season is
from December to March. Rainfall is highly variable between the
wet and dry seasons, ranging from ~120mm in May to ~280mm in
January. The El Niño-Southern Oscillation has a dominant effect on
rainfall patterns. High rainfall consistently follows major El Niño
events. Drought is common, extended periods without rain occur
every other year (Hassall 1994; Hunter, Harris et al. 1994). Increases in
cover of tall woody vegetation could significantly change the latent
heat flux of the terrestrial biome on the island. Evapotranspiration
and photosynthesis occurring in the forest ecosystems will result in 9 9 Introduction & Background Introduction & Background less sensible heat (cf. latent heat) than without the forests. The loss
of forests in a warm country like Nauru therefore increases the
heating of the land surface and contributes to drying and water loss. Soil rehabilitation will be essential for increasing vegetation cover to
mitigate these effects. Freshwater Nauru has limited freshwater resources. A brackish freshwater lens
is supported within the substrate of the island and is replenished by
rainfall infiltration (Ghassemi and Jakeman 1990). This freshwater
resource is threatened by salination due to over-extraction, faecal
coliforms from human waste, cadmium toxicity, and contamination
from cemetery and landfill leachate (Marsh 1994). Hundreds of 10 Introduction & Background wells on Bottomside extract groundwater, a third of which are
drawing water with higher dissolved salts than recommended by
the World Health organisation (Nauru 2003). Infrequent and
inconsistent rainfall is a significant obstacle to revegetation, which
requires significant freshwater resources to ensure plant growth. Currently, most freshwater in Nauru is produced through
desalination (Hunter, Harris et al. 1994). The desalination plant relies
on imported diesel to function (NCCC 1999; Nauru 2004). Increasing
international fuel prices and transport costs limit and are likely to
further restrict freshwater production capacity. MANDATE TO REHABILITATE The Nauru National Sustainable Development Strategy (NSDS) is a
high level guiding document providing an agenda for
governmental reform and economic development in Nauru. The
NSDS was prepared in 2005 through a consultative process with
stakeholders. It emphasises the need for structural reform to enable
Nauru to adjust to its changing economic, financial, social and
environmental circumstances in a progressive manner. Its goals
are listed in Table 1.1 including the components of each goal listed
in order of priority. Because the symptoms of land degradation in
Nauru are pervasive and burdensome almost all of the goal
components listed in Table 1.1 are affected by Topside restoration. Table 1.1 - Goals of the Nauru National Sustainable
Development Strategy. (Emphasis added. Italicised goals
refer specifically to land restoration in Nauru. Those
components in bold are other goal components directly
affected by landscape restoration.). 11 Introduction & Background Stable, trustworthy,
fiscally responsible
government
Transparent and accountable governance
practices
Conducive legislative framework
Efficient and productive public service
Enabling and cooperative international
relations
Efficient and effective law and order system
Increased community role in governance
Provision of
enhanced social,
infrastructure and
utilities services
Broadened educational system
Alternative (including renewable) energy
sources
Improved access to water
Preventative health service
Improved sports and recreational facilities
Viable social welfare systems
Well maintained infrastructure
Development of an
economy based on
multiple sources of
revenue
Phosphate mining
Fisheries resource management
A developed SME sector
Efficient use of resources – people and
natural
Increased job opportunities locally and
regionally
National trust fund Stable, trustworthy,
fiscally responsible
government 12 12 Introduction & Background Rehabilitation of mined land in Nauru is one of the five key goals
identified by the NSDS, and of the remaining four, three are either
dependent upon or positively associated with land restoration (refer
to components of goals in bold above). Progress as defined by the
NSDS was recently reviewed and several key areas, most notably
Topside rehabilitation, were found to have progressed
insufficiently. A series of restoration plans have been developed through the
Nauru Australia Cooperation (NAC) agreement and through
consultants to the Nauru Rehabilitation Corporation (NRC), the
organisation which is mandated with the task of rehabilitating the
mined areas of the island. MANDATE TO REHABILITATE The Rehabilitation and Development Feasibility Study (RDFS) 1994
produced by NAC is a landmark document presenting the work of 13 Introduction & Background a number of academics from Australia, and the Pacific focused
exclusively on the issue of rehabilitation and development in
Nauru. It is an extensive work, comprising seven documents
covering waste management, Topside rehabilitation, forestry,
housing, and human resources development. This document has
played a key role in establishing the current post-restoration land-
use plan. The NSDS states that soil is an essential component for
1236 of the 1405 ha of land which is to be rehabilitated and that the
importation of soil is not a feasible option. Soil is a central and integral factor in terrestrial ecosystem
pathways. Action by the Nauru government to rehabilitate soils on
Nauru will contribute to satisfying their local, regional, and
international commitments. Healthy soils can provide a stable,
dependable platform, contributing to the achievement of goals 1, 3,
4, 5, 6, 7 and 8 of the Millennium Development Goals, as well as the
United Nations Convention to Combat Desertification (UNCCD),
Convention on Biological Diversity (CBD), and climate change
mitigation. The restoration of Nauru’s soils will also support their
regional commitments to the Convention for the Protection of the
Natural Resources and Environment of the South Pacific Region,
and their local commitments to the economic, social, and cultural
well-being of the people of Nauru. ECOLOGICAL RESTORATION Davis and Slobodkin (2004) state that ecological restoration is ‚the
process of restoring one or more valued processes or attributes of a
landscape‛. Accordingly, while it employs scientific methods to 14 Introduction & Background achieve specific objectives, the objectives themselves are socially
defined (Davis & Slobodkin 2004). The natural variability of the ecosystems of Nauru has been lost due
to the extensive and intensive perturbation of phosphate mining. The landscape has been thoroughly altered; it is unlikely that the
historic conditions which preceded the mining can be re-
established. Likewise, the historic environmental conditions of
Nauru are unlikely to be regarded as socially ideal, given the
dramatic cultural changes which have occurred in Nauru. Expectations have changed with regard to resource use,
governance, property ownership, affluence, and maximal
population to name a few, all of which contribute to a transformed
relationship between people and land. The natural variability of the ecosystems of Nauru has been lost due
to the extensive and intensive perturbation of phosphate mining. The landscape has been thoroughly altered; it is unlikely that the
historic conditions which preceded the mining can be re-
established. Likewise, the historic environmental conditions of
Nauru are unlikely to be regarded as socially ideal, given the
dramatic cultural changes which have occurred in Nauru. Expectations have changed with regard to resource use,
governance, property ownership, affluence, and maximal
population to name a few, all of which contribute to a transformed
relationship between people and land. Nauruan society has radically changed in size and composition
since pre-mining days, and as such it is unclear exactly what
constitutes ideal ecological conditions for Nauru. What is clear is
that the current conditions are less than ideal and that acting to
transform the landscape cannot wait for a detailed understanding of
that ideal due to the unsustainability of Nauru’s current situation. Although a specific ecological goal may not be easily defined, there
are a range of general goals that are definable. The author posits
that these goals might be – rehabilitating the mined landscape to a state which is safe for
human habitation increasing vegetative cover increasing soil organic matter (SOM) the preservation and cultivation of culturally significant species 15 Introduction & Background the preservation and cultivation of ecologically significant and
endangered species the development of fertile soils for agriculture. Ecological relevance Soil is an active interface between abiotic and biotic systems
combining geological, atmospheric, organic and living systems in
intricate cycles (Richter & Markewitz 1995; Baskin 2005). The dynamic
properties of soils are largely a consequence of the organisms which
inhabit them. Soil organisms break down organic matter, store and
cycle nutrients, renew soil fertility, filter and purify water, degrade
and detoxify pollutants, and control plant pests and pathogens
(Killham 1994). Soils provide anchorage for roots, oxygen and
mineral nutrients, water, and buffering against the adverse effects
of temperature and pH in an association of mineral particles
dynamically linked with organic matter, water and gases (Donahue
1958; Wild 1993; Matson 1997; Sumner 2000; White 2006). Though
many aspects of the dynamics of soils are still not well understood,
it is clear that they provide essential and diverse services upon
which human societies rely. ECOLOGICAL RESTORATION There is a need for explicit goals, a restoration programme
developed with an awareness of local ecology, ongoing monitoring
to provide quantitative data of change, and analysis of the results in
order to adapt the design according to increasing knowledge of the
ecology of the restoration landscape (Palmer, Falk et al. 2006). One of
the outcomes from this model of experimental design is the
development of ‘knowledge targets’; ecological goals specific to the
local conditions. development of ‘knowledge targets’; ecological goals specific to the
local conditions. This report emphasises knowledge targets corresponding to the
physiological challenges of land rehabilitation in Nauru, employing
analytical research design. This report inevitably touches on many
other ecological restoration knowledge targets as raised in the
discussion (Chapter 5); however, they are not of central importance
to this analysis. Part of the reasoning for the approach employed in
this report is that the abiotic conditions found in Nauru are
particularly severe and require initial attention. The relationship
between soils and vegetation in the environmental conditions found
in Nauru are assessed herein. Environmental cadmium is directly
assessed and the general effects of the environmental conditions are
assessed indirectly through measures of plant productivity and
survival. 16 Introduction & Background Introduction & Background Nauru soil ecology Morrison & Manner (2005) identified four historical soil types on
Nauru – coastal fringe soils, deep and moderately deep Topside
soils, steep-land soils with dolomite influence, and Buada soils. Generally, the soils of Nauru are coarse textured with low water
retention, due to rapid water infiltration and transmission rates. Soil development is almost entirely due to the accumulation of
organic material. The parent material is either resilient to
weathering, in the case of apatite, or easily weathered, in the case of
carbonates. With a substrate low in bioavailable minerals, Nauru’s 17 17 Introduction & Background soil fertility is highly dependent upon organic matter to provide
nutrients, lower soil pH, and retain water (Nauru 2003). Increasing soil quantity and quality Soil development strategies will need to be targeted to specific
outcomes depending on landscape use, with different approaches to
satisfy each outcome. Land uses proposed by the Rehabilitation
and Development Feasibility Study include conservation areas,
housing, agriculture, forestry, a municipal waste site, a cemetery, a
water reservoir, recreational areas, industrial areas, government
facilities, and roads. With the exception of roads, and possibly the
municipal waste site, all other areas will need to have soil to a
greater or lesser extent (Carstairs 1994). Phosphate mining has resulted in the loss of almost all productive
land in Nauru. There is now a paucity of land on which to produce
food-crops on the island. This poses a significant challenge for the
inhabitants who now rely heavily on imported foods. Land suitable
for agricultural purposes requires that specific biophysical systems
such as carbon cycling are functioning to support the production of
useful agricultural products. In all cases increased humus content
will have beneficial effects on the services provided by the soils. Importation of soil
Soil importation to Nauru has been suggested as a method to
replenish diminished soil stocks on Nauru (Carstairs 1994). While
this method may satisfy the need for greater soil quantities, the
costs, both in economic and biological terms, are considerable. 18 Introduction & Background The economic costs associated with the importation of soils include;
sourcing of appropriate soils, purchase of the soil stock from a
willing seller, excavation, transportation to an appropriate port,
loading costs, shipping costs, unloading, and distribution. This
represents a net loss to the local economy of around $1,450,000
AUD per hectare (Carstairs 1994). It would be many years before the
gains would offset the costs. The potential environmental costs to Nauru of imported soils are
also considerable. Soils are ecosystems in their own right,
containing biologically distinct taxa, particular to their place of
origin (Baskin 2005). Interactions within the soil system play a
considerable role in characterising ecosystem biota, thus
importation of soils from outside of Nauru poses a considerable risk
to the biodiversity of native soil organisms, and their mutual
reliance upon indigenous flora and fauna. Sterilisation of imported
soils is potentially costly and will provide a ‘dead’ soil substrate,
with properties which may not be ideally suited to native soil
organisms. Consequently, the author does not consider soil
importation to be a feasible or useful component of soil restoration
on Nauru. Soil development on Nauru Recommendation 13 of the ‘Recommendations used in Steering
Committee Deliberations’ states that ‚It is recommended that soil
generation be achieved by utilising materials available on Nauru,
using simple technology‛ (Hunter, Harris et al. 1994). Soil
rehabilitation is dependent on raising soil volumes, fertility, and
increasing microbial content. Composted organic inputs offer the Recommendation 13 of the ‘Recommendations used in Steering
Committee Deliberations’ states that ‚It is recommended that soil
generation be achieved by utilising materials available on Nauru,
using simple technology‛ (Hunter, Harris et al. 1994). Soil rehabilitation is dependent on raising soil volumes, fertility, and
increasing microbial content. Composted organic inputs offer the 19 Introduction & Background best method for achieving these three outcomes. Under-utilised
organic inputs are currently readily available on Nauru (Marsh
1994). Management systems should focus on sustaining soils
through reintegrating the inputs that are already available. Mineral inputs
Phosphates are an obvious source of locally available mineral
fertiliser, although phosphate levels in Nauru soils are unlikely to
become depleted except under intense agricultural pressure. It has
been proposed that the use of phosphate be weighed against the
opportunity cost of not selling it (Baines 1994). Organic inputs Organic inputs
Organic inputs have been highlighted as the most promising
method to raise soil fertility. Fortunately many sources of organic
inputs already exist on the island. Facilitating their biodegradation
will increase the rate at which organic materials become available in
soils and thereby increase the rate at which the soils and vegetation
can develop. Organic inputs increase the amount of water available
to plants and reduce the density of the soil, facilitating plant growth
(Bhogal, Nicholson et al. 2009). The use of locally available organic
fertilisers offers fewer risks to groundwater quality than mineral
fertilisers and reduces the incidence of stresses associated with
water availability in soils (Duwig, Becquer et al. 1998). Organic inputs
to soils result in significantly greater species richness in both
bacteria and nematode communities (van Diepeningen, de Vos et al. 2006; Bhogal, Nicholson et al. 2009). 20 Introduction & Background The natural accumulation of organic material is a consequence of
production, slope and rate of decomposition. Rainfall is a limiting
factor to vegetative growth, and is highly variable on Nauru,
indicating that organic inputs to soils would vary considerably from
year to year. With high humidity and aeration of soils,
decomposition is likely to be rapid, leading to relatively low
equilibrium levels of organic matter in the soils (Morrison & Manner
2005). Soil organisms and mycorrhizae
Soil microorganisms increase the potential for soil biological activity
and improve physiochemical soil properties, contributing to rapid
humification of fresh organic matter and may be used as indicators
of soil health (Valarini, Alvarez et al. 2002, Valarini 2003). Symbiotic
associations between plants and mycorrhizae greatly increase
growth productivity by enhancing nutrient availability and uptake
and by maintaining and improving the physical, chemical and
biological qualities of soils (Sen 2005; Cardoso & Kuyper 2006). Further research is necessary to identify soil organisms which may
benefit native reforestation and agriculture on Nauru. Activity and
abundance of soil organisms correspond to levels of organic
material in soils (Baldock 2000). Sourcing of organic inputs Organic inputs on Nauru are available from human effluent, green
waste, food and kitchen waste, and the growth of green manures
(Marsh 1994). Cultivation of soil organisms will facilitate nutrient
availability and improve overall soil development and productivity. 21 Introduction & Background Introduction & Background Cover crops and green mulches Cover crops and green mulches Cover crops and green mulches
While the terms are often used interchangeably, cover crops and
green manures differ in their intended use. Cover crops are used,
as the name implies, to cover the soil and thereby protect it, often in
periods where seasonal weather patterns pose risks to exposed soils
(Thurston 1997). Green manures are grown to be integrated into the
soil after a period of growth to increase the organic content of the
soil (Sullivan 2003). The terms are used interchangeably as it is often
the case that both goals are expressed in a single crop. Cover crops Cover crops are primarily planted to cover and protect the soil from
erosion. Other benefits may include – Cover crops are primarily planted to cover and protect the soil from
erosion. Other benefits may include – Potential nitrogen gains where legumes are used Potential nitrogen gains where legumes are used
Organic matter content can significantly increase
Protection from soil erosion
Weed suppression
Low labour costs
Low expenses – seeds produced on site
Potential for animal forage
Reduction of photodegradation of soil organic matter
(Bunch 1994; Rutledge, Campbell et al. 2010) Organic matter content can significantly increase Protection from soil erosion Reduction of photodegradation of soil organic matter
(Bunch 1994; Rutledge, Campbell et al. 2010) Recent research shows that exposure of soil organic matter (SOM) to
solar irradiance in dry environments results in substantial losses of
SOM to the atmosphere by way of CO2, produced by
photodegradation (Rutledge, Campbell et al. 2010). Limiting this
pathway by which organic matter is lost from the soil may be of Recent research shows that exposure of soil organic matter (SOM) to
solar irradiance in dry environments results in substantial losses of
SOM to the atmosphere by way of CO2, produced by Recent research shows that exposure of soil organic matter (SOM) to
solar irradiance in dry environments results in substantial losses of
SOM to the atmosphere by way of CO2, produced by photodegradation (Rutledge, Campbell et al. 2010). Limiting this
pathway by which organic matter is lost from the soil may be of 22 Introduction & Background considerable benefit when seeking to increase the organic content of
soils. Living groundcover reduces photodegradation by limiting
the amount of radiation incident upon soil organic matter. Green manures & mulches Green manures & mulches Green manures & mulches
Green manures are cover crops grown to be reincorporated into the
soil for the purpose of increasing soil fertility (Ghosh 2007; Liu,
Gumpertz et al. 2007). Other advantages of green manures include
soil profile development through root intrusion, protection of soils
from environmental stresses by acting as living mulch (Elfstrand,
Bath et al. 2007). Vegetation growth and productivity of native tree
species such as the Tomago (Calophyllum inophyllum) would be
greatly increased by the use of green manures (Moitra, Ghosh et al. 1994). Green manures are often species that harbour nitrogen fixing
bacteria. The incorporation of green material from nitrogen fixing
species minimises volatilisation of nitrogen (Thurston 1997). Green mulches are grown for the benefits that they provide while
growing (or during growth), including improvements to soil
texture, and structure. Mulches are used to cover the soil, create a
distinct layer between the soil and atmosphere and as such are akin
to cover crops (Lal 1990). Potential positive effects of green manures Potential positive effects of green manures Decrease soil moisture evaporation, protecting against drought
(Buckles, Ponce et al. 1994; Moreno & Sánchez 1994) Increase water infiltration (Ayanlaja & Sanwo 1991) Reduce erosion and water runoff (Ayanlaja & Sanwo 1991; Akobundu 1993; Buckles, Ponce et al. 1994; Moreno & Sánchez 1994) Akobundu 1993; Buckles, Ponce et al. 1994; Moreno & Sánchez 1994) 23 Introduction & Background Reduce temperatures of soil Reduce temperatures of soil Reduce temperatures of soil Increase populations and activity of earthworms (Fragoso, Barois
et al. 1993) Increase SOM (Ayanlaja & Sanwo 1991) Increase soil nutrients(Ayanlaja & Sanwo 1991; Buckles, Ponce et al. 1994; Moreno & Sánchez 1994) Increase soil nutrients(Ayanlaja & Sanwo 1991; Buckles, Ponce et al. 1994; Moreno & Sánchez 1994) 1994; Moreno & Sánchez 1994)
Protect young plants and seedlings from physical stressors such
as wind and rain (Thurston 1992)
Aid in weed management (Akobundu 1993)
May reduce labour (Thurston 1992)
Increase crop yields Protect young plants and seedlings from physical stressors such
as wind and rain (Thurston 1992) Potential negative effects of green manures
Pests - eg. mice, rats, insects (Buckles, Ponce et al. Green manures & mulches 1994)
Environment for some plant pathogens(Moreno & Sánchez 1994)
Effects on C/N ratio (Jordan 1989)
Nitrogen loss in some cases due to formation of ammonia (Costa,
Bouldin et al. 1990) Nitrogen loss in some cases due to formation of ammonia (Costa,
Bouldin et al. 1990) Increased labour costs in some cases (Thurston 1992) Slashed shrubs or trees
While it is common to use grasses or other ground cover plants as
cover-crops, there has been research into the use of shrub and tree
species as sources of green manure. Various methods have been
proposed for mulch produced from shrubs and trees including the
lopping of foliage and coppicing (Brewbaker 1990). Often the land in
question is organised so as to allow for multiple production sources,
whereby trees provide material for integration into the soil, and
protection for crops which are grown between. This has various 24 Introduction & Background names such as alley cropping, hedgerow cropping, avenue
cropping, or hedgerow intercropping. It has been observed that
coppicing is especially useful in the seasonally dry tropics where
other forms of production may suffer (Nyerges 1989). Additional
positive and negative aspects are listed below. Other positive aspects of intercropped tree mulching systems Other positive aspects of intercropped tree mulching systems Other positive aspects of intercropped tree mulching systems
Erosion control (Fernandes 1991; Akobundu 1993; Kang 1993) Erosion control (Fernandes 1991; Akobundu 1993; Kang 1993) Improvement of soil physical properties by addition of SOM
(Kang & Reynolds 1989; Fernandes 1991; Akobundu 1993; Kang 1993) Nutrient provisioning (esp. from legumes) (Kang & Reynolds
1989; Fernandes 1991; Kang 1993; Szott 1993) Firewood, poles, other wood products
Fodder (Kang & Reynolds 1989) Firewood, poles, other wood products Weed suppression – shading and mulch (Kang & Reynolds 1989;
Akobundu 1993) Beneficial microclimate effects Tree crops Negative aspects of intercropped tree mulching systems
Competition with crops for moisture, sunlight and nutrients
Reduction of space available for other activities
Disease issues caused by reduced air flow
Risk of tree species becoming an invasive weed
Labour inputs can be high
Requires intensive management
Phytoremediation of soils
Cadmium liberation Phytoremediation of soils 25 Introduction & Background Introduction & Background An unfortunate consequence of phosphate mining on Nauru has
been the liberation of substantial quantities of cadmium from
subsoil deposits into the wider environment. Cadmium may have
effects on ecological community structure in Nauru. Cadmium has
negative effects on human health, thus cadmium bioavailability is a
critical concern in agricultural soils. Ecological effects of cadmium Cadmium Bioavailability Bioavailability is a complex term. It refers to the amount of
contaminant in the soil which is available to an organism which
may accumulate it. Dynamic equilibria of chemical species and
their interactions with living organisms affect the rate and degree of
bioavailability. Despite these difficulties in quantification, it is a
useful qualitative concept, allowing for meaningful comparisons
between different soil and plant interactions. In the context of this
study, the terms bioavailability and phytoavailability refer to the
heavy metals in the soil which are available for accumulation in
plant tissues. The laboratory method employed in this report to
assess the mobile fraction of cadmium in soil samples utilises
NaNH3, which is a weak extractant, to assess the free ionic
concentration of cadmium in the soils. This fraction of the soil
cadmium consists of a range of chemical species which are highly
mobile and are readily available for uptake by plants. Ecological effects of cadmium Cadmium is classified as a toxic heavy metal. Contaminated areas
are considered to be a threat to the well-being of humans and
animals (Czeczot and Skrzycki, 2010). Cadmium affects the
kidneys, liver, lungs, pancreas, skeleton and testis in humans due to
disruptions of cellular adhesion, the immune system, the cellular
antioxidant system, and cellular communication systems, inhibition
of DNA repair and methylation, promotion of cell proliferation and
apoptosis, and initiation of cell mutagenesis and carcinogenesis
(Czeczot and Skrzycki, 2010; Dufresne et al., 2010; Satarug et al.,
2010; Trzcinka-Ochocka et al., 2010). Cadmium has been
demonstrated to have a wide variety of deleterious effects in
ecological systems, negatively affecting soil microbial community
structure and activities, affecting growth and causing severe
physiological disorders in plants and animals, disrupting renal,
hepatic systems, and endocrine system functioning, skeletal
development, and fat storage in animals (Angenard et al., 2010; Ci
et al., 2010; Czeczot and Skrzycki, 2010; Dufresne et al., 2010; Khan
et al., 2010; Kumar et al., 2009; Lucia et al., 2010; Planelló et al., 2010;
Van Campenhout et al., 2010). Cadmium bioaccumulates in food
webs amplifying the risk to organisms at higher trophic levels (Sato
et al., 2010; van den Brink et al., 2010). 26 Introduction & Background Introduction & Background Introduction & Background Cadmium Bioavailability Cadmium Bioavailability Soil acidity As soil pH affects precipitation of salts it is considered to be the
single most significant factor determining solubility of heavy metals
in the soil. Soil pH and solubility negatively correlate, meaning that
all things being equal, acidic soils will have a greater availability of
heavy metals than alkaline soils. Nauru’s soils are alkaline due to
the calcium carbonate substrate. Productive agricultural soils are
typically more acidic than those found in Nauru, as acidity also
increases the availability of soil nutrients. Accordingly, there will
be conflicts for target pH of agricultural soils in Nauru, between
increased productivity and cadmium bioavailability. Reducing the 27 Introduction & Background Introduction & Background cadmium load in soils intended for agriculture will reduce that
conflict. Phytoremediation It has been suggested that phytoremediation via phytoextraction
may be an economically sensible tool for remediation of soils
contaminated with heavy metals. It is a technique that employs
plants as absorbers of inorganic contaminants for subsequent
removal via harvesting. Advantages of phytoremediation include: Advantages of phytoremediation include: Can be performed in situ Can be performed in situ Less invasive than civil-engineering earth moving projects
(Cunningham & Berti 1993; Cunningham, Berti et al. 1995;
Wiltse, Rooney et al. 1998) Less expensive than civil-engineering earth moving projects
(Cunningham & Berti 1993; Cunningham, Berti et al. 1995;
Wiltse, Rooney et al. 1998; Garbisu & Alkorta 2001) Application over large surface areas (Wiltse, Rooney et al. 1998) App icatio
o e
a ge su ace a eas (
i tse,
oo ey et a
998)
Targets the bioavailable fraction of heavy metals in the soil Targets the bioavailable fraction of heavy metals in the soil Avoids further soil loss Avoids further soil loss Drawbacks include Drawbacks include Long remediation time (years to decades) Perception of uncertainty and limits to performance
predictability associated with biological remediation systems Elevated concentrations of heavy metals in aboveground plants
parts may enter the food chain via herbivory. 28 Introduction & Background Techniques employed in soil phytoremediation include Phytotransformation/phytodegradation The breakdown of contaminants through plant metabolic
processes Phytostimulation The stimulation of fungal and microbial degradation of organic
pollutants by plant exudates or enzymes The immobilisation of soil contaminants by plants limiting
leaching, especially to groundwater The formation and release of volatile forms of pollutants
absorbed by plants
Phytoextraction The formation and release of volatile forms of pollutants
absorbed by plants The extraction and accumulation of contaminants in harvestable
plant parts (Kumar, Dushenkov et al. 1995) Of these, phytoextraction is the primary phytoremediation
technique which will be evaluated as part of this research due to the
relative accessibility of this technique for Nauru. Phytoextraction is
cheap, low-tech and easier to employ over large areas than other
phytoremediation techniques. Phytoextraction There are considerable incentives for the use of phytoextraction,
including non-invasiveness, economics, aesthetics, soil stability, 29 Introduction & Background increased soil organic carbon, and the ability to use the land area
during the rehabilitation process (Cunningham & Berti 1993;
Cunningham, Berti et al. 1995; Salt, Blaylock et al. Phytoremediation 1995; Garbisu & Alkorta
2001). Recent research has focused on increasing extraction efficiency of
heavy metals. Generally, two methods are pursued: Recent research has focused on increasing extraction efficiency of
heavy metals. Generally, two methods are pursued: The use of hyperaccumulating species (Baker, Reeves et al. 1994;
Brown, Chaney et al. 1994; Nanda-Kumar, Dushenkov et al. 1995; Krämer, Smith et al. 1997; Zhao, Lombi et al. 2001) The use of soil amendments to stimulate the uptake and
accumulation of heavy metals (Banuelos, Cardon et al. 1993;
Huang, Chen et al. 1997; Ebbs & Kochian 1998; Huang, M.J. et al. 1998; Cooper, Sims et al. 1999; Lombi, Zhao et al. 2001; Shen, Li
et al. 2002). Other options include evaluating the benefits of genetically
modified species (Gleba, Borisjuk et al. 1999; Kärenlampi, Schat et al. 2000) and rhizospheric associations between plants, mycorrhizae
and bacteria (Killham & Firestone 1983; Khan 2001; Kunito, Saeki et al. 2001; Li, Chen et al. 2002; Bi, Li et al. 2003). This research has focused on identifying locally available plant
species which may act as hyperaccumulators, or at least serve the
purpose of phytoextractors in the conditions found in Nauru. Many
of the academic papers listed above deal with heavy metal
contamination as the only significant limit to species viability;
however, Nauru has various environmental factors other than
heavy metal load which limit the viability of a wide range of 30 Introduction & Background species. Rather than focusing on hyperaccumulating species, or on
soil amendments to increase heavy metal transport into plants, this
research has focused on determining which species provide
maximum yield in situ. Soil amendments to improve heavy metal
transport have been of benefit to a number of soil restoration
projects, however, the focus of this project has been on ascertaining
the most basic and fundamental aspects of soil phytoremediation in
Nauru, largely using the species and resources available on the
island. Typically, the significant restrictions to efficient
phytoextraction are bioavailability, the tolerance of each species to
heavy metals, and translocation; however, the adverse growing
conditions in Nauru pose their own set of physiological difficulties
(Herzig, Rehnert et al. 2000). Translocation Aboveground accumulation of heavy metals in phytoaccumulating
plants is limited by the translocation barrier formed by Casparian
bands that separate the root cortex from the vascular bundle,
forming a physiological barrier to heavy metals being transported
from the roots to the shoots (Huang 1989). Various papers have
described the differences in accumulated concentrations of
cadmium between roots and shoot tissues (Kirkham 1978; Kastori,
Petrovic et al. 1992; Guo & Marschner 1995; Salt, R.C. et al. 1995; Tack,
Esteban-Mozo et al. 1998). Accordingly, analysis of accumulated concentrations of cadmium in
root, stem, and leaf tissue will be compared to determine which
species have the greatest efficiency of transportation of cadmium to
aboveground plant tissues. 31 Introduction & Background Introduction & Background Germination Germination is a critical life process of plants whereby seeds take in
water, rapidly increase respiratory activity, mobilise nutrient
reserves, and initiate embryo growth (Bradbeer 1988; Fenner &
Thompson 2005). Studies have found widely varied effects of
cadmium on germination success describing either increased
germination success with increased levels of cadmium, the
opposite, or no effect (XinHong, YuXiu & RunJin; Zhang et al. 2010;
Lefevre, 2009; Wang & Zheng, 2009; ZhiDe et al. 2009, Kumar et al. 2009;
HuaBing, 2009; Farooqi et al. 2009). Nauru’s rehabilitation project
provides an interesting setting in which to explore the extent to
which cadmium determines germination success. Firstly, the
results may benefit the management of the rehabilitation process. Secondly, Nauru’s cadmium levels are naturally elevated, in
contrast to the artificially elevated levels assessed within the
literature. ADAPTIVE MANAGEMENT Adaptive management (AM) is an approach to management in the
face of uncertainty. AM has an iterative management approach,
requiring that the results of management approaches are monitored
and analysed, that the analysis is used to modify or update the
management approach, and that any such modifications are
subsequently employed and monitored (Walters & Hollings 1990;
Gregory, Ohlson et al. 2006). By learning from management
outcomes, AM systematically improves environmental
management policies and practices (Gregory, Ohlson et al. 2006). 32 Introduction & Background Two forms of AM exist – Active AM and Passive AM. Active
adaptive management requires that several models are employed in
such a way as to provide useful information for subsequent
management. Experimentation is built into the management
framework. Passive adaptive management employs one ‚best
method‛ usually based on historical data. This method is trialled,
the effects are monitored, and an updated management approach is
implemented (Walters & Hollings 1990; Gregory, Ohlson et al. 2006). Active AM is more akin to scientific experimentation than passive
AM and is consequently more likely to determine specific factors
for shaping successful management approaches than Passive AM. This is due to controls and comparable treatments in Active AM to
reduce the likelihood of measurement errors caused by stochastic
effects, which might otherwise confound management and
environmental effects (Walters & Hollings 1990). Typically, Active
AM results in faster accumulation of information useful to
environmental management. However, Active AM usually requires
greater funding, preparation, and attention to the use of
experimental design principles (Gregory, Ohlson et al. 2006). The rehabilitation programme in Nauru cannot be expected to
prescribe exacting methods by which rehabilitation can be achieved. Therefore, to be able to proceed with a reasonable expectation of
success, adaptive management principles will need to be employed. This report explores some aspects of restoration in a manner that is
consistent with an AM approach to restoration in Nauru. It does so
by exploring biomass productivity, cadmium phytoremediation and
germination success according to AM experimental design
principles. In the cases of biomass productivity and cadmium 33 Introduction & Background phytoremediation, the experiments were conducted as field trials
under conditions akin to those which are expected to be found in
the rehabilitation areas. Biomass production in Nauru The environmental conditions found in Nauru are severely limiting
to plant growth. It is useful to determine the biomass output in situ
of potentially useful green mulch species under likely conditions in
Nauru as a guide for nursery work and planting on the island. To
this end, a field trial designed to explore biomass production was
conducted on Nauru from April to October 2009. This aspect of the restoration issue will be evaluated in greater
depth in Chapter 2. Introduction & Background Introduction & Background Introduction & Background Chapters 2, 3 and 4 describe specific experiments that were
performed to explore the issues of biomass accumulation, cadmium
phytoremediation, and the effects of soil cadmium on germination
success. The chapters have been written such that they individually
perform the tasks of academic papers. Consequently there is some
unavoidable repetition. Chapter 5 draws from each of the
experimental chapters and integrates the information they
individually report to inform the Nauru rehabilitation process. What follows are brief outlines of what the reader can expect from
the following chapters. What follows are brief outlines of what the reader can expect from
the following chapters. RESEARCH APPROACH AND THESIS CONTENT The soil stockpiles on Nauru are an essential resource for
rehabilitation which could be better managed than they have
historically been. How best to manage this resource, however, is
not clear. Determining, in greater detail, the potential impact of
different management options would be useful as it will allow for
more informed decision making. Some key concerns are: Some key concerns are: the effects of cadmium on germination success of species in
Nauru the impact of Nauruan growth conditions on various species
that may be used for soil restoration whether phytoremediation techniques may be applicable in
Nauru and to what extent. This thesis aims to provide a preliminary exploration of these
factors for rehabilitation through experimentation in Nauru. It
must be noted that there were significant constraints to this process. Firstly, this is a Master’s thesis and thus the scope of the research
must be commensurate with the expectations of the examining
body, both in terms of size and format. Secondly, it was only
possible to visit Nauru three times and not for the full extent of the
field trial, which meant that it was not possible to mitigate problems
that arose between visits. 34 Cadmium bioremediation Bioremediation of soil cadmium in Nauru may be of considerable
use in ensuring that agricultural soils have levels of bioavailable
cadmium which pose little to no threat to human health and well- 35 Introduction & Background being. A field trial designed to explore phytoremediation was
conducted on Nauru from April to October 2009. This aspect of the restoration issue will be evaluated in greater
depth in Chapter 3. Effects of cadmium on germination success If elevated levels of cadmium are affecting germination rates then
community composition may become altered through time. Testing
the effects of soil cadmium on seed germination would provide
useful information for the restoration process. An experiment
designed to explore this issue was performed in Nauru in April
2009. This aspect of the restoration issue will be evaluated in greater
depth in Chapter 4. REFERENCES Akobundu, I.O. 1993. Integrated weed management techniques to reduce
soil degradation: IITA. Angenard, G., Muczynski, V., Coffigny, H., Pairault, C., Duquenne, C.,
Frydman, R., Habert, R., Rouiller-Fabre, V. & Livera, G. 2010. Cadmium Increases Human Fetal Germ Cell Apoptosis. Environmental Health Perspectives 118, 7. Environmental Health Perspectives 118, 7. Ayanlaja, S.A. & Sanwo, J.O. 1991. Management of soil organic matter in
the farming systems of the low land humid tropics of West Africa. A
review. Soil Technology 4 (3):13. Baines, G. 1994. Environment Part A. Environmental aspects of
rehabilitation of Topside Nauru. In Rehabilitation and Development
Feasibility Study: Nauru Australia Cooperation. 36 Introduction & Background Introduction & Background Baker, A.J.M., Reeves, R.D. & Hajar, A.S.M. 1994. Heavy metal
accumulation and tolerance in British populations of the
metallophyte Thlaspi caerulescens J. & C. Presl. (Brassicaceae). New
Phytologist 127:8. Baldock, J.A. & Nelson, P.N. 2000. Soil organic matter. In Handbook of Soil
Science, edited by Sumner, M.E.: CRC Press LLC. Banuelos, G.S., Cardon, G., Mackey, B., Ben-Asher, J., Wu, L., Beuselinck,
P., Akohoue, S. & Zambrzuski, S. 1993. Boron and selenium
removal in boron-laden soils by four sprinkler irrigated plant species. Journal of Environmental Quality 22:25. Barker, X. 2009. Acting Director, Nauru Branch, University of the South
Pacific. A. Feary. Baskin, Y. 2005. Underground: How creatures of mud and dirt shape our world:
Island Press/Shearwater Books. Bettencourt, S., Croad, R., Freeman, P., Hay, J., Jones, R., King, P., Lai, P.,
Mearns, A., Miller, G., Pswarayi-Riddihough, I., Simpson, A.,
Teuatabo, N., Trotz, U. & Van Aalst, M. Not if but when: Adapting to
natural hazards in the Pacific Islands Region - A policy note. The World
Bank - East Asia and Pacific Region - Pacific Islands Country
Management Unit 2006 [cited]. Bhogal, A., Nicholson, F. A., & Chambers, B. J. 2009. Organic carbon
additions: effects on soil bio-physical and physico-chemical
properties. European Journal of Soil Science 60. Bi, Y.L., Li, X.L., & Christie, P. 2003. Influence of early stages of arbuscular
mycorrhiza on uptake of zinc and phosphorus by red clover from a
low-phosphorus soil amended with zinc and phosphorus. Chemosphere 50:7. Bill, L. 1996. Egade. In Stories from Nauru, edited by Crowl, L. & Garrett, J.:
The University of the South Pacific, Nauru Centre and Institute of
Pacific Studies. 37 37 Introduction & Background Introduction & Background Blake, C. 1992. Environmental distribution of heavy metals on Nauru, Central
Pacific, and possible relationships to human health, University of New
England. Bachelor of Applied Science (Honours): 86. Boivin, P., Schaffer, B. & Sturny, W. 2009. Quantifying the relationship
between soil organic carbon and soil physical properties using
shrinkage modelling. European Journal of Soil Science 60. Bradbeer, J. W. 1988. Seed dormancy and germination, Blackie & Sons Ltd. Brewbaker, J.L. 1990. Nitrogen fixing trees. In Fast Growing Trees and
Nitrogen Fixing Trees, edited by Werner, D. & P. Müller. Stuttgart:
Gustav Fischer Verlag. Brown, S.L., Chaney, R.L., Angle, J.S. & Baker, A.J.M. 1994. Phytoremediation potential of Thlaspi caerulescens and bladder
campion for Zinc- and Cadmium-contaminated soil. Journal of
Environmental Quality 23:7. Buckles, D., Ponce, I., Sain, G. & Medina, G. 1994. Cowardly land becomes
brave. The use and diffusion of fertilizer bean (Mucuna deeringianum)
on the hillsides of Atlantic Honduras. In Tapado Slash/Mulch: How
Farmers Use It, and What Researchers Know About It, edited by H. D. Thurston, M. Smith, G. Abawi & S. Kearl. Ithaca, NY: CIIFAD and
CATIE, Cornell University. Bunch, R. 1994. The potential of slash/mulch for relieving poverty and
environmental degradation. In Tapado Slash/Mulch: How Farmers Use
It, and What Researchers Know About It, edited by H. D. Thurston, M. Smith, G. Abawi & S. Kearl. Ithaca, NY: CIIFAD and CATIE,
Cornell University. Burns, M. 2007. Progressive Rehabilitaion Strategy - Nauru. Global Soil Systems
(GSS). Burns, W.C.G. 2003. Pacific Island Developing Country water resources
and climate change. In The World's Water: The biennial report on
freshwater resources 2002-2003, edited by P. Gleick. Campbell, C. J. 2002. Petroleum and people. Population and Environment 24
(2):193-207. 38 Introduction & Background Cardoso, I. M. & Kuyper, T. W. 2006. Mycorrhizas and tropical soil fertility. Agriculture Ecosystems & Environment 116 (1-2):72-84. Carstairs, B. 1994. Pinnacle land restoration. In Rehabilitation and
Development Feasibility Study: Nauru Australia Cooperation. Cazenave, A. & Nerem, R. S. 2004. Present-day sea level change:
Observations and causes. Reviews of Geophysics 42 (3). Cherian, A. 2007. Linkages between biodiversity conservation and global
climate change in small island developing States (SIDS). Natural
Resources Forum 31 (2):128-131. Ci, D., Jiang, D., Wollenweber, B., Dai, T., Jing, Q. & Cao, W. 2010. Cadmium stress in wheat seedlings: growth, cadmium accumulation
and photosynthesis. Acta Physiologiae Plantarum 32, 9. CIA. 2007. Nauru. Introduction & Background The World Factsheet Retrieved 7 August, 2007, from
https://www.cia.gov/library/publications/the-world-
factbook/print/nr.html. Cooper, E.M., Sims, J.T., Cunningham, S.D., Huang, J.W. & Berti, W.R. 1999. Chelate-assisted phytoextraction of lead from contaminated
soils. Journal of Environmental Quality 28:11. Costa, F.J.S.A., Bouldin, D.R. & Suhet, A.R. 1990. Evaluation of N
recovery from mucuna placed on the surface or incorporated in a
Brazilian oxisol. Plant and Soil 124:6. Cummings, J., Reid, N., Davies, I. & Grant, C. 2005. Adaptive restoration
of sand-mined areas for biological conservation. Journal of Applied
Ecology 42 (1):160-170. Cunningham, S.D. & Berti., W.R. 1993. Remediation of contaminated soils
with green plants: an overview. In Vitro Cellular Development and
Biology 29:6. Cunningham, S.D., Berti, W.R. & Huang, J.W. 1995. Phytoremediation of
contaminated soils. Trends in Biotechnology 13:3. Curran, M. P., Miller, R. E., Howes, S. W., Maynard, D. G., Terry, T. A.,
Heninger, R. L., Niemann, T., van Rees, K., Powers, R. F. &
Schoenholtz, S. H. 2005. Progress towards more uniform assessment 39 Introduction & Background and reporting of soil disturbance for operations, research, and
sustainability protocols. Forest Ecology and Management 220 (1-3):17-
30. Czeczot, H. & Skrzycki, M. 2010. Cadmium - element completely
unnecessary for the organism. Postepy Higieny i Medycyny
Doswiadczalnej 64, 10. Davis, M. A. & Slobodkin, L.B. 2004. "The Science and Values of
Restoration Ecology." Restoration Ecology 12(1): 3. Debao, L. 2009. NRC Operations Report – October 2009. Nauru
Rehabilitation Corporation. Dell'Amico, E., Cavalca, L. & Andreoni, V. 2008. Improvement of
Brassica napus growth under cadmium stress by cadmium-resistant
rhizobacteria. Soil Biology and Biochemistry 40: 11. Demmke, A., Haberkorn, G., Gadabu, I., Rakaseta, V.L., Beccalossi, G. 1999. Nauru population profile: A guide for policy-makers. In
Population/Demography Programme. Noumea, New Caledonia:
Secretariat of the Pacific Community. Donahue, R.L. 1958. Soils: An introduction to soils and plant growth. New
Jersey: Prentice-Hall, Inc. Drummond, E. (1921). Mandate for Nauru, His Majesty's Stationery Office. Dufresne, M. M., Frouin, H., Pillet, S., Lesage, V., De Guise, S. & Fournier,
M. 2010. Comparative sensitivity of harbour and grey seals to several
environmental contaminants using in vitro exposure. Marine Pollution
Bulletin 60, 6. Duncan, R. C. 2003. Three world oil forecasts predict peak oil production. Oil & Gas Journal 101 (21):18-21. Duwig, C., Becquer, T., Clothier, B. E. & Vauclin, M. 1998. Nitrate
leaching through oxisols of the Loyalty Islands (New Caledonia)
under intensified agricultural practices. Geoderma 84 (1-3):29-43. Ebbs, S.D. & Kochian, L.V. 1998. Introduction & Background Phytoextraction of Zinc by oat (Avena
sativa), barley (Hordeum vulgare), and indian mustard (Brassica
juncea). Environmental Science and Technology 32:5. 40 Introduction & Background Introduction & Background Elfstrand, S., Bath, B. & Martensson, A. 2007. Influence of various forms of
green manure amendment on soil microbial community
composition, enzyme activity and nutrient levels in leek. Applied Soil
Ecology 36 (1):70-82. Fagence, M. 1996. Nauru: Substituting one dependency for another? A
challenge to the orthodoxy of sustainable development. Sustainable
Development 4:10. Fageria, N. K. 2007. Soil fertility and plant nutrition research under field
conditions: Basic principles and methodology. Journal of Plant
Nutrition 30 (2):203-223. Fenner, M. & Thompson, K. 2005. The ecology of seeds, Cambridge
University Press. Ferguson, C., & Kasamas, H. (1999). Risk assessment for contaminated sites
in Europe., LQM Press, Nottingham (UK),. Fernandes, E.C.M. 1991. Ensayo de providencias de Gliricidia sepium
(Jacq.) Walp. en un ultisol de la Amazonia Peruana. In Manejo de
Suelos Tropicales in Latinoamerica, edited by J. T. Smith, W. R. Raun
& F. Bertsch. Raleigh, NC: Soil Sci. Dept., North Carolina State
University. Fragoso, C., Barois, G., Gonzalez, G., Arteaga, C. & Patrón, J.C. 1993. Relationship between earthworms and soil organic matter levels in
natural and managed ecosystems in the Mexican tropics. In soil
organic matter dynamics and sustainability of tropical agriculture, edited by
K. Mulongoy & R. Merckx. New York: Wiley. Garbisu, C. & Alkorta, I. 2001. Phytoextraction : a cost-effective plant-based
technology for the removal of metals from the environment. Bioresource Technology 77 (3):8. Ghassemi, F. & Jakeman, A.J. 1990. Mathematical modelling of sea water
intrusion, Nauru Island. Hydrological Processes 4:23. Ghosh, A. 2007. Sustainable impact of in situ leguminous green manuring
on grain yield and N utilization patterns of rainfed lowland rice 41 Introduction & Background (Oryza sativa) grown under different cropping geometries. Journal of
Sustainable Agriculture 30 (3):71-86. Gleba, D., Borisjuk, N.V., Borisjuk, L.G., Kneer, R., Poulev, A.,
Skarzhinskaya, M., Dushenkov, S., Logendra, S., Gleba, Y.Y. &
Raskin. I. 1999. Use of plant roots for phytoremediation and
molecular farming. Proceedings of the National Academy of Science USA
96:5. Gowdy, J.M., & McDaniel, C.N. 1999. The physical destruction of Nauru:
An example of weak sustainability. Land Economics 75 (2):6. Gregory, R., Ohlson & D. Arvai, J. 2006. Deconstructing adaptive
management: Criteria for applications to environmental
management. Ecological Applications 16(6): 15. Guo, Y., & Marschner, H. 1995. Introduction & Background Uptake, distribution, and binding of
cadmium and nickel in different plant species. Journal of Plant
Nutrition 18:16. Han, X. Z., Wang, S. Y., Veneman, P. L. M. & Xing, B. S. 2006. Change of
organic carbon content and its fractions in black soil under long-
term application of chemical fertilizers and recycled organic manure. Communications in Soil Science and Plant Analysis 37 (7-8):1127-1137. Harrington, C. A. 1999. Forests planted for ecosystem restoration or
conservation. New Forests 17 (1-3):175-190. Harrison, D. 2006. Peak oil, climate change, public health and well-being. Journal of the Royal Society for the Promotion of Health 126 (2):62-63. Helmke, P.A. 2000. The chemical composition of soils. In Handbook of Soil
Science, edited by M. E. Sumner: CRC Press LLC. Herzig, R., Rehnert, A., Erismann, K.-H., Müller-Schërer, H. & Guadagnini,
M. 2000. Comparative screening and assessment of the
phytoextraction power of biotechnical improved tobacco clones on a
soil spiked with elevated concentrations of plant available metals –
first results with special emphasis of Cd, Zn and Cu. In COST 837
Conference. Madrid (Spain). 42 Introduction & Background Hirsch, R. L., Bezdek, R. & Wendling, R. 2006. Peaking of world oil
production and its mitigation. Aiche Journal 52 (1):2-8. Huang, C.X. & Van Steveninck. 1989. The role of particular pericycle cells
in the apoplastic transport in root meristems of barley. Journal of
Plant Physiology 135:5. Huang, J.W., Chen, J., Berti, W.R. & Cunningham, S.D. 1997. Phytoremediation of lead-contaminated soils: role of synthetic
chelates in lead phytoextraction. Environmental Science and Technology
31:6. Huang, J.W., Blaylock M.J., Kapulmik, Y., & Ensley B.D. 1998. Phytoremediation of uranium contaminated soils: role of organic
acids in triggering uranium hyperaccumulation in plants. Environmental Science and Technology 32:5. Hunter, G., Harris, L. & Pollock, N. 1994. Overview report. In
Rehabilitation and Development Feasibility Study, edited by G. Hunter:
Nauru Australia Cooperation. IPCC. 2001. Climate Change 2001: Impacts, Adaptation and Vulnerability:
Intergovernmental panel on Climate Change. IPCC. 2001. Climate Change 2001: Impacts, Adaptation and Vulnerability:
Intergovernmental panel on Climate Change. Jordan, C.F., ed. 1989. An Amazonian Rain Forest. The Structure and Function
of a Nutrient Stressed Environment and the Impact of Slash-and-Burn
Agriculture. Park Ridge, NJ: Parthenon Publishing Group. Jordan, C.F., ed. 1989. An Amazonian Rain Forest. The Structure and Function
of a Nutrient Stressed Environment and the Impact of Slash-and-Burn
Agriculture. Park Ridge, NJ: Parthenon Publishing Group. Jordan, C.F., ed. 1989. An Amazonian Rain Forest. Introduction & Background The Structure and Function Kang, B.T. 1993. Alley cropping: past achievements and future directions. Agroforestry Systems 23:15. Kang, B.T. & Reynolds, L. 1989. Alley Cropping in the Humid and Subhumid
Tropics. Ibadan, Nigeria: IDRC. Kang, B.T., Wilson, G.T., & Lawson, T.L. 1981. Alley cropping of maize
(Zea mays L.) and Leucaena (Leucaena leucocephala Lam.) in southern
Nigeria. Plant Soil 63:15. Kärenlampi, S., Schat, H., Vangronsveld, J., Verkleij, J.A.C., Van der Lelie,
D., Mergeay, M. & Tervahauta, A.I. 2000. Genetic engineering in
the improvement for phytoremediation of metal polluted soils. Environmental Pollution 107:7. 43 Introduction & Background Kastori, R., Petrovic, M. & Petrovic, N. 1992. Effects of excess Pb, Cd, Cu
and Zn on water relations in sunflower. Journal of Plant Nutrition
15:13. Khan, A.G. 2001. Relationships between chromium biomagnification ratio,
accumulation factor, and mycorrhizae in plants growing on tannery
effluent-polluted soils. Environment International 26:7. Khan, S., El-Latif Hesham, A., Qiao, Q., Rehman, S. & He, J. 2010. Effects
of Cd and Pb on soil microbial community structure and activities. Environmental Science & Pollution Research 17, 9. Killham, K. 1994. Soil ecology: Cambridge University Press. Killham, K. & Firestone, M.K. 1983. Vesicular arbuscular mycorrhizal
mediation of grass response to acidic and heavy metal depositions. Plant and Soil 72:10. Kingston, P.A. 2004. Surveillance of Drinking Water Quality in the Pacific
Islands: Situation Analysis and Needs Assessment . Country Reports. UNEP UNEP Kirk, A.R. 2000. The Republic of Nauru, The Blue Pacific Series: Capital Letters
Publishing. Kirkham, M.B. 1978. Water relations of cadmium-trated plants. Journal of
Environmental Quality 7:3. Kneeshaw, D. D., Leduc, A., Drapeau, P., Gauthier, S., Pare, D., Carignan,
R., Doucet, R., Bouthillier, L. & Messier, C. 2000. Development of
integrated ecological standards of sustainable forest management at
an operational scale. Forestry Chronicle 76 (3):481-493. Krämer, U., Smith, R.D., Wenzel, W.W., Raskin, I. & Salt, D.E. 1997. The
role of metal transport and tolerance in Nickel hyperaccumulation
by Thlaspi goesingense Halacsy. Plant Physiology 115:10. Kumar, P.B.A.N., Dushenkov, V., Motto, H. & Raskin, I. 1995. Phytoextraction: the use of plants to remove heavy metals from soils. Environmental Science and Technology 29:7. Kumar, N., Bauddh, K., Singh, R., Anand, K., Barman, S. C. &Singh, D. P. 2009. Phytotoxicity of trace metals (Cu & Cd) to Gram (Cicer 44 Introduction & Background arientinum) and Mung (Phaseolus mungo). Journal of Ecophysiology
and Occupational Health 9, 7. Kunito, T., Saeki, K., Nagaoka, K., Oyaizu, H. & Matsumoto, S. 2001. Characterization of copper resistant bacterial community in
rhizosphere of highly copper-contaminated soil. European Journal of
Soil Biology 37:8. Lal, R. 1990. Soil Erosion in the Tropics. New York: McgrawHill. ———. 2007. Soil science and the carbon civilization. Soil Science Society of
America Journal 71 (5):1425-1437. Lasch, P., Lindner, M., Erhard, M., Suckow, F. & Wenzel, A. 2002. Regional impact assessment on forest structure and functions under
climate change - the Brandenburg case study. Forest Ecology and
Management 162 (1):73-86. Li, X., Chen, B., Feng, G. & Christie, P. 2002. Role of arbuscular
mycorrhizal fungi in alleviation of Zn phytotoxicity and mineral
nutrition of host plants. In 17th WCSS World Congress of Soil Science
Bangkok, Thailand. Lin, R., Wang, X., Luo, Y., Du, W., Guo, H. & Yin, D. 2007. Effects of soil
cadmium on growth, oxidative stress and antioxidant systems in
wheat seedlings (Triticum aestivum L.). Chemosphere 69: 10. Liu, B., Gumpertz, M. L., Hu, S. & Ristaino, J. B. 2007. Long-term effects of
organic and synthetic soil fertility amendments on soil microbial
communities and the development of southern blight. Soil Biology &
Biochemistry 39 (9):2302-2316. Lombi, E., Zhao, F.J., Dunham, S.J. & McGrath, S.P. 2001. Phytoremediation of heavy metalcontaminated soils : natural
hyperaccumulation versus chemically enhanced phytoextraction. Journal of Environmental Quality 30:8. UNEP Lucia, M., Andre´, J.-M., Gonzalez, P., Baudrimont, M., Bernadet, M.-D.,
Gontie, K., Maury-Brachet, R., Guy, G. & Davail, S. 2010. Effect of
dietary cadmium on lipid metabolism and storage of aquatic bird
Cairina moschata. Ecotoxicology 19, 8. 45 Introduction & Background Introduction & Background Introduction & Background Macdonald, B. 1988. In Pursuit of the Sacred Trust: Trusteeship and
Independence in Nauru, New Zealand Institute of Internal Affairs. Maksymiec, W. & Krupa, Z. 2006. The effects of short-term exposition to
Cd, excess Cu ions and jasmonate on oxidative stress appearing in
Arabidopsis thaliana. Environmental and Experimental Botany 57: Manner, H. I., Thaman, R. R., Hassall, D. C. 1984. Phosphate mining
induced vegetation changes on Nauru Island. Ecology 65 (5):11. Marsh, J. 1994. Environment B. Waste management. In Rehabilitation and
Development Feasibility Study: Nauru Australia Cooperation. Masters, R. 2007. Proposed Rehabilitation Implementation Strategy – Nauru
Rehabilitation Corporation – Draft report. GSS Environmental. Matson, P. A., Parton, W. J., Power, A. G. & Swift, M. J. 1997. Agricultural
Intensification and Ecosystem Properties. Science 277:6. Meehl, G. A. 1996. Vulnerability of freshwater resources to climate change
in the tropical Pacific region. Water Air and Soil Pollution 92 (1-2):203-
213. Moitra, R., Ghosh, D. C., Majumdar, S. K. & Sarkar, S. 1994. Influence of
tillage andmulching on growth, yield and economics of rain-fed
yellow sarson (Brassica-Rapa var. Glauca) in lateritic acid belt of
West-Bengal. Indian Journal of Agricultural Sciences 64 (3):156-159. Moreno, R.A., & Sánchez, J.F. 1994. The effect of using mulches with
intercropping. In Tapado Slash/Mulch: How Farmers Use It, and What
Researchers Know About It, edited by H. D. Thurston, M. Smith, G. Abawi & S. Kearl. Ithaca, NY: CIIFAD and CATIE, Cornell
University. Morrison, R. J. & Manner, H. I. 2005. Pre-mining pattern of soils on
Nauru, central Pacific. Pacific Science 59 (4):523-540. Nanda-Kumar, P.B.A., Dushenkov, V., Motto, H. & Raskin, I. 1995. Phytoextraction: the use of plants to remove heavy metals from soils. Environmental Science and Technology 29:7. Environmental Science and Technology 29:7. 46 Introduction & Background Nauru, Republic of. 2003. First National Report of the Republic of Nauru
to the United Nations Convention to Combat Desertification:
Department of Economic Development and Environment. Nauru, Republic of. 2003. First National Report of the Republic of Nauru
to the United Nations Convention to Combat Desertification:
Department of Economic Development and Environment. ———. 2004. Ten Year Review of th Barbados Programme of Action. In
National Assessment Review: Republic of Nauru. ———. 2004. Ten Year Review of th Barbados Programme of Action. In
National Assessment Review: Republic of Nauru. NCCC. 1999. Climate Change Response: Nauru National Committee on
Climate Change. Nurse, L. & Moore, R. 2007. Introduction & Background Critical considerations for future action
during the second commitment period: A small islands' perspective. Natural Resources Forum 31 (2):102-110. Nyerges, A.E. 1989. Coppice swidden fallows in tropical deciduous forest:
biological, technological, and sociocultural determinants of
secondary forest successions. Human Ecology 17:22. Palmer, M. A., Falk, D. A. & Zedler, J.B. 2006. Ecological theory and
restoration ecology. Foundations of restoration ecology. D. A. Falk. Washington, Island Press. Planelló, R., Martínez-Guitarte, J. L. & Morcillo, G. 2010. Effect of acute
exposure to cadmium on the expression of heat-shock and hormone-
nuclear receptor genes in the aquatic midge Chironomus riparius. Science of the Total Environment 408, 6. Prasad, R. 2006. Towards sustainable agriculture in India. National Academy
Science Letters-India 29 (1-2):41-44. Quanchi, M. 2007. Troubled times: Development and economic crisis in
Nauru. In Security and development in the Pacific Islands: Social resilience
in emerging States, edited by M. A. Brown: Lynne Rienner Publishers. Quanchi, M. 2007. Troubled times: Development and economic crisis in
Nauru. In Security and development in the Pacific Islands: Social resilience
in emerging States, edited by M. A. Brown: Lynne Rienner Publishers. Ramĺrez, C., Sanchez, G., Kass, D., Viquez, E., Sanchez, J., Vasquez, N., &
Ramirez, G. 1990. Advances in Erythrina research at CATIE. In Fast
Growing Trees and Nitrogen Fixing Trees, edited by D. Werner & P. Müller. Stuttgart: Gustav Fischer Verlag. Richter, D.D. & Markewitz, D. 1995. How deep is soil? Bioscience 45:10. Robin South, G., Skelton, P.A., Veitayana, J., Resture, A., Carpenter, C., Nauru. In Security and development in the Pacific Islands: Social resilience
in emerging States, edited by M. A. Brown: Lynne Rienner Publishers. Ramĺrez, C., Sanchez, G., Kass, D., Viquez, E., Sanchez, J., Vasquez, N., &
Ramirez, G. 1990. Advances in Erythrina research at CATIE. In Fast
Growing Trees and Nitrogen Fixing Trees, edited by D. Werner & P. Müller. Stuttgart: Gustav Fischer Verlag. Ramĺrez, C., Sanchez, G., Kass, D., Viquez, E., Sanchez, J., Vasquez, N., &
Ramirez, G. 1990. Advances in Erythrina research at CATIE. In Fast
Growing Trees and Nitrogen Fixing Trees, edited by D. Werner & P. Müller. Stuttgart: Gustav Fischer Verlag. Richter, D.D. & Markewitz, D. 1995. How deep is soil? Bioscience 45:10. Robin South, G., Skelton, P.A., Veitayana, J., Resture, A., Carpenter, C., Ramĺrez, C., Sanchez, G., Kass, D., Viquez, E., Sanchez, J., Vasquez, N., &
Ramirez, G. 1990. Advances in Erythrina research at CATIE. Introduction & Background In Fast
Growing Trees and Nitrogen Fixing Trees, edited by D. Werner & P. Müller. Stuttgart: Gustav Fischer Verlag. ,
y
Growing Trees and Nitrogen Fixing Trees, edited by D. Werner & P. Müller. Stuttgart: Gustav Fischer Verlag. Müller. Stuttgart: Gustav Fischer Verlag. Richter, D.D. & Markewitz, D. 1995. How deep is soil? Bioscience 45:10. Robin South, G., Skelton, P.A., Veitayana, J., Resture, A., Carpenter, C.,
Pratt, C. & Lawedrau, A. 2004. The Global International Waters 47 Introduction & Background Assessment for the Pacific Islands: Aspects of transboundary, water
shortage, and coastal fisheries issues. Ambio 33 (1-2):9. Rutledge, S., Campbell, D. I., Baldocchi, D. & Schipper, L.A. 2010. Photodegradation leads to increased carbon dioxide losses from
terrestrial organic matter. Global Change Biology 16: 10. Salt, D.E., Blaylock, M., Kumar, N.P.B.A., Dushenkov, V., Ensley, B.D.,
Chet, I. & Raskin I. 1995. Phytoremediation: a novel strategy for the
removal of toxic metals from the environment using plants. Biotechnology 13:7. Salt, D.E., Price R.C., Pickering I.J., & Raskin I. 1995. Mechanisms of
cadmium mobility and accumulation in Indian mustard. Plant
Physiology 109:7. Satarug, S., Garrett, S. H., Sens, M. A. & Sens, D. A. 2010. Cadmium,
Environmental Exposure, and Health Outcomes. Environmental
Health Perspectives 118, 9. Sato, R., Kido, T., Honda, R., Nishijo, M., Nakagawa, H., Kobayashi, E. &
Suwazono, Y. 2010. Seventeen-Year Observation on Urinary
Cadmium and β2-Microglobulin in Inhabitants After Cessation of
Cadmium-Exposure in Japan. Bulletin of Environmental
Contamination and Toxicology 84, 5. Sen, R. 2005. Towards a multifunctional rhizosphere concept: back to the
future? New Phytologist 168 (2):266-268. Shen, Z.-G., Li, X.-D., Wang, C.-C., Chen, H.-M. & Chua, H. 2002. Lead
phytoextraction from a contaminated soil with high-biomass plant
species. Journal of Environmental Quality 31:8. Sullivan, P. 2003. Overview of cover crops and green manures: Fundamentals of
sustainable agriculture, Appropriate Technology Transfer for Rural
Areas (ATTRA). Sumner, M.E. & Wilding, L.P. 2000. Introduction. In Handbook of Soil
Science, edited by M. E. Sumner: CRC Press LLC. 48 Introduction & Background Sun, Y., Zhou, Q. & Diao, C. 2008. Effects of cadmium and arsenic on
growth and metal accumulation of Cd-hyperaccumulator Solanum
nigrum L. Bioresource Technology 99: 8. Szott, L.T. 1993. Fertilizers in agroforestry systems. Agroforestry Systems 23:19. Tack, F.M.G, Esteban-Mozo, J. & Verloo, M.G. 1998. Cadmium uptake by
cucumber plants as affected by fluctuations in nutrient solution
cadmium concentrations during growth. Communications in Soil
Science Analysis 29:7. Thaman, R.R. 1992. Introduction & Background Vegetation of Nauru and the Gilbert Islands - case
studies of poverty, degradation, disturbance and displacement. Pacific Science 46(2): 128-158. Thaman, R.R., Fosberg, F.R., Manner, H.I, & Hassall, D.C. 1994. The flora
of Nauru: a compilation and analysis of the vegetation and flora of
the equatorial Pacific Ocean island of Nauru. Atoll Research Bulletin
390-396(392). Thaman, R.R., Hassall, D.C., & Takeda, S. 2008. Plants of Nauru - Guide to
indigenous and introduced plants of particular cultural importance and
weeds of potential threat to Nauru. Suva, Fiji, Secretariat of the Pacific
Community (SPC) Forests and Trees Programme; South Pacific
Regional Herbarium (SPRH); and School of Geography Faculty of
Islands and Oceans (FIO) The University of the South Pacific (USP). Thurston, H.D. 1992. Sustainable Practices for Plant Disease Management in
Traditional Farming Systems. Boulder, CO: Westview. ———. 1997. Slash/Mulch Systems - Sustainable Methods for Tropical Agriculture. Colorado: Westview Press. Trzcinka-Ochocka, M., Jakubowski, M., Szymczak, W., Janasik, B. &
Brodzka, R. 2010. The effects of low environmental cadmium
exposure on bone density. Environmental Research 110, 8. Tyrer, T.G. 196-? Nauru and Ocean Islands story: New Zealand Dairy Exporter. Valarini, P.J., Alvarez, M.C.D., Gasco, J.M., Guerrero, F. & Tokeshi, H. 2002. Integrated evaluation of soil quality after the incorporation of 49 Introduction & Background organic matter and microorganisms. Brazilian Journal of Microbiology
33 (1):35-40. ———. 2003. Assessment of soil properties by organic matter and EM-
microorganism incorporation. Revista Brasileira De Ciencia Do Solo 27
(3):519-525. Van Campenhout, K., Goenaga Infante, H., Hoff, P. T., Moens, L.,
Goemans, G., Belpaire, C., Adams, F., Blust, R. & Bervoets, L. 2010. Cytosolic distribution of Cd, Cu and Zn, and metallothionein
levels in relation to physiological changes in gibel carp (Carassius
auratusgibelio) from metal-impacted habitats. Ecotoxicology and
Environmental Safety 73, 10. van den Brink, N., Lammertsma, D., Dimmers, W., Boerwinkel, M.-C. &
van der Hout, A. 2010. Effects of soil properties on food web
accumulation of heavy metals to the wood mouse (Apodemus
sylvaticus). Environmental Pollution 158, 7. van Diepeningen, A.D., de Vos, O.J., Korthals, G.W. & van Bruggen, van Diepeningen, A.D., de Vos, O.J., Korthals, G.W. & van Bruggen,
A.H.C. 2006. Effects of organic versus conventional management on
chemical and biological parameters in agricultural soils. Applied Soil
Ecology 31 (1-2):120-135. Vecchi, G.A. & Soden, B.J. 2007. Global warming and the weakening of the
tropical circulation. Journal of Climate 20 (17):4316-4340. Voelkner, H. 1979. Introduction & Background Urgently needed – Ideal green mulch crop for the
Tropics. World Crops 31 (2):76-78. Walters, C.J. & Hollings, C.S. 1990. Large-scale management experiment
and 'learning by doing'. Ecology 71(6): 9. Waqa, B.D. 1999. The Year 2000 Assessment Country report 1999. Country:
Nauru: Education for all (EFA). Weeramantry, C. 1992. Nauru - Environmental damage under international
trusteeship: Oxford University Press. White, I., Falkland, T., Metutera, T., Metai, E., Overmars, M., Perez, P. &
Dray, A. 2007. Climatic and human influences on groundwater in
low atolls. Vadose Zone Journal 6 (3):581-590. 50 Introduction & Background White, R.E. 2006. Principles and Practice of Soil Science - The soil as a natural
resource. 4 ed: Blackwell Publishing. Wild, A. 1993. Soils and the Environment: An introduction. Cambridge:
Cambridge University Press. Wiltse, C.C., Rooney, W.L., Chen, Z., Schwab, A.P. & Banks, M.K. 1998. Greenhouse evaluation of agronomic and crude oil
phytoremediation potential among alfalfa genotypes. Journal of
Environmental Quality 27:5. Zhao, F.J., Lombi, E., McGrath, S.P. 2001. Cadmium hyperaccumulation by
Thlaspi caerulescens: physiology and potential for phytoremediation. Paper read at Proceedings of the 6th International Conference on
the Biogeochemistry of trace metals, ICOBTE, at Glueph (Canada). 51 51 Introduction & Background 52 52 Chapter 2 – Biomass Production ABSTRACT Nauru requires substantial volumes of organic matter to improve soils for
landscape rehabilitation. This paper describes a preliminary field trial
assessing the productivity of a range of species grown in situ in four local
soils, with and without mulch. Total and exchangeable fractions of soil
cadmium were also assessed with regard to effects on biomass production. Overall, productivity was highest - for three species - Cajanus cajan, Leucaena
leucocephala, and Calopogonium mucunoides; for plants grown in the soil
known locally as Black soil; when wood-chip mulch was used; and in soils
containing lower levels of cadmium. INTRODUCTION Healthy functional soil systems are essential for human wellbeing. Soil infertility and toxicity are two examples of threats to the health
of this system. Loss of soil fertility may lead to desertification in
environments where biota are necessary to protect soils from
environmental stresses. Soils containing toxic compounds pose
health risks to humans and all organisms living on or near
contaminated land. The severity of the risks are clearly illustrated
in the Pacific Islands, where infertile or contaminated land can
significantly exacerbate the shortage of available land (Gowdy &
McDaniel 1999; van der Velde, Green et al. 2007). Few places
demonstrate this situation more starkly than Nauru. Nauru is a 21km2 raised limestone island 41km south of the equator
at longitude 166° 56’ E in the Central Pacific (Fig. 1.1). It is 53 Chapter 2 – Biomass Production surrounded by a fringing coral reef that sharply drops away 4,300m
to the Pacific floor. ‚Bottomside‛ is the local name for the thin
coastal plain surrounding the interior of the island, known as
‚Topside‛. Topside is a 1,620ha raised plateau that ranges from 20-
60m in elevation, and is largely divided from Bottomside by a steep
escarpment. A brackish lagoon, called ‚Buada‛ is in a large interior
depression, and is hydraulically connected with the Pacific (Hunter,
Harris et al. 1994; Kingston, P.A. 2004) (See Appendices I & II). surrounded by a fringing coral reef that sharply drops away 4,300m
to the Pacific floor. ‚Bottomside‛ is the local name for the thin
coastal plain surrounding the interior of the island, known as
‚Topside‛. Topside is a 1,620ha raised plateau that ranges from 20-
60m in elevation, and is largely divided from Bottomside by a steep
escarpment. A brackish lagoon, called ‚Buada‛ is in a large interior
depression, and is hydraulically connected with the Pacific (Hunter,
Harris et al. 1994; Kingston, P.A. 2004) (See Appendices I & II). Figure 2.1 – Map of Nauru’s position in the Pacific Figure 2.1 – Map of Nauru’s position in the Pacific Mining of phosphate in Nauru has lead to various environmental
effects, including deforestation, biodiversity loss, soil loss, liberation
of toxic sub-soil cadmium deposits, soil degradation and
desertification (Manner 1984; Morrison & Manner 2005). Nauru is
facing various social, political, economic, and health issues which
are exacerbated by soil degradation (Weeramantry 1992; Fagence 1996;
Gowdy & McDaniel 1999). INTRODUCTION Rehabilitation of mined lands in Nauru
constitutes one of the five goals of the Nauru National Sustainable 54 Chapter 2 – Biomass Production Chapter 2 – Biomass Production Chapter 2 – Biomass Production Development Strategy 2005-2025 (Nauru 2005). The 2009 review of
the NSDS identified that objectives for the rehabilitation of mined
land have thus far not been achieved according to its targets (Nauru
2009). There are various reasons why NSDS targets have not been
achieved: There is little knowledge of how to achieve specific goals in the
ecological setting of Nauru. The amount of information which is potentially relevant to
rehabilitation in this setting is vast. The scale and nature of the project is unlike any previous
restoration project anywhere. Soil moisture is generally low, due to highly variable rainfall
patterns, high rates of evaporation, and ready percolation
through the porous underlying rock. Records of the historical ecology of Nauru, which could inform
management decisions, are fragmentary (Morrison & Manner
2005). In Nauru there is a lack of human capital with experience and
skills appropriate to landscape rehabilitation. A range of goals for restoration were developed under the Nauru
Australia Cooperation Rehabilitation and Development Feasibility
Study 1994 (RDFS), many of which require the rehabilitation of soils
for various land uses including housing, conservation, A range of goals for restoration were developed under the Nauru
Australia Cooperation Rehabilitation and Development Feasibility
Study 1994 (RDFS), many of which require the rehabilitation of soils
for various land uses including housing, conservation,
agroforestry/residential, sports/recreation/parks, an education
complex, a public service complex, a cemetery, a municipal waste A range of goals for restoration were developed under the Nauru
Australia Cooperation Rehabilitation and Development Feasibility
Study 1994 (RDFS), many of which require the rehabilitation of soils
for various land uses including housing, conservation,
agroforestry/residential sports/recreation/parks an education agroforestry/residential, sports/recreation/parks, an education
complex, a public service complex, a cemetery, a municipal waste agroforestry/residential, sports/recreation/parks, an education
complex, a public service complex, a cemetery, a municipal waste 55 Chapter 2 – Biomass Production site, a hospital complex, and an industrial complex (Carstairs 1994;
Hunter, Harris et al. 1994). site, a hospital complex, and an industrial complex (Carstairs 1994;
Hunter, Harris et al. 1994). These land uses require soils, although they require them for
different reasons and accordingly the properties required of the
soils differ in relation to their intended purposes. Chapter 2 – Biomass Production Generally the soil
purposes can be distinguished according to use as: a) substrate, such as for hospital, education, public service and
industrial complexes b) growth media for food crops, as in the agroforestry/residential
areas c) growth media for vegetation cover for various purposes, as in
the conservation, agroforestry/residential and
sports/recreation/parks areas, as well as selected areas of the
education, public service, hospital and industrial complexes and
cemetery d) media for containment and amelioration of biological hazards,
as in the cemetery area and municipal waste site
(Baines 1994; Carstairs 1994). Since 1970 mining operations have stockpiled soil for rehabilitation
purposes (Baines 1994; Gowdy & McDaniel 1999). These soils have
lost considerable amounts of material, organic content and biota
while stockpiled, leaving the soils relatively infertile. Additionally,
cadmium liberated through mining operations has contaminated
the stockpiled soils limiting their value for rehabilitation. Overall, it is required that the volume and fertility of soils available
for landscape rehabilitation be increased. Soil provides plants with 56 Chapter 2 – Biomass Production an anchorage for roots, supply of water, oxygen and mineral
nutrients, and a buffering against the adverse changes of
temperature and pH (Donahue 1958; Wild 1993; Matson, Parton et al. 1997). Increasing the organic carbon content of stockpiled soils is a
viable method for increasing soil volume and fertility and may
provide additional benefits to soils including increased water
retention properties, increased soil biota and thus increased
biological activity, and a carbon sink with potential benefits for the
country with regard to carbon balance and associated carbon
finance opportunities. Of additional concern are the significant concentrations of cadmium
in Nauruan soils. Cadmium is known to induce a range of severe
physiological disorders in plants such as chlorophyll reduction,
changes to phenols, oxidative stress and altered enzyme activity
which ultimately retard plant growth (Maksymiec & Krupa 2006; Lin,
Wang et al. 2007; Dell'Amico, Cavalca et al. 2008; Sun, Zhou et al. 2008; Ci,
Jiang et al. 2009). It must be determined to what cadmium toxicity is
of concern to the rehabilitation project. This paper describes the first part of a two-part experimental field
trial to assist in the developing a successful soil restoration
programme through biomass production and cadmium
phytoaccumulation for Nauru. AIM To assess the biomass production capacity of a range of plant
species for the rehabilitation of soils on Nauru HYPOTHESES Soils with the greatest cadmium content will have the lowest
biomass accumulation rates Mulch will increase biomass accumulation There will be variation in productivity between species Soil type will effect biomass production Chapter 2 – Biomass Production The second paper entitled
‚Cadmium phytoaccumulation in Nauru: A preliminary field trial‛
assesses the cadmium phytoaccumulation capability of various
species in a range of Nauruan soils as well as the impact of the
addition of biomass through a locally produced wood chip mulch
upon cadmium phytoaccumulation. 57 Chapter 2 – Biomass Production In this paper, biomass production capability is assessed for various
species in a range of Nauruan soils as well as the impact of the
addition of biomass through locally produced wood chip mulch. Total biomass accumulated according to soil-type, species,
cadmium level, and use of mulch is described. METHOD A selection of species were sown in a range of soils, watered and
grown for a period of seven months whereupon they were to be
assessed for their biomass production potential. Field plots were established in Nauru within Topside, the elevated
platform which constitutes the centre, and greater part of, the
island. 58 Chapter 2 – Biomass Production Buada soil Buada soil
‘Buada soil’ is soil from the Buada province of Nauru. Buada is a
large depression containing a brackish lagoon and fairly fertile soils. Buada is protected from the hot salt laden winds that sweep across
Nauru so the plants that grow there are less prone to desiccation. Buada soils are undisturbed compared with Black soil, and contain
considerably higher volumes of organic material and soil
organisms. Variables Five species were selected according to their suitability for soil
rehabilitation on Pacific tropical islands– green mulches, drought
tolerant, fast growth, and readily available seed as determined by
the Centre for Tropical Agriculture and Human Resources
(CTAHR) at the University of Hawai’i (Smith & Valenzuela 2002;
Valenzuela & Smith 2002a; Valenzuela & Smith 2002b). The species used were
White oat - Avena sativa
Black oat - Avena strigosa Schreb. Ryegrass - Lolium multiflorum
Barley - Hordeum vulgare
Hairy Vetch - Vicia villosa
Soil types
The soils used were
Black soil
Buada soil
Topsoil
Dolomite The species used were Black soil
‘Black soil’ is the name given locally to soil from the soil stockpiles
on Topside. Black soil is the remaining topsoil from across the
central plateau, removed for phosphate mining access, and left
stockpiled for several decades. This soil has lost significant
amounts of organic material through stockpiling and exposure to 59 Chapter 2 – Biomass Production the sun without substantial cover. Stockpiling also resulted in large
anoxic areas within the piles which, along with desiccation and
losses of soil organic matter, are likely to have reduced the
populations of soil organisms within the soil. The soil used for this
experiment was sourced from Stockpile A which has a greater
variety of plant species growing on it than Stockpile B which is
covered with a monoculture of Leucaena leucocephala. Mulch Growing conditions on the Topside of Nauru are arid. Nauru
experiences highly variable rainfall and large amounts of incident
sunlight due to its location in the dry equatorial zone of the tropical
Pacific Ocean. In these dry conditions, desiccation leads to
increased plant mortality and reduced growth. Mulches can be
used to protect soils from temperature fluctuation, to reduce soil
moisture evaporation, and increase soil carbon content. Local
production of wood-chip mulch from biomass was investigated
within this experiment. Wood-chip was applied to a depth of 4-5cm
on one of the two experimental beds. To ensure sufficient soil
moisture, the beds were watered daily for the first month and then
weekly unless rainfall meant that this was unnecessary. Dolomite The ‘Dolomite’ does not have all of the characteristics necessary to
be accurately described as a soil. It is a mixed aggregate of fine to
course material which has been mined containing a high proportion
of rock phosphate. This material has been proposed as a medium
for developing soil layers by the Nauru Rehabilitation Feasibility
Study and other documents relating to landscape restoration on
Nauru. Topsoil The ‘Topsoil’ is sourced from the Nauru Rehabilitation Corporation
(NRC) nursery area in Topside which is located in an area that has
operated as a sports oval and recreation area; the soil was part of
the track and field area and was tended through use of fertiliser for
many years. It was then used as the site of the Nauru detention
centre for Australia, before becoming the site of the NRC nursery. This soil has been managed to some degree, having received
applications of fertiliser and pesticides. The soil has been covered
by pasture for many years, and is used preferentially in garden
developments by the nursery staff. 60 Chapter 2 – Biomass Production Complications The intention of the experiment was to test the viability of a range
of selected species for phytoaccumulation in Nauru. Accordingly, a
range of species of seeds were planted according to a randomised
plan (Fig. 3.1). However, the resulting seedlings were unsuccessful
as the adverse growing conditions found in the field in Nauru 61 Chapter 2 – Biomass Production combined with the success of adventitious local seed from within
the soils killed off the experimental species. This outcome posed
significant issues for the original experimental design. However,
though an unexpected outcome, this result revealed two factors
important to the rehabilitation project; the significant challenges of
the Nauruan growing environment, and the potential for using local
flora which were evidently better suited for growth in adverse
conditions. So we identified the species, measured their biomass
production under the different treatments, and determined the
cadmium levels in different parts of the plants. Field plots The seeds of a range of species were sown in randomly assigned
mini-plots within larger plots of specific soil types. The larger plots
were arranged into two beds, one with mulch, the other without. (Fig. 2.2). The beds were situated at the Nauru Rehabilitation
Corporation nursery site (See Appendices I & II). Seeds were sown as per standard sowing densities for each species,
as recommended by the seed producers. The seeds were monitored
for germination success for 10 days. For the following six months
research obligations outside of Nauru, meant the beds were left in
the care of the nursery staff. Due to the lack of access to
telecommunications equipment on Nauru at the time, it was not
possible to contact the nursery staff during this absence. Nursery
staff observed during this period that adventitious species were
germinating in the soils, with the Topsoil plots showing the greatest
amount. Field plots 62 Chapter 2 – Biomass Production Key:
1
White Oat
Topsoil
2
Black Oat
Black soil
3
Ryegrass
Dolomite
4
Barley
Buada soil
5
Hairy Vetch
2
4
5
1
2
4
3
3
3
3
1
5
1
1
2
2
1
1
3
3
3
5
5
4
4
5
2
4
4
2
4
1
5
2
4
2
5
3
1
5
5
1
4
3
3
1
2
5
3
5
3
4
5
2
3
2
4
4
5
2
1
3
5
3
1
2
2
5
2
4
4
1
2
3
1
1
4
5
1
4
2
3
3
4
5
2
1
2
1
1
2
5
4
4
3
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5
4
1
2
1
3
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3
5
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1
2
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1
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4
Bed A
Bed B
Figure 2.2 – Field trial plots
= 1.666m
= 2m Key:
1
White Oat
Topsoil
2
Black Oat
Black soil
3
Ryegrass
Dolomite
4
Barley
Buada soil
5
Hairy Vetch 2
4
5
1
2
4
3
3
3
3
1
5
1
1
2
2
1
1
3
3
3
5
5
4
4
5
2
4
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2
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1
5
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1
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1
4
3
3
1
2
5
3
5
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5
2
3
2
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2
1
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2
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3
1
1
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1
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2
1
2
1
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2
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4
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1
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Bed A
Bed B
Figure 2.2 – Field trial plots
= 1.666m
= 2m 2
4
5
1
2
4
3
3
3
3
1
5
1
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4
Bed A
Bed B
Figure 2.2 – Field trial plots
= 1.666m
= 2m Figure 2.2 – Field trial plots 63 Chapter 2 – Biomass Production Preparation for sample collection
At the start of the third trip to Nauru, six months later in October
2008, it was found that weed species had overtopped and killed off
the planted trial species. Field plots This posed several issues to the
experimental design. The statistical power was reduced, as it was
now not possible to produce a full factorial model. Nevertheless, it
was determined that the circumstances could still provide valuable
data for the development of a process for cadmium
phytoaccumulation. At this point, the soils contained a large
amount of seed of local weedy species which successfully
germinated and grew in the adverse conditions found in Topside,
Nauru. Accordingly, the germination and seedling success of the
weedy species provides evidence of their adaptive suitability to the
conditions found on Topside. It was decided to identify and sample
the species which had grown, to assess their suitability for
phytoaccumulation (Table 2.1). Preparation for sample collection
At the start of the third trip to Nauru, six months later in October
2008, it was found that weed species had overtopped and killed off
the planted trial species. This posed several issues to the
experimental design. The statistical power was reduced, as it was
now not possible to produce a full factorial model. Nevertheless, it
was determined that the circumstances could still provide valuable
data for the development of a process for cadmium phytoaccumulation. At this point, the soils contained a large
amount of seed of local weedy species which successfully germinated and grew in the adverse conditions found in Topside,
Nauru. Accordingly, the germination and seedling success of the
weedy species provides evidence of their adaptive suitability to the
conditions found on Topside. It was decided to identify and sample
the species which had grown, to assess their suitability for
phytoaccumulation (Table 2.1). Field plots The species which were identified were –
Table 2.1 – Weed species found in soils
Sampled species
Black soil
Topsoil
Buada soil
Dolomite
Calopogonium
mucunoides
X
X
X
X
Cajanus cajan
X
X
X
Leucaena
leucocephala
X
X
X
X Sampled species
Black soil
Topsoil
Buada soil
Dolomite
Calopogonium
mucunoides
X
X
X
X
Cajanus cajan
X
X
X
Leucaena
leucocephala
X
X
X
X 64 Chapter 2 – Biomass Production Chapter 2 – Biomass Production Indigofera
hirsuta
X
X
X
X
Senna
occidentalis
X
X
Sida acuta
X
X
Indigofera
spicata
X
X
Waltheria indica X
X
Phylanthus
amarus
X
X
Ipomoea
macrantha
X
Scaevola taccada X
X
Unsampled
species
Chamaesyce
hirta
X
Tridax
procumbens
X
Stachytarpheta
jamaicensis
X
Of note was the presence of Pigeon Pea Cajanus cajan which is listed
by the College for Tropical Agriculture and Human Resources,
University of Hawai’i Mānoa as a beneficial green manure for the Indigofera
hirsuta
X
X
X
X
Senna
occidentalis
X
X
Sida acuta
X
X
Indigofera
spicata
X
X
Waltheria indica X
X
Phylanthus
amarus
X
X
Ipomoea
macrantha
X
Scaevola taccada X
X
Unsampled
species
Chamaesyce
hirta
X
Tridax
procumbens
X
Stachytarpheta
jamaicensis
X
Of note was the presence of Pigeon Pea Cajanus cajan which is listed Plant tissue sample collection At the end of the trial period, all plant material from each soil plot
was removed, washed, desiccated and weighed. Samples of leaf,
stem and root tissue were taken for each species in each plot. Sample Preparation and Analysis Soil cadmium concentrations were analysed using Inductively
Coupled Plasma Mass Spectrometry (IC-PMS). Samples were
prepared according to the methodologies described below. Soil samples were digested for exchangeable cadmium using the
NaNO3 extraction method described under Swiss legislation and by
Meers, Samson et al. (VSBo 1986; Meers, Samson et al. 2007). Unsampled
species Of note was the presence of Pigeon Pea Cajanus cajan which is listed
by the College for Tropical Agriculture and Human Resources,
University of Hawai’i, Mānoa as a beneficial green manure for the
Pacific (Valenzuela & Smith 2002c). It is possible that the species
arrived with asylum seekers who were detained on Nauru. This 65 Chapter 2 – Biomass Production species has a global production of around 40,000 km2, and is
cultivated for its protein rich seed, as a cover crop and companion
plant, for fodder and for firewood (Almeida, Furtunato et al. 2010; da
Silveira, Ribeiro da Cunha et al. 2010; Eltayeb, Ali et al. 2010; Linares,
Scholberg et al. 2010; Thampan & Remany 2010). It is a perennial
nitrogen fixing legume used for producing biomass. Cajanus cajan
grew successfully in conditions found on Topside Nauru that were
limiting for other species. Plant tissue sample collection Extraction of total cadmium in soils Extraction of total cadmium in soils
Total cadmium was extracted through total digestion of soil
samples as described by Temminghoff & Houba in 2004. 15mL
Teflon beakers were cleaned by initial triple rinse with milli-Q
water followed by soaking in 5% HNO3 for 2 weeks. The beakers
were treated to additional cleaning using concentrated (6-7 mol) 66 Chapter 2 – Biomass Production sub-boiled HNO3. 3 mL of sample was placed in the beakers which
were then sealed and placed on a hot plate at 120°C for 48 hours. The HNO3 was then removed from the beakers and the process was
repeated a second time. After treatment with HNO3 the samples
were thoroughly rinsed with milli-Q water and treated with
concentrated (6-7 mol) sub-boiled HCl in the same manner as used
with the HNO3 treatment. Following this, the beakers were cleaned
with 3mL concentrated HNO3 and 1mL concentrated HF and left
sealed on a 120°C hotplate for 36 hours. The beakers were then
triple rinsed with milli-Q water and dried. Each sample was individually homogenised and approximately
130mg of each soil sample was placed in the 15mL Teflon containers
and weighed. 4mL of concentrated HNO3 was added to the Teflon
containers and then left on a 120°C hotplate for 24 hours unsealed. This process was repeated and then 50 drops of concentrated HF
and 30 drops of concentrated HNO3 was added and sealed and
placed on a 120°C hotplate for 36 hours. Two additional treatments
with concentrated HNO3 were performed to ensure that all organic
compounds were chemically degraded. The product was then
dissolved in 5mL of 5% HNO3 and then weighed. 1mL was then
extracted and diluted with 3mL of 5%HNO3. Extraction of exchangeable cadmium in soils
15mL Teflon beakers and 10mL c-tubes were cleaned by initial triple
rinse with milli-Q water followed by soaking in 5% HNO3 for 2
weeks. The beakers were treated to additional cleaning using
concentrated (6-7 mol) sub-boiled HNO3. 3mL of sample was
placed in the beakers which were then sealed and placed on a hot 67 Chapter 2 – Biomass Production plate at 120°C for 48 hours. The HNO3 was then removed from the
beakers and the process was repeated a second time. After
treatment with HNO3 the samples were thoroughly rinsed with
milli-Q water and treated with concentrated (6-7 mol) sub-boiled
HCl in the same manner as used with the HNO3 treatment. Extraction of total cadmium in soils The
beakers and tubes were then triple rinsed with milli-Q water and
dried in preparation for extraction of bioavailable cadmium. Each sample was individually homogenised and approximately 1g
of soil sample was measured into the cleaned 15mL reaction tubes,
and weighed on a calibrated Sartorius CP225D balance. 5mL of
0.1M NaNO3 extractant was pipetted into each reaction tube and the
total masses of the extractant and the soil were weighed. The soil
extractant mixture was initially agitated to ensure full contact
between the extractant and soil. The reaction tubes were then
placed on a mixing plate to maintain full exposure of the soil to the
extractant. After 2 hours on the mixing plate, the reaction tubes
were placed in a centrifuge for 6 minutes at 3500rpm to settle out
suspended particles. 5 mL of 5% HNO3 was pipetted into the cleaned 10mL c-tubes,
weighed and 300µL of sample were added to each tube from the
centrifuged reaction tubes and weighed. This product constituted
the material to be analysed by the ICP-MS. This standard procedure was employed for each of the 18 soil
samples and 3 cadmium deposit samples. For error testing, five
samples were made up to three times the volume of the other
samples to provide sufficient quantities for multiple analyses and 68 Chapter 2 – Biomass Production Chapter 2 – Biomass Production data consistency checks. This applied to five samples: four soils and
one cadmium deposit. Additionally, 1ppb and 2ppb standards were made up from a
10ppb standard of toxic metals for calibration of ICP-MS output
data. These standards were made to greater volumes than the
samples and with volumes of 5% HNO3 and 0.1M NaNO3 in
equivalent proportions to that of the samples to ensure that the
viscosity of the standards would be equivalent to that of the
samples. This was done to ensure consistency of analysis in the
ICP-MS as fluid viscosity affects the rate of delivery to the nebuliser
of prepared samples. Inductively Coupled Plasma Mass Spectrometry analysis
Exchangeable fractions of soil cadmium in solution were then
determined using Inductively Coupled Plasma Mass Spectrometry
(ICP-MS). ICP-MS uses inductively coupled plasma to ionise
samples that are then recorded by a mass spectrometer. This
method allows for highly precise measurements of elemental
concentrations (Hokura, Matsuura et al. 2000). Samples in solution are
nebulised into plasma which ionises the elements contained therein. RESULTS The results of the analysis of cadmium concentrations in
experimental soils, and biomass productivity are as follows. Extraction of total cadmium in soils The ions are then passed through a system of electromagnets and
are directed to detectors which register the characteristics of
individual ions. Samples were delivered to the ICP-MS nebuliser for 60 seconds
followed by milli-Q water for 30 seconds; a 5% HNO3 wash for 90
seconds; then 60 seconds of a 1% HNO3 wash, before the next
sample was analysed. 1 ppb standards were used at the start, end 69 Chapter 2 – Biomass Production and at frequent intervals throughout the analyses. 2ppb standards
were used as secondary standards within the analysis to test the
standardisation process. Additionally, multiple analyses of some
samples and ‚blank‛ tubes were performed to ensure that both the
sample preparation and ICP-MS analysis were accurate and
effective. and at frequent intervals throughout the analyses. 2ppb standards
were used as secondary standards within the analysis to test the
standardisation process. Additionally, multiple analyses of some
samples and ‚blank‛ tubes were performed to ensure that both the
sample preparation and ICP-MS analysis were accurate and
effective. Soil cadmium Measurements of total and exchangeable soil cadmium were
statistically analysed to determine differences in cadmium
concentration between soil types Total cadmium The soils differed in total cadmium concentrations (p=0.001, The soils differed in total cadmium concentrations (p=0.001,
F=16.257, (3, 12 df), R2=0.859, adj. R2=0.806). Figure 2.3 illustrates the
grouping of soils according to concentration of total cadmium. The
dolomite gravel has the lowest concentration; Topsoil and Black soil
together form an intermediate group; Buada soil has the greatest
concentration of total cadmium. 70 Chapter 2 – Biomass Production Figure 2.3 – Total cadmium concentrations in Nauruan soils
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
Black soil
Buada soil
Topsoil
Dolomite
Cadmium concentration (ppb) Figure 2.3 – Total cadmium concentrations in Nauruan soils Exchangeable fraction Soils differed significantly in concentration of exchangeable
cadmium (p<0.001, F=64.509, (3, 20 df), R2=0.924, adj. R2=0.909). Soil
exchangeable cadmium differed significantly between soil types
(Fig. 3.4). Black soil, Topsoil and Buada soils contain equal
concentrations of exchangeable cadmium but significantly higher
concentration than dolomite. Figure 2.4 – Concentrations of exchangeable cadmium in
Nauruan soils
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Black soil
Buada soil
Topsoil
Dolomite
Cadmium concentration (ppb) Figure 2.4 – Concentrations of exchangeable cadmium in
Nauruan soils 71 71 Chapter 2 – Biomass Production Chapter 2 – Biomass Production Chapter 2 – Biomass Production Biomass by Total Cd Biomass production was found to not be significantly determined
by total soil cadmium (p=0.546, F=0.366 (1, 181 df.), R2=0.262, adj. R2=0.156). Biomass by exchangeable Cd y
g
Biomass production was found to be significantly determined by
exchangeable soil cadmium (p=0.031, F=4.766 (1, 176 df.), R2=0.377,
adj. R2=0.288). The β value for exchangeable soil cadmium was
found to be -0.043, σ=0.020, indicating that as exchangeable soil
cadmium increases by 1ppb, biomass production is reduced by
0.043g per m2. Biomass productivity Measurements of produced biomass were statistically analysed to
determine which, of a range of predictive factors, were significant. Total biomass per m2 by soil type Total biomass per m2 by soil type This analysis assessed the differences between soil types, according
to the total mass of plant material produced per m2, comparing
between plots. Soil type is a significant determiner of total m2
biomass productivity (p=0.002, F=9.410 (3, 16 df.), R2=0.702, adj. R2=0.627). Topsoil has the greatest productivity and variability,
followed by Black soil, then Buada soil and Dolomite (Fig. 2.5). 72 Chapter 2 – Biomass Production Figure 2.5 – Productivity by soil type – Total m2 productivity
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Black soil
Buada Soil
Topsoil
Dolomite
Biomass productivity (gm2) Dolomite Topsoil Figure 2.5 – Productivity by soil type – Total m2 productivity Mean Biomass per m2 by Soil type This analysis assessed the difference between soil types, according
to the mean mass of plant material per m2, comparing between
plots. Soil type is a significant determiner of biomass productivity
(p<0.001, F=13.493 (1,88 df.), R2=0.892, adj. R2=0.785). Topsoil has the
greatest productivity, followed by Black soil, then Buada soil and
Dolomite (Fig. 2.6). Figure 2.6 – Mean Productivity by soil type
0
50
100
150
200
250
300
Black soil
Buada Soil
Topsoil
Dolomite
Biomass productivity (gm2) Dolomite Figure 2.6 – Mean Productivity by soil type 73 73 Chapter 2 – Biomass Production Biomass by species Species was a significant determinant of biomass production
(p<0.001, F=4.161 (10, 176 df.), R2=0.377, adj. R2=0.288). Cajanus cajan
and Calopogonium mucunoides produce the highest biomass followed
by Leucaena leucocephala, and then Indigofera hirsuta. The remaining
species averaged relatively low levels of biomass production
(Fig.2.7). Figure 2.7 – Mean biomass productivity per species
0
100
200
300
400
500
600 Indigofera hirsutaLeucaena leucocephalaCalopogonium mucunoidesCajanus cajanWaltheria indicaSida acutaSenna occidentalisPhyllanthus amarusIndigofera spicataScaevola taccadaIpomoea macrantha
Biomass productivity (gm2) Figure 2.7 – Mean biomass productivity per species Mean biomass production as affected by use of mulch
Mulch was a significant determinant of biomass production
(p=0.013, F=6.307 (1, 176 df.), R2=0.377, adj. R2=0.288). Use of mulch
produced 330gm-2 of biomass, by comparison with 120gm-2
produced for plots that lacked mulch (Fig.2.8). 74 Chapter 2 – Biomass Production Figure 2.8 – Mean productivity with and without mulch
0
50
100
150
200
250
300
350
400
450
No mulch
Mulch
Biomass productivity (gm2) Figure 2.8 – Mean productivity with and without mulch Biomass productivity of specific soil types Due to significant variation in species and biomass production
between soil types, the significance of species and mulch were
statistically tested for specific soil types. Black soil biomass productivity per species
Species was a significant determinant of biomass production in
Black soil (p<0.001, F=5.757 (8, 27 df.), R2=0.630, adj. R2=0.521),
however mulch use did not produce a significant effect (p=0.682,
F=0.171 (1, 34 df.), R2=0.005, adj. R2=-0.024). In the Black soil, Calopogonium mucunoides was most productive,
followed by Cajanus cajan. Indigofera hirsuta had relatively low
levels of production, while the remaining species produced
negligible biomass (Fig. 2.9). 75 Chapter 2 – Biomass Production Figure 2.9 – Mean Black soil biomass productivity per species
0
200
400
600
800
1000
1200 Indigofera hirsutaLeucaena leucocephalaCalopogonium mucunoidesCajanus cajanWaltheria indicaSida acutaIndigofera spicataScaevola taccadaIpomoea macrantha
Biomass productivity (gm2) Figure 2.9 – Mean Black soil biomass productivity per species Figure 2.9 – Mean Black soil biomass productivity per species Topsoil biomass productivity per species Topsoil biomass productivity per species
Species was not a significant determinant of biomass production in
Topsoil at the 5% level, but was at the 10% level (p=0.077, F=2.147 (7,
24 df.), R2=0.385, adj. R2=0.206), and mulch use did not produce a
significant effect (p=0.164, F=2.038 (1, 30 df.), R2=0.064, adj. R2=0.032). In the Topsoil, Cajanus cajan and Leucaena leucocephala produced the
greatest biomass. Senna occidentalis and Indigofera hirsuta produced
moderate to low levels of biomass, while the remainder were
negligibly productive (Fig. 2.10). 76 Chapter 2 – Biomass Production Figure 2.10 – Mean Topsoil biomass productivity per species
0
200
400
600
800
1000
1200
1400
1600 Indigofera hirsutaLeucaena leucocephalaCalopogonium …Cajanus cajanWaltheria indicaSenna occidentalisIndigofera spicataScaevola taccada
Biomass productivity (gm2) Figure 2.10 – Mean Topsoil biomass productivity per species Buada soil productivity per species Buada soil productivity per species Buada soil productivity per species Species was not a significant determinant of biomass production in
Buada soil (p=0.705, F=0.594 (5, 18 df.), R2=0.142, adj. R2=-0.097),
mulch use also did not produce a significant effect (p=0.187, F=1.858
(1, 22 df.), R2=0.078, adj. R2=0.036). Species was not a significant determinant of biomass production in
Buada soil (p=0.705, F=0.594 (5, 18 df.), R2=0.142, adj. R2=-0.097),
mulch use also did not produce a significant effect (p=0.187, F=1.858
(1, 22 df.), R2=0.078, adj. R2=0.036). mulch use also did not produce a significant effect (p=0.187, F=1.858
(1, 22 df.), R2=0.078, adj. R2=0.036). Biomass productivity of specific soil types In the Buada soil only Sida acuta was significantly productive, and
only to moderate to low levels (Fig. 2.11). Figure 2.11 – Mean Buada soil biomass productivity per
species
0
50
100
150
200
250
300
350
400
450
500 Indigofera hirsutaLeucaena leucocephalaCalopogonium …Sida acutaSenna occidentalisPhyllanthus amarus
Biomass productivity (gm2) Figure 2.11 – Mean Buada soil biomass productivity per
species 77 77 Chapter 2 – Biomass Production Chapter 2 – Biomass Production Dolomite biomass productivity per species
Species was not a significant determinant of biomass production in
Dolomite at the 5% level but was at the 10% level (p=0.057, F=2.917
(4, 15 df.), R2=0.437, adj. R2=0.287), and mulch use did not produce a
significant effect (p=0.698, F=0.155 (1, 20 df.), R2=0.009, adj. R2=-
0.047). Dolomite biomass productivity per species Leucaena leucocephala was the most productive species in dolomite,
however productivity was low for it and all other species (Fig. 12). Leucaena leucocephala was the most productive species in dolomite,
however productivity was low for it and all other species (Fig. 12). Figure 2.12 – Mean Dolomite biomass productivity per species
0
20
40
60
80
100
120
140
Indigofera
hirsuta
Leucaena
leucocephala
Calopogonium
mucunoides
Cajanus cajan Phyllanthus
amarus
Biomass productivity (gm2) Figure 2.12 – Mean Dolomite biomass productivity per species Research limitations In determining the significance of the biomass results three factors
must be taken into consideration. Firstly, the plots consisted of
mixed species, and so the values reflect biomass production with
competition and allelopathic effects. Secondly, each species was not
found in every plot, as the plants grew from seed present in the
soils. This seed distribution was unlikely to be homogenous, as
reflected in the selection of species that grew in each of the soils. Thirdly, there were no controls on seed density or distribution, so
stochastic effects are likely to have played a significant role. Accordingly, the results must be accepted as preliminary. This
research does not fully explore the potential of each of these species
in each of the soils. Accordingly, the results must be accepted as preliminary. This
research does not fully explore the potential of each of these species
in each of the soils. Another factor not tested was the effects of different regimens of
soil moisture and watering on biomass production. As freshwater
is a very limited resource in Nauru, ensuring low water use in
biomass production is of critical concern. It is recommended that
this aspect of rehabilitation be examined. Research outcomes Exchangeable soil cadmium was negatively correlated with biomass
production. This suggests cadmium inhibits plant growth. As
exchangeable soil cadmium increased by 1ppb, biomass production
reduced by 0.043g per m2 (σ=0.020) However, as there is low
variation in exchangeable cadmium concentration between soils
(~2ppm), the size of the effect is insignificant. 78 Chapter 2 – Biomass Production Soil type was a significant predictor of total and mean biomass
production (Figs. 2.5, 2.6). Topsoil had the highest and most varied
productivity, followed by Black soil, Buada soil, and then dolomite. Black soil productivity was 1,740±460gm-2 over a 6 month period. Assuming that this figure can be extrapolated this equates to
productivity of 34.8±9.2 tonnes of biomass per hectare per year. Soil type was a significant predictor of total and mean biomass
production (Figs. 2.5, 2.6). Topsoil had the highest and most varied
productivity, followed by Black soil, Buada soil, and then dolomite. Black soil productivity was 1,740±460gm-2 over a 6 month period. Assuming that this figure can be extrapolated this equates to
productivity of 34.8±9.2 tonnes of biomass per hectare per year. Biomass production differed significantly between species, with
Cajanus cajan and Calopogonium mucunoides producing the highest
biomass followed by Leucaena leucocephala, and then Indigofera
hirsuta. Biomass production increased with use of mulch. Mulch maintains
soil moisture, increases SOM, protects soil from erosion, suppresses
weeds and in doing so supports more robust communities of soil
organisms any or all of which may have promoted biomass
production. This effect was particularly pronounced for Leucaena
leucocephala and Cajanus cajan, which were more than 10 times as
productive when mulch was used. Use of mulch was not a
significant factor with regard to biomass production when the soils
were assessed separately. However, the sample sizes (two for each
factor) were too low to provide certainty. It is more reasonable to
accept the conclusions provided by the aggregated analysis in this
case. Species productivity varied considerably between soils. In Black
soil Calopogonium mucunoides had the highest productivity, followed
by Cajanus cajan. In Topsoil, Cajanus cajan and Leucaena leucocephala 79 Chapter 2 – Biomass Production Chapter 2 – Biomass Production shared the highest productivity. Productivity in the Buada and
Dolomite soils was generally low. Relevance to academic literature Soil organic matter and its interactions with soil organisms
underpin the ecological functions of soil fertility, maintenance of
soil structure and biodegradation of wastes (Hopkins & Gregorich
2005). Over time, the accumulation of carbon in soils is a
consequence of differing rates of production and decomposition 80 Chapter 2 – Biomass Production and is associated with an increase in soil microbial biomass (Hopkins
& Gregorich 2005). Soil organic matter also has a key role in
determining the volume and distribution of moisture in the soil, a
characteristic which significantly affects soil organism function
(Young & Ritz 2005). Pre-mining soil fertility in Nauru was high for a Pacific atoll, but
nevertheless fertility was limited (Morrison & Manner 2005). The
dominant process for soil formation and fertility in Nauru is the
accumulation of organic material (Morrison & Manner 2005). Somewhat remarkably for a Pacific atoll, Nauru had mollisols
(Morrison & Manner 2005). Mollisols have a very rich organic layer
in their dark-coloured A horizon (Gardiner & Miller 2001). Mollisols
are considered the most productive soils for agriculture due to their
structure and high organic content (Gerrard 2000; Dubbin 2001). Mulch has been used effectively to increase soil fertility in the
rehabilitation of phosphate mining areas (Best, Wallace et al. 1988). Buada soil, with its high SOM is locally regarded as a good growth
medium however, biomass productivity in Buada test soils was low. While this may have been due to seed source, both incidentally
occurring seed and seed sown as part of this experiment produced a
low yield in Buada soil. This suggests that the growing conditions
inhibited growth more than competition from weed species. Conclusion Biomass production and integration into soils must be emphasised
as an integral component of soil restoration in Nauru. Protecting
soils with highly productive vegetation will be critical. This 81 Chapter 2 – Biomass Production Chapter 2 – Biomass Production research has identified several species which may be used for the
purposes of biomass production in the difficult growing conditions
on Nauru. Cajanus cajan and Calopogonium mucunoides produced the
highest biomass followed by Leucaena leucocephala, and then
Indigofera hirsuta. research has identified several species which may be used for the
purposes of biomass production in the difficult growing conditions
on Nauru. Cajanus cajan and Calopogonium mucunoides produced the
highest biomass followed by Leucaena leucocephala, and then
Indigofera hirsuta. Biomass produced in the rehabilitation project could be used to
produce mulch, thereby improving subsequent biomass production
rates. An alternative may be the use of biomass as feedstock for
biochar production to produce useful volatile gases and stable
forms of carbon for soil rehabilition. This research provides evidence that wood-chip mulch use has a
significant impact on biomass production and that soil cadmium
has a significant but minimal effect on biomass production. The
fastest and most effective method for rehabilitating Nauruan soils
will be to ensure that vegetation is managed to promote
productivity. Regular crop thinning and mulching of the soil
surface will increase soil organic matter, soil microbial life, and
vegetative productivity. REFERENCES Almeida, D. T. d., Furtunato, D. M. d. N., Andrade, T. J. C. & Feitosa, S. 2010. Nutritional value, antinutritional factors, forms of
consumption, processing and medicinal properties of pigeon pea
(Cajanus cajan). Higiene Alimentar 24(184/185): 70-75. Baines, G. 1994. Environment Part A. Environmental aspects of
rehabilitation of Topside Nauru. Rehabilitation and Development
Feasibility Study, Nauru Australia Cooperation. Baines, G. 1994. Environment Part A. Environmental aspects of
rehabilitation of Topside Nauru. Rehabilitation and Development
Feasibility Study, Nauru Australia Cooperation. 82 Chapter 2 – Biomass Production Chapter 2 – Biomass Production Hopkins, D. W. & Gregorich, E. G. 2005. Carbon as a substrate for soil
organisms. Biological Diversity and Function in Soils. R. D. Bardgett, M. B. Usher and D. W. Hopkins, Cambridge University Press. Hunter, G., Harris, L. & Pollock, N. 1994. Overview report. Rehabilitation
and Development Feasibility Study. G. Hunter, Nauru Australia
Cooperation. Kingston, P.A. 2004. Surveillance of Drinking Water Quality in the Pacific
Islands: Situation Analysis and Needs Assessment . Country Reports. UNEP Chapter 2 – Biomass Production Best, G. R., Wallace, P. M., Dunn, W.J. & Odum, H.T. 1988. Enhanced
Ecological Succession Following Phosphate Mining, Florida Institute of
Phosphate Research, Centre for Wetlands, University of Florida,
USA. Carstairs, B. 1994. Pinnacle land restoration. Rehabilitation and Development
Feasibility Study, Nauru Australia Cooperation. Ci, D., Jiang, D., Wolloenweber, B., Dai, T., Jing, Q. & Cao, W. 2009. Cadmium stress in wheat seedlings: growth, cadmium accumulation
and photosynthesis. Acta Physiologiae Plantarum 32: 9. da Silveira, P. M., Ribeiro da Cunha, P. C., Stone, L.F. & dos Santos, G.G. da Silveira, P. M., Ribeiro da Cunha, P. C., Stone, L.F. & dos Santos, G.G. 2010. Chemical attributes of a soil cultivated with different cover
crops. Pesquisa Agropecuaria Tropical 40(3): 283-290. 2010. Chemical attributes of a soil cultivated with different cover
crops. Pesquisa Agropecuaria Tropical 40(3): 283-290. Dell'Amico, E., Cavalca, L. & Andreoni, V. 2008. Improvement of Brassica
napus growth under cadmium stress by cadmium-resistant
rhizobacteria. Soil Biology and Biochemistry 40: 11. Donahue, R. L. 1958. Soils: An introduction to soils and plant growth. New
Jersey, Prentice-Hall, Inc. Dubbin, W. 2001. Soils. London, The National History Museum. Eltayeb, A. R. S. M., Ali, A. O. & Haron, R. 2010. The chemical
composition of pigeon pea (Cajanus cajan) seed and functional
properties of protein isolate. Pakistan Journal of Nutrition 9(11): 1069-
1073. Gardiner, D. T. & Miller, R. 2001.Soils in our environment, Pearson
Education. Gerrard, J. 2000. Fundamentals of soils, Routledge. Gerrard, J. 2000. Fundamentals of soils, Routledge. Gerrard, J. 2000. Fundamentals of soils, Routledge. Gowdy, J. M. & McDaniel, C. N. 1999. The physical destruction of Nauru:
An example of weak sustainability. Land Economics 75(2): 6. Hokura, A., Matsuura, H., Katsuki, F. & Haraguchi, H.. 2000. Multielement Determination of Major-to-Ultratrace Elements in
Plant Reference Materials by ICP-AES/ICP-MS and Evaluation of
Their Enrichment Factors. Analytical Sciences 16. Gowdy, J. M. & McDaniel, C. N. 1999. The physical destruction of Nauru:
An example of weak sustainability. Land Economics 75(2): 6. Hokura, A., Matsuura, H., Katsuki, F. & Haraguchi, H.. 2000. 83 UNEP Lin, R., Wang, X., Luo, Y., Du, W., Guo, H. & Yin, D. 2007. Effects of soil
cadmium on growth, oxidative stress and antioxidant systems in
wheat seedlings (Triticum aestivum L.). Chemosphere 69: 10. Linares, J., Scholberg, J. M. S., Chase, C. & McSorley, R. 2010. Evaluation
of annual warm-season cover crops for weed management in organic
citrus. Acta Horticulturae (873): 235-242. Maksymiec, W. & Krupa, Z. 2006. The effects of short-term exposition to
Cd, excess Cu ions and jasmonate on oxidative stress appearing in
Arabidopsis thaliana. Environmental and Experimental Botany 57: 8. Manner, H. I., Thaman, R. R. & Hassall, D. C. 1984. Phosphate mining
induced vegetation changes on Nauru Island. Ecology 65(5): 11. Matson, P. A., Parton, W. J., Power, A. G. & Swift, M. J. 1997. Agricultural
Intensification and Ecosystem Properties. Science 277(5325): 504-
509. Meers, E., Samson, R., Tack, F.M.G., Ruttens, A., Vandegehuchte, M.,
Vangronsveld, J. & Verloo, M.G. 2007. Phytoavailability assessment
of heavy metals in soils by single extractions and accumulation by
Phaseolus vulgaris. Environmental and Experimental Botany 60: 12. Morrison, R. J. & Manner, H. I. 2005. Pre-mining pattern of soils on
Nauru, central Pacific. Pacific Science 59(4): 523-540. Nauru, R. o. 2005. Nauru National Sustainable Development Strategy 2005- 84 Chapter 2 – Biomass Production ———.2009. Nauru National Sustainable Development Strategy Review 2005-
2025. ———.2009. Nauru National Sustainable Development Strategy Review 2005-
2025. Smith, J. & Valenzuela, H. 2002. Woollypod Vetch. Sustainable Agriculture
Green Manure Crops, College of Tropical Agriculture and Human
Resources, University of Hawai'i at Mānoa. Smith, J. & Valenzuela, H. 2002. Woollypod Vetch. Sustainable Agriculture
Green Manure Crops, College of Tropical Agriculture and Human
Resources, University of Hawai'i at Mānoa. Sun, Y., Zhou, Q. & Diao, C. 2008. Effects of cadmium and arsenic on
growth and metal accumulation of Cd-hyperaccumulator Solanum
nigrum L., Bioresource Technology 99: 8. Temminghoff, E. J. M. & Houba, V. J. G., Eds. 2004. Plant Analysis
Procedures 2nd Edition, Kluwer Academic Publishers. Thampan, P. K. & Remany, G. 2010. Nitrogen fixing trees in coconut based
mixed cropping system. Indian Coconut Journal 53(4): 2-6. Valenzuela, H. & Smith, J. 2002. Barley. Sustainable Agriculture Green Manure
Crops, College of Tropical Agriculture and Human Resources,
University of Hawai'i at Mānoa. ———. 2002. Common Oats. Sustainable Agriculture Green Manure Crops,
College of Tropical Agriculture and Human Resources University of
Hawai'i at Mānoa. ———. 2002. Pigeonpea. Chapter 2 – Biomass Production
Young, I. M. & K. Ritz 2005. The habitat of soil microbes. Biological Diversity
and Function in Soils. R. D. Bardgett, M. B. Usher and D. W.
Hopkins, Cambridge University Press Chapter 2 – Biomass Production UNEP Sustainable Agriculture Green Manure Crops, College of
Tropical Agriculture and Human Resources, University of Hawai'i at
Mānoa. van der Velde, M., S. R. Green, Vanclooster, M. & Clothier, B.E. 2007. Sustainable development in small island developing states:
Agricultural intensification, economic development, and freshwater
resources management on the coral atoll of Tongatapu. Ecological
Economics 61(2-3): 456-468. VSBo 1986. Verordnung über Schadstoffhegalt im Boden, Swiss ordinance on
pollutants in soils, Swiss Government. Ber. 3000 Bern, Switzerland,
Eidgenössische Drucksachen- und Materialzentrale (EDMZ). Nr 814.12. Wild, A. 1993. Soils and the Environment: An introduction. Cambridge,
Cambridge University Press. 85 Chapter 2 – Biomass Production Young, I. M. & K. Ritz 2005. The habitat of soil microbes. Biological Diversity
and Function in Soils. R. D. Bardgett, M. B. Usher and D. W. Hopkins, Cambridge University Press Young, I. M. & K. Ritz 2005. The habitat of soil microbes. Biological Diversity
and Function in Soils. R. D. Bardgett, M. B. Usher and D. W. Hopkins, Cambridge University Press 86 Chapter 3 – Cadmium phytoaccumulation ABSTRACT This paper reports the findings of a field trial experiment which assessed
how locally grown plants could be used to bioaccumulate soil cadmium in
Nauru. A range of species were grown in different soils, with and without
mulch. Cadmium concentrations in root, stem and leaf tissues were
assessed using ICP-MS. Senna occidentalis and Indigofera hirsuta accumulated
the greatest concentration of cadmium (both around 200ppm) There was
some evidence to suggest that cadmium was found in the greatest
concentration in root tissues, followed by leaf tissues; and that the use of
mulch had a positive effect on cadmium concentration in plant tissues,
however limited data restricted the statistical power of the analysis. INTRODUCTION Healthy functional soil systems are essential for human wellbeing. Soil infertility and toxicity are two examples of threats to the health
of this system Loss of soil fertility may lead to desertification in
environments where biota are necessary to protect soils from
environmental stresses. Soils containing toxic compounds pose
health risks to humans and all organisms living on or near
contaminated land. The severity of the risks are clearly illustrated
in the Pacific Islands, where infertile or contaminated land can
significantly exacerbate the shortage of available land (Gowdy &
McDaniel 1999; van der Velde, Green et al. 2007). Few places
demonstrate this situation more starkly than Nauru. demonstrate this situation more starkly than Nauru. 87 87 Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation Nauru is a 21km2 raised limestone island 41km south of the equator
at longitude 166° 56’ E in the Central Pacific (Fig. 1.1). It is
surrounded by a fringing coral reef that sharply drops away 4,300m
to the Pacific floor. ‚Bottomside‛ is the local name for the thin
coastal plain surrounding the interior of the island, known as
‚Topside‛. Topside is a 1,620ha raised plateau that ranges from 20-
60m in elevation, and is divided from Bottomside by a steep
escarpment in most places. A brackish lagoon, called ‚Buada‛ is in
a large interior depression, and is hydraulically connected with the
Pacific (Hunter, Harris et al. 1994; Kingston, P.A. 2004) (See Appendices
I & II). Figure 3.1 – Map of Nauru’s position in the Pacific Figure 3.1 – Map of Nauru’s position in the Pacific Mining of phosphate in Nauru has lead to various environmental
effects, including deforestation, biodiversity loss, soil loss, liberation
of toxic sub-soil cadmium deposits, soil degradation and
desertification (Manner 1984; Morrison & Manner 2005). Nauru is
facing various social, political, economic, and health issues which
are considerably affected by soil degradation (Weeramantry 1992; 88 Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation Fagence 1996; Gowdy & McDaniel 1999). Rehabilitation of mined
lands in Nauru constitutes one of the five goals of the Nauru
National Sustainable Development Strategy 2005-2025 (Nauru 2005). The 2009 review of the NSDS identified that objectives for the
rehabilitation of mined land have thus far not been achieved
according to its targets (Nauru 2009). There are various reasons why NSDS targets have not been
achieved: There is little knowledge of how to achieve specific goals in the
ecological setting of Nauru. The amount of information which is potentially relevant to
rehabilitation in this setting is vast. The scale and nature of the project is unlike any previous
restoration project anywhere. Soil moisture is generally low, due to highly variable rainfall
patterns, high rates of evaporation, and ready percolation
through the porous underlying rock. Records of the historical ecology of Nauru, which could inform
management decisions, are fragmentary (Morrison & Manner
2005). In Nauru there is a lack of human capital with experience and
skills appropriate to landscape rehabilitation. A range of goals for restoration were developed under the Nauru
Australia Cooperation Rehabilitation and Development Feasibility
Study 1994 (RDFS), many of which require the rehabilitation of soils
for various land uses including housing, conservation,
agroforestry/residential, sports/recreation/parks, an education A range of goals for restoration were developed under the Nauru
Australia Cooperation Rehabilitation and Development Feasibility
Study 1994 (RDFS), many of which require the rehabilitation of soils
for various land uses including housing, conservation,
agroforestry/residential, sports/recreation/parks, an education 89 Chapter 3 – Cadmium phytoaccumulation complex, a public service complex, a cemetery, a municipal waste
site, a hospital complex, and an industrial complex (Carstairs 1994;
Hunter, Harris et al. 1994). complex, a public service complex, a cemetery, a municipal waste
site, a hospital complex, and an industrial complex (Carstairs 1994;
Hunter, Harris et al. 1994). These land uses require soils, although they require them for
different reasons and accordingly the properties required of the
soils differ in relation to their intended purposes. Generally the soil
purposes can be distinguished according to use as: These land uses require soils, although they require them for
different reasons and accordingly the properties required of the
soils differ in relation to their intended purposes. Chapter 3 – Cadmium phytoaccumulation Generally the soil
purposes can be distinguished according to use as: a) substrate, such as for hospital, education, public service and
industrial complexes b) growth media for food crops, as in the agroforestry/residential
areas c) growth media for vegetation cover for various purposes, as in
the conservation, agroforestry/residential and
sports/recreation/parks areas, as well as selected areas of the
education, public service, hospital and industrial complexes and
cemetery d) media for containment and amelioration of biological hazards,
as in the cemetery area and municipal waste site
(Baines 1994; Carstairs 1994). Since 1970 mining operations have stockpiled soil for rehabilitation
purposes (Baines 1994; Gowdy & McDaniel 1999). These soils have
lost considerable amounts of material, organic content and biota
while stockpiled, leaving the soils relatively infertile. Additionally,
cadmium liberated through mining operations has contaminated
the stockpiled soils limiting their value for rehabilitation. 90 90 Chapter 3 – Cadmium phytoaccumulation Cadmium is classified as a toxic heavy metal. Contaminated areas
are considered to be a threat to the well-being of humans and
animals (Czeczot & Skrzycki, 2010). Cadmium affects the kidneys,
liver, lungs, pancreas, skeleton and testis in humans due to
disruptions of cellular adhesion, the immune system, the cellular
antioxidant system, and cellular communication systems, inhibition
of DNA repair and methylation, promotion of cell proliferation and
apoptosis, and initiation of cell mutagenesis and carcinogenesis
(Czeczot & Skrzycki, 2010; Dufresne et al., 2010; Satarug et al., 2010;
Trzcinka-Ochocka et al., 2010). Cadmium has been demonstrated to
have a wide variety of deleterious effects in ecological systems,
negatively affecting soil microbial community structure and
activities, affecting growth and causing severe physiological
disorders in plants and animals, disrupting renal, hepatic, and
endocrine system function; skeletal development; and fat storage in
animals (Angenard et al., 2010; Ci et al., 2010; Czeczot & Skrzycki, 2010;
Dufresne et al., 2010; Khan et al., 2010; Kumar et al., 2009; Lucia et al.,
2010; Planelló et al., 2010; Van Campenhout et al., 2010). Cadmium
bioaccumulates in food webs amplifying the risk to organisms at
higher trophic levels (Sato et al., 2010; van den Brink et al., 2010). Phytoextraction, the utilisation of plants to extract toxic compounds
from soils, is a low technology method which may be used to Phytoextraction, the utilisation of plants to extract toxic compounds
from soils, is a low technology method which may be used to
reduce cadmium concentrations in contaminated soils. AIM AIM To determine the cadmium phytoaccumulation potential of a
range of green mulch species. HYPOTHESES Plant species will accumulate differing concentrations of
cadmium in their roots, stems and leaves Cadmium accumulation will be influenced by soil type and use
of mulch. METHOD A selection of species were sown in a range of soils, watered and
grown for a period of seven months to be assessed for their
cadmium phytoaccumulation potential. Field plots were
established in Nauru on the central plateau known as Topside. Chapter 3 – Cadmium phytoaccumulation Cadmium is
known to induce a range of severe physiological disorders in plants
such as chlorophyll reduction, changes to phenols, oxidative stress
and altered enzyme activity which ultimately retard plant growth
(Maksymiec & Krupa 2006; Lin, Wang et al. 2007; Dell'Amico, Cavalca et
al. 2008; Sun, Zhou et al. 2008; Ci, Jiang et al. 2009). 91 Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation The quality and quantity of stockpiled soil is insufficient for
restoration. Far greater volumes of soil will be needed to restore the
mined areas. The issues of infertility and contamination will need
to be resolved. Increasing the organic carbon content of stockpiled soils is a viable
method for increasing soil fertility and may provide additional
benefits to soils including increased water retention properties,
increased soil biota and thus increased biological activity, and a
carbon sink with potential benefits for the country with regard to
carbon accounting and accreditation. An additional concern is the lack of soils sufficiently low in
cadmium for safe food production. Phytoextraction, the utilisation
of plants to extract toxic compounds from soils, is a low-technology
method which may be used to achieve this outcome. This paper describes the second of a two-part experimental field
trial to assist in developing a successful soil restoration programme
through biomass production and cadmium phytoaccumulation for
Nauru. The first paper entitled ‚Biomass production in Nauru: A
preliminary field trial‛ assesses the biomass production capability
of various species in a range of Nauruan soils as well as the impact
of the addition of biomass through a locally produced wood chip
mulch upon plant productivity. Concentrations of cadmium in soil and plant tissues were assayed
to determine: Concentrations of cadmium in soil and plant tissues were assayed
to determine: What effect soil cadmium has on cadmium phytoaccumulation What effect soil cadmium has on cadmium phytoaccumulation 92 Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation Whether any of a range of factors (use of mulch, tissue type,
species, or soil type) had significant effects on cadmium
phytoaccumulation. Soil cadmium was measured for both total cadmium, and the free
ionic fraction, using NaNH3 solution as the extraction agent. Soil cadmium was measured for both total cadmium, and the free
ionic fraction, using NaNH3 solution as the extraction agent. Species Five species were selected for their suitability for soil rehabilitation
on Pacific tropical islands – drought tolerance, fast growth, on Pacific tropical islands – drought tolerance, fast growth,
appropriateness for use as green mulches, and seed availability, as appropriateness for use as green mulches, and seed availability, as 93 Chapter 3 – Cadmium phytoaccumulation determined by the Centre for Tropical Agriculture and Human
Resources (CTAHR) at the University of Hawai’i (Smith & Valenzuela
2002; Valenzuela & Smith 2002a; Valenzuela & Smith 2002b). The species used were:
White oat - Avena sativa
Black oat - Avena strigosa Schreb. Ryegrass - Lolium multiflorum
Barley - Hordeum vulgare
Hairy Vetch - Vicia villosa
Soil types
The soils used were
Black soil
Buada soil
Topsoil
Dolomite Black soil Black soil
‘Black soil’ is the name given locally to soil from the soil stockpiles
on Topside. Black soil is the remaining topsoil from across the
central plateau, removed for phosphate mining access, and left
stockpiled for several decades. This soil has lost significant
amounts of organic material through stockpiling and exposure to
the sun without substantial cover. Stockpiling also resulted in large
anoxic areas within the piles which, along with desiccation and
losses of soil organic matter, are likely to have reduced the
populations of soil organisms within the soil. The soil used for this
experiment was sourced from Stockpile A which has a greater 94 Chapter 3 – Cadmium phytoaccumulation variety of plant species growing on it than Stockpile B which is
covered with a monoculture of Leucaena leucocephala. variety of plant species growing on it than Stockpile B which is
covered with a monoculture of Leucaena leucocephala. Buada soil
‘Buada soil’ is soil from the Buada province of Nauru. Buada is a
large depression containing a brackish lagoon and fairly fertile soils. Buada is protected from the hot salt laden winds that sweep across
Nauru so the plants that grow there are less prone to desiccation. Buada soils are undisturbed compared with Black soil, and contain
considerably higher volumes of organic material and soil
organisms. Topsoil The ‘Topsoil’ is sourced from the Nauru Rehabilitation Corporation
(NRC) nursery area in Topside which is located in an area that has
operated as a sports oval and recreation area; the soil was part of
the track and field area and was tended through use of fertiliser for
many years. It was then used as the site of the Nauru detention
centre for Australia, before becoming the site of the NRC nursery. This soil has been managed to some degree, having received
applications of fertiliser and pesticides. The soil has been covered
by pasture for many years, and is used preferentially in garden
developments by the nursery staff. Mulch Growing conditions on the Topside of Nauru are arid. Nauru
experiences highly variable rainfall and large amounts of incident
sunlight due to its location in the dry equatorial zone of the tropical
Pacific Ocean. In these dry conditions, desiccation leads to
increased plant mortality and reduced growth. Mulches can be
used to protect soils from temperature fluctuation, to reduce soil
moisture evaporation, and increase soil carbon content. Local
production of wood-chip mulch from biomass was investigated
within this experiment. Wood-chip was applied to a depth of 4-5cm
on one of the two experimental beds. To ensure sufficient soil
moisture, the beds were watered daily for the first month and then
weekly unless rainfall meant that this was unnecessary. Dolomite The ‘Dolomite’ does not have all of the characteristics necessary to
be accurately described as a soil. It is a mixed aggregate of fine to
course material which has been mined containing a high proportion
of rock phosphate. This material has been proposed as a medium 95 Chapter 3 – Cadmium phytoaccumulation for developing soil layers by the Nauru Rehabilitation Feasibility
Study and other documents relating to landscape restoration on
Nauru. for developing soil layers by the Nauru Rehabilitation Feasibility
Study and other documents relating to landscape restoration on
Nauru. Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation the Nauruan growing environment, and the potential for using local
flora which were evidently better suited for growth in adverse
conditions. So we identified the species, measured their biomass
production under the different treatments, and determined the
cadmium levels in different parts of the plants. the Nauruan growing environment, and the potential for using local
flora which were evidently better suited for growth in adverse
conditions. So we identified the species, measured their biomass
production under the different treatments, and determined the
cadmium levels in different parts of the plants. Complications The intention of the experiment was to test the viability of a range
of selected species for phytoaccumulation in Nauru. Accordingly, a
range of species of seeds were planted according to a randomised
plan (Fig. 3.1). However, the resulting seedlings were unsuccessful
as the adverse growing conditions found in the field in Nauru
combined with the success of adventitious local seed from within
the soils killed off the experimental species. This outcome posed
significant issues for the original experimental design. However,
though an unexpected outcome, this result revealed two factors
important to the rehabilitation project; the significant challenges of 96 Field plots The seeds of a range of species were sown in randomly assigned
33cm by 50cm plots within 1.66m by 2.0m plots of specific soil
types. The larger plots were arranged into two beds; Bed A with
mulch, Bed B without (Fig. 3.2). The beds were situated at the
Nauru Rehabilitation Corporation nursery site, (See Appendices I &
II). Seeds were sown as per standard sowing densities for each species,
as recommended by the seed producers. The seeds were monitored
for germination success for 10 days. For the following six months
research obligations outside of Nauru, meant the beds were left in
the care of the nursery staff. Due to the lack of access to
telecommunications equipment on Nauru at the time, it was not
possible to contact the nursery staff during this absence. Nursery
staff observed during this period that adventitious species were
germinating in the soils, with the Topsoil plots showing the greatest
amount. Field plots 97 Chapter 3 – Cadmium phytoaccumulation Key:
1
White Oat
Topsoil
2
Black Oat
Black soil
3
Ryegrass
Dolomite
4
Barley
Buada soil
5
Hairy Vetch
2
4
5
1
2
4
3
3
3
3
1
5
1
1
2
2
1
1
3
3
3
5
5
4
4
5
2
4
4
2
4
1
5
2
4
2
5
3
1
5
5
1
4
3
3
1
2
5
3
5
3
4
5
2
3
2
4
4
5
2
1
3
5
3
1
2
2
5
2
4
4
1
2
3
1
1
4
5
1
4
2
3
3
4
5
2
1
2
1
1
2
5
4
4
3
5
5
4
1
2
1
3
5
4
3
5
4
1
2
5
4
1
4
2
5
3
3
1
2
3
4
4
1
4
2
4
3
1
1
2
2
5
5
5
1
4
3
1
4
1
1
3
5
2
2
3
5
3
3
1
2
5
5
5
3
2
4
2
4
3
1
1
5
2
5
1
3
4
4
4
2
4
3
2
1
2
3
5
4
1
2
5
2
3
2
2
3
5
4
4
4
5
5
3
1
3
1
3
5
1
5
5
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5
3
2
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3
2
3
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4
1
2
1
1
5
2
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5
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1
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1
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1
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1
1
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5
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3
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2
1
1
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1
3
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5
2
4
5
1
1
3
1
1
5
3
2
2
2
2
5
3
4
Bed A
Bed B
Figure 3.2 – Field trial plots
= 1.666m
= 2m Key:
1
White Oat
Topsoil
2
Black Oat
Black soil
3
Ryegrass
Dolomite
4
Barley
Buada soil
5
Hairy Vetch 2
4
5
1
2
4
3
3
3
3
1
5
1
1
2
2
1
1
3
3
3
5
5
4
4
5
2
4
4
2
4
1
5
2
4
2
5
3
1
5
5
1
4
3
3
1
2
5
3
5
3
4
5
2
3
2
4
4
5
2
1
3
5
3
1
2
2
5
2
4
4
1
2
3
1
1
4
5
1
4
2
3
3
4
5
2
1
2
1
1
2
5
4
4
3
5
5
4
1
2
1
3
5
4
3
5
4
1
2
5
4
1
4
2
5
3
3
1
2
3
4
4
1
4
2
4
3
1
1
2
2
5
5
5
1
4
3
1
4
1
1
3
5
2
2
3
5
3
3
1
2
5
5
5
3
2
4
2
4
3
1
1
5
2
5
1
3
4
4
4
2
4
3
2
1
2
3
5
4
1
2
5
2
3
2
2
3
5
4
4
4
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5
3
1
3
1
3
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1
5
5
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5
3
2
4
3
2
3
2
4
4
4
1
2
1
1
5
2
1
3
2
5
4
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3
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5
1
5
1
3
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1
1
2
5
3
4
1
3
1
2
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3
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2
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1
4
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5
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5
2
4
3
2
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5
1
4
5
5
4
1
1
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2
1
5
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5
1
3
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5
2
2
1
1
3
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1
3
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3
2
4
4
3
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4
5
2
4
5
1
1
3
1
1
5
3
2
2
2
2
5
3
4
Bed A
Bed B
Figure 3.2 – Field trial plots
= 1.666m
= 2m Bed A Figure 3.2 – Field trial plots Figure 3.2 – Field trial plots 98 Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation Preparation for sample collection Preparation for sample collection At the start of the third trip to Nauru, six months later in October
2008, it was found that weed species had overtopped and killed off
the planted trial species. This posed several issues to the
experimental design. The randomisation of planting was reduced,
and it was now not possible to produce a full factorial model. Nevertheless, it was determined that the circumstances could still
provide valuable data for the development of a process for
cadmium phytoaccumulation. At this point, the soils contained a
large amount of seed of local weedy species which successfully
germinated and grew in the adverse conditions found in Topside,
Nauru. Accordingly, the germination and seedling success of the
weedy species provides evidence of their adaptive suitability to the
conditions found on Topside. It was decided to identify and sample
the species which had grown, to assess their suitability for
phytoaccumulation (Table 3.1). At the start of the third trip to Nauru, six months later in October
2008, it was found that weed species had overtopped and killed off
the planted trial species. This posed several issues to the
experimental design. The randomisation of planting was reduced,
and it was now not possible to produce a full factorial model. Nevertheless, it was determined that the circumstances could still
provide valuable data for the development of a process for
cadmium phytoaccumulation. At this point, the soils contained a
large amount of seed of local weedy species which successfully
germinated and grew in the adverse conditions found in Topside,
Nauru. Accordingly, the germination and seedling success of the
weedy species provides evidence of their adaptive suitability to the
conditions found on Topside. It was decided to identify and sample
the species which had grown, to assess their suitability for
phytoaccumulation (Table 3.1). Chapter 3 – Cadmium phytoaccumulation The species which were identified were:
Table 3.1 – Weed species found in soils The species which were identified were:
Table 3.1 – Weed species found in soils Sampled species
Black soil
Topsoil
Buada soil
Dolomite
Calopogonium
mucunoides
X
X
X
X
Cajanus cajan
X
X
X
Leucaena
leucocephala
X
X
X
X
Indigofera hirsuta
X
X
X
X
Senna occidentalis
X
X
Sida acuta
X
X X 99 Chapter 3 – Cadmium phytoaccumulation
Indigofera spicata
X
X
Waltheria indica
X
X
Phylanthus amarus
X
X
Ipomoea macrantha
X
Scaevola taccada
X
X
Unsampled
species
Chamaesyce hirta
X
Tridax procumbens
X
Stachytarpheta
jamaicensis
X Chapter 3 – Cadmium phytoaccumulation Of note was the presence of Pigeon Pea Cajanus cajan which is listed
by the College for Tropical Agriculture and Human Resources,
University of Hawai’i, Mānoa as a beneficial green manure for the
Pacific (Valenzuela & Smith 2002c). It is possible that the species
arrived with asylum seekers who were detained on Nauru. This
species has a global production of around 40,000 km2, and is
cultivated for its protein rich seed, as a cover crop and companion
plant, for fodder and for firewood (Almeida, Furtunato et al. 2010; da
Silveira, Ribeiro da Cunha et al. 2010; Eltayeb, Ali et al. 2010; Linares,
Scholberg et al. 2010; Thampan & Remany 2010). It is a perennial
nitrogen fixing legume used for producing biomass. Cajanus cajan
grew successfully in conditions found on Topside Nauru that were
limiting for other species. 100 Chapter 3 – Cadmium phytoaccumulation Plant tissue sample collection At the end of the trial period, all plant material from each soil plot
was removed, washed, desiccated and weighed. Samples of leaf,
stem and root tissue were taken for each species in each plot. Sample Preparation and Analysis Soil and plant cadmium concentrations were analysed using
Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Samples
were prepared according to the methodologies described below. Soil samples were digested for exchangeable cadmium using the
NaNO3 extraction method described under Swiss legislation and by
Meers, Samson et al. (VSBo 1986; Meers, Samson et al. 2007). Extraction of total cadmium in soils ac o
o
o a cad
u
so s
Total cadmium was extracted through total digestion of soil
samples as described by Temminghoff & Houba in 2004. 15mL
Teflon beakers were cleaned by initial triple rinse with milli-Q
water followed by soaking in 5% HNO3 for 2 weeks. The beakers
were treated to additional cleaning using concentrated (6-7 mol)
sub-boiled HNO3. 3 mL of sample was placed in the beakers which
were then sealed and placed on a hot plate at 120°C for 48 hours. The HNO3 was then removed from the beakers and the process was
repeated a second time. After treatment with HNO3 the samples
were thoroughly rinsed with milli-Q water and treated with
concentrated (6-7 mol) sub-boiled HCl in the same manner as used
with the HNO3 treatment. Following this, the beakers were cleaned
with 3mL concentrated HNO3 and 1mL concentrated HF and left 101 Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation sealed on a 120°C hotplate for 36 hours. The beakers were then
triple rinsed with milli-Q water and dried. sealed on a 120°C hotplate for 36 hours. The beakers were then
triple rinsed with milli-Q water and dried. Each sample was individually homogenised and approximately
130mg of each soil sample was placed in the 15mL Teflon containers
and weighed. 4mL of concentrated HNO3 was added to the Teflon
containers and then left on a 120°C hotplate for 24 hours unsealed. This process was repeated and then 50 drops of concentrated HF
and 30 drops of concentrated HNO3 was added and sealed and
placed on a 120°C hotplate for 36 hours. Two additional treatments
with concentrated HNO3 were performed to ensure that all organic
compounds were chemically degraded. The product was then
dissolved in 5mL of 5% HNO3 and then weighed. 1mL was then
extracted and diluted with 3mL of 5%HNO3. Extraction of exchangeable cadmium in soils
15mL Teflon beakers and 10mL c-tubes were cleaned by initial triple
rinse with milli-Q water followed by soaking in 5% HNO3 for 2
weeks. The beakers were treated to additional cleaning using
concentrated (6-7 mol) sub-boiled HNO3. 3mL of sample was placed
in the beakers which were then sealed and placed on a hot plate at
120°C for 48 hours. The HNO3 was then removed from the beakers
and the process was repeated a second time. After treatment with
HNO3 the samples were thoroughly rinsed with milli-Q water and
treated with concentrated (6-7 mol) sub-boiled HCl in the same
manner as used with the HNO3 treatment. The beakers and tubes
were then triple rinsed with milli-Q water and dried in preparation
for extraction of bioavailable cadmium. 102 Chapter 3 – Cadmium phytoaccumulation Each sample was individually homogenised and approximately 1g
of soil sample was measured into the cleaned 15mL reaction tubes,
and weighed on a calibrated Sartorius CP225D balance. 5mL of
0.1M NaNO3 extractant was pipetted into each reaction tube and the
total masses of the extractant and the soil were weighed. The soil
extractant mixture was initially agitated to ensure full contact
between the extractant and soil. The reaction tubes were then
placed on a mixing plate to maintain full exposure of the soil to the
extractant. Chapter 3 – Cadmium phytoaccumulation After 2 hours on the mixing plate, the reaction tubes
were placed in a centrifuge for 6 minutes at 3500rpm to settle out
suspended particles. 5 mL of 5% HNO3 was pipetted into the cleaned 10mL c-tubes,
weighed and 300µL of sample were added to each tube from the
centrifuged reaction tubes and weighed. This product constituted
the material to be analysed by the ICP-MS. This standard procedure was employed for each of the 18 soil
samples and 3 cadmium deposit samples. For error testing, five
samples were made up to three times the volume of the other
samples to provide sufficient quantities for multiple analyses and
data consistency checks. This applied to five samples: four soils and
one cadmium deposit. Additionally, 1ppb and 2ppb standards were made up from a
10ppb standard of toxic metals for calibration of ICP-MS output
data. These standards were made to greater volumes than the
samples and with volumes of 5% HNO3 and 0.1M NaNO3 in
equivalent proportions to that of the samples to ensure that the 103 Chapter 3 – Cadmium phytoaccumulation viscosity of the standards would be equivalent to that of the
samples. This was done to ensure consistency of analysis in the
ICP-MS as fluid viscosity affects the rate of delivery to the nebuliser
of prepared samples. viscosity of the standards would be equivalent to that of the
samples. This was done to ensure consistency of analysis in the
ICP-MS as fluid viscosity affects the rate of delivery to the nebuliser
of prepared samples. Samples of vegetation assessed for cadmium content
Due to time and financial limitations not all samples available for
analysis were analysed. Representative samples were selected from
those available so as to produce sufficient overlap for comparative
analysis. Vegetation samples were cleaned onsite and dried as per
requirements of the New Zealand Department of Conservation
(DoC) allocated importation licence. On arrival in New Zealand,
where the laboratory analysis was performed, the samples were
cleaned once more using milli-Q water and dried in a dehydrator
for 2 hours at 40°C. For each measurement, approximately 200mg
of dry plant tissue was measured into the cleaned 15mL Teflon
beakers, and weighed on a calibrated Sartorius CP225D balance. 3mL of concentrated (6-7 mol) sub-boiled HNO3 was then added to
each beaker to digest the samples. RESULTS The results of the analysis of cadmium concentrations in Nauruan
soils, and cadmium phytoaccumulation are presented. Soil Cadmium Measurements of total and exchangeable cadmium were statistically
analysed to determine differences in cadmium concentrations
between soil types Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation sample preparation and ICP-MS analysis were accurate and
effective. Chapter 3 – Cadmium phytoaccumulation The beakers were placed on a
hotplate at 120°C without lids and monitored for the first 40
minutes to ensure that the reactions were not so volatile as to lead
to cross-contamination. Once reaction rates had settled sufficiently,
the samples were left to evaporate. 3 mL of 1 mol sub-boiled HNO3
was then added to the digested residue and enclosed for a further
period of digestion. The subsequent solution was transferred to
cleaned c-tubes, weighed, and centrifuged at 6000rpm for 10
minutes to remove any sedimentitious contaminants from 104 Chapter 3 – Cadmium phytoaccumulation suspension. 1mL of solution was pipetted into cleaned c-tubes and
weighed. 5mL of milli-Q water was added to dilute the solution
and the final solution was again weighed. Additionally, 1ppb and 2ppb standards were made up from SRM
1575 Pine Needle standard for toxic metals for calibration of ICP-MS
output data (Hokura, Matsuura et al. 2000). The standards were
processed in the same manner as the experimental samples. Inductively Coupled Plasma Mass Spectrometry analysis
Exchangeable fractions of soil cadmium in solution were then
determined using Inductively Coupled Plasma Mass Spectrometry
(ICP-MS). ICP-MS uses inductively coupled plasma to ionise
samples that are then recorded by a mass spectrometer. This
method allows for highly precise measurements of elemental
concentrations (Hokura, Matsuura et al. 2000). Samples in solution are
nebulised into plasma which ionises the elements contained therein. The ions are then passed through a system of electromagnets and
are directed to detectors which register the characteristics of
individual ions. Samples were delivered to the ICP-MS nebuliser for 60 seconds
followed by milli-Q water for 30 seconds; a 5% HNO3 wash for 90
seconds; then 60 seconds of a 1% HNO3 wash, before the next
sample was analysed. 1 ppb standards were used at the start, end
and at frequent intervals throughout the analyses. 2ppb standards
were used as secondary standards within the analysis to test the
standardisation process. Additionally, multiple analyses of some
samples and ‚blank‛ tubes were performed to ensure that both the 105 Total cadmium The soils differed in total cadmium concentrations (p=0.001,
F=16.257, (3, 12 df), R2=0.859, adj. R2=0.806). Figure 3.3 illustrates the
grouping of soils according to concentration of total cadmium. The
dolomite gravel has the lowest concentration; Topsoil and Buada
soil together form an intermediate group; Black soil has the greatest
concentration of total cadmium. Figure 3.3 – Total cadmium concentrations in Nauruan soils
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
Black soil
Buada soil
Topsoil
Dolomite
Cadmium concentration (ppb) Figure 3.3 – Total cadmium concentrations in Nauruan soils 106 Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation Exchangeable fraction
Soils differed significantly in concentration of exchangeable
cadmium (p<0.001, F=64.509, (3, 20 df), R2=0.924, adj. R2=0.909). Soil
exchangeable cadmium differed significantly between soil types
(Fig. 3.4). Black soil, Topsoil and Buada soils contain equal
concentrations of exchangeable cadmium but significantly higher
concentration than the dolomite. Exchangeable fraction Figure 3.4 – Concentrations of exchangeable cadmium in
Nauruan soils
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Black soil
Buada soil
Topsoil
Dolomite
Cadmium concentration (ppb) Dolomite Figure 3.4 – Concentrations of exchangeable cadmium in
Nauruan soils Cadmium phytoaccumulation Measurements of cadmium concentration in plant tissues were
statistically analysed to determine which, of a range of predictive
factors, were significant. Phytoaccumulation by total and exchangeable cadmium
Two general univariate analyses were conducted, using
alternatively, total soil cadmium concentration data, or 107 107 Chapter 3 – Cadmium phytoaccumulation exchangeable cadmium concentration of the acquired plant tissue
data with post hoc Tukey and LSD analysis. This included species,
mulch use, and tissue type as factors, with interactions between
species and tissue type, and species and soil. Both soil total
cadmium (p=0.955, F=0.005 (1, 1 df.), R2=0.998, adj. R2=0.919) and soil
exchangeable cadmium (p= 0.957, F=0.005 (1, 1 df.), R2=0.998, adj. R2=0.911) were found to not be significant predictors of plant tissue
cadmium concentrations. Additionally, soil type was not a significant predictor of plant tissue
cadmium concentrations (p=0.559, F=0.699 (3,33 df.), R2=0.060, adj. R2=-0.026). Cadmium concentration by species
Species type did not significantly predict cadmium concentrations
in plant tissues (p=0.154, F=5.769 (5, 2 df.), R2=0.989, adj. R2=0.794)
(Fig.3.5). Species type did not significantly predict cadmium concentrations
in plant tissues (p=0.154, F=5.769 (5, 2 df.), R2=0.989, adj. R2=0.794)
(Fig.3.5). Figure 3.5 – Cadmium concentrations in tested plant species
0
500
1000
1500
2000
2500
3000
3500 Cajanus cajanIndigofera hirsutaLeucaena leucocephalaCalopogonium mucunoidesSenna occidentalisSida acuta
Cadmium concentration (ppb) Figure 3.5 – Cadmium concentrations in tested plant species 108 Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation Cadmium concentrations by tissue type
Tissue types did significantly differ with regard to cadmium
concentrations (p=0.037, F=26.183 (2, 2 df.), R2=0.989, adj. R2=0.794)
(Fig.3.6). Cadmium concentrations by tissue type
Tissue types did significantly differ with regard to cadmium
concentrations (p=0.037, F=26.183 (2, 2 df.), R2=0.989, adj. R2=0.794)
(Fig.3.6). Figure 3.6 – Cadmium concentrations by tissue type
0
500
1000
1500
2000
2500
3000
3500
Leaf
Stem
Root
Cadmium concentration (ppb) Figure 3.6 – Cadmium concentrations by tissue type Cadmium concentrations by use of mulch There was no significant difference in cadmium concentrations
between plants grown with or without mulch (p=0.425, F=0.986 (1, 2
df.), R2=0.989, adj. R2=0.794) (Fig. 3.7). Cadmium phytoaccumulation 0
500
1000
1500
2000
2500
No Mulch
Mulch
Cadmium concentration (ppb) 0
500
1000
1500
2000
2500
No Mulch
Mulch
Cadmium concentration (ppb) 109 109 Chapter 3 – Cadmium phytoaccumulation Figure 3.7 - Cadmium concentrations as determined by use of
mulch Plant cadmium accumulation by soil type
Plant cadmium concentrations by soil type was not significant
(p=0.334, F=1.190 (3, 25 df.), R2=0.504, adj. R2=0.286) (Fig.3.8). Plant cadmium accumulation by soil type Plant cadmium accumulation by soil type Plant cadmium concentrations by soil type was not significant
(p=0.334, F=1.190 (3, 25 df.), R2=0.504, adj. R2=0.286) (Fig.3.8). Plant cadmium concentrations by soil type was not significant
(p=0.334, F=1.190 (3, 25 df.), R2=0.504, adj. R2=0.286) (Fig.3.8). Figure 3.8 – Plant cadmium concentrations by soil type
0
500
1000
1500
2000
2500
3000
Black soil
Buada soil
Topsoil
Dolomite
Cadmium concentration (ppb) Figure 3.8 – Plant cadmium concentrations by soil type Plant cadmium accumulation by species and tissue type
The interaction of species and tissue type was not significant
(p=0.221, F=3.900 (10, 2 df.), R2=0.989, adj. R2=0.794) (Fig.3.9). 110 Chapter 3 – Cadmium phytoaccumulation Figure 3.9 – Cadmium concentrations in tissues of plant
species
0
1000
2000
3000
4000
5000
6000
7000
Leaf
Stem
Root
Leaf
Stem
Root
Leaf
Stem
Root
Leaf
Stem
Root
Leaf
Stem
Root
Leaf
Stem
Root
Cajanus cajan
Indigofera
hirsuta
Leucaena
leucocephala
Calopogonium
mucunoides
Senna
occidentalis
Sida acuta
Cadmium concentration (ppb) Figure 3.9 – Cadmium concentrations in tissues of plant
species Research outcomes Soil types differed significantly in their total cadmium and
exchangeable fractions. The exchangeable fraction of cadmium was
typically around 3 orders of magnitude less than the concentration
of total cadmium. The levels of exchangeable cadmium differed
significantly across soil type – with the Buada soil, Topsoil and
Black soil containing ~4ppb, and Dolomite ~2ppb. Plant tissues accumulated concentrations of cadmium (1000-
2500ppb) that were considerably greater than the concentration of
exchangeable cadmium available in the soil (2-3.5 ppb). While we 2500ppb) that were considerably greater than the concentration of
exchangeable cadmium available in the soil (2-3.5 ppb). While we 111 Chapter 3 – Cadmium phytoaccumulation may expect bioaccumulation to account for some of this difference,
it is also possible that the NaNH3 extraction method does not
sufficiently account for all of the exchangeable cadmium in the soils. The cadmium levels found in the plants are high (Figs. 3.5, 3.9),
though not sufficiently so to be categorised as hyperaccumulators
(>100ppm) (Kamel & Sakr 2009). The concentration of cadmium in root tissues differed significantly
from cadmium concentrations in stem and leaf tissues (Fig. 3.6). This is unsurprising as one of the effects of the Casparian bands is
to limit the transportation of cadmium into above-ground plant
parts (Yamaguchi, Mori, et al. 2009). Research limitations Due to time and financial constraints it was not possible to analyse a
larger number of samples to increase the statistical power of the
analysis. This is a significant issue with this analysis as insufficient
data limited the outcomes of this analysis considerably. A further limitation was caused by limited equipment in the field. This meant it was not possible to determine the weight of each of
the plant parts (leaf, stem and root) for each species in each plot. Having done so would have allowed for an analysis of the total
amount of cadmium extracted from the soils by the plant species. The experimental design was not intended to accommodate the
drastic changes which occurred due to the success of the ‚weed‛
species. Redesigning this experiment to assess the 112 112 Chapter 3 – Cadmium phytoaccumulation phytoaccumulative capacity of the weed species would allow a
more robust evaluation of their potential. NaNH3 extraction assesses the most mobile chemical species of soil
cadmium. Accordingly, it does not reflect the full extent of the
exchangeable fraction of cadmium in the soil. An analysis to
determine the chemical species in which cadmium occurs in
Nauruan soils may assist in determining a more suitable chemical
extractant. Relevance to academic literature The concentrations of total soil cadmium found in this report differ
significantly from earlier analyses. Blake (1992) recorded stockpiled
Black soil cadmium levels of between 214.9 and 353.4 ppm, and an
NRC (1991) report provides estimates of between 93.8 and 95.3
ppm. These measurements are considerably more than was
recorded in this report (~5.5ppm). The duration between
measurements (12/13 years) may account for this variation. Table
3.3 presents a summary of Blake’s measurements of cadmium in the
Black soil stockpiles. What is unclear from both Blake’s and the
NRC analyses is the methodology employed to assess the cadmium
load in the soils. While Blake describes the methods employed for
sample collection and cadmium extraction, he omits the method of
measurement. The NRC analysis merely states the recorded levels
without any description of methodology. 113 Chapter 3 – Cadmium phytoaccumulation Table 3.3 – Cadmium concentrations in stockpiled soils from
Blake (1992) Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation which cadmium is mobile in the soil system. Changes to cadmium
mobility due to phytoaccumulation may influence soil rehabilitation
management of accumulated biomass. Multi-generational studies
of cadmium bearing chemical species may provide insight into how
phytoaccumulation processes can lower concentrations of
exchangeable cadmium through removal of phytoaccumulated
cadmium and/or reduce the chemical mobility of the toxic element. which cadmium is mobile in the soil system. Changes to cadmium
mobility due to phytoaccumulation may influence soil rehabilitation
management of accumulated biomass. Multi-generational studies
of cadmium bearing chemical species may provide insight into how
phytoaccumulation processes can lower concentrations of
exchangeable cadmium through removal of phytoaccumulated
cadmium and/or reduce the chemical mobility of the toxic element. Conclusion While this research does not provide an exhaustive assessment of
phytoaccumulation in Nauru, there are some research outcomes
which may be of benefit for rehabilitation processes in Nauru. Firstly, the species assessed were successful colonisers of arid,
infertile and cadmium enriched soils. Secondly, cadmium
phytoaccumulation differed between plant parts. Thirdly,
measured cadmium levels in stockpiled soils were dramatically
lower than those found by Blake (1992) and in NRC (1991). This
may indicate that there are methodological differences in cadmium
assessment between researchers and/or changes in cadmium
concentration through transportation away from the cadmium
waste deposits. While this research does not provide an exhaustive assessment of
phytoaccumulation in Nauru, there are some research outcomes
which may be of benefit for rehabilitation processes in Nauru. In six months the experimental species grew in dense clusters up to
~2.4m in height, providing good protection to the soils beneath. 114 Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation Given the success of these species, they may be useful in the
rehabilitation process. They may be grown to achieve specific
rehabilitation outcomes such as biomass production. Understanding the differences in cadmium concentrations between
plant parts may be useful to determine how and where each of
those tissues is best used post-harvest. Due to the association of cadmium with phosphate in Nauru, the
contemporary soil cadmium load is considered to be significantly
higher than in pre-mining soils and represents a significant abiotic
change to the Nauruan environment. Heightened soil cadmium
levels may threaten the health of Nauru’s flora and fauna, in
particular, species currently in decline or with restricted
distributions. Such effects, however, shall remain unresolved until
comparative studies with non-contaminated soils are able to
demonstrate or rule out such ecological effects. Other areas for further research include; Other areas for further research include; Other areas for further research include; Assessment of biodegradable physicochemical agents such as
chelating agents or micronutrients, to increase bioavailability
and subsequent cadmium uptake in hyperaccumulator species Assessment of stimulated microbiological uptake of cadmium Assessment of the chemical species containing cadmium in
Nauruan soils to provide further insight into the chemical
dynamics of the soil system, and better predict the long term
effects of phytoaccumulation on exchangeable cadmium. It is likely that biochemical processes during phytoaccumulation
alter the cadmium chemical species and consequently the degree to 115 REFERENCES Almeida, D. T. d., Furtunato, D. M. d. N., Andrade, T. J. C. & Feitosa, S. 2010. Nutritional value, antinutritional factors, forms of
consumption, processing and medicinal properties of pigeon pea
(Cajanus cajan). Higiene Alimentar 24(184/185): 70-75. Angenard, G., Muczynski, V., Coffigny, H., Pairault, C., Duquenne, C.,
Frydman, R., Habert, R., Rouiller-Fabre, V. & Livera, G. 2010. Cadmium Increases Human Fetal Germ Cell Apoptosis. Environmental Health Perspectives 118, 7. Baines, G. 1994. Environment Part A. Environmental aspects of
rehabilitation of Topside Nauru. Rehabilitation and Development
Feasibility Study, Nauru Australia Cooperation. Blake, C. 1992. Environmental distribution of heavy metals on Nauru, Central
Pacific, and possible relationships to human health, University of New
England. Bachelor of Applied Science (Honours): 86 Carstairs, B. 1994. Pinnacle land restoration. Rehabilitation and Development
Feasibility Study, Nauru Australia Cooperation. Ci, D., Jiang, D., Wolloenweber, B., Dai, T., Jing, Q. & Cao, W. 2009. Cadmium stress in wheat seedlings: growth, cadmium accumulation
and photosynthesis. Acta Physiologiae Plantarum 32: 9. Czeczot, H. & Skrzycki, M. 2010. Cadmium - element completely
unnecessary for the organism. Postepy Higieny i Medycyny
Doswiadczalnej 64, 10. 116 Chapter 3 – Cadmium phytoaccumulation Khan, S., El-Latif Hesham, A., Qiao, Q., Rehman, S. & He, J. 2010. Effects
of Cd and Pb on soil microbial community structure and activities. Environmental Science & Pollution Research 17, 9. Kingston, P.A. 2004. Surveillance of Drinking Water Quality in the Pacific
Islands: Situation Analysis and Needs Assessment . Country Reports. Chapter 3 – Cadmium phytoaccumulation da Silveira, P. M., Ribeiro da Cunha, P. C., Stone, L.F. & dos Santos, G.G. 2010. Chemical attributes of a soil cultivated with different cover
crops. Pesquisa Agropecuaria Tropical 40(3): 283-290. Dell'Amico, E., Cavalca, L. & Andreoni, V. 2008. Improvement of Brassica
napus growth under cadmium stress by cadmium-resistant
rhizobacteria. Soil Biology and Biochemistry 40: 11. Donahue, R. L. 1958. Soils: An introduction to soils and plant growth. New
Jersey, Prentice-Hall, Inc. Dufresne, M. M., Frouin, H., Pillet, S., Lesage, V., De Guise, S. & Fournier,
M. 2010. Comparative sensitivity of harbour and grey seals to several
environmental contaminants using in vitro exposure. Marine Pollution
Bulletin 60, 6. Eltayeb, A. R. S. M., Ali, A. O. & Haron, R. 2010. The chemical
composition of pigeon pea (Cajanus cajan) seed and functional
properties of protein isolate. Pakistan Journal of Nutrition 9(11): 1069-
1073. Fagence, M. 1996. Nauru: Substituting one dependency for another? A
challenge to the orthodoxy of sustainable development. Sustainable
Development 4: 10. Gowdy, J. M. & McDaniel, C. N. 1999. The physical destruction of Nauru:
An example of weak sustainability. Land Economics 75(2): 6. Gowdy, J. M. & McDaniel, C. N. 1999. The physical destruction of Nauru:
An example of weak sustainability. Land Economics 75(2): 6. Hokura, A., Matsuura, H., Katsuki, F. & Haraguchi, H.. 2000. Multielement Determination of Major-to-Ultratrace Elements in Hokura, A., Matsuura, H., Katsuki, F. & Haraguchi, H.. 2000. Multielement Determination of Major-to-Ultratrace Elements in
Plant Reference Materials by ICP-AES/ICP-MS and Evaluation of
Their Enrichment Factors. Analytical Sciences 16. Hunter, G., Harris, L. & Pollock, N. 1994. Overview report. Rehabilitation
and Development Feasibility Study. Hunter, G., Nauru Australia
Cooperation. Kamel, M. M. & Sakr, W. R. 2009. Response of Senna occidentalis, Link
Plants to Fertilization as Well as Citric Acid and Their Role in
Remediating Soil Polluted with Cu and Pb. World Journal of
Agricultural Sciences 5(6): 15. 117 UNEP Kumar, N., Bauddh, K., Singh, R., Anand, K., Barman, S. C. &Singh, D. P. 2009. Phytotoxicity of trace metals (Cu & Cd) to Gram (Cicer
arientinum) and Mung (Phaseolus mungo). Journal of Ecophysiology and
Occupational Health 9, 7. Lin, R., Wang, X., Luo, Y., Du, W., Guo, H. & Yin, D. 2007. Effects of soil
cadmium on growth, oxidative stress and antioxidant systems in
wheat seedlings (Triticum aestivum L.). Chemosphere 69: 10. Linares, J., Scholberg, J. M. S., Chase, C. & McSorley, R. 2010. Evaluation
of annual warm-season cover crops for weed management in organic
citrus. Acta Horticulturae (873): 235-242. Lucia, M., Andre´, J.-M., Gonzalez, P., Baudrimont, M., Bernadet, M.-D.,
Gontie, K., Maury-Brachet, R., Guy, G. & Davail, S. 2010. Effect of
dietary cadmium on lipid metabolism and storage of aquatic bird
Cairina moschata. Ecotoxicology 19, 8. Maksymiec, W. & Krupa, Z. 2006. The effects of short-term exposition to
Cd, excess Cu ions and jasmonate on oxidative stress appearing in
Arabidopsis thaliana. Environmental and Experimental Botany 57: 8. Manner, H. I., Thaman, R. R. & Hassall, D. C. 1984. Phosphate mining
induced vegetation changes on Nauru Island. Ecology 65(5): 11. Matson, P. A., Parton, W. J., Power, A. G. & Swift, M. J. 1997. Agricultural
Intensification and Ecosystem Properties. Science 277(5325): 504-
509. Meers, E., Samson, R., Tack, F.M.G., Ruttens, A., Vandegehuchte, M.,
Vangronsveld, J. & Verloo, M.G. 2007. Phytoavailability assessment
of heavy metals in soils by single extractions and accumulation by
Phaseolus vulgaris. Environmental and Experimental Botany 60: 12. 118 Chapter 3 – Cadmium phytoaccumulation Morrison, R. J. & Manner, H. I. 2005. Pre-mining pattern of soils on
Nauru, central Pacific. Pacific Science 59(4): 523-540. Nauru, R. o. 2005. Nauru National Sustainable Development Strategy 2005-
2025. ———. 2009. Nauru National Sustainable Development Strategy Review 2005-
2025. Planelló, R., Martínez-Guitarte, J. L. & Morcillo, G. 2010. Effect of acute
exposure to cadmium on the expression of heat-shock and hormone-
nuclear receptor genes in the aquatic midge Chironomus riparius. Science of the Total Environment 408, 6. Satarug, S., Garrett, S. H., Sens, M. A. & Sens, D. A. 2010. Cadmium,
Environmental Exposure, and Health Outcomes. Environmental
Health Perspectives 118, 9. Sato, R., Kido, T., Honda, R., Nishijo, M., Nakagawa, H., Kobayashi, E. &
Suwazono, Y. 2010. Seventeen-Year Observation on Urinary
Cadmium and β2-Microglobulin in Inhabitants After Cessation of
Cadmium-Exposure in Japan. Bulletin of Environmental Contamination
and Toxicology 84, 5. Smith, J. & Valenzuela, H. 2002. Woollypod Vetch. Sustainable Agriculture
Green Manure Crops, College of Tropical Agriculture and Human
Resources, University of Hawai'i at Mānoa. Sun, Y., Zhou, Q. & Diao, C. 2008. Effects of cadmium and arsenic on
growth and metal accumulation of Cd-hyperaccumulator Solanum
nigrum L. Bioresource Technology 99: 8. Temminghoff, E. J. M. & Houba, V. J. G., Eds. 2004. Plant Analysis
Procedures 2nd Edition, Kluwer Academic Publishers. Thampan, P. K. & Remany, G. 2010. Nitrogen fixing trees in coconut based
mixed cropping system. Indian Coconut Journal 53(4): 2-6. Trzcinka-Ochocka, M., Jakubowski, M., Szymczak, W., Janasik, B. &
Brodzka, R. 2010. The effects of low environmental cadmium
exposure on bone density. Environmental Research 110, 8.Valenzuela,
H. & Smith, J. 2002. 119 Chapter 3 – Cadmium phytoaccumulation Valenzuela, H. & Smith, J. 2002a. Barley. Sustainable Agriculture Green
Manure Crops, College of Tropical Agriculture and Human
Resources, University of Hawai'i at Mānoa. ———. 2002b. Common Oats. Sustainable Agriculture Green Manure Crops,
College of Tropical Agriculture and Human Resources University of
Hawai'i at Mānoa. ———. 2002c. Pigeonpea. Sustainable Agriculture Green Manure Crops, College
of Tropical Agriculture and Human Resources, University of Hawai'i
at Mānoa. Van Campenhout, K., Goenaga Infante, H., Hoff, P. T., Moens, L.,
Goemans, G., Belpaire, C., Adams, F., Blust, R. & Bervoets, L. 2010. Cytosolic distribution of Cd, Cu and Zn, and metallothionein
levels in relation to physiological changes in gibel carp (Carassius
auratusgibelio) from metal-impacted habitats. Ecotoxicology and
Environmental Safety 73, 10. van den Brink, N., Lammertsma, D., Dimmers, W., Boerwinkel, M.-C. &
van der Hout, A. 2010. Effects of soil properties on food web
accumulation of heavy metals to the wood mouse (Apodemus
sylvaticus). Environmental Pollution 158, 7. van der Velde, M., Green, S. R., Vanclooster, M. & Clothier, B.E. 2007. Sustainable development in small island developing states:
Agricultural intensification, economic development, and freshwater
resources management on the coral atoll of Tongatapu. Ecological
Economics 61(2-3): 456-468. Agricultural intensification, economic development, and freshwater VSBo 1986. Verordnung über Schadstoffhegalt im Boden, Swiss ordinance on
pollutants in soils, Swiss Government. Ber. 3000 Bern, Switzerland,
Eidgenössische Drucksachen- und Materialzentrale (EDMZ). Nr 814.12. Weeramantry, C. 1992. Nauru - Environmental damage under international
trusteeship, Oxford University Press. Wild, A. 1993. Soils and the Environment: An introduction. Cambridge,
Cambridge University Press. 120 Chapter 3 – Cadmium phytoaccumulation Yamaguchi, N., Mori, S., Yada, S., Baba, K., Hokura, A. & Terada, Y. 2009. Comparison of cadmium distribution in the root tissues of Solanum
melongena and Solonum torvum, which have different abilities for
cadmium transport from the root to shoot., in The Proceedings of the
International Plant Nutrition Colloquium XVI, Department of Plant
Sciences, UC Davis 121 Chapter 3 – Cadmium phytoaccumulation Chapter 3 – Cadmium phytoaccumulation 122 Chapter 4 – Germination ABSTRACT This paper assesses the effects of cadmium in native soils on the germination
success of a selection of plant species which have been identified as having
potential roles in soil rehabilitation in Nauru. The soils of Nauru have
historically had relatively high levels of cadmium. Phosphate mining on the
island has liberated substantial amounts of previously buried cadmium
which has further increased the cadmium concentration in soils. Germination is a critical stage of plant development which may be affected
by bioavailable cadmium in growth media. The seeds of ten species were
planted in a selection of native soils, and native soils with altered cadmium
levels, and then monitored for germination success. Germination varied
between species and was also significantly affected by soil type but not
cadmium concentration. INTRODUCTION The soils of Nauru contain relatively high levels of cadmium due to
liberation of subterranean cadmium through phosphate mining
(Blake 1992). It is useful to determine the ecological effects of soil
cadmium in order to develop rehabilitation guidelines suited to the
conditions found on Nauru. Germination is a critical stage in the
life cycle of plants and thus in community assemblage (Fenner &
Thompson 2005). This paper seeks to determine whether cadmium
in Nauruan soils has effects on the germination success of various
native and non-native species. 123 Chapter 4 – Germination Studies have found widely varied effects of cadmium on
germination success describing either increased germination
success with increased levels of cadmium, the opposite, or no effect
(XinHong, YuXiu & RunJin 2009; Zhang et al. 2010; Lefevre, 2009; Wang
& Zheng, 2009; ZhiDe et al. 2009, Kumar et al. 2009; HuaBing, 2009;
Farooqi et al. 2009). Cadmium in soils can exist in a wide range of chemical forms. When used to refer to cadmium and other toxic metals found in soil
the terms ‘bioavailable’, ‘bioavailable fraction’, ‘phytoavailable’ are
inadequate as the complexity of dynamics between toxic metals, soil
chemistry and species effects prevent us from defining a single
figure which defines the mobile proportion of toxic material within
the soil system (Meers, Unamuno et al. 2005; Meers, Samson et al. 2007). However, the terms are useful as they describe a concept of
importance, namely that a proportion of the total amount of the
toxic material is mobile in the soil system and it is this exchangeable
fraction which is potentially problematic for plant life. Accordingly
I have used a method of assessment of the exchangeable fraction of
soil cadmium (hereafter referred to as Exchangeable Cadmium)
which is used under Swiss legislation (VSBo 1986; Meers, Samson et al. 2007). This method uses 0.1M Sodium Nitrate as a leaching agent to
extract Cadmium from prepared soil samples. Cadmium in soils can exist in a wide range of chemical forms. However, the terms are useful as they describe a concept of
importance, namely that a proportion of the total amount of the
toxic material is mobile in the soil system and it is this exchangeable
fraction which is potentially problematic for plant life. AIM To determine the effect of soil cadmium on germination success
for a range of species in Nauruan soils. INTRODUCTION Accordingly
I have used a method of assessment of the exchangeable fraction of
soil cadmium (hereafter referred to as Exchangeable Cadmium)
which is used under Swiss legislation (VSBo 1986; Meers, Samson et al. 2007). This method uses 0.1M Sodium Nitrate as a leaching agent to
extract Cadmium from prepared soil samples. Currently the effects of soil cadmium on ecological processes in
Nauru are unknown. Determining the effects of soil cadmium on
seed germination may provide information of significant value to
the land rehabilitation programme in Nauru. 124 Chapter 4 – Germination Chapter 4 – Germination The experimental design employed utilised locally available soils
which are intended for use in the restoration of the Nauruan
landscape. The soils used are listed below. They are listed
according to the names which were most commonly used on the
island by the Nauru Rehabilitation Corporation (NRC) Plant
Nursery staff. Black soil with Dolomite rock mix. HYPOTHESES Cadmium content in soils will have a negative effect on seed
germination of species selected for their potential use in land
rehabilitation. Soil type will influence germination success of species selected
for their potential use in land rehabilitation. Seeds of a range of species were sown in a selection of soils in
Nauru with either natural or increased cadmium levels and
monitored for germination success. Black soil Black soil ‘Black soil’ is the name given locally to soil from the soil stockpiles
on Topside. Previous to mining this soil was spread across the
landscape and was accumulated into the stockpiles as mining
progressed. This soil has lost significant amounts of organic
material through stockpiling and exposure to the sun without
substantial cover. Stockpiling also resulted in large anoxic areas
within the piles which, along with desiccation and losses of soil
organic matter, are likely to have reduced the populations of soil
organisms within the soil. The soil used for this experiment was
sourced from Stockpile A which has a greater variety of plant
species growing on it than Stockpile B which is covered with a
monoculture Leucaena leucocephala. Variables Black soil with Dolomite rock mix. 125 Chapter 4 – Germination Topsoil The ‘Topsoil’ is sourced from the NRC nursery area Topside which
is located on an area that has operated as a sports oval and
recreation area; the soil was part of the track and field area and was
tended through use of fertiliser for many years. It was then used as
the site of the Nauru detention centre for Australia, before
becoming the site of the NRC nursery. This soil has been managed
to some degree, having received applications of fertiliser and
pesticides. The soil has been covered by pasture for many years. This soil is used preferentially in garden developments by the
nursery staff. Black soil with Dolomite rock mix. Black soil with Dolomite rock mix. Due to insufficient volumes of stockpiled soil available for
landscape rehabilitation, relatively fine dolomite rock aggregate, 126 126 Chapter 4 – Germination Chapter 4 – Germination which is readily available from mine sites, has been proposed as a
resource for bulking out the available soils, either as a substrate, or
directly incorporated into the soil. This trial utilised a 50:50 blend
of Black soil with Dolomite rock to determine whether the use of
this material would affect germination success. Added Cadmium Cadmium levels were manipulated by the addition of cadmium
waste sourced from a cadmium dump site in Buada to produce two
levels - natural cadmium concentration and raised cadmium. The
cadmium waste is a distinctive, silty, blue-grey substance produced
as a by-product of the phosphate manufacturing process. While the
volumes of soil and cadmium waste used to prepare the cadmium
enriched soils were estimated due to the variability of inputs and
the lack of laboratory resources on Nauru, cadmium concentrations
of the resulting soils were measured using Inductively Coupled
Plasma Mass Spectrometry (ICP-MS). Species Ten species of plants were selected for trial based on their
availability and suitability according to various characteristics. Species A through F were selected due to suitability for use as green
mulch species. The species used come from a list of species
identified as appropriate for use in Pacific islands by the College of
Tropical Agriculture and Human Resources at the University of
Hawai’i, Mānoa (Smith & Valenzuela 2002; Valenzuela & Smith 2002;
Valenzuela & Smith 2002). Species G through I were selected with guidance from nursery
workers employed by NRC according to availability and growth
characteristics, including success as ruderal species in the mined
areas known as the Topside. Species J was selected due to seed availability to nursery staff. The
Japanese government had supplied large volumes of seed to Nauru
to assist the land rehabilitation process. The seed had been stored
on Nauru for a period of at least two years, and as such was not in
ideal condition for assessment. 127 Chapter 4 – Germination A - Barley - Hordeum vulgare
B - White Oat - Avena sativa
C - Black Oat - Avena strigosa Schreb. D - Ryegrass - Lolium multiflorum
E - Fiji Feathers Pea - Pisum sativum
F - Hairy Vetch - Vicia villosa
G - Wild Tamarind – Leucaena leucocephala
H - Red Bean – Adenanthera pavonina
I - Flame Tree - Delonix regia
J - Millet - Pennisetum glaucum Issues Millet was excluded from the data as it was assumed to be non-
viable as in all circumstances no seed germinated. The seed had
been stored in the Nursery for at least two years prior to the
experiment. Its history prior to this was unclear. Experimental Process Soils and cadmium waste were collected from various locations
around Nauru. Mixed soil and cadmium waste treatments were
prepared. Soils were put into seed raising trays in preparation for
seed. Scarification of the seed testa of Delonix regia was performed
to improve germination success by cutting back the testa with a 128 Chapter 4 – Germination Chapter 4 – Germination knife. Seeds of the selected species were sown in seedling trays in
experimental soils to provide 15 repetitions of each treatment. They
were watered twice daily, and monitored for germination success
for a period of 30 days. Germination success was recorded. Sample Analysis Soil cadmium concentrations were analysed using Inductively
Coupled Plasma Mass Spectrometry (ICP-MS). Samples were
prepared according to the methodologies described below. Soil samples were digested for exchangeable cadmium using the
NaNO3 extraction method described under Swiss legislation and in
various research papers (VSBo 1986; Meers, Samson et al. 2007). Extraction of total cadmium in cadmium waste
Total cadmium was extracted through total digestion of cadmium
waste samples as described by Temminghoff & Houba in 2004. 15mL Teflon beakers were cleaned by initial triple rinse with milli-
Q water followed by soaking in 5% HNO3 for 2 weeks. The beakers
were treated to additional cleaning using concentrated (6-7 mol)
sub-boiled HNO3. 3 mL of sample was placed in the beakers which
were then sealed and placed on a hot plate at 120°C for 48 hours. Extraction of total cadmium in cadmium waste
Total cadmium was extracted through total digestion of cadmium
waste samples as described by Temminghoff & Houba in 2004. The HNO3 was then removed from the beakers and the process was The HNO3 was then removed from the beakers and the process was The HNO3 was then removed from the beakers and the process was 129 Chapter 4 – Germination repeated a second time. After treatment with HNO3 the samples
were thoroughly rinsed with milli-Q water and treated with
concentrated (6-7 mol) sub-boiled HCl in the same manner as used
with the HNO3 treatment. Following this, the beakers were cleaned
with 3mL concentrated HNO3 and 1mL concentrated HF and left
sealed on a 120°C hotplate for 36 hours. The beakers were then
triple rinsed with milli-Q water and dried. Each sample was individually homogenised and approximately
130mg of each cadmium sample was placed in the 15mL Teflon
containers and weighed. 4mL of concentrated HNO3 was added to
the Teflon containers and then left on a 120°C hotplate for 24 hours
unsealed. This process was repeated and then 50 drops of
concentrated HF and 30 drops of concentrated HNO3 was added
and sealed and placed on a 120°C hotplate for 36 hours. Two
additional treatments with concentrated HNO3 were performed to
ensure that all organic compounds were chemically degraded. The
product was then dissolved in 5mL of 5% HNO3 and then weighed. 1mL was then extracted and diluted with 3mL of 5%HNO3. Sample Analysis Each sample was individually homogenised and approximately
130mg of each cadmium sample was placed in the 15mL Teflon
containers and weighed. 4mL of concentrated HNO3 was added to
the Teflon containers and then left on a 120°C hotplate for 24 hours
unsealed. This process was repeated and then 50 drops of Extraction of Exchangeable Cd in Soils
15mL Teflon beakers and 10mL c-tubes were cleaned by initial triple
rinse with milli-Q water followed by soaking in 5% HNO3 for a
minimum of 2 weeks. The beakers were treated to additional
cleaning using concentrated (6-7 mol) sub-boiled HNO3, 3 mL of
which was placed in the beakers which were then sealed and placed
on a hot plate at 120°C for 48 hours. The HNO3 was then removed
from the beakers and the process was repeated a second time. After
treatment with HNO3 the samples were thoroughly rinsed with 130 Chapter 4 – Germination milli-Q water and treated with concentrated (6-7 mol) sub-boiled
HCl in the same manner as used with the HNO3 treatment. The
beakers and tubes were then triple rinsed with milli-Q water and
dried in preparation for extraction of bioavailable cadmium. Approximately 1g of soil was measured into the cleaned 15mL
reaction tubes, and weighed on a calibrated Sartorius CP225D
balance. 5mL of 0.1M NaNo3 extractant was pipetted into each
reaction tube and the total masses of the extractant and the soil were
weighed. The soil-extractant mixture was initially agitated to
ensure full contact between the extractant and soil. The reaction
tubes were then placed on a mixing plate to maintain full exposure
of the soil to the extractant. After 2 hours on the mixing plate, the
reaction tubes were placed in a centrifuge for 6 minutes at 3500 rpm
to settle out suspended particles. 5 mL of 5% HNO3 was pipetted into the cleaned 10mL c-tubes,
weighed and 300µL of sample were added to each tube from the
centrifuged reaction tubes and weighed. This product constituted
the material to be analysed by the ICP-MS. This standard procedure was employed for each of the 18 soil
samples and 3 cadmium deposit samples. For error testing, a
selection of samples was made up to three times the volume of the
other samples to provide sufficient quantities for multiple analyses
and data consistency checks. This applied to five samples: four soils
and one cadmium deposit. RESULTS The results of the analysis of cadmium concentrations in
experimental soils, and germination success are presented. Sample Analysis 131 Chapter 4 – Germination Additionally, 1ppb and 2ppb standards were made up from a
10ppb standard of toxic metals for calibration of ICP-MS output
data. These standards were made to greater volumes than the
samples but with volumes of 5% HNO3 and 0.1M NaNO3 in
equivalent proportions to that of the samples to ensure that the
viscosity of the standards would be equivalent to that of the
samples. This was done to ensure consistency of analysis in the
ICP-MS, as fluid viscosity affects the rate of delivery of the sample
fluid through the nebuliser. Inductively Coupled Plasma Mass Spectrometry analysis
Exchangeable fractions of soil Cd in solution were then determined
using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). ICP-MS uses inductively coupled plasma to ionise samples that are
then recorded by a mass spectrometer. This method allows for
precise measurements of elemental concentrations (Hokura, Matsuura
et al. 2000). Samples in solution are nebulised into plasma which
ionises the elements contained therein. The ions are then passed
through a system of electromagnets and are directed to detectors
which register the characteristics of individual ions. Samples were delivered to the ICP-MS nebuliser for 60 seconds
followed by milli-Q water for 30 seconds; a 5% HNO3 wash for 90
seconds; then 60 seconds of a 1% HNO3 wash, before another
sample was analysed. 1 ppb standards were used at the start, end
and between every sample. 2ppb standards were used as
secondary standards within the analysis to test the standardisation
process. Additionally, multiple analyses of some samples and 132 Chapter 4 – Germination ‚blank‛ tubes were performed to ensure that both the sample
preparation and ICP-MS analysis were accurate and effective. Soil Cadmium Measurements of total and exchangeable cadmium were statistically
analysed to determine differences in cadmium concentration
between soil types Exchangeable Cadmium in Experimental Soils Exchangeable Cadmium in Experimental Soils
The exchangeable fractions of cadmium in the soils differed
significantly (p=0.002, F=10.732 (2, 12 d.f.), R2 = 0.956, adj. R2 = 0.938). The concentration of bioavailable Cd in each soil type is shown in The exchangeable fractions of cadmium in the soils differed
significantly (p=0.002, F=10.732 (2, 12 d.f.), R2 = 0.956, adj. R2 = 0.938). The concentration of bioavailable Cd in each soil type is shown in Figure 4.1
Figure 4.1 – Concentrations of exchangeable cadmium in soils
0
2
4
6
8
10
12
14
16
Black soil
Topsoil
Black soil and
dolomite
Cadmium concentration (ppb) Black soil and
dolomite Figure 4.1 – Concentrations of exchangeable cadmium in soils 133 Chapter 4 – Germination Chapter 4 – Germination Soils with Cadmium Added Soils with Cadmium Added The addition of cadmium waste had a significant effect on the
concentration of exchangeable cadmium in the soil (p<0.001,
F=214.599 (1, 12 d.f.) R2 = 0.956, adj. R2 = 0.938). When averaged
across soils, those with added cadmium contained nearly five times
the amount of exchangeable cadmium (17.4 ± 1.4 ppb for soils with
added cadmium, 3.3 ± 1.4 ppb for soils without) (Fig.4.2). Figure 4.2 – Mean concentrations of exchangeable cadmium
between samples with and without added cadmium. 0
2
4
6
8
10
12
14
16
18
20
No Cd added
Cd added
Cadmium concentration (ppb) Cd added Figure 4.2 – Mean concentrations of exchangeable cadmium
between samples with and without added cadmium. Interaction between Soil Type and Added Cadmium
There was a significant interaction between the factors of soil type
and cadmium addition (p=0.001, F=13.398 (2, 12 d.f.) R2 = 0.956, adj. R2 = 0.938) (Fig.4.3). Interaction between Soil Type and Added Cadmium
There was a significant interaction between the factors of soil type
and cadmium addition (p=0.001, F=13.398 (2, 12 d.f.) R2 = 0.956, adj. R2 = 0.938) (Fig.4.3). 134 Chapter 4 – Germination 0
5
10
15
20
25
30
No Cd
added
Cd added
No Cd
added
Cd added
No Cd
added
Cd added
Black soil
Topsoil
Black soil and dolomite
Cadmium concentration (ppb) Figure 4.3 – Concentration of exchangeable cadmium in
three soils with and without added cadmium. Exchangeable Cadmium in Experimental Soils Error bars
indicate two standard errors
0
5
10
15
20
25
30
No Cd
added
Cd added
No Cd
added
Cd added
No Cd
added
Cd added
Black soil
Topsoil
Black soil and dolomite
Cadmium concentration (ppb) Figure 4.3 – Concentration of exchangeable cadmium in Figure 4.3 – Concentration of exchangeable cadmium in
three soils with and without added cadmium. Error bars
indicate two standard errors. Analysis of Germination Success A stepwise forward likelihood binary logistic regression was
performed on the germination data. Soil cadmium concentration
was not significant (p=0.458), so was excluded as a factor and
replaced with categorical factors for soil and added cadmium. The
categorical cadmium factor was not significant (p=0.719). Species
(p<0.001) and soil type (p<0.001) were significant factors in
determining germination success. Univariate analysis determined
that germination success of the trial species differed significantly
(Table 4.1). Topsoil had the highest germination success rate at
95%, as shown in Table 4.2. A test of the full model versus an
intercept only model showed that species and soil type were
statistically significant factors ( 2(10, N = 900) = 347.27, p < 0.001). The model was able to correctly classify 74% of seeds which failed 135 Chapter 4 – Germination to germinate and 86.5% of seeds which successfully germinated, for
an overall success rate of 81.6%. to germinate and 86.5% of seeds which successfully germinated, for
an overall success rate of 81.6%. Table 4.1 – Germination success of experimental species
Species
Percent germination Table 4.1 – Germination success of experimental species
Species
Percent germination Barley - Hordeum vulgare
93
Black Oat - Avena strigosa Schreb. 62
Flame Tree - Delonix regia
54
Fiji Feathers Pea - Pisum sativum
94
Red Bean - Adenathera pavonina
9
Ryegrass - Lolium multiflorum
98
Tamarind - Leucaena leucocephala
83
Vetch - Vicia villosa
46
White Oat - Avena sativa
95
Table 4.2 – Germination success in Soils
Soil type
Percent germination
Black soil with Dolomite
86
Black soil
89
Topsoil
95
Table 4.3 shows the logistic regression coefficient, Wald test, and
significance for each of the predictors. Using a 0.05 criterion of
significance, all soil types and six of the nine species types had
significant effects. White Oat was the reference variable for species Table 4.3 shows the logistic regression coefficient, Wald test, and
significance for each of the predictors. Using a 0.05 criterion of
significance, all soil types and six of the nine species types had
significant effects. White Oat was the reference variable for species
type. Black Oat, Flame Tree, Red Bean, Tamarind and Vetch were 136 Chapter 4 – Germination Chapter 4 – Germination all significantly different with respect to White Oat. Both Black Soil
and Topsoil were significantly different with respect to Black Soil
with Dolomite. Analysis of Germination Success Table 4.3 – Logistic regression predicting decisions from
Species and Soil Type
Predictor
B
Wald 2
p
Species
Barley
-0.43
0.83
0.360
Black Oat
-2.47
35.00
<0.001
Flame Tree
-2.81
44.48
<0.001
Fiji Feathers Pea
-0.23
0.23
0.633
Red Bean
-5.23
78.89
<0.001
Ryegrass
0.88
1.99
0.158
Tamarind
-1.33
9.76
0.002
Vetch
-3.12
53.34
<0.001
Predictor
B
Wald 2
p
Soil
Black soil
-1.14
22.89
<0.001
Topsoil
-0.86
13.25
<0.001
Constant
2.95
56.18
<0.001 Table 4.3 – Logistic regression predicting decisions from
Species and Soil Type 137 Chapter 4 – Germination Figure 4.4 - Probability of germination success of seeds grown
in three soils
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 Hordeum vulgareAvena strigosa Schreb.Delonix regiaPisum sativumAdenathera pavoninaLolium multiflorumLeucaena leucocephalaVicia villosaAvena sativa
Probability of germination
Blacksoil with Dolomite
Blacksoil
Topsoil Figure 4.4 - Probability of germination success of seeds grown
in three soils
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 Hordeum vulgareAvena strigosa Schreb.Delonix regiaPisum sativumAdenathera pavoninaLolium multiflorumLeucaena leucocephalaVicia villosaAvena sativa
Probability of germination
Blacksoil with Dolomite
Blacksoil
Topsoil 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 Hordeum vulgareAvena strigosa Schreb.Delonix regiaPisum sativumAdenathera pavoninaLolium multiflorumLeucaena leucocephalaVicia villosaAvena sativa
Probability of germination
Blacksoil with Dolomite
Blacksoil
Topsoil Blacksoil with Dolomite
Blacksoil
Topsoil Figure 4.4 - Probability of germination success of seeds grown
in three soils Cadmium concentrations in cadmium waste
Measured values of cadmium concentrations in cadmium waste are
listed in Table 4.4. Cadmium concentrations in the cadmium waste
ranged from ~19 - ~21 ppm. Cadmium concentrations in cadmium waste Research outcomes The results of this report confirm that for the species tested, and
with the treatments employed, cadmium concentration does not
have a statistically significant effect on germination success. This
finding was reinforced by the statistical insignificance of added
cadmium on germination success. Soil type was a significant factor affecting germination success,
indicating that soil characteristics other than cadmium
concentration play a role. Black soil, which is available for
restoration in far greater quantities than Topsoil, showed
significantly lower germination rates than Topsoil across all species
and the addition of dolomite rock further reduced germination
success (Table 4.2, Figure 4.4). Species was a significant factor affecting germination success, which
is to be expected as it is known that species have differing
germination success rates (Bradbeer 1988; Fenner & Thompson 2005). The analysis showed that the order of rates of germination success
from highest to lowest were – Ryegrass > White Oat > Fiji Feathers
Pea > Barley > Tamarind > Black Oat > Flame Tree > Vetch > Red
Bean (Table 4.1). Cadmium concentrations in cadmium waste Measured values of cadmium concentrations in cadmium waste are
listed in Table 4.4. Cadmium concentrations in the cadmium waste
ranged from ~19 - ~21 ppm. Measured values of cadmium concentrations in cadmium waste are
listed in Table 4.4. Cadmium concentrations in the cadmium waste
ranged from ~19 - ~21 ppm. Table 4.4 – Cadmium concentration in Cadmium waste. Sample
Cd concentration
(ppb)
1
21360 ± 30
2
18880 ± 60
3
19420 ± 30 138 Chapter 4 – Germination Chapter 4 – Germination Time Constraints Limits to time in the field as well as other financial and temporal
restrictions, to some extent, restricted this research. The duration of
the experiment was shorter than the germination periods of Delonix 139 Chapter 4 – Germination Chapter 4 – Germination regia and Adenathera pavonina which is likely to have affected
recorded rates of germination (Alam, Basak et al. 1996; Vozzo Unpubl.). We would expect to have higher rates of germination for these
species if the experiment had run longer. Sample analysis limitations
High operational costs, lengthy sample processing times and
logistical constraints using ICP-MS reduced the data available for
analysis, thereby limiting the robustness of the statistical analysis. Due to this, the total cadmium concentrations of the experimental
soils were not measured nor the significance of their effects. It is
tempting to assume, as the exchangeable fraction of cadmium did
not significantly influence germination success, and the
concentrations of total and exchangeable soil cadmium are likely to
be proportionate across the experimental soils, that measures of
total soil cadmium would also produce statistically insignificant
results. Species knowledge limitations Species knowledge limitations Knowledge of appropriate seed pre-treatment may have reduced
germination times for Leucaena leucocephala and Adenathera pavonina,
which may be improved by scarification; for example, immersion in
very hot water for a short period of time, followed by soaking in
cold water for an extended period. This information was not
known by the NRC nursery staff and will be valuable if either of
these species is used in the rehabilitation process. 140 Chapter 4 – Germination Methodological limitations It is possible that the method for measuring concentrations of
exchangeable cadmium used may not accurately reflect the
exchangeable fraction of the soils. As soil characteristics play a
significant role in determining cadmium mobility, the measured
exchangeable fraction may not reflect a proportion of the available
soil cadmium which is consistent when relating between the
different soils and may thus have produced a falsely negative
significance for the effect of cadmium concentration on germination
success. Logistical constraints limited this experiment to 10 species. It
cannot be concluded that the effects found for the tested species will
hold true for all species of interest or value for Nauru. It may be
worth assessing the influence of cadmium on germination success
for other species relevant for rehabilitation and/or conservation. Cadmium waste Cadmium waste
Levels of cadmium in cadmium waste deposits measured in 1992 by
Blake ranged from 169.7ppm to 781.9ppm, down from original
estimates of 2,000ppm when the cadmium waste was deposited in
the cadmium slimes dam (Baines 1994). Measurements of cadmium
concentrations in cadmium waste for this report were around
20ppm (Table 4.4). This may represent methodological differences
in cadmium assessment and/or changes in cadmium concentration
through transportation away from the cadmium waste deposits. If
cadmium has been lost from the cadmium waste, its absence is of
concern as it is unclear where the transported cadmium has gone. If
cadmium has been transported away from the waste deposits it is
likely that water has been the method of transportation. As
overland flow is unlikely in Nauru, cadmium is likely to have
moved down in the soil profile, possibly into the porous underlying
rock, or even into the freshwater lens. It is recommended that there
be ongoing monitoring of cadmium concentrations in cadmium
waste and groundwater. Relevance to other studies Researchers have found positive, negative and an absence of effects
of cadmium on germination success (XinHong, YuXiu & RunJin 2009;
Zhang et al., 2010; Lefevre, 2009; Wang & Zheng, 2009; ZhiDe et al., 2009,
Kumar et al. 2009; HuaBing, 2009; Farooqi et al. 2009). This experiment
found no significant effect of cadmium on germination success. In
the context of the academic literature, this result indicates that the
effects of cadmium on germination success vary depending on
circumstances, or that stochastic effects are influencing
experimental outcomes. It is known that cadmium does have
effects on other aspects of plant growth and development
(Maksymiec and Krupa 2006; Lin, Wang et al. 2007; Dell'Amico, Cavalca et 141 Chapter 4 – Germination Chapter 4 – Germination al. 2008; Sun, Zhou et al. 2008; Ci, Jiang et al. 2009). In the associated
paper assessing biomass production (Chapter 2), exchangeable
cadmium had a significant but minimal negative impact on plant
biomass production. Impacts for restoration Soil type had an effect on germination success for all species
assessed. Topsoil had the highest rates, followed by Black soil,
followed by the lowest rates in the Black soil Dolomite mix. It is
evident that biophysical properties other than cadmium 142 Chapter 4 – Germination concentration are influencing germination success. Soil moisture
retention may be playing a role. The experiments were conducted
in the NRC nursery in a roofed, open walled structure affording no
humidity control. The nursery is situated in the midst of the
phosphate mine karrenfield. The location results in a nursery
climate that is very hot and dry, elevating the potential for soil
desiccation. A new enclosed plant nursery has recently been built
which provides better control over the ambient conditions of
cultivated plants. It may be of use to experiment with the different
soils in the new nursery to determine whether the differences in
germination success between soil types persist. The differences in germination success between soil types are
relevant to landscape rehabilitation in Nauru due to the relative
availability of the soils and the current rehabilitation plan. The
rehabilitation plan intends to use Black soil due to the volume
available in soil stockpiles. The Black soil in the soil stockpiles will
be used, along with dolomite gravels as media for developing soils
on rehabilitation areas. The Nursery is currently using Topsoil as
the preferred medium for raising seedlings from seed, largely due
to the Topsoil available at the nursery site. Given the availability of
the soil, and greater germination success, it is recommended that
this practice is continued. Costs associated with raising seedlings in
the nursery require that germination success be taken into
consideration to ensure that the work is performed efficiently. Black soil, with dolomite rock, is to be used in the rehabilitation
areas. The reduced germination success of seed in the rehabilitation
areas may not be of significant concern, given sufficient viable seed. 143 Chapter 4 – Germination While the effect of cadmium on germination success was absent for
the assessed species, this may not be the case for other ecologically
significant species. There will be value in monitoring seed
production and community composition to determine larger scale
effects. The dramatically lower measured concentrations of total cadmium
in the cadmium waste in this study compared with Blake (1992) and
Baines (1994) require reconciliation. Impacts for restoration It is recommended that
additional, and ongoing assays of cadmium concentrations in the
cadmium waste be performed to determine whether this study,
Blake’s, and/or Baines’ present inaccurate measurements, whether
methods of analysis of cadmium differed significantly between
studies, and/or whether cadmium concentrations in the cadmium
waste are dropping as rapidly as these studies suggest. If cadmium
levels in the cadmium waste are dropping, as suggested by the
measured data, determining the transport mechanisms and
destinations of the cadmium is strongly recommended. To protect
the health and safety of the people of Nauru it is necessary to
ensure that vectors for exposure to dangerous levels of cadmium
are understood and mitigated. REFERENCES Alam, M.R., Basak, N.C., Bheemaiah, G., Bino, B., Biswal, A.K., Dagar,
J.C., Daniel, J.N. Ejercito, M.G.A., Gill, A.S., Gunasena, H.P.M. Gutteridge, R.C., Hengel, T.O., Kato, T., Khandale, D.Y., Korwar,
G.R., Lesseps, S.J.R., Liyanage, M. de S., Mutty, P.K., Norton, B.W.,
Novaho, S., Paterson, R.T., Pathak, N.N., Pathak, P.S., Patil, F.B.,
P
A N F P
i S R
h tk
J M Si gh R V St
t J L & 144 Chapter 4 – Germination Chapter 4 – Germination Takewale, P.S. 1996. Nitrogen fixing trees for fodder production – A field
manual. Winrock International. Takewale, P.S. 1996. Nitrogen fixing trees for fodder production – A field
manual. Winrock International. Baines, G. 1994. Environmental Component – Part A. In Rehabilitation and
Development Feasibility Study: Nauru Australia Cooperation. Blake, C. 1992. Environmental distribution of heavy metals on Nauru, Central
Pacific, and possible relationships to human health, University of New
England. Bachelor of Applied Science (Honours): 86. g
pp
Bradbeer, J. W. 1988. Seed dormancy and germination, Blackie and Sons Ltd. Bradbeer, J. W. 1988. Seed dormancy and germination, Blackie and Sons Ltd. Ci, D., Jiang, D., Wolloenweber, B., Dai, T., Jing, Q. & Cao, W. 2009. Cadmium stress in wheat seedlings: growth, cadmium accumulation
and photosynthesis. Acta Physiologiae Plantarum 32: 9. Dell'Amico, E., Cavalca, L. & Andreoni, V. 2008. Improvement of Brassica
napus growth under cadmium stress by cadmium-resistant
rhizobacteria. Soil Biology and Biochemistry 40: 11. Farooqi, Z. R., Iqbal, M. Z., Kabir, M. & Shafiq, M., 2009, Toxic effects of
lead and cadmium on germination and seedling growth of Albizia
lebbeck (L.) Benth., in Pakistan Journal of Botany, Vol.41, Iss.1, pp.27-
33. Fenner, M. Thompson, K. 2005. The Ecology of Seeds. Cambridge,
Cambridge University Press. Hokura, A., Matsuura, H., Katsuki, F. & Haraguchi, H.. 2000. Multielement Determination of Major-to-Ultratrace Elements in
Plant Reference Materials by ICP-AES/ICP-MS and Evaluation of
Their Enrichment Factors. Analytical Sciences 16. HuaBing, M., ShuJin, H., Shamsi, I. H., Jilani, G., YuanLong, L. & Lixi, L. 2009. Cadmium-induced stress on the seed germination and
seedling growth of Brassica napus L., and its alleviation through
exogenous plant growth regulators. In Plant Growth Regulation,
Vol.58, Iss.1, pp.47-59. Kumar, N., Bauddh, K., Singh, R., Anand, K., Barman, S. C. & Singh, D. P. 2009 Ph
f
l (C & Cd)
G
(C Kumar, N., Bauddh, K., Singh, R., Anand, K., Barman, S. C. & Singh, D. P. 2009. REFERENCES Phytotoxicity of trace metals (Cu & Cd) to Gram (Cicer 145 Chapter 4 – Germination arientinum) and Mung (Phaseolus mungo), in Journal of Ecophysiology and
Occupational Health, Vol.9, Iss.1-2, pp.59-65 Lefevre, I., Marchal, G., Correal, E., Zanuzzi, A. & Lutts, S. 2009, Variation
in response to heavy metals during vegetative growth in Dorycnium
pentaphyllum Scop., in Plant Growth Regulation, Vol.59, Iss.1, pp.1-11. Lin, R., Wang, X., Luo, Y., Du, W., Guo, H. & Yin, D. 2007. Effects of soil
cadmium on growth, oxidative stress and antioxidant systems in
wheat seedlings (Triticum aestivum L.). Chemosphere 69: 10. LiYan, W. & ShiYing, Z. 2009. Effects of cadmium and lead and their
combined pollution on seed germination of wheat. in Journal of
Triticeae Crops, Vol.29, Iss.1, pp.146-148. Maksymiec, W. & Krupa, Z. 2006. The effects of short-term exposition to
Cd, excess Cu ions and jasmonate on oxidative stress appearing in
Arabidopsis thaliana. Environmental and Experimental Botany 57: 8. Meers, E., R. Samson, R., Tack, F.M.G., Ruttens, A., Vandegehuchte, M.,
Vangronsveld, J. & Verloo, M.G. 2007. Phytoavailability assessment
of heavy metals in soils by single extractions and accumulation by
Phaseolus vulgaris. Environmental and Experimental Botany 60: 12. Meers, E., Unamuno, V., Vandegehuchte, M., Vanbroekhoven, K.,
Geebelin, W. Samson, R., Vangronsveld, J., Diels, L., Ruttens, A., Du Laing, G. &
Tack, F.M.G. 2005. Soil-solution speciation of Cd as affected by soil
characteristics in unpolluted and polluted soils. Environmental
Toxicology and Chemistry 24(3): 11. Sun, Y., Zhou, Q. & Diao, C. 2008. Effects of cadmium and arsenic on
growth and metal accumulation of Cd-hyperaccumulator Solanum
nigrum L. Bioresource Technology 99: 8. Vozzo, J.A. Unpublished. Delonix regia (Bojer ex Hook.) Raf. Flamboyán. In
Woody Plant Seed Manual, U.S. Forest Service, Department of
Agriculture. 146 Chapter 4 – Germination Chapter 4 – Germination VSBo 1986. Verordnung über Schadstoffhegalt im Boden, Swiss ordinance on
pollutants in soils Ber. 3000 Bern, Switzerland, Eidgenössische
Drucksachen- und Materialzentrale (EDMZ). Nr 814.12. XinHong, C., YuXiu, Y. RunJin, P. 2009. Effects of Cd, Pb on seed
germination and seedling growth of cucumber. In China Vegetables
Issue 8, pp.18-22. Zhang, X., Fan, X., Li, C. & Nan, Z. 2010, Effects of cadmium stress on seed
germination, seedling growth and antioxidative enzymes in
Achnatherum inebrians plants infected with a Neotyphodium
endophyte, in Plant Growth Regulation, Vol.60, Iss.2, pp.91-97. ZhiDe, C., WeiGong, Z., Jun, W., Jie, Y. & HongSheng, Z. 2009. Seedbank Nauruan soils contain seed. Some of the species of seed found in
the soils were able to grow in the taxing conditions found on
Topside. Calopogonium mucunoides, Leucaena leucocephala, and
Indigofera hirsuta were successful in Black soil, Topsoil, Buada soil
and dolomite. As none of these were among the five trial species,
This indicates that the soils contained their seed, and that the soils
were adequate to promote their germination and growth. INTRODUCTION This chapter is a summary of the results of the biomass, cadmium
phytoaccumulation and germination experiments which were
performed as part of this research. The implications of these results
for the soil rehabilitation process are discussed within the
conceptual frameworks of ecological restoration and adaptive
management. REFERENCES Effects of
cadmium stress on seed germination and seedling growth of various
rice varieties., in Jiangsu Journal of Agricultural Sciences, Vol.25, Iss.1,
pp.19-23. 147 Chapter 5 – Discussion 148 Chapter 5 – Discussion Chapter 5 – Discussion Chapter 5 – Discussion Cadmium Analysis of cadmium phytoaccumulation by a range of plant
species in different Nauruan soils revealed that, in the conditions of
the field trial: Total cadmium concentrations in the tested soils differed
significantly; Buada soils contained the highest concentrations at
4ppm, followed by Black soil and Topsoil with 2ppm, and the
lowest concentrations were found in the dolomite at 1ppm Plant tissue concentrations of cadmium differed according to
tissue type; concentrations of cadmium were highest in root
tissues. Biomass Analysis of biomass production of a range of plant species in
different Nauruan soils revealed that, in the conditions of the field
trial: Soil types differed significantly in biomass productivity; Black
soil and Topsoil were substantially more productive than Buada
soil or dolomite Black soil productivity was 1,740±460gm-2 over a 6 month
period. Assuming that this figure can be extrapolated this 149 Chapter 5 – Discussion equates to productivity of 34.8±9.2 tonnes of biomass per hectare
per year. equates to productivity of 34.8±9.2 tonnes of biomass per hectare
per year. equates to productivity of 34.8±9.2 tonnes of biomass per hectare
per year. equates to productivity of 34.8±9.2 tonnes of biomass per hectare
per year. Calopogonium mucunoides and Cajanus cajan were the most
productive species in Black soil Cajanus cajan and Leucaena leucocephala were the most productive
species in Topsoil Use of woodchip mulch had a large positive effect on biomass
production Exchangeable soil cadmium had a small negative effect on
biomass productivity. Germination Analysis of germination success of seed from a range of plant
species in different Nauruan soils revealed that, in the conditions of
the germination trial: Exchangable soil cadmium levels did not determine
germination success 150 Chapter 5 – Discussion Soil type had a significant effect on germination success; Topsoil
had the highest success rate, followed by Black soil. The lowest
rates of success were for the Black soil dolomite mix Species was a significant factor determining germination
success. Seedbank The success of species which grew from seed in the experimental
soils provides information which is valuable for rehabilitation
planning. Firstly, there must be recognition of the soil seedbank;
the soils cannot be regarded as being devoid of vegetation
propagules. Secondly, within the seedbank are seed of species
which can successfully germinate and develop into mature plants in
rehabilitation conditions. Thirdly, judging from their germination
and growth rates, the species present in the seedbank appear to be
well adapted to the conditions found in Topside, Nauru. The best
use of stockpiled soils requires a management approach that
considers land use. The stockpiled soils should not be used for land
uses where the presence of viable seed stock is problematic. considers land use. The stockpiled soils should not be used for land
uses where the presence of viable seed stock is problematic. Allowing the seed to germinate and systematically removing plants
prior to seed set will be a suitable option in many cases. In soils
that contain large amounts of undesired seed other management
options may be more appropriate. Calopogonium mucunoides, Leucaena leucocephala, and Indigofera hirsuta
successfully germinated and grew in all four soils. Their success
indicates their potential to be widespread pest species. However, it
may also be the case that these coloniser species will establish 151 Chapter 5 – Discussion Chapter 5 – Discussion environmental conditions which promote the successional
establishment of a wider range of species, significantly benefiting
rehabilitation. Accordingly, monitoring of developing vegetation
patterns in rehabilitation areas should be conducted to determine to
what extent these species may assist or hinder the rehabilitation
process. Biomass There were significant differences in biomass production between
soil types. Topsoil and Black soil were far more productive than
either the Buada soil or dolomite. This is a positive outcome as the
Black soil is to be used for land rehabilitation, and the Topsoil is
used as a seed raising and potting medium for plants in the nursery. The low rates of biomass production in the Buada soil may be due
to lower levels of seed in the soil, especially of species suited to
growing on Topside. The use of woodchip mulch doubled biomass
production. This provides some support for the idea that increasing
soil organic matter will improve the fertility of Nauruan soils
through the various mechanisms discussed in Chapter 1. Likewise,
biomass produced in rehabilitation areas could be used for
woodchip mulch production, to be returned to the site for
subsequent accumulation of organic matter to provide further
benefits for biomass production. Black soil productivity was 1,740±460gm-2 over a 6 month period. Assuming that this figure can be extrapolated this equates to
productivity of 34.8±9.2 tonnes of biomass per hectare per year. It is
likely that, as soil fertility improves, biomass production will
increase. 152 Chapter 5 – Discussion Calopogonium mucunoides and Cajanus cajan were the most
productive species in Black soil. Both species are likely candidates
for use in the rehabilitation area for increasing biomass available for
mulch production to improve soil fertility. Calopogonium mucunoides
is a twining or trailing short-lived perennial herb which grows in a
dense mat up to 50cm thick. It is a valuable leguminous pioneer
species, grown as a cover crop to reduce erosion and improve soil
fertility. It will associate with a wide range of mycorrhizae in its
rhizobia, and cowpea inoculants can be used where soils are sterile
(Chen & Aminah 1992; Veasey et al. 1999). Cajanus cajan is a
leguminous shrub that can grow to heights of 5m. It is hardy, widely adaptable and tolerant of drought and high temperatures. It
produces peas which are used throughout the world for food, and is
an important source of biomass for household fuel in many parts of
the world (Sheldrake & Narayanan 1979; Willey et al. 1981; Sheldrake
1984). The growth forms of Calopogonium mucunoides, and Cajanus
cajan are such that they may be complementary crops, with low
levels of competition. Biomass In the trial plots they formed a robust
vegetative cover for the soil. Given their success in the field trial, it
is recommended that their use as colonisers in the rehabilitation
project be assessed. Cadmium Buada soils contained the highest concentrations of cadmium. This
may indicate that the soil poses a greater risk than either Black soil
or Topsoil when used for agricultural purposes. Agriculture is
currently practiced in the Buada area, in kitchen gardens and at the
Taiwanese agricultural trial facility. It would be prudent to test for 153 Chapter 5 – Discussion cadmium concentrations in agricultural products grown in Buada
soil for comparison with equivalent products grown in other
Nauruan soils to determine whether those agricultural products
pose an unnecessary risk. Measured variations in cadmium concentration in plant tissues
allow us to make both general and specific statements. Generally,
root tissues contain significantly higher concentrations of cadmium
than above ground tissues. This can be understood as a
consequence of the effect of the Casperian bands which significantly
reduce cadmium transport out of the root system. More
specifically, we can state that, within the context of comparison
between the analysed species, the root tissues of Cajanaus cajan
accumulate particularly high concentrations of cadmium; and that
Sida acuta accumulates comparably high levels of cadmium in all
tissues. These are important considerations when harvesting plant
biomass where cadmium phytoaccumulation is an objective. It is
likely that for soils intended for agriculture, increasing soil organic
matter (SOM) and reducing the cadmium burden will be desired
outcomes. Accordingly, as adding woodchip mulch is a relatively
easy method for increasing SOM, selectively adding low cadmium
biomass would be advised. For species containing relatively low
concentrations of cadmium in leaf and stem tissues low cadmium
biomass could be produced on soils containing significant levels of
cadmium. Coppicing of suitable species may be a useful technique
for harvesting biomass; however, due to the low levels of cadmium
in leaf and stem tissue, it is unlikely that this technique will be
useful for the phytoremediation of soils with the test species. Harvesting root tissues will be necessary for the phytoextraction of Measured variations in cadmium concentration in plant tissues
allow us to make both general and specific statements. Generally,
root tissues contain significantly higher concentrations of cadmium
than above ground tissues. This can be understood as a
consequence of the effect of the Casperian bands which significantly
reduce cadmium transport out of the root system. Cadmium More
specifically, we can state that, within the context of comparison
between the analysed species, the root tissues of Cajanaus cajan
accumulate particularly high concentrations of cadmium; and that Sida acuta accumulates comparably high levels of cadmium in all
tissues. These are important considerations when harvesting plant
biomass where cadmium phytoaccumulation is an objective. It is
likely that for soils intended for agriculture, increasing soil organic
matter (SOM) and reducing the cadmium burden will be desired
outcomes. Accordingly, as adding woodchip mulch is a relatively
easy method for increasing SOM, selectively adding low cadmium
biomass would be advised. For species containing relatively low
concentrations of cadmium in leaf and stem tissues low cadmium
biomass could be produced on soils containing significant levels of
cadmium. Coppicing of suitable species may be a useful technique
for harvesting biomass; however, due to the low levels of cadmium
in leaf and stem tissue, it is unlikely that this technique will be
useful for the phytoremediation of soils with the test species. Harvesting root tissues will be necessary for the phytoextraction of 154 Chapter 5 – Discussion cadmium from soils to occur at a maximal rate. Harvesting the root
tissue is a considerably more involved process than harvesting
above ground tissues as it involves mechanical removal of root
tissue from the soil medium. It cannot be expected that all root
tissue will be removed and in doing so the structure of the soil will
inevitably be disturbed. Due to this it is likely that efforts to
phytoremediate cadmium by removing cadmium contaminated
root tissue will be most effective in highly managed areas, for the
purposes of generating a low cadmium soil for use in agricultural
areas, rather than in situ phytoremediation. Scheid et al. described how cadmium in leaf litter became relatively
fixed in the leaf material of alder (Alnus glutinosa) and poplar
(Populus tremula). Cadmium in leaf material was largely bound two
years after leaf fall and thus was not returned to the soil (Scheid et al. 2009). This may also be the case for species grown in Nauru. If
cadmium fixation in leaf litter does occur, we would expect that as a
greater proportion of exchangeable cadmium is phytoaccumulated
the dynamics of exchangeable cadmium will alter. Cadmium While it is
unlikely to be a priority at this stage in the rehabilitation process,
assessing the degree to which cadmium is fixated in the tissues of
species in Nauru may provide valuable insights into the future
dynamics of cadmium in the Nauruan landscape, as affected by
increasing proportions of organic material. Germination While soil cadmium had no effect on germination success, soil type
did. Currently the nursery staff use Topsoil for raising seedlings as
its presence in the nursery site makes it convenient to do so. Given 155 Chapter 5 – Discussion that the highest rates of germination success were consistently
found in the Topsoil, it makes sense for this practice to continue to
ensure that the return on effort invested in seedlings is high. The
reduced rates of germination success in Black soil may not be cause
for concern assuming that, when used for land rehabilitation, the
availability of seed in the rehabilitation areas is sufficiently high to
ensure that recruitment rates allow for continued development of
the vegetated ecosystems. In contrast to this, an overabundance of
seed of some species may lead to them outcompeting other plants in
such a way as to lead to low levels of plant biodiversity, as can be
seen occurring with Red Bean in parts of the Buada district. Germination rates of Red Bean were very low at around nine per
cent. The addition of dolomite to the Black soil resulted in reduced
rates of germination for all experimental species. This result raises
questions about the consequences of using dolomite gravel in the
rehabilitation process. The mechanisms by which seed germination
rates were affected by the trialled soil types are currently unclear. Possible mechanisms include soil pH and soil moisture. To ensure
that germination success rates in the nursery can be optimised to
produce large numbers of vigorous plants suitable for landscape
restoration further experimentation and analysis is necessary. Germination rates of Red Bean were very low at around nine per
cent. The addition of dolomite to the Black soil resulted in reduced
rates of germination for all experimental species. This result raises
questions about the consequences of using dolomite gravel in the
rehabilitation process. The mechanisms by which seed germination
rates were affected by the trialled soil types are currently unclear. Possible mechanisms include soil pH and soil moisture. To ensure
h
i
i
i
h
b
i
i
d Soil stockpiles Of central importance to the rehabilitation programme are the soil
stockpiles. Managing them in their current state so as to maximise
their productive potential and limit the negative effects of
stockpiling will pay significant dividends for subsequent land
rehabilitation. Ideally, prior to use in rehabilitation areas,
stockpiled Black soil will have been managed to increase organic 156 156 Chapter 5 – Discussion content and microbial life to help ensure that the subsequently
planted vegetation has a good chance of becoming established. Given the cyclical relationship between vegetation and soil, failure
to quickly establish vegetation on Black soil in the rehabilitation
areas is likely to result in further losses in soil fertility. It is
recommended that a programme is established which attends to
this concern as soon as possible, in order to ensure the availability
of higher quality soils for rehabilitation and so provide a better
chance of rehabilitation success. Also of potential concern are the species present in the Black soil
seedbank. Stockpile A is currently covered by a monoculture of
Leucaena leucocephala. It is highly likely that the seed in the
seedbank of stockpile A are almost exclusively wild tamarind seed. The field trial employed Black soil sourced from Stockpile B, which
supports a wide range of species, as reflected in the species which
successfully germinated and grew from the Black soil seedbank
(Table 2.1). Leucaena leucocephala was one of the species which
successfully germinated from the Black soil seedbank, and
successfully developed into mature plants. Leucaena leucocephala
was one of three species which successfully grew from the
seedbanks of all four trialled soils. In all soils it was a high biomass
producer. These factors indicate that it is well suited to the
environmental conditions found in Topside. Given that Leucaena
leucocephala has successfully established itself as a monoculture on
Stockpile A, it is prudent to develop a plan for managing the species
to ensure that it does not become a problem in the rehabilitation
areas. 157 Chapter 5 – Discussion Leucaena leucocephala has a reputation in many other parts of the
world for its uses in intercropped tree mulching systems. Leucaena
leucocephala handles repeated prunings and regenerates vigorously,
is deep rooted which reduces competition with other less deeply
rooted plants, and produces large amounts of useful wood, has well
balanced N composition in pruned biomass (Kang et al. 1981). Soil stockpiles Biannual lopping of seedless Leucaena leucocephala clones on
Balinese coffee plantations resulted in nitrogen fertilisation of 30-
80kg/ha (Brewbaker 1990). Cultivating seedless cloned Leucaena
leucocephala may provide a method by which the species can be
employed in rehabilitation efforts without the risk of it becoming a
pest species. Cadmium waste The dramatically lower measured concentrations of total cadmium
in the cadmium waste in this study compared with Blake (1992) and
Baines (1994) require reconciliation (Table 5.1). It is recommended
that additional, and ongoing assays of cadmium concentrations in
the cadmium waste be performed to determine whether this study,
Blake’s, and/or Baines’ present inaccurate measurements, whether
methods of analysis of cadmium differed significantly between
studies, and/or whether cadmium concentrations in the cadmium
waste are dropping as rapidly as these studies suggest. If cadmium
levels in the cadmium waste are dropping, as suggested by the
measured data, determining the transport mechanisms and
destinations of the cadmium is strongly recommended. To protect
the health and safety of the people of Nauru it is necessary to
ensure that vectors for exposure to dangerous levels of cadmium
are understood and mitigated. 158 Chapter 5 – Discussion Table 5.1 – Comparison of measured concentrations of
cadmium in cadmium waste Table 5.1 – Comparison of measured concentrations of
cadmium in cadmium waste
Study
Year
Cd conc. in Cd waste
Recorded in Baines (1994)
1990
~ 2000ppm
Blake
1992
169.7ppm – 781.9ppm
Current study
2008
18.9ppm – 21.4ppm Study
Year
Cd conc. in Cd waste
Recorded in Baines (1994)
1990
~ 2000ppm
Blake
1992
169.7ppm – 781.9ppm
Current study
2008
18.9ppm – 21.4ppm Ecological Restoration Ecological Restoration Typically an ecological restoration project might involve one of the
following restoring a highly degraded site such as a mine to ensure the
return of vegetation improving the productive capabilities of soils for agriculture enhancing and protecting the conservation values of landscapes enhancing and protecting the conservation values of landscapes The situation in Nauru involves all three, as well as complex social,
historical, cultural, technical, aesthetic, economic, and political
contexts (Hobbs & Norton 1996; Harris & van Diggelen 2006). It must
be emphasised that the rehabilitation project is unlike any before
attempted. The situation in Nauru involves all three, as well as complex social,
historical, cultural, technical, aesthetic, economic, and political
contexts (Hobbs & Norton 1996; Harris & van Diggelen 2006). It must
be emphasised that the rehabilitation project is unlike any before
attempted. In the introduction to this thesis (Chapter 1) I posited that the goals
of ecological restoration in Nauru might be – rehabilitating the
mined landscape to a state which is safe for human habitation,
increasing vegetative cover, increasing soil organic matter (SOM),
the preservation and cultivation of culturally significant species, the
preservation and cultivation of ecologically significant and 159 Chapter 5 – Discussion endangered species, and the development of fertile soils for
agriculture. This research provides a preliminary assessment of the
relationship between soils and vegetation in the environmental
conditions found in Topside, Nauru. Environmental cadmium was
directly assessed and the general effects of the environmental
conditions were assessed indirectly through measures of plant
productivity and survival. It is unclear what role environmental cadmium will have on
ecological restoration. Germination rates were unaffected, and
while cadmium did have a deleterious effect on plant productivity,
the effect was very small. Assessments involving a larger range of
species, specific life cycle stages such as seed set, and/or community
effects, especially those involving trophic interactions, may reveal
specific challenges posed by cadmium for the rehabilitation process. This research has been limited to determining the impacts of
specific environmental stressors which pose physiological
challenges to the ecological restoration of rehabilitation land. A
large number of ecological factors other than physiological
challenges are relevant to the land rehabilitation process (Table 5.1). Attending to these factors will aid in ensuring the successful
rehabilitation of Topside. Ecological Restoration 2006) Locally adapted phenotypes Bottlenecks and founder effects Physiological challenges Physiological limits with regard to
survival and reproduction Site specific phenotypes 161 Chapter 5 – Discussion
Community ecology
Community composition
Trophic structure
Dispersal
Environmental filters
Disturbance regimes
Mutualisms
Ecological dynamics
Resilience
Ecological thresholds
Linear and non-linear dynamics
Biodiversity and
ecosystems
Functional diversity
Redundancy
Invasive species
Community invisibility
Alteration of ecosystem processes
Resistance and resilience
Competition
Disturbance theory
Research design
Replication
Sample size
Statistical framework
Repeated measures through time
Climate
Climate and relationship with vegetation
Natural variability
In order for the rehabilitation programme to benefit from ecological
restoration principles it is recommended that rehabilitation
management develop a clear understanding of how the knowledge
targets proposed in Table 5.2 relate to the ecology of Nauru. Adaptive Management
It is likely that even with comprehensive planning unexpected Chapter 5 – Discussion In order for the rehabilitation programme to benefit from ecological
restoration principles it is recommended that rehabilitation
management develop a clear understanding of how the knowledge
targets proposed in Table 5.2 relate to the ecology of Nauru. Ecological Restoration Generally, abiotic factors play a more significant role in the early
phases of land restoration, while biotic factors become more
pronounced as communities become established (Sänger & Jetschke
2004). In these circumstances we would expect ruderal and stress
tolerant species to be successful as colonizers, although other 160 Chapter 5 – Discussion studies have indicated that this is not always the case (Sänger &
Jetschke 2004). Table 5.2 lists a range of information targets which are valuable for
ecological restoration. This report does not attempt to resolve the
majority of these information targets and instead recognises that
they are information ideals which may be resolved through the
process of land rehabilitation in Nauru. This report emphasises
aspects of the physiological challenges of land rehabilitation in
Nauru, employing analytical research design. This report has
touched on many of the other ecological restoration knowledge
targets; however, they were not of central importance to this
analysis. Part of the reasoning for the approach employed in this
report is that the abiotic conditions found in Nauru are particularly
severe and require initial attention. The relationship between soils
and vegetation in the environmental conditions found in Nauru are
assessed herein. Environmental cadmium is directly assessed and
the general effects of the environmental conditions are assessed
indirectly through measures of plant productivity and survival. Table5.2 - Knowledge targets for ecological restoration
(adapted from Palmer, Falk et al. 2006)
Propagule sources
Locally adapted phenotypes
Bottlenecks and founder effects
Physiological challenges
Stress tolerance
Physiological limits with regard to
survival and reproduction
Site specific phenotypes Table5.2 - Knowledge targets for ecological restoration
(adapted from Palmer, Falk et al. 2006)
Propagule sources
Locally adapted phenotypes
Bottlenecks and founder effects
Physiological challenges
Stress tolerance
Physiological limits with regard to
survival and reproduction
Site specific phenotypes Table5.2 - Knowledge targets for ecological restoration
(adapted from Palmer, Falk et al. Adaptive Management It is likely that even with comprehensive planning unexpected
outcomes will occur. Rehabilitation management must be prepared
to accept this, and contend with the information that such surprises
provide. In accepting and preparing to contend with the inevitable
uncertainty of how rehabilitation is to proceed, the NRC will enable
itself to progress with land rehabilitation despite it. 162 Chapter 5 – Discussion Land rehabilitation in Nauru needs to progress to achieve Nauru’s
development goals. Progress may be measured in successful
rehabilitation, but given the paucity of ecological information
currently available to guide rehabilitation procedure a more
valuable metric may be the acquisition of information about how to
proceed. An adaptive management approach will allow for this
progress to occur. There is a need for explicit goals, a restoration design developed
with an awareness of local ecology, ongoing monitoring to provide
quantitative data of change and analysis of the results in order to
adapt the design according to increasing knowledge of the ecology
of the restoration landscape (Walters & Holling 1990; Gregory et al. 2006; Palmer et al. 2006). In order to implement an adaptive management approach it will be
necessary to ensure that there are stable institutions in place which
will ensure that the program will persist long term. Particularly in
the short term, there needs to be an understanding among high
level managers that measures of success under an adaptive
management framework should be found in acquisition of
knowledge about restoration management based on hypothesis
testing (Walters & Holling 1990; Gregory et al. 2006). Two arenas exist in which adaptive management principles can be
employed within the established rehabilitation framework, within
the rehabilitation area and within the nursery supplying plants to
the programme. The trial rehabilitation areas Pit 6 is an ideal 163 Chapter 5 – Discussion setting in which to ‘learn by doing’. It is part of the rehabilitation
area, and the landscape has been resurfaced as per the rehabilitation
plan. The current plan is to spread Black soil and plant a wide
range of species cultivated in the nursery. Lists of species to be
used in rehabilitation have been compiled, but there are no
documents guiding the species composition of specific areas,
planting densities and patterns, soil preparation, watering, or how
planting will be temporally organised. The absence of these
documents comes as no surprise given that there is so little known
about how restoration should proceed. Adaptive Management These knowledge gaps are
all opportunities for the application of experimental assessment
under an adaptive management framework. Other avenues for
field trial experimentation include assessing the effects of different
soil depths, use of different mulches, use of living ground covers,
use of mycorrhizae, coppicing, pruning, and successional processes. The other arena for adaptive management is the NRC nursery
which provides a more laboratory like setting in which to explore
aspects of the rehabilitation process which are relevant to the
establishment of viable plant stock, and aspects of restoration
requiring greater control over environmental conditions. The
nursery conditions may be used to disentangle potentially relevant
variables from environmental inconsistency, and test hypotheses
which require more rigorously controlled conditions than are
available in the field. Avenues for nursery based experimentation
include variations in soil organic content, soil additives such as
vermiculite, watering regimes, companion planting, mycorrhizal
inoculations, seed treatments, and the effects of pruning on plant
development. 164 Chapter 5 – Discussion Ideally, several management methods will be trialled at the same
time within well defined land units. Management methods will
need to be replicated several times in a randomised pattern to
ensure the validity of statistical analysis. Establishing other trial
areas employing the same, similar, contrasting, or different
management approaches will allow for useful comparisons of
management techniques between sites. The benefit of trialling the
same group of management techniques in another area is that it
increases the number of repetitions of any treatment to improve
statistical power and stochastic difference can be better accounted
for in the statistical model. An additional benefit may be the
formation of an ecosystem chronosequence of rehabilitation areas,
providing more information for comparison between rehabilitation
areas as the ecosystems within them develop through time. Freshwater The lack of availability of large amounts of fresh water in Nauru is
likely to be one of the most limiting factors for land rehabilitation. Soil moisture deficits in the rehabilitation areas will restrict the
diversity of species, and their growth rates. Field trials to determine
optimum and minimum functional levels of soil moisture will help
to determine the freshwater demands of the rehabilitation process. A management priority should be the collection and storage of
rainfall to ensure that rehabilitation does not further reduce
freshwater availability. Once vegetation is well established soils
will be able to retain greater soil moisture, reducing the impact of
drought conditions, and reducing the freshwater needs of the
rehabilitation sites. An additional benefit of healthy soils will be the
protection of the freshwater lenses beneath Nauru. Soils are
valuable assets in protecting water quality, as they can be effective
as repositories and biodegraders of toxins which might otherwise
enter the freshwater lenses (Lal 2007). There are large water storage
tanks on Nauru which are currently unused and have fallen into
disrepair. It is recommended that the existing infrastructural assets
be assessed to determine whether they could be used in a rainwater
capture and storage system. The lack of availability of large amounts of fresh water in Nauru is
likely to be one of the most limiting factors for land rehabilitation. Soil moisture deficits in the rehabilitation areas will restrict the
diversity of species, and their growth rates. Field trials to determine
optimum and minimum functional levels of soil moisture will help
to determine the freshwater demands of the rehabilitation process. A management priority should be the collection and storage of
rainfall to ensure that rehabilitation does not further reduce
freshwater availability. Once vegetation is well established soils
will be able to retain greater soil moisture, reducing the impact of
drought conditions, and reducing the freshwater needs of the
rehabilitation sites. An additional benefit of healthy soils will be the
protection of the freshwater lenses beneath Nauru. Soils are
valuable assets in protecting water quality, as they can be effective
as repositories and biodegraders of toxins which might otherwise
enter the freshwater lenses (Lal 2007). There are large water storage
tanks on Nauru which are currently unused and have fallen into
disrepair. It is recommended that the existing infrastructural assets
be assessed to determine whether they could be used in a rainwater
capture and storage system. CONCLUDING REMARKS Most restoration projects are not as complex as Topside, Nauru. p
j
p
p
There are a wide variety of uncertainties, and it is likely that there
will be surprises. Accordingly, it is essential that the management
of rehabilitation is prepared to learn and adapt. There is a need for
experimentation with management techniques based on sound
hypotheses, monitoring of the outcomes and adaptation of
management to suit the developing knowledge of how
rehabilitation management should proceed. The remainder of this
section will introduce aspects of the restoration project which were
not directly addressed in the body of this report but are relevant to
its success. 165 Chapter 5 – Discussion Community restoration Nauru is not a large island. Human impacts are widespread and
pervasive. For land rehabilitation on Nauru to be successful there is
a need for strong Nauruan community dedicated to the sustainable
management of Nauruan soils. Including community in mine
rehabilitation may assist in this (Leigh 2005). Community based
restoration can facilitate stronger relationships between land and 166 Chapter 5 – Discussion culture by reconnecting communities with their landscapes,
empowering citizens through participation, and promoting
enduring awareness of the relationship between healthy
environments and healthy communities (Harris & van Diggelen
2006). While it is uncertain how community based restoration may
contribute to the rehabilitation effort, it is an avenue which may
provide significant benefits and should be explored. Tension between mining and rehabilitation Mining in Nauru has resulted in extensive ecological degradation. The ethos which resulted in these losses cannot be expected to be
appropriate for guiding the rehabilitation process. The Nauru
Rehabilitation Corporation (NRC) has emerged from the Nauru
Phosphate Corporation, utilising the same or similar staff,
equipment, and management. This is to be expected due to
convenience; however, there is the potential for management
conflicts to emerge. A potential example of such a conflict is with
the current status of mined lands. Currently, almost all mined
lands are not classified as mined-out due to the significant volumes
of phosphate remaining in the landscape post-mining. Global
phosphate prices are trending upwards, making secondary mining
increasingly attractive. Much of the mined land is thus in an
interim state, neither mined-out nor un-mined. The current
proposal is that land rehabilitation coincides with secondary
phosphate mining, to generate revenue through phosphate exports
and to mechanically resurface the landscape prior to soil deposition
and planting. While secondary mining and rehabilitation will
benefit from a combined effort, circumstances may rise where their
interests do not perfectly coincide. In such a case compromises will and planting. While secondary mining and rehabilitation will
benefit from a combined effort, circumstances may rise where their
interests do not perfectly coincide. In such a case compromises will 167 Chapter 5 – Discussion need to be made. It is recommended that rehabilitation
management policy contain provisions for deciding which objective
is prioritised. The author of this report emphasises that to ensure
that the rehabilitation of mined lands effectively progresses it will
be necessary to ensure that the operational procedures of secondary
phosphate mining do not supersede those of land rehabilitation. Buada lagoon Water quality in Buada lagoon is currently degraded due to
feedbacks between high dissolved nutrients and algae. Eutrophication is a serious concern for the lagoon. The lagoon is a
valuable resource, providing an environment which has
traditionally been used for aquaculture. The lagoon is a potential
source of nutrients for soil rehabilitation, both from aquatic plants
and algae, and from dredged sediments. However, material
removed from the lagoon may contain levels of cadmium
contamination which are too high for use in areas where cadmium
contamination is a concern. Water quality in Buada lagoon is currently degraded due to
feedbacks between high dissolved nutrients and algae. Eutrophication is a serious concern for the lagoon. The lagoon is a
valuable resource, providing an environment which has Biochar Biochar is a manufactured product which is used as a soils
amendment to improve soil quality. It is produced from organic
waste products by low-temperature pyrolysis which also produces
volatile compounds such as bio-oil and synthesis gas which can be
used as a source of energy. Biochar improves various properties of
soils such as water retention, soil volume, microbial mass, soil
structure, and nutrient retention. 168 Chapter 5 – Discussion Biochar is produced from waste organic material. This
characteristic can be especially useful in circumstances where
disposal of organic wastes such as animal manures and human
body waste may be problematic due to risks of contaminating the
environment with pathogens. The pyrolysis process destroys
pathogens, producing a biochemically useful substance without
contamination risks. Biochar can also be produced using cultivated
organic material. By converting organic material into black carbon
through the pyrolysis process, carbon is stabilised in a form which
has greater longevity within the soil system than organic material
normally would. A significant issue with fertility in Nauru soils are
the impermanence of soil organics, the high temperatures and
humidity result in relatively rapid loss of soil organics through
microbial processes. Stabilising carbon in the soil system through
the use of biochars may provide long-term benefits for the fertility
of Nauruan soils. Biochar is known to absorb toxic heavy metals, substantially
reducing their availability in soils. This quality, along with the
other soil benefits listed, suggests that biochar may be an ideal soil
amendment material for Nauru if sufficient biomass feedstock can
be sourced. Composting toilets Composting toilets, while lacking the energy production
possibilities of a centralised sewerage system, have a number of
advantages making them a practical solution for managing Nauru’s
human waste. Functioning composting toilets remove or destroy
pathogens in human excrement and produce soil organism rich 169 Chapter 5 – Discussion humus as an end product much. They can be built from local
materials according to local needs; have negligible freshwater
inputs by comparison with sewerage systems; lack the need for
expensive reticulation systems, which is particularly beneficial in
facilitating waste management in isolated locales; and greatly
reduces the risk of soil and groundwater contamination due to
structural failures in reticulated pipe and subsurface storage and
treatment infrastructure (Del Porto & Steinfeld 2000). Added plant
material is an essential input to maintain the high Carbon:Nitrogen
ratio needed for proper functioning of composting toilets (Crennan
1996). Human resources A wide range of skills will be required for the rehabilitation
programme in Nauru. It will be important to identify required and
useful skills, recognise which of these skills are available in the NRC
staff, and methods for acquiring those skills currently lacking. It is
likely that many of the skills required can be attained for the
rehabilitation project by supporting skills development in NRC
staff. The Nauruan branch of the University of the South Pacific
(USP) is available to NRC for skills development. A mutually
supportive relationship based in open communication between
NRC and USP will foster both organisations to achieve their goals. RECOMMENDATIONS -
Black soil seedbank species -
Black soil seedbank species o It will be necessary to categorise the species according
to their desirability in rehabilitation areas. 170 Chapter 5 – Discussion o It will be necessary to identify which species pose
risks as invasive species and establish appropriate
management techniques eg. - Calopogonium mucunoides, Leucaena
leucocephala, and Indigofera hirsuta -
Biomass production -
Biomass production o Calopogonium mucunoides and Cajanus cajan are
promising biomass production crops which can be
used to colonise, cover, protect and improve Black
soils. o Black soil productivity was determined to be 34.8±9.2
tonnes of biomass per hectare per year. -
Cadmium o Management of biomass production should recognise
that Sida acuta biomass and root tissues of Cajanus
cajan may contain significant concentrations of
cadmium where grown in cadmium contaminated
soils. o Ongoing monitoring of cadmium concentrations in
cadmium waste dump sites should be performed to
determine whether the dramatic losses of cadmium
suggested in the literature accurately reflect changes
in cadmium concentration. If significant amounts of
cadmium are being lost from the cadmium waste, it
will be important to identify where the cadmium is
going to ensure that the people of Nauru are not
unnecessarily exposed to excess cadmium. o Ongoing monitoring of cadmium concentrations in
cadmium waste dump sites should be performed to
determine whether the dramatic losses of cadmium
suggested in the literature accurately reflect changes
in cadmium concentration. If significant amounts of
cadmium are being lost from the cadmium waste, it
will be important to identify where the cadmium is
going to ensure that the people of Nauru are not
unnecessarily exposed to excess cadmium. o Monitoring of cadmium concentrations in
groundwater is advised. 171 Chapter 5 – Discussion -
It is recommended that adaptive management principles be
adopted to allow rehabilitation to progress in the face of
considerable uncertainty. o Management will need to proceed by testing clearly
specified hypotheses. o Management should expect that successful
rehabilitation is very unlikely in the short term. Rehabilitation success should be measured in the
acquisition of information relevant to rehabilitation
knowledge targets. -
Stockpiles o The vegetation on the stockpiles should be managed
to reduce weed numbers, and increase soil organic
matter in preparation for use in rehabilitation areas. -
Ecological restoration o Rehabilitation management should proceed in a
manner that achieves ecological information targets
(listed in Table 5.2). REFERENCES Baines, G. 1994. Environmental Component – Part A. In Rehabilitation and
Development Feasibility Study: Nauru Australia Cooperation. Baines, G. 1994. Environmental Component – Part A. In Rehabilitation and
D
l
F
ibili
S d
N
A
li C
i Baines, G. 1994. Environmental Component – Part A. In Rehabilitation and
Development Feasibility Study: Nauru Australia Cooperation. Baines, G. 1994. Environmental Component – Part A. In Rehabilitation and
Development Feasibility Study: Nauru Australia Cooperation. Development Feasibility Study: Nauru Australia Cooperation. Blake, C. 1992. Environmental distribution of heavy metals on Nauru, Central
Pacific, and possible relationships to human health, University of New
England. Bachelor of Applied Science (Honours): 86. Blake, C. 1992. Environmental distribution of heavy metals on Nauru, Central
Pacific, and possible relationships to human health, University of New
England. Bachelor of Applied Science (Honours): 86. Brewbaker, J. L. 1990. Nitrogen fixing trees. In Fast Growing Trees and
Nitrogen Fixing Trees, D. Werner, and P. Müller, eds., Stuttgart:
Gustav Fischer Verlag, p. 9. Chen, C.P. & Aminah, A. 1992. Calopogonium mucunoides . In: 't
Mannetje, L. and Jones, R.M. (eds) Plant Resources of South-East Asia
No. 4. Forages. pp. 72-74. (Pudoc Scientific Publishers, Wageningen,
the Netherlands). 172 Chapter 5 – Discussion Chapter 5 – Discussion Crennan, L. 1996. Case study 4: Composting toilet trial on Kirimati,
Kiribati. In Technology for augmenting freshwater resources in small island
developing states, United Nations Environment Programme (UNEP). Del Porto, D. & Steinfeld, C. 2000. The composting toilet system book: A
practical guide to choosing, planning and maintaining composting toilet
systems, a water saving, pollution-preventing alternative: The Center for
Ecological Pollution Prevention (CEPP). Gregory, R., Ohlson, D. & Arvai, J. 2006. Deconstructing adaptive
management: Criteria for applications to environmental
management. In Ecological Applications, 16(6), pp.2411-2425. Harris, J. A. & van Diggelen, R. 2006. Ecological restoration as a project for
global society. In Restoration Ecology: The New Frontier, J. van Andel,
and J. Aronson, eds. (Blackwell Science Ltd.). Hobbs, R.J., Norton, D.A. 1996. Towards a conceptual framework for
RestorationEcology, in Restoration Ecology, Vol.4, No.2, pp.93-110. Kang, B. T., Wilson, G. T. & Lawson, T. L. 1981. Alley cropping of maize
(Zea mays L.) and Leucaena (Leucaena leucocephala Lam.) in southern
Nigeria. Plant Soil 63, 15. Lal, R. 2007. Soil science and the carbon civilization. Soil Science Society of
America Journal 71, 1425-1437. Leigh, P. 2005. Willey, R.W., Rao, M.R. & Natarajan, M. 1981. Traditional cropping
systems with pigeonpeas and their improvement. In Proceedings of the
International Workshop on Pigeonpeas, Volume 1. 15-19. December
1980, ICRISAT, India. REFERENCES The ecological crisis, the human condition, and commumity-
based restoration as an instrument for its cure, in Ethics in Science
and Environmental Politics(ESEP), 3-15. Palmer, M. A., Falk, D. A. & Zedler, J. B. 2006. Ecological theory and
restoration ecology. In Foundations of restoration ecology, D.A. Falk,
ed., Washington: Island Press. Sänger, H. & Jetschke, G. 2004. Are Assembly Rules Apparent in the
Regeneration of a Former Uranium Mining Site? In Assembly Rules
and Restoration Ecology: Bridging the gap between theory and Practice,
V.M. Temperton, R.J. Hobbs, T. Nuttle, and S. Halle, eds., Island
Press. Scheid, S., Gūnthardt-Goerg, Schulin, R. & Nowack, B. 2009. Accumulation and solubility of metals during leaf litter
decomposition an non-polluted and polluted soil. European Journal of
Soil Science 60, 9. Sheldrake, A.R. 1984. Pigeonpea. in Goldsworthy & Fisher, eds., The
Physiology of Tropical Field Crops, Wiley , London Sheldrake, A.R. & Narayanan, A. 1979. Growth, development & nutrient
uptake in pigeonpeas. Journal of Agricultural Science, Cambridge. 92:513-526. Veasey, E.A., Werner, J.C., Colozza, M.T., Freitas, J.C.T. de, Lucena,
M.A.C. de, Beisman, D.A. & Gerdes, L. 1999. Evaluation of
morphological, phenological and agronomic characters of
tropical forage legumes in relation to seed production. Boletim de
Industria Animal, 56, 109-125. Vinneras, B., Bjorklund, A. & Jonsson, H. 2003. Thermal composting of
faecal matter as treatment and possible disinfection method - 173 Chapter 5 – Discussion laboratory-scale and pilot-scale studies. Bioresource Technology 88, 47-
54. laboratory-scale and pilot-scale studies. Bioresource Technology 88, 47-
54. Walters, C.J., Holling, C.S. 1990. Large-scale management experiments and
‘learning by doing’, in Ecology, 71(6), pp.2060-2068. Walters, C.J., Holling, C.S. 1990. Large-scale management experiments and
‘learning by doing’, in Ecology, 71(6), pp.2060-2068. Willey, R.W., Rao, M.R. & Natarajan, M. 1981. Traditional cropping
systems with pigeonpeas and their improvement. In Proceedings of the
International Workshop on Pigeonpeas, Volume 1. 15-19. December
1980, ICRISAT, India. Willey, R.W., Rao, M.R. & Natarajan, M. 1981. Traditional cropping
systems with pigeonpeas and their improvement. In Proceedings of the
International Workshop on Pigeonpeas, Volume 1. 15-19. December
1980, ICRISAT, India. 174 174
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Shared care in the follow-up of early-stage melanoma: a qualitative study of Australian melanoma clinicians’ perspectives and models of care
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* Correspondence: lucie.rychetnik@sydney.edu.au
1Screening and Test Evaluation Program, School of Public Health, The
University of Sydney, Sydney, Australia
8School of Public Health, The University of Sydney, Edward Ford Building
(A27), Camperdown, NSW 2006, Australia
Full list of author information is available at the end of the article RESEARCH ARTICLE Open Access Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 Shared care in the follow-up of early-stage
melanoma: a qualitative study of Australian
melanoma clinicians’ perspectives and models
of care Lucie Rychetnik1,8*, Rachael L Morton2, Kirsten McCaffery1, John F Thompson3,4,5,6, Scott W Menzies3,7
and Les Irwig1 © 2012 Rychetnik et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Abstract Background: Patients with early stage melanoma have high survival rates but require long-term follow-up to detect
recurrences and/or new primary tumours. Shared care between melanoma specialists and general practitioners is an
increasingly important approach to meeting the needs of a growing population of melanoma survivors. Methods: In-depth qualitative study based on semi-structured interviews with 16 clinicians (surgical oncologists,
dermatologists and melanoma unit GPs) who conduct post-treatment follow-up at two of Australia’s largest
specialist referral melanoma treatment and diagnosis units. Interviews were recorded, transcribed and analysed to
identify approaches to shared care in follow-up, variations in practice, and explanations of these. Methods: In-depth qualitative study based on semi-structured interviews with 16 clinicians (surgical oncologists,
dermatologists and melanoma unit GPs) who conduct post-treatment follow-up at two of Australia’s largest
specialist referral melanoma treatment and diagnosis units. Interviews were recorded, transcribed and analysed to
identify approaches to shared care in follow-up, variations in practice, and explanations of these. Results: Melanoma unit clinicians utilised shared care in the follow-up of patients with early stage melanoma. Schedules were determined by patients’ clinical risk profiles. Final arrangements for delivery of those schedules (by
whom and where) were influenced by additional psychosocial, professional and organizational considerations. Four
models of shared care were described: (a) surgical oncologist alternating with dermatologist (in-house or local to
patient); (b) melanoma unit dermatologist and other local doctor (e.g. family physician); (c) surgical oncologist and
local doctor; or (d) melanoma physician and local doctor. noma unit clinicians utilised shared care in the follow-up of patients with early stage melanoma. Schedules were determined by patients’ clinical risk profiles. Final arrangements for delivery of those schedules (by
whom and where) were influenced by additional psychosocial, professional and organizational considerations. Four
models of shared care were described: (a) surgical oncologist alternating with dermatologist (in-house or local to
patient); (b) melanoma unit dermatologist and other local doctor (e.g. family physician); (c) surgical oncologist and
local doctor; or (d) melanoma physician and local doctor. Conclusions: These models of shared care offer alternative solutions to managing the requirements for long-term
follow-up of a growing number of patients with stage I/II melanoma, and warrant further comparative evaluation of
outcomes in clinical trials, with detailed cost/benefit analyses. Keywords: Melanoma, Follow-up, Shared care, Models of care Background number of ‘shared care’ approaches have been described,
including patients alternating follow-up visits between
the oncologist and their local GP [8-10], or attending
‘specialist’ or ‘shared care’ GPs [11-13]. Post-treatment follow-up is an important component of
cancer care [1]. In many countries there is growing de-
mand for oncology services and physician assistants and
nurse practitioners have evolved in response to this de-
mand [2-4]. There is also growing awareness of the im-
portant role of general practitioners, both in cancer
management and in post-treatment follow-up [5-7]. A Melanoma is a growing burden worldwide and the
fourth
most
common
cancer
in
Australia
[14-16]. Patients with AJCC stage I/II melanoma have high sur-
vival rates [17], but require long-term follow-up to de-
tect recurrences and/or new primary tumours [18,19]. Melanoma patients also experience significant anxiety
related to their disease [20]. Routine follow-up can pro-
vide reassurance but attending for follow-up can itself
be a source of anxiety, and a burden in terms of time, Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 Page 2 of 9 Table 1 Specialty and gender of study participants
Clinician characteristics (Total n= 16)
Specialty
Surgical Oncology
7
Dermatology
5
Primary Care, with focus on melanoma follow-up
4
Gender
Male
12
Female
4 Table 1 Specialty and gender of study participants Table 1 Specialty and gender of study participants
Clinician characteristics (Total n= 16)
Specialty
Surgical Oncology
7
Dermatology
5
Primary Care, with focus on melanoma follow-up
4
Gender
Male
12
Female
4 Table 1 Specialty and gender of study participants
Clinician characteristics (Total n= 16) travel and cost [21]. Sharing follow-up between melan-
oma specialists and local doctors is one solution to
meeting the care needs of a growing population of mel-
anoma survivors [22]. Some GPs may feel hesitant due
to concerns about skills and capacity [23,24], but trials
of GP-led care have been well received by GPs and
patients in the UK [13,23,25-27]. In Australia, oncology
specialists working in the field of breast cancer have also
reported high levels of willingness to share care with
other health professionals, but only 15% of their patients
attended GPs for post-treatment follow-up [28]. The
proportion of melanoma follow-up conducted as shared
care is currently unknown. And while GP and patient
experiences of shared care in melanoma follow-up have
been described [26,27], no studies have examined shared
care from the perspective of melanoma clinicians. Background The interviews were conducted by three researchers
(LR, RM or KM) and based on an interview schedule
(Appendix 1). This schedule was developed following a
systematic review of the literature [21] and consultations
with melanoma clinicians. The interviews, each lasting
30–60 minutes, were conducted face-to-face (n=12) or
by telephone (n=4) and all were recorded and tran-
scribed. Analysis was conducted as a group process in
which the researchers (LR, RM and KM) read all tran-
scripts and independently prepared analytical notes that
were discussed in regular meetings where the develop-
ment of key categories was revised and refined [30,31]. The practice of shared care in melanoma follow-up was
identified as an important category in the initial stages
of analysis, and the commonalities and variations in
practice, and potential explanations of these, were subse-
quently explored in the data [30,31]. Two participating
clinicians were invited to provide feedback on the valid-
ity of these findings. The purpose of this study was to examine specialist
melanoma clinicians’ perspectives on the provision of
post-treatment follow-up for patients with early stage
melanoma in order to understand and inform future re-
search on optimal models of care. In this paper we de-
scribe approaches to shared care in the follow-up of
patients with AJCC stage I/II melanoma among melan-
oma specialists (surgical oncologists, dermatologists)
and melanoma unit GPs (i.e. GPs based in a specialist
melanoma unit and trained in melanoma follow-up); and
outline four models of shared care as practiced in two of
Australia’s largest tertiary referral melanoma diagnostic
and treatment units. Melanoma specialists views on the
overall functions of follow-up and follow-up intervals
have been reported separately [29]. Shared responsibilities in melanoma follow-up Shared responsibilities in melanoma follow-up
Long term routine follow-up for stage I/II melanoma
was often conducted as a form of shared care in which
patients alternated between different clinicians at the
melanoma units and/or their local / referring GP or skin
cancer clinic.a The melanoma unit clinicians noted a
paucity of evidence on best practice in melanoma fol-
low-up, and therefore follow-up schedules were primar-
ily based on each patient’s expected risk of recurrence
and of developing a new primary tumour, as well as on
clinical guidelines [18]. A patient’s need for reassurance
or further education to reinforce skin self-examination
or sun-protective behaviours also influenced the recom-
mended frequency of visits - especially in the first two
years. Methods This qualitative study was conducted in collaboration
with two melanoma units in NSW, Australia. Melanoma
Institute Australia (MIA) is one of the largest melanoma
treatment units in the world and hosts the clinics of sur-
gical oncologists and dermatologists, as well as melan-
oma unit GPs who conduct follow-up in some of its
surgeons’ practices. The Sydney Melanoma Diagnostic
Centre (SMDC) provides dermatology services and long-
term monitoring of patients at high risk of primary mel-
anoma, including those with previously treated disease. All clinicians at these units involved in post-treatment
follow-up of patients with stage I/II melanoma were
invited to participate in an in-depth, semi-structured
interview about the nature and provision of follow-up
care. All those invited (n=17) consented but one inter-
view did not eventuate due to difficulties in finding a
suitable time and 16 interviews were completed. The
specialty and gender of participants are reported in
Table 1. The study was approved by the Sydney South
West Area Health Service Ethics Review Committee
(Protocol No X09-0364). There was variation in whether, and for how long, sur-
gical oncologists participated in long-term follow-up of
patients with stage I/II melanoma; and for those who
did, whether they themselves regularly conducted full
body skin examinations as part of follow-up visits. Some
surgical oncologists routinely referred patients with early
stage melanoma to other clinicians for long-term follow-
up e.g. to dermatologists at the melanoma unit (or if Page 3 of 9 Page 3 of 9 Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 Table 2 Factors considered in melanoma follow-up that determined the use of shared care-a summary of melanoma
clinicians’ perspective
‘CONTINUING CARE’ factors
Variables
‘COMMUNITY REFERRAL’ factors
Inclined melanoma unit clinicians towards specialist or ‘in-
house’ follow-up e.g. by surgeon or melanoma unit
dermatologist or melanoma GP
Inclined melanoma unit clinicians towards enabling follow-up
by community doctors e.g. dermatologist, local GP or skin
cancer clinic
▪Higher risk of recurrence or new primary disease (prior
melanoma, tumor thickness, ulceration, mitotic rate, family
history, skin type, number of moles etc)
Clinical
▪Lower risk of recurrence or new primary disease
▪Indications for extended post-surgical monitoring e.g. Methods pain,
hematomas, lymphodema, affected functioning
▪Patient request for ‘in-house’ follow-up by someone with
identified melanoma expertise
Patient
Psychosocial
▪Proximity and travel to unit pose significant burdens; potential
barrier for patient attending scheduled visits (live far away, have
poor mobility etc)
▪Patient allegiance to specialist with preference for attending
with them personally
▪Patient prefers follow-up with own family physician or local
referring doctor, or happy to participate in shared care
▪Patient very anxious; requires high emotional support and
reassurance
▪Patient organizes and coordinates follow-up with preferred
providers and follow-up consistent with recommended
schedule
▪Patient uncomfortable with referral to local doctor for
follow-up
▪Patient knowledgeable, confident and conscientious in
conducting skin self-examination
▪Patient lackadaisical about skin surveillance and needs
ongoing education and reinforcement of self examination
▪Patient lives close by or is able and willing to travel to unit for
appointments
▪Emphasis on specialisation in follow-up; ie specialist training
and/or location in melanoma unit to facilitate early detection of
disease 1
Melanoma
Clinician
▪Professionally comfortable with sharing follow-up with non-
specialist clinicians; especially when preferred by patient and/or
addresses other psychosocial needs
▪Sense of overall responsibility for ones patients; professional
obligation to provide ongoing care or oversee quality of skin
surveillance provided by others
▪Sense of obligation to expand capacity of one’s practice to
accommodate new melanoma patients
▪Value of health system efficiency and maximizing benefits for
greatest number of patients i.e. focusing specialist care for those
at greatest need / highest risk
▪Value of knowing patient well and patient-doctor rapport to
facilitate education, early diagnosis and treatment ie doctor is
familiar with patients’ skin, character, lifestyle, preferences; and
patient comfortable to ask questions or return if worried
▪Value of efficient care for individual patients i.e. Methods Other surgical oncologists re-
ferred to one of several melanoma unit GPs, who are co-
located in the melanoma unit facilities and to whom the
surgical oncologists provide specialist support. weighing factors in these two categories, the melanoma
unit clinicians determined whether follow-up was best
shared ‘in-house’ or with other clinicians – or indeed if
and when patients were referred for sole follow-up with
their local doctor. The important variables that impact on shared care
are further sub-divided as follows: patient clinical and
psychosocial
variables;
melanoma
clinician variables;
community doctor variables; and organizational (melan-
oma unit) variables. For example, patient characteristics
included: patients’ overall risk of recurrence and new
primary disease, patient preferences for follow-up, their
level of anxiety and need for information and reassur-
ance, and overall confidence in the alternative follow-up
options. The melanoma clinician variables included dif-
ferent perspectives regarding their own and other clini-
cians’ roles and responsibilities in long-term melanoma
follow-up. Community doctor variables related to the
availability and reliability (sometimes based on feedback
from patients) of the follow-up by the patient’s local or
referring doctor. Finally, organizational factors were also
identified by clinicians as important considerations in
follow-up, including the finite capacity of a specialist
melanoma unit to cope with ever-increasing numbers of
patients requiring follow-up, and its additional functions
in melanoma research (including
accurate database
maintenance). Surgical oncologists who preferred to retain greater,
ongoing personal involvement in patients’ follow-up usu-
ally opted for shared care in the form of alternating vis-
its with either a melanoma unit dermatologist or the
patient’s local doctor. Although surgical oncologists pre-
ferred to focus on the wound site and detecting recur-
rences, they also described conducting full-body skin
examinations if their patient had not received a compre-
hensive skin check elsewhere. Finally, surgical oncolo-
gists also described determining their own continued
involvement in follow-up by a weighing patient’s clinical
risks against logistic considerations. Thus they per-
formed skin checks for patients who lived in remote
country towns without access to a skin specialist, or
organized follow-up with a local doctor for those with
limited capacity for travel. The latter option was espe-
cially valued if the local doctor already had a good rela-
tionship with the melanoma unit and/or the patient and
expressed interest in contributing to their follow-up. Methods reducing
burdens of travel and cost of follow-up relative to clinical
returns for those with lowest risk of disease
▪Clinical interest in observing surgical and clinical outcomes
over the long-term; being able to personally monitor
developments
▪Enjoyment of psychosocial aspects of follow-up ie regular
contact with ‘well’ patients
▪Professional courtesy and goodwill towards referring doctor;
inclined to offer continued contribution to follow-up even if
specialist in-put not clinically necessary
▪Alternative follow-up with community doctor not available or
accessible to patient
Community
Doctor
▪Local doctor perceived to be knowledgeable, skilled and
competent in providing melanoma follow-up 1
▪Local doctor’s skills and interest in follow-up unknown;
specialist feels need to supervise follow-up more closely
▪Local doctor known to melanoma unit; eg has other
successful shared care arrangements with specialist clinicians
▪Patient has no or poor relationship with local doctors
▪Local doctor known to be interested and motivated to
conduct melanoma follow-up
▪Specialist or patient perceive local doctor not to have the
knowledge, skills, capacity or interest to conduct melanoma
follow-up
▪Patient has established good and trusting relationship with
local doctor
▪Value of research roles and responsibilities of specialist unit;
benefits of longitudinal data on patient outcomes
Organizational
(melanoma
unit)
▪Limited capacity of specialist melanoma unit clinicians
(surgical oncologists in particular) to provide long-term routine
skin surveillance for patients at low risk of recurrence or new
disease
▪Institutional benefits of constituency and support-base for a
specialist unit from maintaining ongoing relationships with
current and past patients follow-up that determined the use of shared care-a summary of melanoma ▪Professionally comfortable with sharing follow-up with non-
specialist clinicians; especially when preferred by patient and/or
addresses other psychosocial needs
▪Sense of obligation to expand capacity of one’s practice to
accommodate new melanoma patients
▪Value of health system efficiency and maximizing benefits for
greatest number of patients i.e. focusing specialist care for those
at greatest need / highest risk
▪Value of efficient care for individual patients i.e. reducing
burdens of travel and cost of follow-up relative to clinical
returns for those with lowest risk of disease Page 4 of 9 Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 Page 4 of 9 Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 available one local to the patient) or to the patient’s GP
or other referring doctor. Methods The melanoma unit GPs and dermatologists identified
long-term routine skin surveillance of patients with stage
I/II melanoma as their primary responsibility, however
they often also shared follow-up with other community-
based doctors and two main approaches to shared care
were identified. Some considered shared care as a way of
providing for the psychosocial needs of anxious patients
i.e. providing additional follow-up and reassurance for
those who wanted more regular skin checks than was
indicated by their risk profile. But others were more
comfortable to alternate the clinically recommended
schedule of skin checks with the patients’ local doctor. This partly depended on feedback from patients i.e. by
instructing patients on what to look for, melanoma clini-
cians felt able to trust the capacity of many of their
patients to make an assessment of skin examinations
received elsewhere. Four models of shared care The four main models of shared care for early stage mel-
anoma, as reported by the melanoma unit clinicians in
this study, are summarized in Figure 1. These models
were established through formal doctor-to-doctor refer-
ral or arranged by patients themselves. It is important to
note that while the models reflect the most commonly
discussed shared care arrangements, they do not reflect
all possible variations of practice, and are not mutually
exclusive. For example, shared care arrangements would
sometimes change due to a patient’s altered clinical or
social circumstances, or over time as the risk of recur-
rence decreased. Model (a) comprised alternating visits with the surgi-
cal oncologist and a dermatologist, who was either based
at the melanoma unit or local to the patient. This model
of shared care could continue for different lengths of
time (e.g. between 2 to 5 years post-treatment) after
which on-going skin surveillance would be fully trans-
ferred to the dermatologist. Alternatively, model (b)
comprised early handover by the surgical oncologists
(e.g. after 1 or 2 visits) for ongoing post-treatment fol-
low-up with a dermatologist. The dermatologist may
then subsequently also share follow-up care with the
patient’s own GP or skin cancer clinic who had originally
referred the patient. To conclude, recommended schedules for the follow-
up of individual patients were primarily determined by
their risk profile, but final arrangements for the delivery
of those schedules (i.e. by whom and where) were influ-
enced by many other considerations. Table 2 provides a
summary of the multiple and competing variables that
were reported by melanoma clinicians to influence the
practice of shared care. The factors listed under ‘Con-
tinuing Care’ inclined melanoma specialists to either
conduct follow-up themselves or refer to ‘in-house’ mel-
anoma unit GPs. ‘Community Referral’ factors inclined
melanoma unit clinicians to refer patients to attend
follow-up
with
their
local
or
referring
doctor. In Page 5 of 9 Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 Model (c) comprised alternating visits with a surgical
oncologist and the patient’s own local GP or skin cancer
clinic. The duration of the shared care arrangements
represented in model (c) was highly variable; some surgi-
cal oncologists reported alternating follow-up with the
local doctor for a few years only post-treatment, but
others described long-term or ongoing involvement in
follow-up (e.g. Four models of shared care for 10 or 15 years post-diagnosis), espe-
cially for those patients who specifically requested that
they continue to see them. (a) Surgical oncologist and dermatologist
Alternating visits with surgical oncologist and dermatologist (at melanoma unit or local to patient), with shared arrangement
ongoing for variable duration (e.g. 2-5 years). (b) Dermatologist and local GP
Dermatologist (at melanoma unit or local to patient) conducts ongoing follow-up; they may also share care with the patient’s
local / referring doctor (e.g. GP or skin cancer clinic). (c) Surgical oncologist and local GP
Alternating visits with surgical oncologist and patient’s local / referring doctor (e.g. GP or skin cancer clinic), with variable
duration of shared arrangements (from a few years to lifelong). (d) Melanoma unit GP and local GP
Surgical oncologist referral to ‘in-house’ melanoma unit GP for ongoing follow-up; the latter may then also share care with
patients local / referring doctor (e.g. family GP or skin cancer clinic)
Figure 1 Four described models of shared care. (a) Surgical oncologist and dermatologist ( )
g
g
g
Alternating visits with surgical oncologist and dermatologist (at melanoma unit or local to patient), with shared arrangement
ongoing for variable duration (e.g. 2-5 years). g
g
g
ing visits with surgical oncologist and dermatologist (at melanoma unit or local to patient), with shared arrangement
for variable duration (e.g. 2-5 years). (b) Dermatologist and local GP (b) Dermatologist and local GP
Dermatologist (at melanoma unit or local to patient) conducts ongoing follow-up; they may also share care with the patient’s
local / referring doctor (e.g. GP or skin cancer clinic). (c) Surgical oncologist and local GP
Alternating visits with surgical oncologist and patient’s local / referring doctor (e.g. GP or skin cancer clinic), with variable
duration of shared arrangements (from a few years to lifelong). (b) Dermatologist and local GP (b) Dermatologist and local GP (b) Dermatologist and local GP ( )
g
Dermatologist (at melanoma unit or local to patient) conducts ongoing follow-up; they may also share care with the patient’s
local / referring doctor (e.g. GP or skin cancer clinic). (c) Surgical oncologist and local GP (c) Surgical oncologist and local GP (c) Surgical oncologist and local GP (c) Surgical oncologist and local GP rgical oncologist and local GP
ting visits with surgical oncologist and patient’s local / referring doctor (e.g. Four models of shared care GP or skin cancer clinic), with variable
n of shared arrangements (from a few years to lifelong). ( )
g
g
Alternating visits with surgical oncologist and patient’s local / referring doctor (e.g. GP or skin cancer clinic), with variable
duration of shared arrangements (from a few years to lifelong). (d) Melanoma unit GP and local GP
Surgical oncologist referral to ‘in-house’ melanoma unit GP for ongoing follow-up; the latter may then also share care with
patients local / referring doctor (e.g. family GP or skin cancer clinic) (d) Melanoma unit GP and local GP ) Melanoma unit GP and local GP
gical oncologist referral to ‘in-house’ melanoma unit GP for ongoing follow-up; the latter may then also share care w
ents local / referring doctor (e.g. family GP or skin cancer clinic) Figure 1 Four described models of shared care. local doctor for a few years only post-treatment, but
others described long-term or ongoing involvement in
follow-up (e.g. for 10 or 15 years post-diagnosis), espe-
cially for those patients who specifically requested that
they continue to see them. Model (c) comprised alternating visits with a surgical
oncologist and the patient’s own local GP or skin cancer
clinic. The duration of the shared care arrangements
represented in model (c) was highly variable; some surgi-
cal oncologists reported alternating follow-up with the Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 Page 6 of 9 Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 Model (d) comprised early handover by the treating
surgeon (e.g. after 1 or 2 visits) for ongoing post-
treatment follow-up with one of the melanoma unit
GPs. Sometimes
the
preferred
arrangement
was
a
slightly longer period of alternating visits between the
two before the melanoma unit GP took over. For many
patients, particularly those for whom travel was a signifi-
cant burden, the melanoma unit GPs then also shared
subsequent follow-up visits with the patient’s local /re-
ferring dermatologist, GP or skin cancer clinic. Model
(d) enabled participating surgical oncologists and melan-
oma unit GPs to accommodate many of the ‘Continued
Care’ factors in Table 2, but within a framework that re-
lied on a less intensive use of specialists. the provision of post-treatment follow-up for a growing
population of melanoma survivors. Four models of shared care As described in the
study, melanoma specialists valued shared care in the
follow-up of patients with stage I/II melanoma to ac-
commodate the needs of their patients, and to manage
the finite capacity of specialised units to provide routine
skin surveillance. Patients’ clinical risk factors influenced
follow-up options, as well as psychosocial and logistic
considerations, such as patients’ anxiety or the distance
between home and the melanoma unit and the patients’
capacity to travel. We describe the nature and range of
factors that may be considered and weighed by melan-
oma specialists in their practice of shared care, and
present four models of shared care from two of Austra-
lia’s largest melanoma units. It should be acknowledged
however, that the perspectives reported in this study
may differ from those clinicians who provide post-
treatment melanoma follow-up in other settings, such as
dermatologists not affiliated with melanoma units, GPs
based in skin cancer clinics, or other GPs. Other melan-
oma units may also adopt alternative or additional mod-
els of shared care. While model (a) was described as providing the high-
est level of specialist care, it was also recognized as
highly resource intensive. The prolonged involvement of
a surgical oncologist was also perceived by some melan-
oma unit clinicians as more than absolutely required for
the majority of patients with stage I/II melanoma. As
outlined in models (a) and (c), however, some surgical
oncologists had ongoing involvement in follow-up, de-
scribing their sense of duty to continue to see patients
they had treated if that was what those patients particu-
larly requested. Shared care with local doctors relied on
less intensive use of melanoma units, and for those liv-
ing further away also reduced financial and travel
burdens. Many patients with AJCC stage I/II melanoma have a
greater risk of developing a new primary melanoma than
a recurrence [33]. In Australia the 10 year risk of mortal-
ity for melanomas with a Breslow thickness ≤1 mm is
less than half the 10 year risk of developing a second pri-
mary melanoma, but for tumours with a Breslow thick-
ness >1 mm the 10 year risk of mortality is substantially
greater [34,35]. Although responsibility for detecting re-
currence is not restricted to melanoma unit clinicians,
improved treatment options may incline melanoma units
more towards identifying options to provide continued
‘in-house’ follow-up for patients with thicker tumours. Four models of shared care The described model (d) of specialists referring follow-
up to melanoma unit GPs is a relatively new approach,
which expands the capacity of a melanoma unit to pro-
vide follow-up by GPs who have greater access to spe-
cialist support than is available to most community GPs. Although this model does not overcome the burdens of
time and travel that are reported by some patients as
barriers to adherence with schedules [21], this was partly
addressed by the melanoma unit GPs also sharing
follow-up with other community-based doctors. Model (d) required the least involvement of surgical
oncology and dermatology specialists. Yet views on this
arrangement varied, and a few specialists expressed un-
certainty about the appropriateness of routine skin sur-
veillance in a specialist tertiary referral unit being
undertaken by the melanoma unit GPs, rather than only
by surgical oncologists or dermatologists. Alternatively,
those supporting model (d) said that because patients
were being seen by GPs with an expressed interest and
in-house training in melanoma follow-up, and with
ready access to support from specialist colleagues, it pro-
vided better quality assurance for long term monitoring
than referrals elsewhere. Model (d) was also recognized
to provide accurate information for the unit’s database
of patient records, which supports longitudinal melan-
oma research. Patients also value GP participation in cancer fol-
low-up, particularly for navigating the health system
and providing information and psychosocial support
[27,36,37]. Other models of shared care reported else-
where have included community-based ‘shared care’
GPs who conduct follow-up for all cancers, including
melanoma [13], and local GP-led melanoma follow-up
supported by additional training and improved sys-
tems of referral to melanoma specialists when suspi-
cious lesions are found [25]. All forms of shared care Endnote 12. If there was good evidence that recurrence-
detection rates and clinical outcomes were not
affected by increasing the time interval between
follow-up appointments (say from 6monthly to
yearly) what other factors would you and your
patients want to consider when reviewing intervals
between appointments? aAustralia has a growing number of skin cancer clinics
which are mostly staffed by general practitioners. They
offer skin examinations and skin cancer diagnostic and
treatment services, as well as referrals to specialists and
specialist melanoma units Discussion Australia has one of the highest rates of cutaneous mel-
anoma in the world, with a growing population of
patients requiring long-term post-treatment follow-up
[14,18,32]. The majority of melanoma specialists are
based in metropolitan areas and there is increasing de-
mand for their services. This qualitative study offers
valuable insights into the practice of shared care by mel-
anoma unit clinicians, which is an important aspect of Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 Page 7 of 9 Page 7 of 9 similarities and differences between SMDC and the
MIA? similarities and differences between SMDC and the
MIA? rely on clarity about roles and responsibilities of the
clinicians, and these must also be understood by
patients themselves [38]. Australian patient perspec-
tives on the benefits and challenges of long term
follow-up of stage I/II melanoma have also been
reported [39]. Melanoma follow-up requires long-term
commitment to regular skin surveillance and many
patients may benefit from increased opportunities to
plan in advance how this can be achieved. Explicit
discussion of alternative options for post-treatment
follow-up could become a formal part of patients’
considerations of treatment referral options. Printed
patient information describing all available models for
their follow-up, including who would conduct each
aspect, qualifications and training of the practitioners,
and any associated costs, could greatly assist this
process. Conclusion 9. From your experience, what would you say are the
main things the patients want from coming to see
you for regular follow-up? Melanoma clinicians consider many competing factors
when they determine an appropriate model of follow-
up care for their patients. A number of alternative
models of shared care have been described, which rely
on
different
levels
of
contribution
from
surgical
oncologists, dermatologists and general practitioners. These models of shared care offer solutions to man-
aging the requirements for long-term follow-up of a
growing number of patients with AJCC stage I/II mel-
anoma, and warrant further comparative evaluation of
outcomes in clinical trials, with detailed cost/benefit
analyses. 10. Do patients ever talk about how they felt before or
after their appointment, how they are feeling while
they are here? A.2. Clinicians own needs, experiences and motivations
for follow-up 5. What about yourself – what would you say are some
of the main benefits for you personally as a
practitioner from having the opportunity to regularly
see your follow-up patients? 6. How does it compare to some of the other clinical
work that you do in terms of job satisfaction or
enjoyment? j
7. Do you ever worry about missing a melanoma? 7. Do you ever worry about missing a melano 8. Do you think there are any medico-legal
implications? A.4. Factors influencing follow-up intervals and any
implications for change 11. In terms of the time interval between appointments
– how do you determine how often a patient should
attend? References 24. Dancey A, Rayatt S, Courthold J, Roberts J: Views of UK melanoma patients
on routine follow-up care. Br J Plast Surg 2005, 58(2):245–250. 1. Hewitt M, Greenfield S, Stovall E: From Cancer Patient to Cancer Survivor; Lost
in Transition. (Committee on Cancer Survivorship: Improving Care and Quality
of Life. National Cancer Policy Board). Washington DC: The National
Academies Press; 2005. 25. Murchie P, Nicolson MC, Hannaford PC, Raja EA, Lee AJ, Campbell NC:
Patient satisfaction with GP-led melanoma follow-up: a randomised
controlled trial. Br J Cancer 2010, 102(10):1447–1455. 2. James ND, Guerrero D, Brada M: Who should follow up cancer patients? Nurse specialist based outpatient care and the introduction of a phone
clinic system. Clin Oncol 1994, 6(5):283–287. 2. James ND, Guerrero D, Brada M: Who should follow up cancer patients? Nurse specialist based outpatient care and the introduction of a phone
clinic system. Clin Oncol 1994, 6(5):283–287. 26. Murchie P, Delaney EK, Campbell NC, Hannaford PC: GP-led melanoma
follow-up: the practical experience of GPs. Fam Pract 2009,
26(4):317–324. 3. Leshin B, Hauser D: The role of a physician assistant in dermatologic
surgery. Dermatol Surg 1999, 25(2):148–150. 3. Leshin B, Hauser D: The role of a physician assistant in dermatologic
surgery. Dermatol Surg 1999, 25(2):148–150. 27. Murchie P, Delaney EK, Campbell NC, Hannaford PC: GP-led melanoma
follow-up: views and feelings of patient recipients. Support Care Cancer
2010, 18(2):225–233. 4. Ross AC, Polansky MN, Parker PA, Palmer JL: Understanding the role of
physician assistants in oncology. J Oncol Pharm Pract 2010, 6(1):26–30. 4. Ross AC, Polansky MN, Parker PA, Palmer JL: Understanding the role of
physician assistants in oncology. J Oncol Pharm Pract 2010, 6(1):26–30. 28. Brennan ME, Butow P, Spillane AJ, Boyle FM: Survivorship care after
breast cancer: follow-up practices of Australian health professionals
and attitudes to a survivorship care plan. Asia Pac J Clin Oncol 2010,
6(2):116–125. 5. Weller DP, Harris MF: Cancer care: what role for the general practitioner? Med J Aust 2008, 189(2):59–60. 5. Weller DP, Harris MF: Cancer care: what role for the general practitioner? Med J Aust 2008, 189(2):59–60. 6. Emery JD: Cancer care: what role for the general practitioner? Med J Aust
2008, 189(9):535. 6. Emery JD: Cancer care: what role for the general practitioner? Med J Aust
2008, 189(9):535. 29. A.5. Monitoring undertaken by others 13. Are there any aspects of follow-up care that are
currently being done by others at SMDC or in the
future could be done just as well or more efficiently
for your patients? 1. If we could start quite broadly, what would you say
are your main aims in undertaking regular follow-up
and monitoring for patients who have had a stage 1
or 2 primary melanoma? 14. Do you ever refer patients for follow up or skin
checks with a local GP or a dermatologist other
than at SMDC? 14. Do you ever refer patients for follow up or skin
checks with a local GP or a dermatologist other
than at SMDC? 2. What do you see as your main responsibility in
relation to follow-up and monitoring for your
melanoma patients? 15. What are your thoughts on other possible benefits
from following up patients over the long term, such
as research benefits? 3. What would you consider to be more within the
scope / role / responsibility of other clinicians? 16. Do you refer any of your follow-up patients for skin
photography? 4. In terms of follow-up of people with stage 1 or 2
melanoma, what would you say are some of the Page 8 of 9 Page 8 of 9 Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 A.6. Patient education 12. Hickman M, Drummond N, Grimshaw J: A taxonomy of shared care for
chronic disease. J Public Health Med 1994, 16(4):447–454. 13. Hall SJ, Samuel LM, Murchie P: Toward shared care for people with
cancer: developing the model with patients and GPs. Fam Pract 2011,
0:1–11. 17. Are you involved in follow-up patient education? What types of patient education do you do? 18. Is there anything else that you would like to see
included in the follow-up or monitoring process? 17. Are you involved in follow-up patient education? What types of patient education do you do? 17. Are you involved in follow-up patient education? What types of patient education do you do? 14. Giblin A, Thomas J: Incidence, mortality and survival in cutaneous
melanoma. J Plast Reconstr Aesthet Surg 2007, 60:32–40. 18. Is there anything else that you would like to see
included in the follow-up or monitoring process? 18. Is there anything else that you would like to see
included in the follow-up or monitoring process? 15. Australian Institute of Health and Welfare: Australia's Health 2012. In
Australia's health no 13 Cat no AUS 156. Canberra: AIHW; 2012. References Rychetnik L, McCaffery K, Morton RL, Thompson JF, Menzies SW, Irwig L:
Follow-up of early stage melanoma: Specialist clinician perspectives on
the functions of follow-up and implications for extending follow-up
intervals. J Surg Oncol 2012, doi:10.1002/jso.23278. Published ahead of print
on 22 Oct. 7. Brennan M, Jefford M: General practitioner-based models of post-
treatment follow up. In Cancer Forum. 33rd edition: Cancer Council
Australia; 2009. http://www.cancerforum.org.au/Issues/2009/November/
Forum/General_practitionerbased_models_of_posttreatment_follow_up. htm. 7. Brennan M, Jefford M: General practitioner-based models of post-
treatment follow up. In Cancer Forum. 33rd edition: Cancer Council
Australia; 2009. http://www.cancerforum.org.au/Issues/2009/November/
Forum/General_practitionerbased_models_of_posttreatment_follow_up. htm. 30. Charmaz K: Constructing Grounded Theory: a practical guide through
qualitative analysis. London: Sage Publications; 2006. 8. Oeffinger KC, McCabe MS, Oeffinger KC, McCabe MS: Models for delivering
i
hi
J Cli O
l 2006 24(32) 5117 5124 8. Oeffinger KC, McCabe MS, Oeffinger KC, McCabe MS: Models for delivering
survivorship care. J Clin Oncol 2006, 24(32):5117–5124. 8. Oeffinger KC, McCabe MS, Oeffinger KC, McCabe MS: Models for delivering
survivorship care. J Clin Oncol 2006, 24(32):5117–5124. 9. Australian Cancer Survivorship Centre: Models for Survivorship Care. 2010, 8. Oeffinger KC, McCabe MS, Oeffinger KC, McCabe MS: Models for delivering
survivorship care. J Clin Oncol 2006, 24(32):5117–5124. 9. Australian Cancer Survivorship Centre: Models for Survivorship Care. 2010,
http://www.petermac.org/cancersurvivorship/DesignofFollow-upCare. 31. Pope C, Mays N: Reaching the parts other methods cannot reach - an
introduction to qualitative methods in health and health-services
research. Br Med J 1995, 311(6996):42–45. 9. Australian Cancer Survivorship Centre: Models for Survivorship Care. 2010,
http://www.petermac.org/cancersurvivorship/DesignofFollow-upCare. 10. Malinowski T, Adams P: Shared Care Models; A high-level literature
review. In Discussion paper. Sydney: Cancer Institute NSW; Feb 2009. http://
www.cancerinstitute.org.au/incite/issue-1/shared-care-models-a-high-level-
literature-review. 32. Australian Institute for Health and Welfare: Cancer in Australia 2010: an
overview. In Cancer series no 60 Cat no CAN 56. Canberra: AIHW; 2010. 33. Turner RM, Bell KJL, Morton R, Hayen A, Brecht Francken A, Howard K,
Armstrong B, Thompson JF, Irwig L: Optimizing the frequency of follow
up visits for patients treated for localised primary cutanous melanoma. J Clin Oncol 2011, 29(35):4641–4646. 11. Chomik T: A Report on Shared Care (Part of the Primary Health Care Shared
Care Network Development Initiative). Province of British Columbia: Report
prepared for Provincial Health Services Authority; 2005. http://www.phsa.ca/
NR/rdonlyres/6A84F609-CCAA-40DC-BD62-B2FAC7BE2356/0/
SharedCareReport2005.pdf. 11. Chomik T: A Report on Shared Care (Part of the Primary Health Care Shared
Care Network Development Initiative). Acknowledgements
f This study was funded by a Program Grant (nos. 402764 and 633033) from
the Australian National Health and Medical Research Council. This study was funded by a Program Grant (nos. 402764 and 633033) from
the Australian National Health and Medical Research Council. 19. Francken AB, Bastiaannet E, Hoekstra HJ: Follow-up in patients with
localised primary cutaneous melanoma. Lancet Oncol 2005, 6(8):608–621. 20. Kasparian NA, McLoone JK, Butow PN, Kasparian NA, McLoone JK, Butow
PN: Psychological responses and coping strategies among patients with
malignant melanoma: a systematic review of the literature. Arch Dermatol
2009, 145(12):1415–1427. Authors’ contributions 17. Balch CM, Gershenwald JE, Soong SJ, Thompson JF, Atkins MB, Byrd DR,
Buzaid AC, Cochran AJ, Coit DG, Ding S, et al: Final version of 2009 AJCC
melanoma staging and classification. J Clin Oncol 2009, 27(36):6199–6206. All authors contributed to the conception and design of the study and
acquisition of the data. LR, RM and KM collaborated on the analysis and
interpretation of data, with substantive contributions from LI, JT and SM. LR
drafted and revised the manuscript, with all other authors providing critical
feedback on important intellectual content resulting in substantial revisions. All authors read and approved the final manuscript. 18. Australian Cancer Network Melanoma Guidelines Revision Working Party:
In Clinical Practice Guidelines for the Management of Melanoma in Australia
and New Zealand (Follow-up, Chapter 19). Edited by National Health and
Medical Research Council. Wellington: Cancer Council Australia and
Australian Cancer Network, Sydney and New Zealand Guidelines Group;
2008:121–126. Competing interests 16. Siegel R, Ward E, Brawley O, Jemal A: Cancer statistics, 2011: the impact of
eliminating socioeconomic and racial disparities on premature cancer
deaths. CA Cancer J Clin 2011, 61(4):212–236. The authors have no financial, commercial or other competing interests. The authors have no financial, commercial or other competing interests. Author details
1 1Screening and Test Evaluation Program, School of Public Health, The
University of Sydney, Sydney, Australia. 2School of Public Health, The
University of Sydney, Sydney, Australia. 3Sydney Medical School, The
University of Sydney, Sydney, Australia. 4Melanoma Institute Australia
(formerly Sydney Melanoma Unit), Sydney, Australia. 5The Mater Hospital,
North Sydney, Australia. 6Royal Prince Alfred Hospital, Camperdown, Australia. 7Sydney Melanoma Diagnostic Centre, Royal Prince Alfred Hospital, Sydney,
Australia. 8School of Public Health, The University of Sydney, Edward Ford
Building (A27), Camperdown, NSW 2006, Australia. 21. Rychetnik L, McCaffery K, Morton RL, Irwig L: Psychosocial aspects of
routine follow-up for stage I/II melanoma: a systematic review of the
literature. Psychooncology 2012, doi:10.1002/pon.3060. Published ahead of
print on 12 March. 22. Sondak VK, Leachman SA: Individualizing follow-up for patients with
early-stage melanoma. J Clin Oncol 2011, 29(35):4606–4608. 23. Murchie P, Hannaford PC, Wyke S, Nicolson MC, Campbell NC: Designing
an integrated follow-up programme for people treated for cutaneous
malignant melanoma: a practical application of the MRC framework for
the design and evaluation of complex interventions to improve health. Fam Pract 2007, 24(3):283–292. Received: 6 June 2012 Accepted: 11 December 2012
Published: 19 December 2012 Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 References Province of British Columbia: Report
prepared for Provincial Health Services Authority; 2005. http://www.phsa.ca/
NR/rdonlyres/6A84F609-CCAA-40DC-BD62-B2FAC7BE2356/0/
SharedCareReport2005.pdf. 34. Doubrovsky A, Menzies SW: Enhanced survival in patients with multiple
primary melanoma. Arch Dermatol 2003, 139(8):1013–1018. Page 9 of 9 Page 9 of 9 Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 Rychetnik et al. BMC Health Services Research 2012, 12:468
http://www.biomedcentral.com/1472-6963/12/468 35. Pollack LA, Li J, Berkowitz Z, Weir HK, Wu XC, Ajani UA, Ekwueme DU, Li CY,
Pollack BP: Melanoma survival in the United States, 1992 to 2005. J Am
Acad Dermatol 2011, 65(5):S78–S86. 36. Kendall M, Boyd K, Campbell C, Cormie P, Fife S, Thomas K, Weller D, Murray
SA: How do people with cancer wish to be cared for in primary care? Serial discussion groups of patients and carers. Fam Pract 2006,
23(6):644–650. 37. Brennan M, Butow P, Spillane AJ, Marven M, Boyle FM: Follow up after
breast cancer Views of Australian women. Aust Fam Physician 2011,
40(5):311–316. 38. Cheung WY, Neville BA, Cameron DB, Cook EF, Earle CC: Comparisons of
patient and physician expectations for cancer survivorship care. J Clin
Oncol 2009, 27(15):2489–2495. 39. Morton RL, Rychetnik L, McCaffery K, Thompson JF, Irwig L: Patients’
perspectives of long-term follow-up for localised cutaneous melanoma. Eur J Surg Oncol 2012, in press, accepted 18 Dec 2012. doi:10.1186/1472-6963-12-468
Cite this article as: Rychetnik et al.: Shared care in the follow-up of early-
stage melanoma: a qualitative study of Australian melanoma clinicians’
perspectives and models of care. BMC Health Services Research 2012
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Dosen Tetap Prodi Pendidikan Bahasa Inggris PENGARUH PENGGUNAAN STRATEGI QUANTUM LEARNING
TERHADAP KEMAMPUAN SISWA DALAM SPEAKING Resty Wahyuni
Surel : Resty_shenie@yahoo.com ABSTRAK Penelitian ini bertujuan untuk mendeskripsikan kemampuan mahasiswa
dalam hal keterampilan berbicara dengan menggunakan Strategi Quantum
Learning. Dari hasil penelitian dapat dilihat dari 72 orang siswa, dalam
mendeskripsikan hasil penelitian ini, peneliti membagi dalam 3 tahap yaitu:
Deskripsi hasil perhitungan kemampuan berbicara mahasiswa fakultas keguruan
dan ilmu pendidikan jurusan bahasa Inggris tanpa strategi Quantum Learning. Deskripsi hasil perhitungan kemampuan berbicara mahasiswa fakultas keguruan
dan ilmu pendidikan jurusan bahasa Inggris dengan menggunakan strategi
Quantum Learning. Deskripsi hasil hubungan antara pengaruh penggunaan
strategi Quantum Learning terhadap kemampuan berbicara mahasiswa FKIP
jurusan bahasa Inggris. Kata Kunci: Pengaruh, Pembelajaran, Strategi, Prestasi, Berbicara, Quantum METODE PENELITIAN dikembangkan di dalam kelas. Di
dalam
mata
kuliah
speaking,
mahasiswa diharapkan untuk dapat
menikmati materi yang diberikan
langsung kepada mereka. Seseorang
yang dikatakan mampu berbahasa
inggris yang aktif apabila ia memiliki
keempat skill dalam bahasa. Penelitian ini dilakukan di
Universitas
Muhammadiyah
Sumatera Utara Fakultas Keguruan
dan Ilmu Pendidikan Semester IV
tahun akademik 2016 – 2017, Jalan
Kapten
Muchtar
Basri,
Medan. Populasi dari penelitian ini adalah
mahasiswa Fakultas Keguruan dan
Ilmu Pendidikan Jurusan Bahasa
Inggris Tahun Pelajaran 2016 – 2017
yang berjumlah 286 orang. Sampel
adalah sebagian dari populasi yang
memiliki karakteristik dasar yang
dapat mewakili populasi tersebut. Dalam pengambilan sampel, peneliti
didasarkan atas pendapat Arikunto
(1993 : 107) yang mengatakan
“Apabila jumlah sampel lebih dari
100 orang maka diambil persentase
10 – 15 % atau 20 – 25%. Karena
jumlah populasi 286 orang, maka
penulis
menetapkan
sampel
berjumlah 72 orang yang terdiri dari
dua kelas. Orang – orang belajar bahasa
asing dengan berbagai alasan, dan
salah
satu
diantaranya
adalah
komunikasi. Alasan lain, karena
keberagaman
manusia
di
dunia
seperti
perbedaan
budaya
dan
bahasa. Setiap orang harus saling
mengetahui satu sama lainnya, hal
ini dapat terjadi dengan adanya
komunikasi. Orang – orang belajar bahasa
asing dengan berbagai alasan, dan
salah
satu
diantaranya
adalah
komunikasi. Alasan lain, karena
keberagaman
manusia
di
dunia
seperti
perbedaan
budaya
dan
bahasa. Setiap orang harus saling
mengetahui satu sama lainnya, hal
ini dapat terjadi dengan adanya
komunikasi. Di
zaman
modern,
komunikasi sebagai bahasa kedua
memiliki peranan yang penting. Semua hal menggunakan bahasa
inggris sebagai petunjuknya, seperti
teknologi,
ekonomi,
bahkan
pendidikan. Banyak cara yang dapat
dilakukan
untuk
meningkatkan
kemampuan siswa berbicara bahasa
inggris anatara lain dengan cara:
debat, diskusi, percakapan, story
telling. Di
zaman
modern,
komunikasi sebagai bahasa kedua
memiliki peranan yang penting. Semua hal menggunakan bahasa
inggris sebagai petunjuknya, seperti
teknologi,
ekonomi,
bahkan
pendidikan. Banyak cara yang dapat
dilakukan
untuk
meningkatkan
kemampuan siswa berbicara bahasa
inggris anatara lain dengan cara:
debat, diskusi, percakapan, story
telling. PENDAHULUAN mengajar. Selain itu dosen dan guru
juga sering menggunakan metode
konvensional, hal ini membuat siswa
bosan dan tidak berminat belajar. Akibatnya hasil pembelajaran tidak
tercapai. Salah satu contoh masalah
yang dihadapi peneliti yaitu metode
atau strategi yang tepat diterapkan di
perguruan
tinggi
dalam
mengembangkan
minat
belajar
mahasiswa dalam speaking. Keterampilan berbahasa ada
empat
yang
setiap
saat
dikembangkan dari tingkat Sekolah
Dasar hingga tingkat yang lebih
tinggi
yaitu tingkat
Universitas. Keterampilan
tersebut
yaitu
keterampilan membaca, berbicara,
menyimak
dan
keterampilan
menulis. Ketika
menerapkan
keterampilan tersebut, guru atau
dosen menggunakan model, metode,
strategi atau teknik yang berbeda. Keterampilan
berbicara
sebagai salah satu aspek sangat
penting dalam berkomunikasi secara
lisan, harus dikuasai betul – betul
oleh para siswa karena jika kurang
terampil berbicara, maka komunikasi
tidak akan berjalan dengan lancar. Berbicara
dalam
bahasa
inggris
berbeda dengan berbicara dalam
bahasa indonesia karena bahasa
inggris adalah bahasa asing. Persoalan
menggunakan
metode,
model
atau
strategi
pembelajaran
yang tepat
adalah
persoalan yang sering dibahas para
guru, dosen atau calon guru, karena
metode adalah salah satu komponen
yang penting dalam menciptakan
proses pembelajaran dikelas. Dalam
proses belajar mengajar sering dosen
dan guru mendapat kesulitan mencari
metode atau model yang tepat untuk
disajikan
pada
kegiatan
belajar Speaking
merupakan
kemampuan yang paling penting
dalam
bahasa
inggris
untuk Speaking
merupakan
kemampuan yang paling penting
dalam
bahasa
inggris
untuk 419 Resty Wahyuni : Pengaruh Penggunaan Strategi ... Prosedur Penelitian Surakhmad (1982 : 131)
menyatakan
“metode
merupakan
cara yang utama untuk mencapai
tujuan, misalnya untuk menguji
serangkaian dengan mempergunakan
teknik, serta alat – alat tertentu. Cara
utama
itu
digunakan
setelah
penyelidikkan
serta
situasi
penyelidikkan”. Berdasarkan
penjelasan
diatas
untuk
meningkatan
keterampilan
berbahasa,
peneliti
tertarik
membahas
salah
satu
masalah
pengembangan
strategi
pembelajaran speaking di Perguruan
Tinggi. Dalam
hal
ini
yang
mendorong
peneliti
berminat
meneliti tentang inovasi strategi
pembelajaran speaking. Sesuai
dengan
tujuan
penelitian
maka
metode
yang
digunakan
adalah
metode
eksperimen yaitu metide penelitian
yang dilakukan dengan mengadakan
pengamatan secara teliti terhadap p-ISSN : 2355-1720
e-ISSN`: 2407-4926 420 SEJ VOLUME 7 NO. 4 DESEMBER 2017 objek penelitian serta adanya kontrol
terhadap objek penelitian tersebut. Variabel kontrol maksutnya adalah
variabel yang digunakan sebagai
bandingan terhadap variabel yang
diteliti, yaitu hasil tes kemampuan
berbicara
mahasiswa
dengan
menggunkan
startegi
Quantum
learning . tidak
terhadap
dua
kelompok
perlakuan. Uji homogenitas dihitung
dengan menggunakan rumus fisher,
setelah
dilakukan
perhitungan
normalitas dan homogenitas maka
dilakukan
analisis
data
untuk
menguji
hipotesis
yang
telah
diajukan, uji ini dilakukan untuk
mengetahui ada tidaknya perbedaan
yang signifikan antara mahasiswa
yang dberi perlakuan teknik jigsaw
dengan perlakuan teknik STAD. Didalam
penelitian,
eksperimen
sampel
dibagi
dua
kelompok yaitu kelompok kelas
eksperimen dan kelompok kelas
kontrol. Pada kelas eksperimen akan
diajarkan
speaking
dengan
menggunakan
strategi
Quantum
learning
didalam
proses
pembelajran. Sebelum
proses
pembelajaran
berlangsung
kedua
kelompok ini akan diberi pre – test
mempraktikkan
percakapan
(speaking). Pre test dilakukan untuk
mengukur
kemampuan
siswa
sebelum diberikan post – test agar
dapat melihat hasil kemampuan
siswa yang telah diberi treatment. Teknik
yang
digunakan
dalam menganalisis data penelitian
ini adalah teknik
analisis data
deskriptif dan inferensial. Statistic
inferensial
diarahkan
untuk
uji
hipotesis. Rumus yang digunakan
adalah Uji t. PEMBAHASAN Sebagaimana
telah
dikemukukakan pada bagian metode
penelitian,
bahwa
alat
yang
digunakan
dalam
mengumpulkan
data adalah melalui tes. Dalam
mendeskripsikan hasil penelitian ini,
peneliti membaginya kedalam tiga
tahapan pengelolaan data, yaitu : Teknik Analisa Data Setelah data – data diperoleh
maka sebelumnya terlebih dahulu
dilakukan uji prasyaratan analisis
yaitu
uji
normalitas
dan
uji
homogenitas. Uji normalitas data
dilakukan untuk mengetahui apakah
data yang diperoleh dari populasi
yang berdistribusi normal atau tidak
perhitungan dengan menggunakan
rumus lilifors. Dan uji homogenitas
dilakukan untuk menguji variasi dari
populasi homogen, uji normalitas
dilakukan untuk mengetahui apakah
data yang diperoleh homogen atau a. Deskripsi
hasil
perhitungan
kemampuan
berbicara
mahasiswa fakultas keguruan
dan ilmu pendidikan
jurusan bahasa Inggris TA
2016 – 2017 tanpa strategi
Quantum learning. jurusan bahasa Inggris TA
2016 – 2017 tanpa strategi
Quantum learning. Deskripsi
hasil
perhitungan
kemampuan
berbicara
mahasiswa fakultas keguruan
dan ilmu pendidikan jurusan
bahasa Inggris TA 2016 – 2017 p-ISSN : 2355-1720
e-ISSN`: 2407-4926 421 Resty Wahyuni : Pengaruh Penggunaan Strategi ... dengan menggunakan strategi
Quantum learning. dengan menggunakan strategi
Quantum learning. Darwis,
dkk. 2011. Terampil
Berbahasa. Bandung: Alfabeta. c. Deskripsi
hasil
hubungan
antara pengaruh penggunaan
strategi
Quantum
Learning
terhadap kemampuan berbicara
mahasiswa
FKIP
jurusan
bahasa Inggris TA 2016 – 2017 De Porter, Bobbie, Mark Readrdon,
and Sarah Singer Nourine. 2004. Quantum
Teaching. Mempraktikkan
Quantum
Learning di Ruang – ruang
Kelas. Bandung: Kaifa. De
Porter,
Bobbie
and
Mike
Hernacki. 2003. Quantum
Learning: Membiasakan Belajar
Nyaman dan Menyenangkan. Bandung : Kaifa. SIMPULAN Berdasarkan hasil penelitian
tentang
penggunaan
strategi
Quantum Learning Freeman, Dianne, Larsen. 2002. Techniques and principle in
language teaching. Oxford :
Oxford University Press. - Mengetahui
perbandingan
peningkatan
hasil
belajar
mahasiswa
yang
menggunakan
Quantum
learning strategi dengan hasil
belajar mahasiswa yang tidak
menggunakan
strategi
Quantum
Learning
tahun
ajaran 2016 – 2017 Harmer, Jeremy. 2003. The practice
of English Language Teaching. Londodn : Longman. Slamet, S. Y. 2008. Dasar-dasar
Pembelajaran
Bahasa
dan
Sastra Indonesia di Sekolah
Dasar. Surakarta: UNS Press. Quantum
Learning
tahun
ajaran 2016 – 2017 Mengetahui
perbedaan
pencapaian
hasil
belajar
mahasiswa
yang
menggunakan
Quantum
learning strategi dengan hasil
belajar mahasiswa yang tidak
menggunakan
strategi
Quantum
Learning
tahun
ajaran 2016 – 2017. Solehan,
T. W,
dkk. 2008. Pendidikan Bahasa Indonesia di
SD. Jakarta: Universitas Terbuka Solehan,
T. W,
dkk. 2008. Pendidikan Bahasa Indonesia di
SD. Jakarta: Universitas Terbuka
Sudjono,
A. 2010. Pengantar
Statistik
Pendidikan. Jakarta:
RajaGrafindo Persada. Sudjono,
A. 2010. Pengantar
Statistik
Pendidikan. Jakarta:
RajaGrafindo Persada. Syah, M. 2008. Psikologi Pendidikan
dengan
Pendekatan
Baru. Bandung: Remaja Rosdakarya. DAFTAR RUJUKAN
Arikunto,
S. 2010. Prosedur
Penelitian. Jakarta:
Rineka
Cipta. DAFTAR RUJUKAN p-ISSN : 2355-1720
e-ISSN`: 2407-4926 422
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https://openalex.org/W3015203085
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https://europepmc.org/articles/pmc7323539?pdf=render
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English
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Severe Acute Respiratory Syndrome Coronavirus 2 Infection among Returnees to Japan from Wuhan, China, 2020
|
Emerging infectious diseases
| 2,020
|
cc-by
| 3,266
|
Author affiliations: National Institute of Infectious Diseases,
Tokyo, Japan (Y. Arima, T. Shimada, M. Suzuki, T. Suzuki,
Y. Kobayashi, Y. Tsuchihashi, H. Nakamura, K. Matsumoto,
A. Takeda, K. Kadokura, T. Sato, Y. Yahata, N. Nakajima,
M. Tobiume, I. Takayama, T. Kageyama, S. Saito, N. Nao,
T. Matsui, T. Sunagawa, H. Hasegawa, M. Ohnishi, T. Wakita);
National Center for Global Health and Medicine, Tokyo
(S. Kutsuna, K. Hayakawa, S. Tsuzuki, Y. Asai, T. Suzuki, S. Ide,
K. Nakamura, Y. Moriyama, N. Kinoshita, Y. Akiyama, Y. Miyazato,
H. Nomoto, T. Nakamoto, M. Ota, S. Saito, M. Ishikane,
S. Morioka, K. Yamamoto, M. Ujiie, M. Terada, H. Sugiyama,
N. Kokudo, N. Ohmagari) DISPATCHES DISPATCHES Severe Acute Respiratory
Syndrome Coronavirus 2 Infection
among Returnees to Japan
from Wuhan, China, 2020 Yuzo Arima,1 Satoshi Kutsuna,1 Tomoe Shimada, Motoi Suzuki, Tadaki Suzuki, Yusuke Kobayashi,
Yuuki Tsuchihashi, Haruna Nakamura, Kaoru Matsumoto, Asuka Takeda, Keisuke Kadokura, Tetsuro Sato,
Yuichiro Yahata, Noriko Nakajima, Minoru Tobiume, Ikuyo Takayama, Tsutomu Kageyama, Shinji Saito,
Naganori Nao, Tamano Matsui, Tomimasa Sunagawa, Hideki Hasegawa, Kayoko Hayakawa,
Shinya Tsuzuki, Yusuke Asai, Tetsuya Suzuki, Satoshi Ide, Keiji Nakamura, Yuki Moriyama, Noriko Kinoshita,
Yutaro Akiyama, Yusuke Miyazato, Hidetoshi Nomoto, Takato Nakamoto, Masayuki Ota, Sho Saito,
Masahiro Ishikane, Shinichiro Morioka, Kei Yamamoto, Mugen Ujiie, Mari Terada, Haruhito Sugiyama,
Norihiro Kokudo, Norio Ohmagari, Makoto Ohnishi, Takaji Wakita, the COVID-19 Response Team 28 symptomatic passengers were transferred to se
lect hospitals for isolation. The remaining 538 were
transported to a designated hospital, where another
35 were found to be symptomatic and were hospital
ized there or transferred to other hospitals, leaving
503 asymptomatic persons for observation in quar
antine (Figure). In early 2020, Japan repatriated 566 nationals from Chi
na. Universal laboratory testing and 14-day monitoring
of returnees detected 12 cases of severe acute respira
tory syndrome coronavirus 2 infection; initial screening
results were negative for 5. Common outcomes were
remaining asymptomatic (n = 4) and pneumonia (n = 6). Overall, screening performed poorly. W W
ith the emergence of severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) in Wu
han, China, several countries, including Japan, repa
triated their nationals (1–3). During January 29–31,
2020, a total of 566 Japanese nationals were repatri
ated via 3 chartered flights from Wuhan (206, 210,
and 150 passengers). After passengers disembarked
in Tokyo, Japan, quarantine officials assessed them
for signs/symptoms (e.g., fever, respiratory illness)
of coronavirus disease (COVID-19) (4). A total of 1These authors contributed equally to this article. DOI: https://doi.org/10.3201/eid2607.200994 The Study We conducted day 1 entry screening by testing oro
pharyngeal swab samples collected from all 566
returnees at the hospitals to which they were ini
tially transported for SARS-CoV-2 (4); all tests were
based on the real-time reverse transcription PCR
developed by the National Institute of Infectious
Diseases (5). Hospitalized patients in isolation and
asymptomatic returnees in quarantine were moni
tored daily for 14 days. If any signs/symptoms de
veloped in a quarantined person, that person was
transported to a designated hospital and oropha
ryngeal swab samples were collected for testing. We conducted exit screening for quarantined per
sons who remained illness-free by collecting oro
pharyngeal swab samples on day 14. The National
Institute of Infectious Diseases Ethics Committee
approved the study (registration no. 1096), and all
566 returnees who provided specimens gave writ
ten informed consent. Among the 63 passengers who were symptom
atic at entry screening, 2 (3.2%) were positive by PCR 1These authors contributed equally to this article. 1596 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 7, July 2020 SARS-CoV-2 Among Returnees from Wuhan, China (Figure); test results were subsequently positive for 2
more. For 1 of these patients, pneumonia was diag
nosed on day 1 and a sputum sample was positive
on day 3; the other patient had fever and cough on
day 1, pneumonia diagnosed on day 2, and a posi
tive oropharyngeal swab sample on day 6. Excluding
1 patient who remained hospitalized for stroke, the
remaining 58 patients were transferred to designated
quarantine facilities after confirmation of good health
and negative PCR results; all 58 remained asymptom
atic after discharge, and PCR results were negative at
exit screening. in 1 facility-quarantined and 1 home-quarantined
person on day 10; both were confirmed positive
by PCR, and pneumonia subsequently developed
in both. The facility-quarantined case-patient was
in a single room; no other person from this facility
acquired COVID-19 or had a positive test result at
exit screening. One person who remained asymp
tomatic had a positive test result at exit screening. Exit-screening results are pending for the patient
hospitalized for stroke and the remaining 13 home-
quarantined persons. Among the 566 returnees, 12 cases of SARS-CoV-2
infection were detected; 540/541 facility-quarantined
persons were confirmed negative by PCR performed
on days 1 and 14 (197/197, 199/199, and 144/145 for
the 3 flights). The Study Entry screening detected 7 infections,
for an infection point prevalence of 1.2%; infection
period prevalence was 2.2% (12/552 returnees with
complete follow-up). Despite universal testing, entry
screening captured only 7/12 cases (58.3% sensitivity). g
For the 503 asymptomatic/subclinical passen
gers, entry-screening PCR results were positive for 5
(1.0%) (Figure); 3 remained asymptomatic, but mild
signs/symptoms (fever, headache, sore throat) de
veloped for 2 persons (1 on day 2, 1 on day 4). Of the
remaining 498 persons with negative PCR results,
484 were quarantined at designated facilities and
14 at home. During quarantine, fever developed Figure. Results of testing 566 Japanese returnees from Wuhan, China, for severe acute respiratory syndrome coronavirus 2 by real-time
reverse transcription PCR, January–February 2020. *Two persons were sampled on day 3, when they provided informed consent. Figure. Results of testing 566 Japanese returnees from Wuhan, China, for severe acute respiratory syndrome coronavirus 2 by real-time
reverse transcription PCR, January–February 2020. *Two persons were sampled on day 3, when they provided informed consent. 6 Japanese returnees from Wuhan, China, for severe acute respiratory syndrome coronavirus 2 by real-tim
anuary–February 2020. *Two persons were sampled on day 3, when they provided informed consent. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 7, July 2020 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 7, July 2020 1597 DISPATCHES Table. Distribution of severe acute respiratory syndrome coronoavirus 2 infections and clinical outcomes, by age group, among 566
Japanese returnees from Wuhan, China, January–February 2020 Age group, y
No. returnees*
No. (%) infected†
Outcome
No. asymptomatic
No. with
mild illness
No. with
pneumonia
No. deaths
<10
6
0
0
0
0
0
10–19
4
0
0
0
0
0
20–29
90
0
0
0
0
0
30–39
138
2 (1.4)
2
0
0
0
40–49
168
4 (2.4)
0
1
3
0
50–59
119
5 (4.2)
1
1
3
0
60–69
26
1 (3.8)
1
0
0
0
70–79
1
0
0
0
0
0
*Day-14 exit-screening test results pending for 14 persons. †Testing by reverse transcription PCR. Although screening symptomatic passengers (3.2%)
was more efficient than screening all passengers
(1.2%), screening only symptomatic passengers
missed 5/7 prevalent infections at entry. Among
symptomatic passengers, with 2 initially negative
persons subsequently testing positive, entry-screen
ing sensitivity was 2/4 (50%). Among asymptomatic
passengers, with 3 initially negative persons subse
quently testing positive, entry-screening sensitivity
was 5/8 (62.5%). The Study atic and presymptomatic cases. Even with universal
screening, nearly half of cases were missed. Because
an asymptomatic case was detected at exit screening,
limiting testing of quarantined persons to those with
signs/symptoms would have missed such a case;
with exit-screening results pending for 14 returnees,
sensitivity could be lower. The poor sensitivity of sin
gle-point testing highlights the challenges of detect
ing SARS-CoV-2 infections. g
The potentially long incubation period of CO
VID-19 was consistent with that recently reported
(8,9) and contributed to the large proportion of
missed cases. Active daily monitoring ensured that
specific illness-onset times were captured, protected
from the limitations associated with patient recall of
symptom onset (10). Although exposure to SARS-
CoV-2 occurred at some time before quarantine (i.e.,
left-censore d), our setting enabled us to estimate
the minimum incubation period for each incident
symptomatic case by taking the return date as the
exposure time. Determining the specific exposure
time can be difficult and is conditional according to
the definition of contact. Given such qualifications, a
conservative minimum incubation period of 10 days
obtained prospectively in a clean quarantine setting,
without recall or assumptions regarding transmis
sion modes, is noteworthy. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 7, July 2020 SARS-CoV-2 Among Returnees from Wuhan, China SARS-CoV-2 Among Returnees from Wuhan, China of COVID-19 (2,12–14); infection and clinical attack
rates were lower among younger persons. Shed
ding light on the severity pyramid among those
infected—not only among those who sought care—
provides an evidence base for risk communication,
healthcare planning, and public health response. Combined with reports suggesting transmissibil
ity of SARS-CoV-2 from asymptomatic/subclinical
case-patients (1,10,15,16), our findings suggest that
controlling COVID-19 through the usual tools of
syndrome-based surveillance and contact tracing
alone may be difficult. Acknowledgments We acknowledge the dedicated work of the frontline clinicians
and the staff at the quarantine facilities, the workers at the local
public health centers and prefectural and municipal public health
institutes, and the Japan Ministry of Health, Labour and Welfare. We also thank the quarantined returnees for their patience
and participation. This study was supported in part by a grant-in-aid from the Japan
Agency for Medical Research and Development (grant nos. JP19fk0108104 and JP19fk0108110). Dr. Arima is an affiliate researcher at the Infectious Disease
Surveillance Center, National Institute of Infectious Diseases
in Tokyo, Japan. His research interests include infectious
disease epidemiology and surveillance in the context of public
health practice. yi
When confronted with an emerging pathogen,
researchers can generate critical epidemiologic in
formation by studying quarantined populations. As
with the First Few X study (7), our design is protected
from the usual biases of passively reported surveil
lance data. Aggregating high-quality data from these
types of investigations can build a larger severity
pyramid, enabling reliable estimation of various se
verity measures (e.g., symptomatic proportion of in
fected case-patients, case severity proportion among
those who are symptomatic). We recommend using
similar assessments to help elucidate the epidemiolo
gy of SARS-CoV-2 and inform public health response. References 1. Hoehl S, Rabenau H, Berger A, Kortenbusch M, Cinatl J,
Bojkova D, et al. Evidence of SARS-CoV-2 infection in
returning travelers from Wuhan, China. N Engl J Med. 2020;382:1278–80. https://doi.org/10.1056/NEJMc2001899 1. Hoehl S, Rabenau H, Berger A, Kortenbusch M, Cinatl J,
Bojkova D, et al. Evidence of SARS-CoV-2 infection in
returning travelers from Wuhan, China. N Engl J Med. 2020;382:1278–80. https://doi.org/10.1056/NEJMc2001899 p //
g/
/
J
2. Jernigan DB; CDC COVID-19 Response Team. Update:
public health response to the coronavirus disease 2019
outbreak—United States, February 24, 2020. MMWR Morb
Mortal Wkly Rep. 2020;69:216–9. https://doi.org/10.15585/
mmwr.mm6908e1 p //
g/
/
2. Jernigan DB; CDC COVID-19 Response Team. Update:
public health response to the coronavirus disease 2019
outbreak—United States, February 24, 2020. MMWR Morb
Mortal Wkly Rep. 2020;69:216–9. https://doi.org/10.15585/
mmwr.mm6908e1 3. Ng OT, Marimuthu K, Chia PY, Koh V, Chiew CJ,
De Wang L, et al. SARS-CoV-2 infection among travelers
returning from Wuhan, China. N Engl J Med. 2020;382:1476–
8. https://doi.org/10.1056/NEJMc2003100 3. Ng OT, Marimuthu K, Chia PY, Koh V, Chiew CJ,
De Wang L, et al. SARS-CoV-2 infection among travelers
returning from Wuhan, China. N Engl J Med. 2020;382:1476–
8. https://doi.org/10.1056/NEJMc2003100 Members of the COVID-19 response team: Akira Ainai,
Minetaro Arita, Tomoko Arita, Hideki Asanuma, Seiichiro
Fujisaki, Itsuki Hamamoto, Yuichi Harada, Shinichiro
Hirai, Shun Iida, Yoshiyuki Ishii, Shigeyuki Itamura,
Naoko Iwata-Yoshikawa, Takayuki Kanno, Harutaka
Katano, Fumihiro Kato, Hirofumi Kato, Hiroshi Katoh,
Minoru Kidokoro, Noriko Kishida, Osamu Kotani, Toru
Kubota, Iwao Kukimoto, Madoka Kuramitsu, Tomoko
Kuwahara, Shutoku Matsuyama, Seiichiro Mori, Yoshio
Mori, Koichi Murakami, Tsutomu Murakami, Noriyo
Nagata, Seishiro Naito, Kazuya Nakamura, Yuichiro
Nakatsu, Mina Nakauchi, Eri Nobusawa, Kiyoko
Okamoto, Kazu Okuma, Noriyuki Otsuki, Masumichi
Saito, Koji Sakai, Kouji Sakai, Masafumi Sakata, Kaori
Sano, Kayoko Sato, Yuko Sato, Fumio Seki, Noriko
Shimasaki, Hiroyuki Shimizu, Masayuki Shirakura,
Kazuya Shirato, Kenji Someya, Ryosuke Suzuki, Yasushi
Suzuki, Maino Tahara, Hitoshi Takahashi, Kenta
Takahashi, Emi Takashita, Makoto Takeda, Takamasa
Takeuchi, Michiko Tanaka, Kenta Tezuka, Kenzo
Tokunaga, Yuji Wada, Kana Watanabe, Shinji Watanabe,
Aya Zamoto-Niikura, (National Institute of Infectious
Diseases); Tomoko Date (National Center for Global
Health and Medicine); Takashi Chiba, Mami Nagashima,
Kenji Sadamasu, Kazuhisa Yoshimura (Tokyo
Metropolitan Institute of Public Health); Manami
Yanagawa (Ministry of Health, Labour and Welfare). p
g
4. Kutsuna S, Suzuki T, Hayakawa K, Tsuzuki S, Asai Y, Suzuki T,
et al. SARS-CoV-2 screening test for Japanese returnees 1 from
Wuhan, China. 2 January 2020. Open Forum Infectious
Diseases. Conclusions Testing all returnees—with follow-up for disease
onset and course—enabled us to evaluate the spec
trum of severity for SARS-CoV-2 infections (Table). From least to most severe, 4 patients experienced
asymptomatic infection, 2 mild illness, and 6 pneu
monia. Prospective monitoring proved essential be
cause of the 7 prevalent infections at entry, 5 were
asymptomatic, 1 mild, and 1 pneumonia. Even with
potential underascertainment of asymptomatic cas
es because of a lack of serologic assessment (6,7) (i.e.,
interval-censoring during screening tests), it is note
worthy that 4/12 persons with infections were as
ymptomatic. Although numbers are small, severity
seemed to be age dependent (Table). No infections
were detected among the 100 persons <30 years of
age; of the 2 infections detected among the 138 per
sons 30–39 years of age, both persons were asymp
tomatic. Although no person in this study died, only
1 was >69 years of age. Regarding sex, excluding
1 returnee for whom sex was unknown and 14 for
whom exit-screening results are pending, of the re
maining 551 returnees, 9 (1.8%) of the 506 male pas
sengers (2 asymptomatic, 2 mild, 5 pneumonia) and
3 (6.7%) of the 45 female passengers (2 asymptom
atic, 1 pneumonia) were infected.i y
Testing and follow-up of all returnees provided
valuable information about the spectrum of SARS-
CoV-2 infection. Most reported data have been
from medically attended patients, skewed toward
symptomatic patients and more severe cases, limit
ing our knowledge of the clinical spectrum of in
fection (6,11). In our setting, we could remove the
influence of patients’ health-seeking behaviors and
clinicians’ diagnostic practices and found that 4/12
case-patients were asymptomatic. At the same time,
of the 8 case-patients who experienced symptoms,
pneumonia developed in 6. Our findings were also
consistent with the reported age-dependent nature p
)
Our findings have public health implications. As
recently reported (1), we found that symptom-based
screening performed poorly, missing asymptom 1598 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 7, July 2020 References N Engl J Med. 2020;382:970–1. Address for correspondence: Yuzo Arima, Infectious Disease
Surveillance Center, National Institute of Infectious Diseases,
1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan;
email arima@niid.go.jp
EID’s spotlight topics highlight the latest articles
and information on emerging infectious
disease topics in our global community
https://wwwnc.cdc.gov/eid/page/spotlight-topics
Emerging Infectious Diseases
Spotlight Topics
Antimicrobial resistance • Ebola
Etymologia • Food safety • HIV-AIDS
Influenza • Lyme disease • Malaria
MERS • Pneumonia • Coronavirus
Rabies • Tuberculosis • Ticks • Zika DISPATCHES References In press 2020. p
g
4. Kutsuna S, Suzuki T, Hayakawa K, Tsuzuki S, Asai Y, Suzuki T,
et al. SARS-CoV-2 screening test for Japanese returnees 1 from
Wuhan, China. 2 January 2020. Open Forum Infectious
Diseases. In press 2020. p
5. Shirato K, Nao N, Katano H, Takayama I, Saito S, Kato F,
et al. Development of genetic diagnostic methods for novel
coronavirus 2019 (nCoV-2019) in Japan. Jpn J Infect Dis. 2020
Feb 18 [Epub ahead of print]. https://doi.org/10.7883/
yoken.JJID.2020.061i 6. Lipsitch M, Swerdlow DL, Finelli L. Defining the
epidemiology of COVID-19–studies needed. N Engl J Med. 2020;382:1194–6. https://doi.org/10.1056/NEJMp2002125 7. World Health Organization. The First Few X (FFX) cases and
contact investigation protocol for 2019-novel coronavirus
(2019-nCoV) infection [cited 2020 Mar 23]. https://www.who. int/publications-detail/the-first-few-x-(ffx)-cases-and-
contact-investigation-protocol-for-2019-novel-coronavirus-
(2019-ncov)-infection 8. Morens DM, Daszak P, Taubenberger JK. Escaping
Pandora’s box—another novel coronavirus. N Engl J Med. 2020;382:1293–5. https://doi.org/10.1056/NEJMp2002106 p
g
p
9. Backer JA, Klinkenberg D, Wallinga J. Incubation period
of 2019 novel coronavirus (2019-nCoV) infections among
travellers from Wuhan, China, 20–28 January 2020. Euro
Surveill. 2020;25. https://doi.org/10.2807/1560-7917. ES.2020.25.5.2000062 10. Tong ZD, Tang A, Li KF, Li P, Wang HL, Yi JP, et al. Potential presymptomatic transmission of SARS-CoV-2, Zhe
jiang Province, China, 2020. Emerg Infect Dis. 2020;26:1050–2. https://doi.org/10.3201/eid2605.200198 1599 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 7, July 2020 DISPATCHES
11. Munster VJ, Koopmans M, van Doremalen N, van Riel D,
de Wit E. A novel coronavirus emerging in China—key
questions for impact assessment. N Engl J Med. 2020;382:692–4. https://doi.org/10.1056/NEJMp2000929
12. World Health Organization. Report of the WHO-China Joint
Mission on Coronavirus Disease 2019 (COVID-19) [cited 2020 Mar
23]. https://www.who.int/docs/default-source/coronaviruse/
who-china-joint-mission-on-covid-19-final-report.pdf
13. Li Q, Guan X, Wu P, Wang X, Zhou L, Tong Y, et al. Early
transmission dynamics in Wuhan, China, of novel
coronavirus-infected pneumonia. N Engl J Med. 2020;382:1199–207. https://doi.org/10.1056/NEJMoa2001316
14. Guan WJ, Ni ZY, Hu Y, Liang WH, Ou CQ, He JX, et al.;
China Medical Treatment Expert Group for COVID-19. Clini
cal characteristics of coronavirus disease 2019 in
China. N Engl J Med. 2020 Feb 28 [Epub ahead of print]. https://doi.org/10.1056/NEJMoa2002032
15. Zou L, Ruan F, Huang M, Liang L, Huang H, Hong Z, et al. SARS-CoV-2 viral load in upper respiratory specimens of infected
patients. N Engl J Med. 2020;382:1177–9. 16. Rothe C, Schunk M, Sothmann P, Bretzel G, Froeschl G,
Wallrauch C, et al. Transmission of 2019-nCoV infection
from an asymptomatic contact in Germany. DISPATCHES China. N Engl J Med. 2020 Feb 28 [Epub ahead of print]. https://doi.org/10.1056/NEJMoa2002032 11. Munster VJ, Koopmans M, van Doremalen N, van Riel D,
de Wit E. A novel coronavirus emerging in China—key
questions for impact assessment. N Engl J Med. 2020;382:692–4. https://doi.org/10.1056/NEJMp2000929 p
g
15. Zou L, Ruan F, Huang M, Liang L, Huang H, Hong Z, et al. SARS-CoV-2 viral load in upper respiratory specimens of infected
patients. N Engl J Med. 2020;382:1177–9. p
g
15. Zou L, Ruan F, Huang M, Liang L, Huang H, Hong Z, et al. SARS-CoV-2 viral load in upper respiratory specimens of infected
patients. N Engl J Med. 2020;382:1177–9. p
g
p
12. World Health Organization. Report of the WHO-China Joint
Mission on Coronavirus Disease 2019 (COVID-19) [cited 2020 Mar
23]. https://www.who.int/docs/default-source/coronaviruse/
who-china-joint-mission-on-covid-19-final-report.pdf p
g
p
12. World Health Organization. Report of the WHO-China Joint
Mission on Coronavirus Disease 2019 (COVID-19) [cited 2020 Mar
23]. https://www.who.int/docs/default-source/coronaviruse/
who-china-joint-mission-on-covid-19-final-report.pdf 16. Rothe C, Schunk M, Sothmann P, Bretzel G, Froeschl G,
Wallrauch C, et al. Transmission of 2019-nCoV infection
from an asymptomatic contact in Germany. N Engl J Med. 2020;382:970–1. ji
p
p
13. Li Q, Guan X, Wu P, Wang X, Zhou L, Tong Y, et al. Early
transmission dynamics in Wuhan, China, of novel
coronavirus-infected pneumonia. N Engl J Med. 2020;382:1199–207. https://doi.org/10.1056/NEJMoa2001316 Address for correspondence: Yuzo Arima, Infectious Disease
Surveillance Center, National Institute of Infectious Diseases,
1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan;
email arima@niid.go.jp p //
g/
/
J
14. Guan WJ, Ni ZY, Hu Y, Liang WH, Ou CQ, He JX, et al.;
China Medical Treatment Expert Group for COVID-19. Clini
cal characteristics of coronavirus disease 2019 in f
/
EID’s spotlight topics highlight the latest articles
and information on emerging infectious
disease topics in our global community
https://wwwnc.cdc.gov/eid/page/spotlight-topics
Antimicrobial resistance • Ebola
Etymologia • Food safety • HIV-AIDS
Influenza • Lyme disease • Malaria
MERS • Pneumonia • Coronavirus
Rabies • Tuberculosis • Ticks • Zika EID’s spotlight topics highlight the latest articles
and information on emerging infectious
disease topics in our global community
https://wwwnc.cdc.gov/eid/page/spotlight-topics 1600 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 7, July 2020
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Structural and morphological properties of sol-gel ZnO:Ni films
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Published under licence by IOP Publishing Ltd This content was downloaded from IP address 130.238.141.100 on 06/12/2018 at 15:05 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Conf Series: Journal of Physics: Conf Series 992 (2018) 012044
doi:10 1088 th International Summer School on Vacuum, Electron and Ion Technologies 5 To whom any correspondence should be addressed. E-mail: tativan@phys.bas.bg Abstract. Ni doping induces modifications and considerable changes in the optical, electrical
and magnetic properties of ZnO films. In this work, the influence is discussed of Ni-doping
(two nickel concentrations) and annealing temperature (ranging from 300 o C to 800 oC) on the
structural and optical properties of sol-gel derived ZnO:Ni films. Uniform and smooth films
were obtained by spin-coating on quartz and Si substrates. The ZnO:Ni films were crystallized
in wurtzite phase with no impurity phases found for annealing temperatures up to 600 oC. The
size of the crystallites was strongly affected by the Ni content and the heat treatment. Furthermore, the Ni doping improved the optical transparency of the sol-gel films, while the
AFM studies showed that the film morphology and the roughness were influenced by the
nickel doping. T Ivanova1,5, A Harizanova1, T Koutzarova2, B Vertruyen3 and B Stefanov4 1Central Laboratory of Solar Energy and New Energy Sources,
Bulgarian Academy of Sciences, 72 Tzarigradsko Chaussee, 1784 Sofia, Bulgaria
2 E. Djakov Institute of Electronics, Bulgarian Academy of Sciences,
72 Tzarigradsko Chaussee, 1784 Sofia, Bulgaria
3LCIS/SUPRATECS, Institute of Chemistry B6, University of Liege,
Sart-Tilman, B-4000 Liege, Belgium
4Department of Engineering Sciences, The Ångström Laboratory, Uppsala University
P.O. Box 534, Uppsala, Sweden 1Central Laboratory of Solar Energy and New Energy Sources,
Bulgarian Academy of Sciences, 72 Tzarigradsko Chaussee, 1784 Sofia, Bulgaria
2 E. Djakov Institute of Electronics, Bulgarian Academy of Sciences,
72 Tzarigradsko Chaussee, 1784 Sofia, Bulgaria
3LCIS/SUPRATECS, Institute of Chemistry B6, University of Liege,
Sart-Tilman, B-4000 Liege, Belgium
4Department of Engineering Sciences, The Ångström Laboratory, Uppsala University
P.O. Box 534, Uppsala, Sweden 1Central Laboratory of Solar Energy and New Energy Sources,
Bulgarian Academy of Sciences, 72 Tzarigradsko Chaussee, 1784 Sofia, Bulgaria
2 E. Djakov Institute of Electronics, Bulgarian Academy of Sciences,
72 Tzarigradsko Chaussee, 1784 Sofia, Bulgaria
3LCIS/SUPRATECS, Institute of Chemistry B6, University of Liege,
Sart-Tilman, B-4000 Liege, Belgium
4Department of Engineering Sciences, The Ångström Laboratory, Uppsala University
P.O. Box 534, Uppsala, Sweden 4Department of Engineering Sciences, The Ångström Laboratory, Uppsala University, 2. Experimental The preparation of sols for ZnO and nickel-doped ZnO deposition were described previously [9]. Briefly, 0.4 M Zn sol and 0.2 M Ni sol were mixed in two volume ratios: ZnO:Ni 0.4 (19.6 ml Zn
sol:0.4 ml Ni sol) and ZnO:Ni 1 (19 ml Zn sol :1 ml Ni sol). The films were obtained by spin coating
at 4000 rpm on Si and quartz substrates. The layer deposition procedure was repeated five times with
pre-heating of 300 oC/10 min between layers. The ZnO:Ni films were annealed at temperatures
ranging from 300 oC to 800 oC. g g
A Bruker D8 XRD diffractometer (grazing angle 2o, step time 8 s) was used for the structural
study. The FTIR spectra were taken by an IRPrestige-21 Shimadzu FTIR Spectrophotometer. The
optical measurements were carried out by a Shimadzu 3600 UV-VIS spectrophotometer. The atomic
force microscopy (AFM) observation was performed by employing a PSIA XE150 SPM/AFM
instrument equipped with an ACTA silicon cantilever (AppNano), a tip radius 6 10 nm operating in
non-contact mode. 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 20th International Summer School on Vacuum, Electron and Ion Technologies 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :1 1234567890 ‘’“”
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/ downwards based on its interaction with an oxidizing or reducing gas, Ni can enhance the gas sensing
properties [4]. ZnO films doped with Co, Ni, Fe have been widely studied as diluted magnetic
semiconductors (DMS) [5]. Doped ZnO films exhibit magnetic behavior due to doping with transition
metals and/or manifest magnetic properties through structural defects caused during the doped films‘
synthesis. The origin of ferromagnetism in nanostructured DMO׳s is still being explored and discussed [6]. downwards based on its interaction with an oxidizing or reducing gas, Ni can enhance the gas sensing
properties [4]. ZnO films doped with Co, Ni, Fe have been widely studied as diluted magnetic
semiconductors (DMS) [5]. Doped ZnO films exhibit magnetic behavior due to doping with transition
metals and/or manifest magnetic properties through structural defects caused during the doped films‘
synthesis. The origin of ferromagnetism in nanostructured DMO׳s is still being explored and discussed [6]. ZnO doped films are deposited by pulse laser deposition, r.f. magnetron sputtering, molecular-
beam epitaxy, CVD and ALD [7]. Among these methods, the sol-gel deposition has several
advantages, such as inexpensive equipment, precise microstructural and chemical control,
reproducibility, possibilities of engineering the films‘ properties. Preparation of high-quality ZnO-
based nanostructured films by the sol-gel technology has been reported earlier [8]. In the present work, the effect is studied of adding Ni (two Ni concentrations) and varying the
annealing temperature (300 oC 800 oC) on the structural, optical and morphological properties of sol-
gel Ni-doped ZnO films. 1. Introduction
i
id ZnO is considered as being one of the most important semiconductors due to its wide bandgap
(3.37 eV), large exciton energy (60 meV), thermal and chemical stability and hexagonal crystal
structure. The ZnO nanostructured films possess desirable optical, structural and electronic properties
for applications in thin-film transistors, solar cells, gas sensors, UV light emitting diodes, field
emitters, piezoelectric devices, photo detectors, flat screen displays, sun screens, spintronic devices, etc. [1]. Doping the ZnO films modulates and changes their morphology, density of states, transmittance,
bandgap, electrical conductivity, etc. [2]. Nickel is an important dopant, as Ni2+ has an ionic radius of
0.069 nm, which is slightly smaller than that of the Zn2+ ion (0.074 nm); thus, Ni2+ ions should be able
to substitute Zn2+ in a ZnO matrix. ZnO:Ni films are being studied for potential applications in solar
cells and memory devices [3]. Ni doping is a viable method of improving the sensing ability of ZnO
by tuning the electronic band structure and modifying its energy levels and surface states. Since gas
sensing is a surface phenomenon, where the sensing element‘s energy band bends upwards or 1 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 films annealed at 300 oC (figure 1), the smaller Ni concentration induces a film crystallization
comparable to that of the ZnO film, as the ZnO:Ni 1 films manifest weaker and broader XRD peaks, a
sign of a less crystallized structure and possible traces of an amorphous phase. After annealing at
600 oC, the ZnO:Ni 0.4 and ZnO:Ni 1 films show lower crystallization compared to undoped ZnO
films. The ZnO:Ni 0.4 and ZnO:Ni 1 films are polycrystalline, with all XRD peaks observed in their
XRD patterns attributed to a hexagonal wurtzite phase of ZnO (according to JCPDS 01-07-8070)
regardless of the annealing temperature, from 300 oC to 600 oC. No diffraction reflections assignable
to Ni oxides or mixed Zn-Ni fractions are registered within the XRD detection limits. The ZnO:Ni 1
film treated at 800 oC reveals a broad peak at 2θ = 43.2o related to NiO bunsenite (JCPDS 00-004-
0835) [9]. The presence of a NiO fraction indicates that the solubility of Ni in ZnO matrix is limited
and dependent on the deposition method [10]. 300
400
500
600
700
800
0
100
200
300
400
500
600
ZnO
ZnO:Ni 0.4
Intensity [arb.u.]
Annealing temperature [oC]
ZnO:Ni 1
XRD peak 002
300
400
500
600
700
800
5
10
15
20
25
30
35
40
45
50
ZnO:Ni 0.4
ZnO:Ni 1
Crystallite size [nm]
Annealing temperature [oC]
ZnO
Figure 2. Intensity of the 002
main XRD peak and estimated
crystallite size for ZnO and
ZnO:Ni films depending on
the annealing temperatures. Figure 2 presents the annealing dependence of the 002 peak’s XRD intensity and of the average
crystallite size as estimated from the XRD peaks (100 002 and 101 reflections) by using Scherrer’s 300
400
500
600
700
800
0
100
200
300
400
500
600
ZnO
ZnO:Ni 0.4
Intensity [arb.u.]
Annealing temperature [oC]
ZnO:Ni 1
XRD peak 002
300
400
500
600
700
800
5
10
15
20
25
30
35
40
45
50
ZnO:Ni 0.4
ZnO:Ni 1
Crystallite size [nm]
Annealing temperature [oC]
ZnO
Figure 2. Intensity of the 002
main XRD peak and estimated
crystallite size for ZnO and
ZnO:Ni films depending on
the annealing temperatures. Figure 2 presents the annealing dependence of the 002 peak’s XRD intensity and of the average
crystallite size as estimated from the XRD peaks (100, 002 and 101 reflections) by using Scherrer’s
formula. 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 It is seen that the XRD intensity depends strongly on the annealing temperature. The
crystallites grow bigger as the annealing temperature is raised up to 600 oC for both the ZnO and
ZnO:Ni films. After annealing at 700 oC and 800oC, the ZnO:Ni crystallites grow smaller, their sizes
become comparable to those of the ZnO films treated at these temperatures. The XRD analysis leads
to the conclusion that Ni doping results in improved film crystallinity. The higher doping
concentration (ZnO:Ni 1 films) leads to narrower XRD lines i e
greater crystallites compared to 300
400
500
600
700
800
0
100
200
300
400
500
600
ZnO
ZnO:Ni 0.4
Intensity [arb.u.]
Annealing temperature [oC]
ZnO:Ni 1
XRD peak 002 Figure 2. Intensity of the 002
main XRD peak and estimated
crystallite size for ZnO and
ZnO:Ni films depending on
the annealing temperatures. gure 2 presents the annealing dependence of the 002 peak’s XRD intensity and of the averag Figure 2 presents the annealing dependence of the 002 peak’s XRD intensity and of the average
crystallite size as estimated from the XRD peaks (100, 002 and 101 reflections) by using Scherrer’s
formula. It is seen that the XRD intensity depends strongly on the annealing temperature. The
crystallites grow bigger as the annealing temperature is raised up to 600 oC for both the ZnO and
ZnO:Ni films. After annealing at 700 oC and 800oC, the ZnO:Ni crystallites grow smaller, their sizes
become comparable to those of the ZnO films treated at these temperatures. The XRD analysis leads
to the conclusion that Ni doping results in improved film crystallinity. The higher doping
concentration (ZnO:Ni 1 films) leads to narrower XRD lines, i.e., greater crystallites compared to
those of the ZnO and ZnO:Ni 0.4 films. g
p
g
p
e
pe
s
y
v
g
crystallite size as estimated from the XRD peaks (100, 002 and 101 reflections) by using Scherrer’s
formula. It is seen that the XRD intensity depends strongly on the annealing temperature. The
crystallites grow bigger as the annealing temperature is raised up to 600 oC for both the ZnO and
ZnO:Ni films. After annealing at 700 oC and 800oC, the ZnO:Ni crystallites grow smaller, their sizes
become comparable to those of the ZnO films treated at these temperatures. The XRD analysis leads
to the conclusion that Ni doping results in improved film crystallinity. 3. Results and discussions The XRD patterns of ZnO and ZnO:Ni films annealed at 300 oC and 600 oC are presented in figure 1;
as seen, the annealing temperature influences significantly the crystallization behavior of ZnO-based
films. The undoped ZnO films improve their crystallization gradually as the annealing temperature is
raised. The Ni-doped ZnO films exhibit a more complicated crystallization evolution. ZnO:Ni 0.4 and
ZnO:Ni 1 films manifest an enhanced crystallinity for the annealing temperatures of 300 oC, 400 oC
and 500oC; then the crystallization degree deteriorates after the high-temperature treatment at 600 oC. y
g
g
p
Our earlier studies [9] revealed that the ZnO:Ni 0.4 films achieved the best crystallization after
treatment at 700 oC, while the ZnO:Ni 1 films exhibited the most intense XRD peaks after annealing at
800 oC,; or, doping with Ni affects significantly the crystallization behavior of ZnO:Ni films. For the 30
40
50
60
70
0
50
100
150
112
103
110
102
101
002
ZnO:Ni 1
ZnO:Ni 0.4
Intensity [arb. u.]
2 degrees
300oC
ZnO
100
30
32
34
36
38
40
101
002
100
ZnO:Ni 1
ZnO:Ni 0.4
300oC
ZnO
30
35
40
45
50
55
60
65
70
0
100
200
300
* ZnO2
101
002
112
103
110
102
ZnO
ZnO:Ni 1
ZnO:Ni 0.4
Intensity [arb.u.]
2degrees
600oC
100
30
32
34
36
38
40
101
002
100
ZnO
ZnO:Ni 1
ZnO:Ni 0.4
600oC
* ZnO2
Figure 1. XRD patterns of ZnO, ZnO:In 0.4 and ZnO:Ni 1 films treated at 300 and 600oC. 30
40
50
60
70
0
50
100
150
112
103
110
102
101
002
ZnO:Ni 1
ZnO:Ni 0.4
Intensity [arb. u.]
2 degrees
300oC
ZnO
100
30
32
34
36
38
40
101
002
100
ZnO:Ni 1
ZnO:Ni 0.4
300oC
ZnO 30
35
40
45
50
55
60
65
70
0
100
200
300
* ZnO2
101
002
112
103
110
102
ZnO
ZnO:Ni 1
ZnO:Ni 0.4
Intensity [arb.u.]
2degrees
600oC
100
30
32
34
36
38
40
101
002
100
ZnO
ZnO:Ni 1
ZnO:Ni 0.4
600oC
* ZnO2 Figure 1. XRD patterns of ZnO, ZnO:In 0.4 and ZnO:Ni 1 films treated at 300 and 600oC. 2 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 The higher doping
concentration (ZnO:Ni 1 films) leads to narrower XRD lines, i.e., greater crystallites compared to
those of the ZnO and ZnO:Ni 0.4 films. We further conducted FTIR measurements to examine the effect of Ni doping on the vib
properties of sol-gel ZnO films; the results are shown in figure 3. 500
1000
1500
2000
2500
3000
3500
4000
ZnO
ZnO:Ni 1
ZnO:Ni 0.4
Transmittance [arb.u.]
Wavenumber [cm-1]
300oC
400
500
600
700
800
900
ZnO
ZnO:Ni 1
ZnO:Ni 0.4
300oC
500
1000
1500
2000
2500
3000
3500
4000
ZnO:Ni 1
ZnO:Ni 0.4
Transmittance [arb.u.]
Wavenumber [cm-1]
600oC
ZnO
400
500
600
700
800
900
ZnO:Ni 1
ZnO:Ni 0.4
600oC
ZnO
Figure 3. FTIR spectra of
ZnO and ZnO:Ni films
treated at 300 oC and
600 oC. Metal-oxygen characteristic absorptions are exhibited in the fingerprint region below 1000 cm-1. Absorptions due to organic residues and water inclusions appear above 1000 cm-1. The ZnO:Ni films
annealed at 300 oC show clear absorptions at 3780 cm-1 (adsorbed water molecules [11]) and broad
bands near 3450 cm-1 (O-H stretching vibrations [12]). The ZnO film reveals a weak line at 3450 cm-1. These bands vanish after high-temperature annealing. The weak band at 1640 cm−1 is assigned to the 500
1000
1500
2000
2500
3000
3500
4000
ZnO:Ni 1
ZnO:Ni 0.4
Wavenumber [cm-1]
600oC
ZnO
400
500
600
700
800
900
ZnO:Ni 1
ZnO:Ni 0.4
600oC
ZnO 500
1000
1500
2000
2500
3000
3500
4000
ZnO
ZnO:Ni 1
ZnO:Ni 0.4
Transmittance [arb.u.]
Wavenumber [cm-1]
300oC
400
500
600
700
800
900
ZnO
ZnO:Ni 1
ZnO:Ni 0.4
300oC
Transmittance [arb u ] 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 400
600
800
1000
1200
1400
1600
1800
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Reflectance [%]
300oC
ZnO:Ni 0.4
ZnO:Ni 1
Transmittance [%]
Wavelength [nm]
ZnO
300
400
500
600
700
800
900
0
10
20
30
40
50
60
70
80
90
100
300oC
ZnO:Ni 0.4
ZnO:Ni 1
Transmittance [%]
Wavelength [nm]
ZnO 400
600
800
1000
1200
1400
1600
1800
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Reflectance [%]
600oC
ZnO
ZnO:Ni 0.4
ZnO:Ni 1
Transmittance [%]
Wavelength [nm]
300
400
500
600
700
800
900
0
10
20
30
40
50
60
70
80
90
100
600oC
ZnO
ZnO:Ni 0.4
ZnO:Ni 1
Transmittance [%]
Wavelength [nm] 800
1000
1200
1
Wavelength [nm] 800
1000
1200
1
Wavelength [nm] 800
1000
1200
1
Wavelength [nm] Figure 4. Transmittance and reflectance spectra of ZnO, ZnO:Ni films treated at 300 oC and 6 Figure 4 presents a comparison of the optical spectra of ZnO and ZnO:Ni films. The estimated
values of the optical bandgap (Eg) and the transmittance at 550 nm are given in table 1. Increasing the
Ni doping improves the transparency, the reflectance being below 15 % for the ZnO:Ni films. The
transmittance reaches up to 80 84 % for the sol-gel ZnO:Ni 1 films. The ZnO films’ thickness was found to be 160 nm. The film thicknesses of the ZnO:Ni 0.4 and
ZnO:Ni 1 films are 150 nm and 140 nm, respectively. The difference in the thickness of the films
studied was small and thus, the optical properties could be compared. The transparency of ZnO and ZnO:Ni films worsened with the temperatures, which became more
pronounced after the treatments at 700 oC and 800 oC. The optical bandgap values diminished as
the annealing temperatures was increased, due to the bigger crystallite sizes. The optical bandgaps of Table 1. Optical bandgap values, transmittance at λ = 550 nm for ZnO and ZnO:Ni films as a
of the annealing temperatures. 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 20th International Summer School on Vacuum, Electron and Ion Technologies 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP C
f S i
J
l f Ph
i
C
f S i
992 (2018) 012044
d i 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 1234567890 ‘’“”
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/ bending δ(HOH) vibration (seen in ZnO, ZnO:Ni films). The peak (seen in all spectra) at 2370 cm-1 is
due to CO2 absorbed from the atmosphere [13]. The absorption bands below 1000 cm-1 of the ZnO
films annealed at 300 oC are located at 760 cm-1 (due to Zn-O), 520 cm-1 (corresponding to wurtzite
ZnO), and a very strong and broad line at 407 cm-1 with a shoulder at 370 cm-1. The absorption bands
at 407 -cm-1 and 520 cm-1 are active infrared modes that have been confirmed theoretically for wurtzite
ZnO [14]. The main absorption line of the ZnO:Ni 0.4 film centered at 402 cm-1 is very strong and
broad with an additional peak at 360 cm-1. Increasing the nickel content (ZnO:Ni 1) results in a
modification of the main absorption feature: the band is located at 372 cm-1 with two lines at 396 cm-1
and 367 cm-1. Annealing at 600 oC induces stronger main absorption bands, shifted to 395 cm-1 (ZnO)
and 376 cm-1 for the ZnO:Ni films. Weak lines at 680 cm-1 and 520 cm-1 are detected for all films. Adding Ni clearly results in a broadening of the absorption bands. The shifting to the infrared
frequencies as the amount of the Ni2+ dopant is increased may be due to a difference in the bond
length, which confirms the incorporation of Ni2+ in the ZnO lattice [15]. The absorption bands
broadness can also point to a nanocrystalline nature of the films [12]. The FTIR studies did not
indicate a presence of Ni oxide phases. This conclusion is in accordance with the XRD
characterization. bending δ(HOH) vibration (seen in ZnO, ZnO:Ni films). The peak (seen in all spectra) at 2370 cm-1 is
due to CO2 absorbed from the atmosphere [13]. 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 The absorption bands below 1000 cm-1 of the ZnO
films annealed at 300 oC are located at 760 cm-1 (due to Zn-O), 520 cm-1 (corresponding to wurtzite
ZnO), and a very strong and broad line at 407 cm-1 with a shoulder at 370 cm-1. The absorption bands
at 407 -cm-1 and 520 cm-1 are active infrared modes that have been confirmed theoretically for wurtzite
ZnO [14]. The main absorption line of the ZnO:Ni 0.4 film centered at 402 cm-1 is very strong and
broad with an additional peak at 360 cm-1. Increasing the nickel content (ZnO:Ni 1) results in a
modification of the main absorption feature: the band is located at 372 cm-1 with two lines at 396 cm-1
and 367 cm-1. Annealing at 600 oC induces stronger main absorption bands, shifted to 395 cm-1 (ZnO)
and 376 cm-1 for the ZnO:Ni films. Weak lines at 680 cm-1 and 520 cm-1 are detected for all films. Adding Ni clearly results in a broadening of the absorption bands. The shifting to the infrared
frequencies as the amount of the Ni2+ dopant is increased may be due to a difference in the bond
length, which confirms the incorporation of Ni2+ in the ZnO lattice [15]. The absorption bands
broadness can also point to a nanocrystalline nature of the films [12]. The FTIR studies did not
indicate a presence of Ni oxide phases. This conclusion is in accordance with the XRD
characterization. 400
600
800
1000
1200
1400
1600
1800
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Reflectance [%]
300oC
ZnO:Ni 0.4
ZnO:Ni 1
Transmittance [%]
Wavelength [nm]
ZnO
300
400
500
600
700
800
900
0
10
20
30
40
50
60
70
80
90
100
300oC
ZnO:Ni 0.4
ZnO:Ni 1
Transmittance [%]
Wavelength [nm]
ZnO
400
600
800
1000
1200
1400
1600
1800
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Reflectance [%]
600oC
ZnO
ZnO:Ni 0.4
ZnO:Ni 1
Transmittance [%]
Wavelength [nm]
300
400
500
600
700
800
900
0
10
20
30
40
50
60
70
80
90
100
600oC
ZnO
ZnO:Ni 0.4
ZnO:Ni 1
Transmittance [%]
Wavelength [nm]
Figure 4. Transmittance and reflectance spectra of ZnO, ZnO:Ni films treated at 300 oC and 600 oC. Transmittance [arb.u.] Transmittance [arb.u.] Figure 3. FTIR spectra of
ZnO and ZnO:Ni films
treated at 300 oC and
600 oC. Wavenumber [cm-1] Wavenumber [cm-1] Wavenumber [cm-1] Metal-oxygen characteristic absorptions are exhibited in the fingerprint region below 1000 cm-1. Absorptions due to organic residues and water inclusions appear above 1000 cm-1. The ZnO:Ni films
annealed at 300 oC show clear absorptions at 3780 cm-1 (adsorbed water molecules [11]) and broad
bands near 3450 cm-1 (O-H stretching vibrations [12]). The ZnO film reveals a weak line at 3450 cm-1. These bands vanish after high-temperature annealing. The weak band at 1640 cm−1 is assigned to the Metal-oxygen characteristic absorptions are exhibited in the fingerprint region below 1000 cm-1. Absorptions due to organic residues and water inclusions appear above 1000 cm-1. The ZnO:Ni films
annealed at 300 oC show clear absorptions at 3780 cm-1 (adsorbed water molecules [11]) and broad
bands near 3450 cm-1 (O-H stretching vibrations [12]). The ZnO film reveals a weak line at 3450 cm-1. These bands vanish after high-temperature annealing. The weak band at 1640 cm−1 is assigned to the 3 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 The
inset images are 3D
micrographs. Figure 5 presents AFM micrographs of the ZnO:Ni 0.4 and ZnO:Ni 1 films treated at 600 oC (scan
area of 5×5 μm). The AFM study of a ZnO film performed at a scan area of 2×2 μm reveals a smooth
and uniform morphology with a grained film structure and an uneven size distribution. The grains are
well-packed, which results in a small r.m.s. roughness Rq = 5.115 nm for the undoped ZnO film (not
shown here). It is known that the roughness parameters are higher when the scan area is increased,
because longer-wavelength topography components are then sampled [17]. Thus, direct comparison
with the results for the ZnO:Ni films cannot be done. The AFM micrographs of the ZnO:Ni films
show different morphological features. Their average and r.m.s. roughness is given in Table 2. The
ZnO:Ni 0.4 film shows bigger grains with greater heights compared to the ZnO:Ni 1 films (see 3D
images, figure 5). On the other hand, the XRD analysis reveals that the ZnO:Ni 0.4 film annealed at
600 oC possesses smaller crystallites compared to ZnO:Ni 1, which can be a sign of micro-strain
broadening of the XRD peaks. Figure 5 presents AFM micrographs of the ZnO:Ni 0.4 and ZnO:Ni 1 films treated at 600 oC (scan
area of 5×5 μm). The AFM study of a ZnO film performed at a scan area of 2×2 μm reveals a smooth
and uniform morphology with a grained film structure and an uneven size distribution. The grains are
well-packed, which results in a small r.m.s. roughness Rq = 5.115 nm for the undoped ZnO film (not
shown here). It is known that the roughness parameters are higher when the scan area is increased,
because longer-wavelength topography components are then sampled [17]. Thus, direct comparison
with the results for the ZnO:Ni films cannot be done. The AFM micrographs of the ZnO:Ni films
show different morphological features. Their average and r.m.s. roughness is given in Table 2. The
ZnO:Ni 0.4 film shows bigger grains with greater heights compared to the ZnO:Ni 1 films (see 3D
images, figure 5). On the other hand, the XRD analysis reveals that the ZnO:Ni 0.4 film annealed at
600 oC possesses smaller crystallites compared to ZnO:Ni 1, which can be a sign of micro-strain
broadening of the XRD peaks. Table 2. 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 AFM parameters of sol-gel ZnO, ZnO:Ni 0.4 and ZnO:Ni 1 films treated at 600oC. AFM parameter
ZnO (2x2 μm)
ZnO:Ni 0.4 (5x5 μm)
ZnO:Ni 1 (5x5 μm)
Average roughness
4,215
8,246
7,00
r.m.s. rougnness
5,115
10,368
8,455 Table 2. AFM parameters of sol-gel ZnO, ZnO:Ni 0.4 and ZnO:Ni 1 films treated at 600oC. ZnO:Ni 1 shows lower roughness parameters values, indicating a smoother surface with a closely-
packed granular structure. This may explain its improved transparency. On the other hand, due to its
rougher surface and, hence, more intensive light scattering, the ZnO:Ni 0.4 films exhibit a lower
transmittance in the visible range. The conclusion we draw from the AFM study is that Ni-doping
affect substantially the film surface morphology. 4. Conclusions The work presented demonstrates that the sol-gel approach is a successful simple technology for
depositing Ni-doped ZnO films. The ZnO:Ni films thus prepared are uniform, smooth, and
polycrystalline with a wurtzite crystal phase. The FTIR analysis shows that Ni-doping changes the
characteristic light absorption features. The transparency is improved by Ni incorporation and the
optical bandgap of the ZnO:Ni films is slightly wider than that for ZnO films. 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 TAnnealing [oC]
ZnO
ZnO/NiO 0.4
ZnO/NiO 1
ZnO
ZnO/NiO 0.4
ZnO/NiO 1
Eg [eV]
T [%] at λ=550 nm
300oC
3,29
3,31
3,32
78,4
81,9
85,2
400oC
3,29
3,29
3,29
80,2
82,0
84,1
500oC
3,28
3,27
3,28
75,3
78,1
82,5
600oC
3,27
3,28
3,28
72,3
75,5
78,1
700oC
3,28
3,28
3,28
67,0
72,1
77,9
800oC
3,27
3,28
3,28
65,6
73,6
74,6 4 20th International Summer School on Vacuum, Electron and Ion Technologies 1234567890 ‘’“”
20th International Summer School on Vacuum, Electron and Ion Technologies
IOP Publishing
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 ZnO:Ni films are slightly higher than the corresponding ZnO values. The estimated Eg values are
within the range of the data reported previously [4, 16]. Figure 5. AFM micro-
graphs of ZnO:Ni 0.4
and ZnO:Ni 1 films
treated at 600 oC. The
inset images are 3D
micrographs. Figure 5 presents AFM micrographs of the ZnO:Ni 0.4 and ZnO:Ni 1 films treated at 600 oC (scan
area of 5×5 μm). The AFM study of a ZnO film performed at a scan area of 2×2 μm reveals a smooth
and uniform morphology with a grained film structure and an uneven size distribution. The grains are
well-packed, which results in a small r.m.s. roughness Rq = 5.115 nm for the undoped ZnO film (not
shown here). It is known that the roughness parameters are higher when the scan area is increased,
because longer-wavelength topography components are then sampled [17]. Thus, direct comparison
with the results for the ZnO:Ni films cannot be done. The AFM micrographs of the ZnO:Ni films
show different morphological features. Their average and r.m.s. roughness is given in Table 2. The
ZnO:Ni 0.4 film shows bigger grains with greater heights compared to the ZnO:Ni 1 films (see 3D
images, figure 5). On the other hand, the XRD analysis reveals that the ZnO:Ni 0.4 film annealed at
600 oC possesses smaller crystallites compared to ZnO:Ni 1, which can be a sign of micro-strain
broadening of the XRD peaks. ZnO:Ni films are slightly higher than the corresponding ZnO values. The estimated Eg values are
within the range of the data reported previously [4, 16]. Figure 5. AFM micro-
graphs of ZnO:Ni 0.4
and ZnO:Ni 1 films
treated at 600 oC. The
inset images are 3D
micrographs. Figure 5. AFM micro-
graphs of ZnO:Ni 0.4
and ZnO:Ni 1 films
treated at 600 oC. [3]
Kayani Z N, Naz F, Riaz S and Naseem Sh 2017 J. Saudi Chem. Soc. 21 425 e e e ces
[1]
Muniyandi I, Mani G K, Shankar P and Rayappan J B B 2014 Ceramics Inter. 40 7993
[2]
Chaitra U, Kekuda D and Rao K M 2017 Ceramics Inter. 43 7115
[3]
Kayani Z N, Naz F, Riaz S and Naseem Sh 2017 J. Saudi Chem. Soc. 21 425 [ ]
y
y pp
[2]
Chaitra U, Kekuda D and Rao K M 2017 Ceramics Inter. 43 7115 [2]
Chaitra U, Kekuda D and Rao K M 2017 Ceramics Inter. 43 7115
[3]
Kayani Z N, Naz F, Riaz S and Naseem Sh 2017 J. Saudi Chem. Soc. 21 425 ]
Muniyandi I, Mani G K, Shankar P and Rayappan J B B 2014 Ceramics Inter. 40 7993 References 5 20th International Summer School on Vacuum, Electron and Ion Technologies 1234567890 ‘’“”
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi :10.1088/1742-6596/992/1/012044 1234567890 ‘’“”
IOP Conf. Series: Journal of Physics: Conf. Series 992 (2018) 012044
doi : [4]
Mani G K and Rayappan J B B 2014 Appl. Surf. Sci. 311 405 y pp
pp
f
[5]
Ramesh J, Pasupathi G, Mariappan R, Kumar V S and Ponnuswamy V 2013 Optik 124 2 [6]
Montes-Valenzuela I, Romero-Paredes G, Vázquez-Agustín M A, Baca-Arroyo R and Peña-
Sierra R 2015 Mater. Sci. Semicond. Processing 37 185 g
[7]
Siddheswaran R, Netrvalová M, Savková J, Novák P, Očenášek P, Šutta P, Kováč Jr J and
Jayavel R 2015 J. Alloys Comp. 636 85 y
y
p
[8]
Srinet G, Kumar R and Sajal V 2013 J. Appl. Phys. 114 033912 [ ]
,
j
pp
y
[9]
Ivanova T, Harizanova A, Koutzarova T and Vertruyen B 2015 Proc. 15th Int. Conf. Nanotechnol. (IEEE-NANO 2015) 7388970 250 [9]
Ivanova T, Harizanova A, Koutzarova T and Ver
Nanotechnol. (IEEE-NANO 2015) 7388970 250 [10] Goswami N and Sahai A 2013 Mat. Res. Bulletin 48 346 [11] Tang A, Li X, Zhou Zh, Ouyang J and Yang H 2014 J. Alloys Comp. 600 204 [
]
g
y
g
g
y
p
[12] El-Kemary M, Nagy N and El-Mehasse I 2013 Mater. Sci. Semicond. Processing 16 174 [
]
y
gy
g
[13] Malika A N, Reddy A R, Babu K S and Reddy K V 2014 Ceramics Int. 40 12171 [14] Iordanescu C, Tenciu D, Feraru I, Kiss A, Bercu M, Savastru D, Notonier R and Grig
2001 Digest J. Nanomater. Biostr. 6 863 [15] Shinde K P, Pawar R C, Sinha B B, Kim H , Oh S and Chung K C 2014 Ceramics Int. 4 [16] Xu K, Liu C, Chen R, Fang X, Wu X and Liu J 2016 Physica B: Cond. Matter 502 155 [17] Elam J W, Sechrist Z A and George S M 2012 Thin Solid Films 414 43 17] Elam J W, Sechrist Z A and George S M 2 6 6
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https://openalex.org/W4309900092
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https://link.springer.com/content/pdf/10.1007/s12187-022-09993-7.pdf
|
English
| null |
Geographic, Socio-Demographic and School Type Variation in Adolescent Wellbeing and Mental Health and Links with Academic Competence in the United Arab Emirates
|
Child indicators research
| 2,022
|
cc-by
| 17,256
| ERROR: type should be string, got "https://doi.org/10.1007/s12187-022-09993-7\nChild Indicators Research (2023) 16:797-836 https://doi.org/10.1007/s12187-022-09993-7\nChild Indicators Research (2023) 16:797-836 Keywords Wellbeing · Life Satisfaction · Mental Health · Adolescence · United \nArab Emirates Geographic, Socio‑Demographic and School Type Variation \nin Adolescent Wellbeing and Mental Health and Links \nwith Academic Competence in the United Arab Emirates Jose Marquez1 · Louise Lambert2 · Megan Cutts1 Accepted: 6 November 2022 \n© The Author(s) 2022\n/ Published online: 23 November 2022 Accepted: 6 November 2022 \n© The Author(s) 2022\n/ Published online: 23 November 2022 *\t Jose Marquez \n\t\njose.marquez@manchester.ac.uk\nExtended author information available on the last page of the article *\t Jose Marquez \n\t\njose.marquez@manchester.ac.uk Extended author information available on the last page of the article *\t Jose Marquez 1 Introduction Global attention to the wellbeing and mental health of children and young people \nhas grown (Diener et al., 2018; Thapar et al., 2021). This is partly due to con-\ncerns about negative trends observed in many countries over the last two decades \n(Marquez & Long, 2020; McManus et al., 2019; Mojtabai et al., 2016) and more \nrecent concerns about the impact of the COVID-19 pandemic (Gavin et al., 2021; \nRacine et al., 2021; UNICEF, 2021). In the case of wellbeing, interest was originally driven by an increasing global \nrecognition that children have the right to be heard in all aspects affecting their \nlives (art. 12 UNCRC, United Nations, 1989). This interest was also driven by \nthe need to move beyond “well-becoming” approaches that are concerned with \nchildren’s future outcomes and towards wellbeing approaches that consider \nchildhood as a stage of significance in its own right and not as a mere journey to \nadulthood (Ben-Arieh, 2007). Still, future outcomes remain a key element motivating interest in child well-\nbeing, with concerns often focusing on the wellbeing of adolescents. A reason \nfor this is that adolescence has been identified as a window of opportunity for \nintervention as this is the time when wellbeing begins to decline (Casas and \nGonzález-Carrasco, 2019) and the prevalence of mental health issues increases \n(NHS Digital, 2018). Evidence indicates that most lifetime cases of mental ill \nhealth manifest by the age of 24, with a peak age of onset of 14.5 years (Colizzi \net al., 2020; Solmi et al., 2021). Further evidence indicates that wellbeing in \nadolescence is predictive of adult wellbeing, mental health, physical health and \nhealth behaviour, relational, as well as labour market and socioeconomic out-\ncomes (Cavioni et al., 2021; DeNeve & Oswald, 2012; Goodman et al., 2015; \nGuzmán et al., 2020; Kansky et al., 2016; Marquez et al., 2022a; Richards & \nHuppert, 2011). Research on the absence of a trade-off between wellbeing and \nacademic outcome (Gutman & Vorhaus, 2012; Lindorff, 2021; Shoshani et al., \n2016) has also helped advanced wellbeing promotion agendas in school contexts.i )\np\ng p\ng\nWellbeing has traditionally been conceptualised in two ways. In the first, \nhedonic or subjective wellbeing comprises a cognitive component (i.e., life sat-\nisfaction (LS)) and an affective component (i.e., experiencing positive and neg-\native moods, feelings and emotions) (Diener et al., 2002). Abstract Interest in adolescents’ wellbeing and mental health is growing worldwide, but little \nresearch in this area has been conducted in certain world regions and countries such \nas the United Arab Emirates (UAE). Geographic, socio-demographic and school \ntype differences in adolescent wellbeing and mental health are commonly observed \nin the field, and the UAE is a diverse country where these types of differences have \nbeen found for other outcomes (notably, academic). Yet, no prior national study has \nexplored these differences in terms of wellbeing and mental health in the nation. We address this gap by investigating differences across emirates, gender, socio-\neconomic status, immigrant status, school sector and school curriculum for overall \nlife satisfaction, positive affect, negative affect, meaning and purpose in life, and \ninternalizing difficulties. We use linear regression to analyse cross-sectional data \nfrom the Programme for International Student Assessment (PISA) study from \n2015 and 2018. We find substantial geographic, socio-demographic and school \ntype differences in levels (2018) of wellbeing and mental health -which vary \nacross distinct domains- and declines (2015–2018) of wellbeing. Better wellbeing \nand mental health are observed in the northern emirates and among boys. Better \nwellbeing and poorer mental health are observed among nationals (compared to \nexpatriates) and in public schools (compared to private schools). Despite presenting \nthe best academic outcomes, British schools present the worst wellbeing and \nmental health outcomes. However, results show the absence of a trade-off between \nacademic competence and wellbeing and mental health, with evidence of a small \npositive association with wellbeing. Keywords Wellbeing · Life Satisfaction · Mental Health · Adolescence · United \nArab Emirates 0123456789)\n1 3 456789)\n3 456789)\n3 J. Marquez et al. 798 1 Introduction This view concerns \nitself with how individuals experience and appraise their own lives. In the sec-\nond, eudaimonic or psychological wellbeing has been conceptualised in terms of \npurpose in life, environmental mastery, autonomy, personal growth, self-accept-\nance, positive relations with others, and optimism (Ryff et al., 2021) and is con-\ncerned with how individuals function and relate to others. In sum, although we \nacknowledge that the term wellbeing/well-being can be used to refer to a range of \noutcomes in different domains (including also objective measures of well-being \nsuch as health status, academic competence, etc.), in this study, we use the term \nwellbeing to refer to subjective/hedonic and psychological/eudaimonic wellbeing. Wellbeing is increasingly being studied in combination with mental health \nin research that, in most cases, defines this in terms of the presence of mental 3 1 3 Geographic, Socio‑Demographic and School Type Variation… 799 ill-health symptoms of internalizing difficulties (depression, anxiety, social anxi-\nety, somatic complaints, etc.) or, less commonly, externalizing difficulties (under-\ncontrolled, impulsive, or aggressive behaviour) (Otto et al., 2021; Petersen et al., \n2018; Trotta et al., 2020). Wellbeing and mental health have customarily been \nconceptualized as two ends of the same spectrum, but research suggests these are \nindependent constructs that, although related, have different correlates and may \nfollow different trajectories over the life course (Bohlmeijer & Westerhof, 2021; \nKinderman et al., 2015; Patalay & Fitzsimons, 2016, 2018; Sharpe et al., 2016; \nWesterhof & Keyes, 2010). y\nStudying wellbeing and mental health together can provide a more complete \npicture of the emotional and mental states of individuals. Yet, while much has \nbeen learned in the last decades about adolescent wellbeing and mental health in \nmany parts of the world, there is little in others. This is the case of the UAE, a high-\nincome nation in the Gulf Cooperation Council (GCC) region comprised of seven \nemirates and characterised by a diverse socio-demographic profile (See Fig. 1). The national population represents only around 10% of the nation, who differ from \nexpatriates (around 90%) in terms of linguistic, religious and cultural characteristics \n(Chan et al., 2021; Maalouf et al., 2019). This diversity is reflected in outcomes \nfor children and young people, including academic outcomes among a student \npopulation with almost three in four students enrolled in private schools (Buckner, \n2018; Marquez et al., 2022b). 1.1 \u0007Regional, Socio‑Demographic and School Type Variation in Adolescent \nWellbeing and Mental Health Research exploring geographic variation in adolescent wellbeing and mental health \nhas grown in recent years. Although there is a large number of studies investigat-\ning within-country variation (i.e., Islam et al., 2021; Schoon & Henseke, 2022; Van \nPhan and O’Brien, 2019), most research in this area focuses on cross-national dif-\nferences. Evidence from cross-national studies indicate substantial variation across \nsocio-cultural contexts and languages (Campbell et al., 2021; Marquez & Main, \n2020; Rees et al., 2015). For instance, in an international study of youth wellbeing \nacross 29 nations, Mutumba and Schulenberg (2020) found that national wellbeing \nvariations were in part explained by place of residence, finding higher subjective \nwellbeing in rural areas. Urban–rural differences in mental health and wellbeing \noutcomes have been well documented, with poorer mental health and wellbeing out-\ncomes noted in urban areas (i.e., Buttazzoni et al., 2022; Murphy et al., 2020). Wellbeing and mental health also vary across socio-demographic groups. For \nexample, there is evidence of wellbeing and mental health inequalities during child-\nhood and adolescence in relation to gender, however, the direction of the inequal-\nity shifts throughout development. Boys tend to report higher rates of mental health \ndifficulties than girls in both Western and Middle Eastern countries until puberty \n(Cyranowski, et al., 2000; NHS Digital, 2021; Patton & Viner, 2007), at which time \ngirls record double the rate until the age of 35 to 40 (Cyranowski et al., 2000; Pata-\nlay and Fitzsimons, 2018). In terms of gender differences in adolescent wellbeing, \ngirls tend to present worse outcomes than boys (Bradshaw et al., 2013; Campbell \net al., 2021; Kaye-Tzadok et al., 2017), but differences across domains are com-\nmon. For example, a meta-analysis found that overall LS (OLS) tends to be higher \namong boys, but satisfaction in some domains is higher among girls (e.g., satisfac-\ntion with family and school) and higher among males in others (e.g., satisfaction \nwith time-use, self) (Chen et al., 2019). Using PISA data from 73 nations, Campbell \net al. (2021) found that females reported worse outcomes on eudaimonic measures \nof meaning and purpose in life, psychological distress, OLS, and hedonic measures \nof positive affect. f\nLower socio-economic status (SES) has also been associated with lower LS \n(i.e., Chzhen et al., 2016; Elgar et al., 2017; Knies, 2022; Marquez, 2021; Patalay \n& Fitzsimons, 2018; Zaborskis et al., 2019a, b), although how it is measured \nmatters. 1 Introduction The current study advances our understanding of adolescent wellbeing and \nmental health inequalities in the UAE across emirates, socio-demographic groups \nand school types. Such studies are critical in developing and refining education Fig. 1 The UAE’s seven emirates Fig. 1 The UAE’s seven emirates Fig. 1 The UAE’s seven emirates 3 3 J. Marquez et al. 800 policies, making decisions around resource allocation and responsibilities. This can \nfacilitate that all young people enjoy life with the fewest mental health concerns and \nthe highest wellbeing possible, and that no region, school type or socio-demographic \ngroup is left behind. This study focuses on differences in levels of overall LS (OLS), \npositive affect, negative affect, meaning and purpose in life, and mental health \nsymptoms of internalizing difficulties in 2018, as well as declines in OLS between \n2015 and 2018. We also examine the association between academic competence and \nwellbeing and mental health outcomes. 1.1 \u0007Regional, Socio‑Demographic and School Type Variation in Adolescent \nWellbeing and Mental Health Associations tend to be more robust in studies using measures of material 1 3 Geographic, Socio‑Demographic and School Type Variation… 801 deprivation compared to family income (Gross-Manos, 2017; Main, 2014; Rees \net al., 2011; Sarriera et al., 2014; Zaborskis et al., 2019b) and even more so in \nstudies that use child-derived measures of material well-being compared to those \nderived by adults (Lau and Bradshaw, 2018; Main, 2014). Researchers have found \nthat children and young people with lower SES also have more mental health issues. A review of 52 studies by Reiss (2013) found a two-to-three-time higher likelihood \nof developing mental health issues at lower SES levels, with problems rising as SES \ndeclined. Meta-analyses (i.e., Peverill et al., 2021; Reiss et al., 2019) have confirmed \nthe same, although to varying degrees and for more granular reasons, i.e., parent \neducation (Merz et al., 2018), parent mental health issues (Amone-P’Olak et al., \n2011). Migration status is another variable associated with wellbeing, although the \nevidence is mixed. Most find that immigrant adolescents report lower wellbeing \nthan native students (Bradshaw et al., 2011; Casas et al., 2013; Marquez & Main, \n2020; Tang, 2019), but others find no differences or even differences in the opposite \ndirection (Filion et al., 2018). Contrasting findings seem to be explained by \ndifferences in the host country considered and what aspect of wellbeing is the focus \nof the study. For example, studies exploring academic and wellbeing outcomes have \nfound that native students have better academic performance, but higher (Wang, \n2021) or lower (Rodríguez et al., 2020) scores in positive affect, as well as equal \n(Alivernini et al., 2020; Rodríguez et al., 2020) or slightly lower (Wang, 2021) \nscores in LS. Where immigrant adolescents report lower wellbeing, differences \nare often explained by socio-economic characteristics, as well as friendships and \nloneliness, discrimination, religiosity, social participation, acculturation, and school \nrelated factors (e.g., language, bullying, school-related anxiety, fear of failure, grade \nrepetition, etc.) (Berry & Hou, 2017; Dryden-Peterson, 2018; Lee, 2019; Paparusso, \n2021; Schwartz et al., 2013). Differences in LS have been similarly explained: using \ndata from PISA 2015 across 48 countries and economies, Tang, (2019) found that \nimmigrant students reported lower LS, but the gap could be partly explained by \nfactors such as talking to parents, bullying and schoolwork-related anxiety. 1.1 \u0007Regional, Socio‑Demographic and School Type Variation in Adolescent \nWellbeing and Mental Health Research \nexploring migrant differences in adolescent wellbeing in the UAE is non-existent, \nand to the best of our knowledge, only one study has examined this for mental health \n(Shah et al., 2020; described below). Studying this question in the UAE is vital as \nthis is a nation where migrant gaps, particularly in academic outcomes (Marquez \net al., 2022b), emerge in the opposite direction compared to most countries.f Finally, wellbeing and mental health differences across types of schools is \nsubstantial. This is not surprising considering the role school factors play in \nexplaining variation in wellbeing (Marquez & Main, 2020; Marquez et al., 2022c; \nTaylor et al., 2022). In an international comparative study using data from PISA 2015, \nMarquez and Main (2020) found that in 15 of 31 countries, the LS of students differed \nacross types of schools (public, semi-private, private). After controlling for SES and \nother socio-demographic and school characteristics, LS was higher in private or \nsemi-private schools in six countries and lower in nine countries, suggesting that the \nwellbeing impact of attending private schools may differ by country. Studies in the \nUK have found that private schools offer no mental health and wellbeing advantages \n(Henderson et al., 2020; Sullivan et al., 2021), with evidence indicating that attending 1 3 J. Marquez et al. 802 private schools is associated with more psychological distress at age 16 for females \nand predicts it at the age of 42 (Sullivan et al., 2021). Liu and Zhao (2016) found that \nChinese youth attending private schools also reported lower LS, experiencing more \nracial discrimination than their public-school peers.f private schools is associated with more psychological distress at age 16 for females \nand predicts it at the age of 42 (Sullivan et al., 2021). Liu and Zhao (2016) found that \nChinese youth attending private schools also reported lower LS, experiencing more \nracial discrimination than their public-school peers.f While research suggests there is no trade-off between academic and wellbe-\ning outcomes (Gutman & Vorhaus, 2012; Lindorff, 2021; Shoshani et al., 2016), a \nstrong focus on academic performance may undermine wellbeing and mental health. 1.1 \u0007Regional, Socio‑Demographic and School Type Variation in Adolescent \nWellbeing and Mental Health A greater emphasis on exams and performance appears to contribute to lower mental \nhealth in girls (Giota & Gustafsson, 2017; Högberg et al., 2020; Long et al., 2021; \nSonmark et al., 2016) and youth from upper middle-class families in high achieving \nschools (Cho & Chan, 2020; Ebbert et al., 2019a, 2019b; Heller-Sahlgren, 2018; \nLuthar et al., 2020). Fear of failure in high-achieving youth from economically pros-\nperous nations has also been linked to lower LS and wellbeing (Borgonovi & Han, \n2021; Govorova et al., 2020; Wang, 2021). 1 3 1.2 \u0007Mental Health and Wellbeing in the United Arab Emirates Few studies exist relative to youth mental health and wellbeing in the UAE and Gulf \nregion (Alzahrani, 2020; Chan et al., 2021; Lansford et al., 2019; Maalouf et al., \n2019), however, a picture is emerging. Across the Eastern Mediterranean Region \n(EMR), the WHO classification for the Middle East/North Africa, Charara et al., \n(2017, 2018) estimated that depression and anxiety were the third and ninth lead-\ning cause of nonfatal burden of disease. Most nations had higher burdens of mental \ndisorders than global means, particularly for women, with anxiety disorders peak-\ning between 15 and 19 years of age, conduct disorders and ADHD at 10 to 14, and \ndepressive disorders at 40 to 44 years. The increase in burden of mental health was \nmost marked in the Gulf nations, including the UAE. Recently, Chan et al. (2021) \ncalculated pooled prevalence rates for depression (between 26 and 46%) and anxi-\nety (between 17 and 57%) in the Gulf region’s youth, noting their upper limits were \nabove global rates. A study conducted in the UAE’s city of Al Ain on 600 students between the \nages of 12 and 18 found that depressive symptoms were reported in 17% of them \n(Shah et al., 2020), with rates highest in South Asian expatriates, where one in three \nreported depressive symptoms, followed by one in five Emirati nationals. Western \nexpatriate students reported half the rates, but all groups had higher than global \naverages. In another UAE study, Al-Yateem et al., (2020) found that females had \ndouble the rate of anxiety and that low to middle SES youth had greater anxiety \nthan those at the higher end. In terms of wellbeing in childhood or adolescence, \nrecent international studies (OECD, 2017, 2019; Marquez & Main, 2020; Marquez \n& Long, 2020) have provided insights with evidence indicating that between 2015 \nand 2018, the UAE was among the top third countries where the OLS of 15-year-old \nadolescents had declined the most. Potentially related, research on academic outcomes reveal important socio-demo-\ngraphic inequalities in adolescents outcomes in the UAE. For instance, Buckner \n(2018) found that low-income Emirati nationals had worse academic performance 1 3 Geographic, Socio‑Demographic and School Type Variation… 803 on the 2015 and 2018 PISA exams (OECD, 2019) than their higher income peers, \nwith the influence of SES on academic achievement stronger in the UAE than \nelsewhere. 1.2 \u0007Mental Health and Wellbeing in the United Arab Emirates In addition to the three-year private–public academic performance gap \n(Marquez et al., 2022a, b, c), there is also a gender gap. Nationally, girls have out-\nperformed boys in all PISA subjects since 2012 and this gap is also larger than any-\nwhere else (OECD, 2019).i The present analysis, the first of its kind, has special relevance and supports \nthe UAE’s dedication to promoting higher wellbeing across the nation. This is \nespecially relevant for national prosperity as difficulties in childhood predict similar \nissues in adulthood (Auerbach et al., 2018; Johnson et al., 2018; Otto et al., 2021; \nTrotta et al., 2020). Thus, to lay the groundwork for the present analysis, as well as \nfuture studies on how existing inequalities (i.e., wealth, gender) potentially influence \nwellbeing and mental health, we address the following questions: 1. How does the wellbeing and mental health of adolescents in the UAE differ across \nthe nation’s emirates (geographical regions within the country), socio-demographic \ngroups and school types? 2. Do these inequalities vary across distinct domains of wellbeing and mental \nhealth?f 3. Is there a trade-off between academic competence and wellbeing and mental \nhealth outcomes in the UAE? 4. Did the decline in wellbeing between 2015 and 2018 manifest similar group \ninequalities? 2.1 \u0007Participants The sample included 19,277 15-year-old students who participated in the PISA \nstudy 2018 in the UAE. The PISA 2015 sample, used only in Sect. 3.4 (research \nquestion 4), included 13,643. Information about the number of participants across \neach socio-demographic group is reported in Sect. 2.2. For information about the \nPISA study, see PISA’s technical report (OECD, 2018). 2.2 \u0007Measures Our dependent variables included measures of cognitive subjective/hedonic well-\nbeing (OLS), affective subjective/hedonic wellbeing (positive affect, and negative \naffect), psychological/eudaimonic wellbeing (meaning and purpose in life), and \nmental health (internalizing difficulties). Specifically: •\t OLS. Students were asked to rate their satisfaction with life overall on a scale \nwith 0 representing the lowest satisfaction possible and 10, the highest. Data \nwere missing for 9.39% of participants in PISA 2018 (3.98% in PISA 2015). •\t OLS. Students were asked to rate their satisfaction with life overall on a scale \nwith 0 representing the lowest satisfaction possible and 10, the highest. Data \nwere missing for 9.39% of participants in PISA 2018 (3.98% in PISA 2015). 1 3 J. Marquez et al. 804 •\t Positive affect. Participants were asked how often (never, rarely, sometimes, \nalways) they felt happy, lively, proud, joyful, cheerful. A scale was derived sum-\nming up scores from these five items (Cronbach’s alpha 0.8266; 11.38% missing \ndata).f •\t Negative affect. Students were asked how often (never, rarely, sometimes, \nalways) they felt scared, miserable, afraid, sad, cheerful. A scale was derived \nsumming up scores from these four items (Cronbach’s alpha 0.7912; 11.82% \nmissing data). •\t Meaning and purpose in life (Eudaimonic/Psychological well-being). This was \nmeasured by asking about the level of agreement (strongly disagree, disagree, \nagree, strongly agree) with the following: My life has clear meaning or purpose; \nI have discovered a satisfactory meaning in life; I have a clear sense of what \ngives meaning to my life. A scale was created by summing up scores (Cron-\nbach’s alpha 0.8611; 8.86% missing data).fi •\t mental health (internalizing difficulties). Participants were asked to report how \noften over the preceding six months (rarely or never, about every month, about \nevery week, more than once a week, about every day) they had experienced \nsomatic symptoms and symptoms of mental health difficulties: headache, stom-\nach pain, back pain, feeling depressed, irritability of bad temper, feeling nervous, \ndifficulties in getting to sleep, feeling dizzy, feeling anxious. A scale was created \nby summing up scores in each item (Cronbach’s alpha 0.8957; 16.71% missing \ndata). Our independent variables included the following socio-demographic groups: Our independent variables included the following socio-demographic groups •\t Gender. This is a binary variable (0 male, 1 female). The sample includes 49% \nboys and 51% girls in both PISA 2018 and PISA 2015. •\t Gender. This is a binary variable (0 male, 1 female). 2.3 \u0007Analytical Approach To answer our research questions, we conducted linear regression analyses using \nmaximum likelihood estimation. This is the recommended approach to analyse PISA \ndata (OECD, 2009). We also followed recommendations with regards to PISA’s \ncomplex design (OECD, 2018) and the need to account for the probability of the \nstudent being selected to participate in the study. To do this, we used PISA’s rep-\nlicate weights. A sequential approach to estimating these linear regression models \nwas used to examine geographic (emirate) differences, socio-demographic (gender, \nnational/expatriate, SES) differences, and differences across types of schools. This \napproach involved the following steps. For each dependent variable, we ran a series \nof linear regression models, sequentially introducing each independent variable. For \nOLS, first, we used the original PISA 0–10 scale first (Table 1 in Sect. 3.1). Next, in \nSect. 3.2, in order to compare across different wellbeing and mental health scales, we \ncreated a standardized version of the scales on OLS, positive affect, negative affect, \nmeaning and purpose in life, and internalizing difficulties (Table 2 in Sect. 3.2. and \nTables 6, 7, 8, 9 and 10 in Appendix 1). Next, in Sect. 3.3, to consider academic \ncompetence (i.e. average PISA 2018 score in reading, maths, and science) and its \nlinks to wellbeing and mental health, we created an additional model incorporating \nthis variable (Table 3 in Sect. 3.3., and Tables 6, 7, 8, 9 to 10 in Appendix 1). Finally, \nto estimate changes in OLS between 2015 and 2018 in Sect. 3.4, we used a sample \nincluding all the UAE students participating in PISA 2015 and 2018. For each socio-\ndemographic group, we ran a regression model with OLS as the dependent variable \nand cohort (i.e., the year in which the PISA test was taken) as the only independent \nvariable in the model (Table 5 in Sect. 3.4). OLS was the only wellbeing and mental \nhealth variable collected in 2015 and 2018. Levels of missing data were relatively \nlow (see details Sect. 2.2.). We use listwise deletion to deal with missing data, which \nis common in analyses using this data set (Schirripa et al., 2020; Tsai et al., 2018; van \nHek et al., 2018). Analyses were conducted in STATA 15 (StataCorp, 2017). 2.2 \u0007Measures This is a categorical variable including Abu Dhabi (33.46% in PISA \n2018; 25.07% in PISA 2015), Dubai (31.82%; 41.76%), Sharjah (10.98%; \n7.30%), Ajman (7.13%; 5.51%), Umm Al Quwain (UAQ, 2.71%; 3.19%), Ras al \nKhaimah (RAK, 8.56%; 8.33%), Fujairah (5.34%; 8.83%). •\t Emirate. This is a categorical variable including Abu Dhabi (33.46% in PISA \n2018; 25.07% in PISA 2015), Dubai (31.82%; 41.76%), Sharjah (10.98%; \n7.30%), Ajman (7.13%; 5.51%), Umm Al Quwain (UAQ, 2.71%; 3.19%), Ras al \nKhaimah (RAK, 8.56%; 8.33%), Fujairah (5.34%; 8.83%). 2.2 \u0007Measures The sample includes 49% \nboys and 51% girls in both PISA 2018 and PISA 2015. •\t Socio-economic status. PISA’s index of family wealth (see PISA’s technical \nreport: OECD, 2018) was standardized, so that 0 indicates the mean in the UAE \nand 1, the standard deviation. •\t Immigrant background. We created this binary variable by assigning a value of \n0 to nationals (i.e., students born in the UAE and whose father and mother were \nalso born in the UAE) and a value of 1 to expatriates (students who were not \nborn in the UAE or whose father or mother was not born in the UAE). In our \nsample, 44% of participants are nationals and 56% are expatriates (45% and 55% \nrespectively in PISA 2015). •\t School sector. We derived a binary variable that assigns a value of 0 if the stu-\ndent is attending a public school (i.e., publicly operated and funded; 40% of par-\nticipants in PISA 2018, 42% in PISA 2015) and 1 if the student is attending a \nprivate school (i.e., privately operated and funded; 60% of participants in PISA \n2018, 58% in PISA 2015). •\t School curriculum. This is a categorical variable with the following categories: \nMinistry of Education (MoE, 50.08% in PISA 2018; 49.82% in PISA 2015), \nBritish (14.03%; 11.38%), American (13.20%; no data in PISA 2015), Indian \n(12.09%; 4.76%), Technical and Vocational (TVET, 4.30%; no data in PISA \n2015), Other (6.30%; 34.04%). The proportion of students attending private 1 3 1 3 Geographic, Socio‑Demographic and School Type Variation… 805 schools in each type of school is: MoE (22.22%; 28.29%), British (96.33%; \n100%), American (98.73%; no data in PISA 2015), Indian (100%; 82.04%), \nTVET (0%; no data in PISA 2015) Other (97 08%; 95 95%) schools in each type of school is: MoE (22.22%; 28.29%), British (96.33%; \n100%), American (98.73%; no data in PISA 2015), Indian (100%; 82.04%), \nTVET (0%\nd t i PISA 2015) Oth\n(97 08% 95 95%) schools in each type of school is: MoE (22.22%; 28.29%), British (96.33%; \n100%), American (98.73%; no data in PISA 2015), Indian (100%; 82.04%), \nTVET (0%; no data in PISA 2015), Other (97.08%; 95.95%). schools in each type of school is: MoE (22.22%; 28.29%), British (96.33%; \n100%), American (98.73%; no data in PISA 2015), Indian (100%; 82.04%), \nTVET (0%; no data in PISA 2015), Other (97.08%; 95.95%). •\t Emirate. 3.1 \u0007Socio‑Demographic Differences in Overall Life Satisfaction The average OLS score of 15-year-old students in the UAE in 2018 was 6.88, \nbut results from Table 1 show significant geographic variation. Compared to Abu 3 3 806 J. Marquez et al. 3.1 \u0007Socio‑Demographic Differences in Overall Life Satisfaction 3\nTable 1 Output from linear regression models: socio-demographic differences in OLS from 0 to 10\nStandard errors (se); ***p<0 01 **p<0 05 *p<0 1\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nB\nse\nB\nse\nb\nse\nB\nse\nb\nse\nb\nse\nConstant\n6.842***\n0.059\n7.052***\n0.073\n7.228***\n0.082\n7.142***\n0.088\n7.269***\n0.099\n7.440***\n0.077\nEmirate (ref.category: Abu Dhabi): Dubai\n-0.123\n0.083\n-0.130\n0.077\n-0.072\n0.078\n-0.101\n0.080\n0.001\n0.087\n0.131\n0.079\nEmirate (ref.category: Abu Dhabi): Sharjah\n-0.128\n0.172\n-0.107\n0.159\n-0.075\n0.156\n-0.054\n0.160\n-0.023\n0.155\n-0.034\n0.122\nEmirate (ref.category: Abu Dhabi): Ajman\n0.420**\n0.133\n0.421**\n0.141\n0.432**\n0.142\n0.486***\n0.140\n0.516***\n0.153\n0.360**\n0.135\nEmirate (ref.category: Abu Dhabi): UAQ\n0.455**\n0.157\n0.466*\n0.184\n0.369\n0.194\n0.373*\n0.179\n0.311\n0.190\n0.320\n0.191\nEmirate (ref.category: Abu Dhabi): RAK\n0.652***\n0.187\n0.645***\n0.191\n0.581**\n0.191\n0.587**\n0.190\n0.517**\n0.197\n0.387*\n0.182\nEmirate (ref.category: Abu Dhabi): Fujairah\n0.808***\n0.172\n0.803***\n0.187\n0.725***\n0.189\n0.727***\n0.187\n0.646***\n0.193\n0.606***\n0.176\nFemale\n-0.402***\n0.082\n-0.422***\n0.082\n-0.437***\n0.083\n-0.473***\n0.085\n-0.462***\n0.063\nExpatriate\n-0.310***\n0.067\n-0.148*\n0.073\n0.063\n0.076\n0.118\n0.071\nSocio-economic status\n0.226***\n0.038\n0.230***\n0.038\n0.331***\n0.035\nPrivate school\n-0.425***\n0.095\nSchool type (ref.category: MoE): British\n-1.382***\n0.132\nSchool type (ref.category: MoE): American\n-0.884***\n0.096\nSchool type (ref.category: MoE): Indian\n-0.400**\n0.129\nSchool type (ref.category: MoE): TVET\n-1.656***\n0.140\nSchool type (ref.category: MoE): Other\n-0.665***\n0.139\nObservations\n17,467\n17,467\n16,960\n16,941\n16,326\n16,941 Standard errors (se); ***p < 0.01, **p < 0.05, *p < 0.1 1 Geographic, Socio‑Demographic and School Type Variation… 807 1 3\nTable 2 Output from linear regression analysis (models 5 and 6): socio-demographic differences in OLS, positive affects, negative affects, meaning and purpose in life, \nand internalizing difficulties (standardized scales)\nStandard errors (se); ***p<0 01 **p<0 05 *p<0 1\nVariable\nOLS\nPositive affects\nNegative affects\nMeaning and purpose \nin life\nInternalizing dif-\nficulties\nB\nSe\nB\nSe\nb\nSe\nb\nse\nb\nse\nConstant\n0.173***\n0.027\n-0.024\n0.025\n-0.408***\n0.031\n0.123***\n0.027\n0.006\n0.035\nEmirate (ref.category: Abu Dhabi): Dubai\n0.046\n0.028\n0.050*\n0.025\n-0.034\n0.027\n0.032\n0.031\n-0.009\n0.031\nEmirate (ref.category: Abu Dhabi): Sharjah\n-0.012\n0.043\n0.063\n0.039\n-0.017\n0.049\n0.071\n0.043\n0.003\n0.038\nEmirate (ref.category: Abu Dhabi): Ajman\n0.126**\n0.047\n0.141***\n0.039\n-0.109**\n0.037\n0.141***\n0.040\n-0.135***\n0.038\nEmirate (ref.category: Abu Dhabi): UAQ\n0.112\n0.067\n0.091\n0.056\n-0.020\n0.066\n0.096\n0.063\n-0.048\n0.038\nEmirate (ref.category: Abu Dhabi): RAK\n0.136*\n0.064\n0.107*\n0.049\n-0.061\n0.057\n0.093*\n0.047\n-0.149**\n0.053\nEmirate (ref.category: Abu Dhabi): Fujairah\n0.213***\n0.062\n0.179***\n0.047\n-0.087\n0.053\n0.263***\n0.050\n-0.159**\n0.058\nFemale\n-0.162***\n0.022\n0.071***\n0.020\n0.348***\n0.023\n0.005\n0.022\n0.383***\n0.023\nExpatriate\n0.041\n0.025\n0.063*\n0.028\n-0.015\n0.024\n0.089***\n0.025\n-0.073**\n0.028\nSocio-economic status\n0.117***\n0.012\n0.148***\n0.012\n-0.081***\n0.014\n0.100***\n0.013\n-0.025\n0.013\nPrivate school\n-0.149***\n0.033\n-0.082**\n0.028\n0.354***\n0.036\n-0.259***\n0.034\n-0.177***\n0.032\nSchool type (ref.category: MoE): British\n-0.486***\n0.046\n-0.331***\n0.044\n0.606***\n0.057\n-0.616***\n0.059\n-0.118**\n0.038\nSchool type (ref.category: MoE): American\n-0.311***\n0.034\n-0.173***\n0.033\n0.483***\n0.034\n-0.407***\n0.036\n-0.138***\n0.040\nSchool type (ref.category: MoE): Indian\n-0.141**\n0.045\n0.033\n0.040\n0.519***\n0.035\n-0.233***\n0.038\n-0.497***\n0.042\nSchool type (ref.category: MoE): TVET\n-0.582***\n0.049\n-0.308***\n0.068\n0.462***\n0.064\n-0.477***\n0.058\n-0.017\n0.050\nSchool type (ref.category: MoE): Other\n-0.234***\n0.049\n-0.124**\n0.042\n0.513***\n0.048\n-0.473***\n0.048\n-0.297***\n0.037\nObservations\n16,941\n16,579\n16,512\n17,034\n15,589 J. 1 3 3.2 \u0007Socio‑Demographic Differences Across Wellbeing and Mental Health \nDomains To compare socio-demographic variations across wellbeing and mental health \ndomains, we conducted the same analysis as in Sect. 3.1. for standardized scales of \nOLS, positive affect, negative affect, meaning and purpose in life, and mental health \n(internalizing difficulties). A summary of this comparative analysis is presented in \nTable 2, which shows the results of the final model (i.e., model 5 for public/private \nschool and model 6 for all other independent variables) for each wellbeing and men-\ntal health variable. Results for models 1 to 6 for each of these standardized scales is \npresented in tables 6, 7, 8, 9 to 10 in Appendix 1. The northern, less-urbanized emirates of Ajman, RAK and Fujairah presented the \nbest wellbeing and mental health outcomes, although for negative affect, this advan-\ntage becomes less clear in RAK and Fujairah when controlling for school sector and \ncurriculum. There are no statistically significant differences in wellbeing and men-\ntal health between Abu Dhabi and Sharjah, whereas compared to Abu Dhabi, UAQ \npresented higher meaning and purpose in life, positive affect and OLS (in model 4, \n0.143, 0.110, and 0.131 s.d. respectively), but these differences become statistically \nnon-significant after controlling for school sector and school curriculum in models \n5 and 6. Finally, compared to Abu Dhabi, Dubai presented lower levels of meaning \nand purpose in life as well as higher levels of negative affect and internalizing dif-\nficulties, although these differences reduce substantially or become non-statistically \nsignificant when controlling for school sector and curriculum. i\nFurther, females reported similar levels of meaning and purpose in life to males, \nslightly higher positive affect (0.071 s.d. in model 6), lower OLS (-0.162 s.d. in \nmodel 6), and much higher levels of negative affect (0.348 s.d. in model 6) and \ninternalizing difficulties (0.383 s.d. in model 6). The gender gap in wellbeing and \nmental health hardly changes after controlling for socio-economic status, immigrant \nstatus and school sector/curriculum.fi As to SES, the relationship with mental health (internalizing difficulties) was not \nstatistically significant, but links with wellbeing emerged. An increase of one stand-\nard deviation in the socioeconomic status scale was associated with higher OLS \n(0.117 s.d. in model 6), higher meaning and purpose in life (0.100 s.d. in model 6), \nhigher positive affect (0.148 s.d. in model 6), and lower negative affect (-0.081 s.d. in model 6). 3.1 \u0007Socio‑Demographic Differences in Overall Life Satisfaction 1 3 1 3 Geographic, Socio‑Demographic and School Type Variation… 809 Compared to schools following the MoE curriculum, OLS was substantially lower \nin TVET schools (-1.656 points), British schools (-1.382 points), American schools \n(-0.884 points), schools following other curriculums (-0.665 points), and Indian \nschools (-0.400 points). 3.1 \u0007Socio‑Demographic Differences in Overall Life Satisfaction Marquez et al. 808 Table 3 Group differences in the average PISA 2018 score (readings, maths, science)\nStandard errors (se); ***p < 0.01, **p < 0.05, *p < 0.1\nVariable\nAverage PISA 2018 score \n(reading, maths, science)\nDifference\nb\nse\nGroup comparison\nAbu Dhabi\n410\nDubai\n484\n74***\n7.1\nAbu Dhabi—Dubai\nSharjah\n442\n32***\n8.6\nAbu Dhabi—Sharjah\nAjman\n400\n-10\n7.8\nAbu Dhabi—Ajman\nUAQ\n397\n-13\n10.1\nAbu Dhabi—UAQ\nRAK\n402\n-8\n7.2\nAbu Dhabi—RAK\nFujairah\n398\n-12\n9.0\nAbu Dhabi—Fujairah\nMale\n418\nFemale\n449\n31***\n6.1\nMale—Female\nNational\n389\nExpatriate\n476\n87***\n3.5\nNational—Expatriate\nSocio-economic status\n-\n-1\n1.5\nSocio-economic status\nPublic school\n379\nPrivate school\n469\n90***\n4.8\nPublic school—Private school\nMoE\n388\nBritish\n510\n122***\n5.6\nMoE—British\nAmerican\n445\n57***\n8.6\nMoE—American\nIndian\n494\n106***\n6.8\nMoE—Indian\nTVET\n397\n9\n11.6\nMoE—TVET\nOther\n484\n96***\n13.7\nMoE—Other Table 3 Group differences in the average PISA 2018 score (readings, maths, science) Dhabi, OLS was not statistically different in Dubai and Sharjah, but it was higher \nin the northern, less-urbanized emirates of Ajman, UAQ, RAK and Fujairah. Such differences decrease slightly (and become non-statistically significant for \nUAQ) after controlling for national/expatriate status, and again after controlling \nfor school sector (public/private) and curriculum, although they remain substan-\ntial, ranging from 0.360 points in Ajman to 0.606 points in Fujairah. Further, females reported almost a half-point lower OLS than males (i.e., \n-0.462 points lower in the final model 5 controlling for all other socio-demo-\ngraphic variables). Expatriates reported lower OLS than nationals (-0.310 points \nin model 3), but this gap is half the size after controlling for socio-economic sta-\ntus (-0.148 points in model 4) and becomes non-statistically significant after con-\ntrolling for school sector and curriculum. Socioeconomic status was positively \nassociated with OLS. In the final model 6 with all the control variables included \nan increase of 1 standard deviation in the index of socioeconomic status was \nassociated with an increase of 0.331 points in the 0 to 10 OLS scale. Students attending private schools also reported lower OLS (-0.425 points) than \ntheir peers attending public schools after controlling for all socio-demographic \ncharacteristics (model 6). Differences across school curriculums were even greater. 3.3 \u0007Wellbeing and Mental Health and Academic Outcomes: Is there a Trade‑Off? Considering the results reported in Sect. 3.2 with findings on academic competence \nin the UAE (scores in PISA 2018) reported in Marquez et al. (2022b), some groups \nof students who do better academically seem to present worse wellbeing outcomes. To illustrate, Table 3 presents PISA 2018 score inequalities (average for reading, \nmaths, and science) across our groups of interest. The average PISA score across \nthe UAE was 434 points, and the s.d. 100 points. Table 3 shows that academic \ncompetence is highest in Dubai (484 points on average) and lowest in the northern \nemirates (around 400 points); higher among females (449 points) compared to \nmales (418 points); higher among expatriate students (476 points) compared to \nnational students (389 points); and higher in private schools (469 points) versus \npublic schools (379 points). Academic competence is highest in British schools \n(510 points), followed by Indian schools (494 points), Other schools (484 points), \nAmerican schools (445 points), TVET schools (397), and MoE schools (388 points). Considering the results reported in Sect. 3.2 with findings on academic competence \nin the UAE (scores in PISA 2018) reported in Marquez et al. (2022b), some groups \nof students who do better academically seem to present worse wellbeing outcomes. To illustrate, Table 3 presents PISA 2018 score inequalities (average for reading, \nmaths, and science) across our groups of interest. The average PISA score across \nthe UAE was 434 points, and the s.d. 100 points. Table 3 shows that academic \ncompetence is highest in Dubai (484 points on average) and lowest in the northern \nemirates (around 400 points); higher among females (449 points) compared to \nmales (418 points); higher among expatriate students (476 points) compared to \nnational students (389 points); and higher in private schools (469 points) versus \npublic schools (379 points). Academic competence is highest in British schools \n(510 points), followed by Indian schools (494 points), Other schools (484 points), \nAmerican schools (445 points), TVET schools (397), and MoE schools (388 points). Given that some groups present higher academic competence but lower wellbeing \noutcomes (e.g. females, expatriates, students from Dubai, students attending private \nschools, especially British schools, etc.), we are compelled to ask whether there is a \ntrade-off between academic competence and wellbeing in the UAE? We explored \nthis question estimating an additional model 7 (Tables 6, 7, 8, 9 to 10 in Appendix 1). 3.2 \u0007Socio‑Demographic Differences Across Wellbeing and Mental Health \nDomains The size of the association between socio-economic status and these \nwellbeing variables are similar in models 4 and 5 (i.e., after accounting for school \nsector), but increase substantially in model 6 (i.e., after accounting for school \ncurriculum). Compared to national students, expatriate students reported lower wellbeing (in \nmodel 3, the gaps are -0.109 s.d. for OLS, 0.226 s.d. for negative affect, -0.067 s.d. 1 3 810 J. Marquez et al. for positive affect, and -0.108 s.d. for meaning and purpose in life), but also lower \nlevels of internalizing difficulties (in model 3, -0.196 s.d.). Yet, after controlling for \nsocio-economic status, the wellbeing gaps reduce substantially (and become non-\nstatistically significant in the case of positive affect), and after controlling for school \nsector and curriculum, they become non-statistically significant for OLS and nega-\ntive affect, and reverse direction for meaning and purpose in life (0.089 s.d. in model \n6) and positive affect (0.063 s.d. in model 6). For internalizing difficulties, the gap \nremains similar after controlling for SES and reduces substantially when controlling \nfor school sector and curriculum. Finally, attending private schools is associated with lower OLS (-0.149 s.d.), \nlower meaning and purpose in life (-0.259 s.d.), higher negative affect (0.354 s.d.) \nand lower positive affect (-0.082 s.d.), but also lower internalizing difficulties \n(-0.177 s.d.). Differences across school curriculum are notable. Overall, MoE \nschools present better wellbeing outcomes compared to any other type of school \n(gaps are especially large for negative affect and meaning and purpose in life) and \nsomewhat worse outcomes for internalizing difficulties (although differences are not \nstatistically significant for TVET). In terms of wellbeing, British schools and TVET \nschools present the poorest outcomes, although these differences are less clear for \nnegative affect. In terms of internalizing difficulties, Indian schools present better \noutcomes than any other type. 3.3 \u0007Wellbeing and Mental Health and Academic Outcomes: Is there a Trade‑Off? Model 7 contains the same independent variables as model 6 but adds academic \ncompetence (i.e. average PISA 2018 score in reading, maths, and science). The results \nare summarized in Table 4. (\np\n)\n(\n)\n(\np\n)\nGiven that some groups present higher academic competence but lower wellbeing \noutcomes (e.g. females, expatriates, students from Dubai, students attending private \nschools, especially British schools, etc.), we are compelled to ask whether there is a \ntrade-off between academic competence and wellbeing in the UAE? We explored \nthis question estimating an additional model 7 (Tables 6, 7, 8, 9 to 10 in Appendix 1). Model 7 contains the same independent variables as model 6 but adds academic \ncompetence (i.e. average PISA 2018 score in reading, maths, and science). The results \nare summarized in Table 4. 1 3 Geographic, Socio‑Demographic and School Type Variation… 811 Results show that an increase of 100 points (1 s.d.) on average in the three core \nPISA subjects (reading, maths, and science) is associated with small increases in OLS \n(0.024 s.d.) and positive affect (0.075 s.d.), as well as a small decrease in negative affect \n(-0.047 s.d.). Effects are not statistically significant for meaning and purpose in life and \ninternalizing difficulties. A comparisson between results in models 6 in Table 2 and \nmodels 7 in Table 4 also show that, when adding academic competence to the mod-\nels, the other estimates barely change in most cases, although some small changes are \nobserved in the positive affect and negative affect models for school type and emirate. 3.4 \u0007Socio‑Demographic Differences in Changes to Overall Life Satisfaction \nbetween 2015 and 2018 In view of socio-demographic differences in levels of wellbeing and mental health \nin 2018, we explored whether declines in student OLS between 2015 and 2018 also \nfollowed different patterns across distinct socio-demographic groups. OLS was the only \nwellbeing variable collected in both PISA waves. Table 5 shows how OLS changed on \naverage for each socio-demographic group between 2015 and 2018. On average across \nthe UAE, OLS declined by 0.42 points on the 0 to 10 scale, but this decline was more \npronounced for some groups. The decline was largest in Sharjah (-0.55 points) and \nAbu Dhabi (-0.50 points), followed by RAK (-0.42 points) and Dubai (-0.37 points). Changes were not statistically significant elsewhere. i\nThe decline in OLS was also greater among females (-0.49 points) compared to \nmales (-0.35 points), nationals (-0.48 points) compared to expatriates (-0.41 points), \nand students of higher socio-economic status. With respect to the latter, differences \nwere not statistically significant for those in the first, second, and third quintile, but \nwere substantial for those in the fourth (-0.51 points) and fifth quintile (-0.58 points). i\nFinally, the decline in OLS was similar in size in public schools (-0.43 points) \nand private schools (-0.45 points), but differences across schools following distinct \ncurriculums were indeed substantial. Changes were not statistically significant in MoE \nschools, but the decline in OLS was very large in British schools (-1.06 points). In Indian \nschools (-0.47 points) and schools following other curriculums (-0.45 points), this decline \nwas slightly above the national average. Information about American and TVET schools \nwas not included in PISA 2015, so no comparisons were possible for these schools. 4.1 \u0007Findings The present study revealed important geographic, socio-demographic, and school \ntype differences in trends and levels of adolescent wellbeing and mental health \nin the UAE. First, the northern emirates of Ajman, RAK and Fujairah presented \nbetter wellbeing and mental health outcomes, although these differences became \nsomewhat smaller when controlling for school sector and school type. In these \nemirates, there are fewer expatriate residents, private schools, and schools overall 3 812 J. Marquez et al. 4.1 \u0007Findings 3\nTable 4 Output from linear regression analysis (model 7): socio-demographic differences in OLS, positive affects, negative affects, meaning and purpose in life, and inter-\nnalizing difficulties (standardized scales), after adding academic competence as an additional independent variable\nVariable\nOLS\nPositive affects\nNegative affects\nMeaning and purpose \nin life\nInternalizing dif-\nficulties\nB\nSe\nB\nSe\nb\nSe\nb\nse\nb\nse\nConstant\n0.189***\n0.028\n0.026***\n0.025\n0.439\n0.032\n0.125\n0.027\n0.010\n0.034\nEmirate (ref.category: Abu Dhabi): Dubai\n0.039\n0.028\n0.028\n0.026\n-0.021\n0.028\n0.032\n0.031\n0.003\n0.030\nEmirate (ref.category: Abu Dhabi): Sharjah\n-0.014\n0.043\n0.055\n0.039\n-0.012\n0.048\n0.071\n0.043\n-0.006\n0.038\nEmirate (ref.category: Abu Dhabi): Ajman\n0.129*\n0.047\n0.147***\n0.038\n-0.112**\n0.036\n0.141***\n0.040\n0.137***\n0.038\nEmirate (ref.category: Abu Dhabi): UAQ\n0.112\n0.068\n0.089\n0.057\n-0.019\n0.065\n0.096\n0.063\n0.047\n0.037\nEmirate (ref.category: Abu Dhabi): RAK\n0.135*\n0.064\n0.102*\n0.048\n-0.058\n0.057\n0.092*\n0.046\n0.147**\n0.053\nEmirate (ref.category: Abu Dhabi): Fujairah\n0.212**\n0.062\n0.175***\n0.045\n-0.085\n0.053\n0.263***\n0.050\n0.158**\n0.058\nFemale\n-0.168***\n0.022\n0.054*\n0.020\n0.359***\n0.023\n0.005\n0.022\n-0.388***\n0.023\nExpatriate\n0.029\n0.026\n0.023\n0.029\n0.010\n0.025\n0.088***\n0.025\n0.061*\n0.027\nSocio-economic status\n0.114***\n0.012\n0.142***\n0.012\n-0.077***\n0.014\n0.100***\n0.013\n0.022\n0.013\nPrivate school\nSchool type (ref.category: MoE): British\n-0.505***\n0.046\n-0.391***\n0.043\n0.643***\n0.056\n-0.618***\n0.058\n-0.099*\n0.039\nSchool type (ref.category: MoE): American\n-0.321***\n0.034\n-0.205***\n0.035\n0.503***\n0.035\n-0.408***\n0.036\n-0.128**\n0.041\nSchool type (ref.category: MoE): Indian\n-0.157**\n0.046\n-0.018\n0.040\n0.551***\n0.035\n-0.234***\n0.039\n-0.481***\n0.043\nSchool type (ref.category: MoE): TVET\n-0.588***\n0.050\n-0.326***\n0.063\n0.472***\n0.062\n-0.478***\n0.058\n-0.012\n0.049\nSchool type (ref.category: MoE): Other\n-0.249***\n0.051\n-0.173***\n0.046\n0.544***\n0.051\n-0.474***\n0.050\n-0.281***\n0.038\nAcademic competence (Mean PISA score)\n0.024*\n0.012\n0.075***\n0.013\n-0.047***\n0.012\n0.002\n0.014\n0.024\n0.012\nObservations\n16,941\n16,941\n16,512\n17,034\n15,589 1 Geographic, Socio‑Demographic and School Type Variation… Geographic, Socio‑Demographic and School Type Variation… 813 Table 5 Output from linear \nregression models: socio-\ndemographic differences in \nchanges in OLS (from 0 to 10) \nbetween 2015 and 2018\nSatisfaction with life as a whole \n(0–10 LS scale)\nDifference\n2015\n2018\nB\ns.e\nUAE\n7.31\n6.88\n-0.42\n***\n0.05\nEmirate\nAbu Dhabi\n7.34\n6.84\n-0.50\n***\n0.08\nDubai\n7.09\n6.72\n-0.37\n***\n0.06\nSharjah\n7.27\n6.71\n-0.55\n**\n0.20\nAjman\n7.45\n7.26\n-0.18\n0.19\nUAQ\n7.32\n7.30\n-0.03\n0.21\nRAK\n7.91\n7.49\n-0.42\n***\n0.12\nFujairah\n7.66\n7.65\n-0.01\n0.14\nGender\nMale\n7.45\n7.10\n-0.35\n***\n0.07\nFemale\n7.18\n6.69\n-0.49\n***\n0.07\nImmigrant status\nNational\n7.60\n7.12\n-0.48\n***\n0.07\nExpatriate\n7.12\n6.71\n-0.41\n***\n0.07\nSES\nLowest SES\n6.68\n6.60\n-0.09\n0.13\nMid-low SES\n6.90\n6.71\n-0.19\n0.11\nMid SES\n6.87\n6.87\n-0.01\n0.11\nMid-high SES\n7.26\n6.74\n-0.51\n***\n0.11\nHighest SES\n7.64\n7.07\n-0.58\n***\n0.07\nSchool sector\nPublic school\n7.65\n7.22\n-0.43\n***\n0.06\nPrivate school\n7.12\n6.66\n-0.45\n***\n0.08\nSchool curriculum\nMoE\n7.44\n7.34\n-0.09\n0.05\nBritish\n7.03\n5.97\n-1.06\n***\n0.16\nAmerican\nIndian\n7.30\n6.83\n-0.47\n*\n0.20\nTVET\nOther\n7.24\n6.79\n-0.45\n***\n0.12 Table 5 Output from linear \nregression models: socio-\ndemographic differences in \nchanges in OLS (from 0 to 10) \nbetween 2015 and 2018 Satisfaction with life as a whole \n(0–10 LS scale) (Sheikh Saud bin Saqr Al Qasimi Foundation for Policy Research, 2020); hence, it \nmay be that schools suffer from fewer social comparisons to other schools like those \nranked in bigger urban centres. 4.1 \u0007Findings They may have a more homogenous student body \nand be able to instil a greater sense of belonging and/or identity. Their lead may also \nreflect the geographical advantage of rural areas to mid-size cities (i.e., Buttazzoni \net al., 2022; Murphy et al., 2020; Tomyn et al., 2017), the impact of lower urban \ndensity or quality of place and neighbourhood, or greater access to nature (Carmona, \n2019; Gorski-Steiner et al., 2022; Rentfrow, 2018; Silva et al., 2016; Vanaken & \nDanckaerts, 2018; Ward et al., 2016). In contrast, the emirate of Dubai presented notably lower scores for meaning and \npurpose, higher scores for negative affect and the greatest mental health issues of all \nthe emirates. This is a striking finding given that it is the only emirate to officially 1 3 J. Marquez et al. 814 endorse school wellbeing (OECD, 2021). Its education authority has promoted \nwellbeing as the cornerstone of its efforts and is an item against which schools are \ninspected and classed (see OECD, 2021). Perhaps its highly competitive, perfor-\nmance focused and publicized school ranking scheme which allows for parents to \nmake informed choices as well as school fees to be raised accordingly, has created \nunintended effects on wellbeing (Sheikh Saud bin Saqr Al Qasimi Foundation for \nPolicy Research, 2020; El-Sholkamyl & Al-Saleh, 2017; OECD, 2021). Still, the \nemirate has the highest number of students, private schools and diversity of all, mak-\ning its challenges greater. It also has higher than average rates of bullying (KHDA, \n2021; OECD, 2021), which may be driving the higher rates of mental health issues. Ranking best for academic performance, student anxiety and parental pressure is \nhigher than in other OECD countries and may also contribute (OECD, 2021). Tak-\ning stronger leadership in guiding wellbeing and mental health policy and program-\nming may be beyond its legislative reach and account for the disparity between its \nendorsement of wellbeing versus implementation. Females also reported more mental health issues, lower OLS, higher negative \naffect and somewhat higher positive affect. These findings are consistent with \nprior studies (Al-Yateem et al., 2020; Bradshaw et al., 2013; Campbell et al., 2021; \nCharara et al., 2017, 2018; Cyranowski et al., 2000; Hamama & Hamama-Raz, \n2021; Kaye-Tzadok et al., 2017). However, that no gender differences were observed \nfor meaning and purpose in life is inconsistent with findings by Campbell et al. 1 3 4.1 \u0007Findings The latter finding contrasts with a \nprevious UAE study highlighting higher rates of internalizing disorders (i.e., \ndepression) in South Asian students in the city of Al Ain (Shah et al., 2020). The \nlowest wellbeing is observed in TVET and British schools, and the highest in MoE \nschools, followed by Indian schools. American schools and schools following other \ncurriculums are somewhere in the middle in terms of wellbeing and mental health \nrelative to other types of schools. The low wellbeing of TVET schools may be due \nto their poor public image and status as students and their parents consider them \noptions for low performers (Khan et al., 2018). Why mental health issues are higher in lower performing schools (here, MoE \nschools) suggests school, community, or cultural factors might play a role. Mental \nhealth issues may be unidentified and hence, overly represented (Lansford et al., \n2019; Maalouf et al., 2019). Alternatively, they may be better tolerated. In Western \ncultures, there is pressure to reduce negative emotional experiences, whereas in \nmany Eastern cultures, distress is accepted and less “psychologized” (e.g., Eloul \net al., 2009; Huang et al., 2020). Thus, mental health issues are construed as an \ninconvenience and reflect a dialectical emotional state where they co-exist with and \nare less impactful on wellbeing than they might in other cultural contexts (Lomas, \n2016). Similarly, it may be a third force that is not being captured in the Western \nmeasures being used in PISA (although they have been regionally validated); \nthat is, the unity between mind, body, community, and spiritual aspects of the \nregion (Joshanloo et al., 2021), which has not yet been captured or investigated \nby education authorities either (OECD, 2021). School factors that produce low \nacademic performance may also fuel mental health concerns, especially as one \ndrives the other (Agnafors et al., 2021; Deighton et al., 2018; Pagerols et al., 2022; \nRiglin et al., 2014). Alternatively, issues identified in UAE public schools, i.e., non-\nnational teachers, low status of teaching, low teacher wellbeing, few resources, and \nlittle parent support (Marquez et al., 2022b) may contribute. In terms of wellbeing, how can the high wellbeing observed in low-performing \nMoE schools be explained? And why is wellbeing so low in high-performing Brit-\nish schools? This question deserves further exploration; however, our study suggests \nthat this is not related to academic outcomes. 4.1 \u0007Findings (2021), who find that in most countries, meaning and purpose in life among 15-year-\nold adolescents is higher in males. Gender differences were larger with regards to \nnegative affect and internalizing difficulties compared to positive affect and OLS.ii ffif\nOur findings also confirm a long-standing truism that for every increase in SES \ngradient, there are parallel increases in wellbeing (Chzhen et al., 2016; Elgar et al., \n2017; Knies, 2022; Marquez, 2021; Patalay & Fitzsimons, 2018; Zaborskis et al., \n2019a, b). However, higher SES was associated with more mental health difficulties, \nwhich contradicts findings from previous literature reviews (Reiss, 2013) and \nmeta-analyses (Peverill et al., 2021; Reiss et al., 2019), and seem to suggest that \nas resources stemming from greater income can buy experiences and time with \nwhich to enjoy life and find it satisfying, the same cannot always be said of those \nsame resources defending against mental health issues, like depression, anxiety, etc. (Killingsworth, 2021), which present a particularly high incidence (above global \nrates) in the GCC region (Chan et al., 2021). Findings around the national/expatriate gaps in wellbeing and mental health \nare compelling. First, expatriates reported lower wellbeing, but also fewer mental \nhealth issues than nationals. Second, after controlling for SES, the gap in wellbeing \nreduces, but the mental health gap remains. The fact that the national/expatriate gap \nin wellbeing, but not mental health, is partly explained by differences in SES aligns \nwith the point discussed above that SES (material well-being) tends to be associated \nwith better wellbeing outcomes but may not necessarily be associated with better \nmental health (see Mogilner et al., 2018), especially in a context of high incidence of \npopulation levels of mental health difficulties (Chan et al., 2021). After controlling \nfor school type (i.e., school sector, curriculum), the wellbeing gaps almost disappear \nfor OLS and negative affect, and reverse direction for meaning and purpose in life. 1 3 815 Geographic, Socio‑Demographic and School Type Variation… The mental health gap reduces as well. This suggests that schools indeed play a role \nin wellbeing and mental health as they explain part of the gaps across the emirates \nand a large proportion of the national/expatriate gaps. Compared to private schools, public schools present higher wellbeing but poorer \nmental health outcomes, and this holds true after controlling for socio-demographic \ncharacteristics. Similarly, the poorest mental health levels are observed in MoE and \nTVET schools, and the best in Indian schools. 4.1 \u0007Findings We found no evidence for a trade-off \nbetween academic and wellbeing and mental health outcomes. In fact, we found a \nsmall positive association with subjective wellbeing, especially with the affective \ncomponent. Higher academic competence was associated with higher OLS, higher \npositive affect, and lower negative affect –although for OLS, the size of the effect \nwas rather small and could potentially be the result of a method effect. Overall, this \nfinding is consistent with previous research reporting (Gutman & Vorhaus, 2012; \nLindorff, 2021; Shoshani et al., 2016). As happier students make better learners, 1 3 816 J. Marquez et al. schools and education authorities should promote both wellbeing and academic out-\ncomes (Taylor et al., 2022). To promote adolescent wellbeing, more research is needed to understand what pol-\nicy-relevant factors influence it. Research highlights factors such as high-stakes test-\ning, fear of failure, performance anxiety, and sense of belonging to school (Borgonovi \n& Han, 2021; Cho & Chan, 2020; Högberg et al., 2020; Long et al., 2021; Marquez & \nMain, 2020; Sonmark et al., 2016; Wang, 2021) are associated with lower wellbeing, \nwhich might explain some of our results. Further, British schools in the UAE achieve \nthe best education inspection ratings, which are largely defined in terms of academic \noutcomes, and consequently demand the highest school fees (El-Sholkamyl & Al-\nSaleh, 2017). However, our study shows that they also present the worst wellbeing \noutcomes and relatively poor mental health outcomes. To provide the right incentives \nfor wellbeing and mental health promotion, we recommend that education authorities \nincorporate wellbeing and mental health outcomes in their school inspections and that \nthese be made transparent in the rankings. This is vital given the null or positive asso-\nciation between wellbeing and academic outcomes described and the predictive asso-\nciation between wellbeing and mental health in adolescence and later life outcomes \n(Cavioni et al., 2021; DeNeve & Oswald, 2012; Goodman et al., 2015; Guzmán et al., \n2020; Kansky et al., 2016; Richards & Huppert, 2011). Finally, understanding trends can help better understand geographic, socio-demo-\ngraphic and school type differences in adolescents’ wellbeing. As noted in Marquez and \nLong (2020), 15-year-old adolescents’ OLS declined in the UAE between 2015 and 2018, \nas it did in most countries, and the UAE was among the top third of countries presenting \nthe largest decline. 4.1 \u0007Findings Yet, in the present study we found that this decline was larger among \nfemales, national students, students of higher SES and in British schools, as well as in the \nemirates of Sharjah, Abu Dhabi, RAK and Dubai. This may be explained by rising mental \nhealth issues, which serve to drag OLS scores downward (Badri et al., 2022; Lombardo \net al., 2018). Declines in youth wellbeing have been especially pronounced in countries \nthat are more egalitarian, with higher GDP per capita, and where concurrent increases in \nacademic pressure are observed (Borgonovi & Han, 2021; Cosma et al., 2020). Further-\nmore, in a recent study, Marquez et al. (2022c) examined factors associated with declines \nin adolescent OLS between 2015 and 2018 in the UK, the United States, France, Ireland \nand Japan, finding that this decline may be largely due to changes in school well-being \nand, to a lesser extent, material well-being and the use of Information and Communica-\ntion Technologies. They also found differences by gender and across countries, notably \nbetween Western nations and Japan. This highlights again the importance of national stud-\nies on adolescent wellbeing and mental health that account for the specific context and \nsocio-demographic profile of the country as levels and changes over time –and the factors \nassociated with these- not only differ across countries but potentially also across different \nsocio-demographic groups (e.g. gender, ethnicity, migrant status, etc.) within nations. 1 3 4.2 \u0007Limitations and Future Directions We analyzed pre-Covid-19 data. The next PISA edition (2022) will be revelatory \nfor the status of youth wellbeing and mental health, especially as the effects of 1 3 Geographic, Socio‑Demographic and School Type Variation… 817 the pandemic will linger beyond its end (Gavin et al., 2021; Racine et al., 2021; \nUNICEF, 2021). Also, due to the cross-sectional nature of the data and the research \ndesign used, causality cannot be established. Moreover, although levels of miss-\ning data were generally low for all the wellbeing outcomes, these were somewhat \nhigher for mental health symptoms of internalising difficulties (see Sect. 2.2.); thus, \nthe results involving this variable should be interpreted with more caution. Another \nimportant limitation to keep in mind refers to the validity of the Western-derived \nmeasures of wellbeing included in PISA in a non-Western nation such as the UAE \nwhere adolescents may understand and respond to questions on their wellbeing in \nsubstantially different ways compared to their Western peers due to cultural and lin-\nguistic issues. Although the body of research assessing the cross-cultural validity \nof adolescent wellbeing measures is growing (Casas & Gonzalez-Carrasco, 2021), \nmore research is needed to examine the validity of these specific measures of ado-\nlescent wellbeing in the UAE. Overall, while most of our findings coincided with \nthe literature, the picture within the UAE is complex and confirms the need for con-\ntinued broad-based assessments of this sort, using a range of measures to understand \nthe wellbeing and mental health landscape for young people in the country. This is \nespecially the case in the public-school students, which tend to be mostly nationals, \nwhere concurrent mental health issues and high wellbeing operate together and cre-\nate a student profile worth further investigation, in addition to confirming the two-\ncontinua model of wellbeing (Keyes, 2005). We finally echo the OECD’s (2021) \nrecommendation for education authorities to share their data. The KHDA collects \nlongitudinal student wellbeing data in a highly granular form, which can be used to \nunderstand what is driving school, familial and community factors that support well-\nbeing and mental health in the emirate and nation. However, this has not been made \npublic, belabouring efforts and delaying progress. 4.3 \u0007Conclusion In adolescence, wellbeing and mental health should not be considered less important \nthan academic outcomes given their capacity to predict important outcomes later \nin life, including academic outcomes (Cavioni et al., 2021; DeNeve & Oswald, \n2012; Goodman et al., 2015; Guzmán et al., 2020; Kansky et al., 2016; Richards \n& Huppert, 2011). This stance was further supported by our finding that in the \nUAE, students who reported greater wellbeing also performed better academically. Consequently, schools, parents and relevant stakeholders should take students \nwellbeing and mental health seriously. Our findings on geographic, socio-\ndemographic and school type inequalities in adolescent wellbeing and mental health \nhelp the identification of the more vulnerable groups in the UAE and provide a \nbaseline against which future changes can be measured. Education authorities at the \nnational and emirate level must invest more heavily in public education to address \nacademic performance, while concurrently addressing mental health across all \nschools, as well as improving wellbeing outcomes uniformly. These aims will ensure \nthat the ability of young people to effectively make a living, as well as live happy, \nsatisfactory lives, is maximized. 1 3 818 J. Marquez et al. 4.3 \u0007Conclusion Table 6 Output from linear regression models: socio-demographic differences in OLS (standardized scale)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nB\nse\nConstant\n-0.037\n0.021\n0.037\n0.026\n0.098***\n0.029\n0.068*\n0.031\n0.113**\n0.035\n0.173***\n0.027\n0.189***\n0.028\nEmirate (ref.category: Abu \nDhabi): Dubai\n-0.043\n0.029\n-0.046\n0.027\n-0.025\n0.027\n-0.036\n0.028\n0.000\n0.031\n0.046\n0.028\n0.039\n0.028\nEmirate (ref.category: Abu \nDhabi): Sharjah\n-0.045\n0.060\n-0.038\n0.056\n-0.026\n0.055\n-0.019\n0.056\n-0.008\n0.055\n-0.012\n0.043\n-0.014\n0.043\nEmirate (ref.category: Abu \nDhabi): Ajman\n0.148**\n0.047\n0.148**\n0.049\n0.152**\n0.050\n0.171***\n0.049\n0.181***\n0.054\n0.126**\n0.047\n0.129*\n0.047\nEmirate (ref.category: Abu \nDhabi): UAQ\n0.160**\n0.055\n0.164*\n0.065\n0.130\n0.068\n0.131*\n0.063\n0.109\n0.067\n0.112\n0.067\n0.112\n0.068\nEmirate (ref.category: Abu \nDhabi): RAK\n0.229***\n0.066\n0.227***\n0.067\n0.204**\n0.067\n0.206**\n0.067\n0.182**\n0.069\n0.136*\n0.064\n0.135*\n0.064\nEmirate (ref.category: Abu \nDhabi): Fujairah\n0.284***\n0.060\n0.282***\n0.066\n0.255***\n0.066\n0.256***\n0.066\n0.227***\n0.068\n0.213***\n0.062\n0.212**\n0.062\nFemale\n-0.141***\n0.029\n-0.148*** 0.029\n-0.154*** 0.029\n-0.166***\n0.030\n-0.162***\n0.022\n-0.168***\n0.022\nExpatriate\n-0.109*** 0.024\n-0.052*\n0.026\n0.022\n0.027\n0.041\n0.025\n0.029\n0.026\nSocio-economic status\n0.079***\n0.013\n0.081***\n0.014\n0.117***\n0.012\n0.114***\n0.012\nPrivate school\n-0.149***\n0.033\nSchool type (ref.category: \nMoE): British\n-0.486***\n0.046\n-0.505***\n0.046\nSchool type (ref.category: \nMoE): American\n-0.311***\n0.034\n-0.321***\n0.034 Geographic, Socio‑Demographic and School Type Variation… 819 Geographic, Socio‑Demographic and School Type\nTable 6 (continued)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nB\nse\nSchool type (ref.category: \nMoE): Indian\n-0.141**\n0.045\n-0.157**\n0.046\nSchool type (ref.category: \nMoE): TVET\n-0.582***\n0.049\n-0.588***\n0.050\nSchool type (ref.category: \nMoE): Other\n-0.234***\n0.049\n-0.249***\n0.051\nAcademic competence (Mean \nPISA score)\n0.024*\n0.012\nObservations\n17,467\n17,467\n16,960\n16,941\n16,326\n16,941\n16,941 Table 6 (continued)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nB\nse\nSchool type (ref.category: \nMoE): Indian\n-0.141**\n0.045\n-0.157**\n0.046\nSchool type (ref.category: \nMoE): TVET\n-0.582***\n0.049\n-0.588***\n0.050\nSchool type (ref.category: \nMoE): Other\n-0.234***\n0.049\n-0.249***\n0.051\nAcademic competence (Mean \nPISA score)\n0.024*\n0.012\nObservations\n17,467\n17,467\n16,960\n16,941\n16,326\n16,941\n16,941\nStandard errors (se); ***p < 0.01, **p < 0.05, *p < 0.1 Table 6 (continued)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nB\nse\nSchool type (ref.category: \nMoE): Indian\n-0.141**\n0.045\n-0.157**\n0.046\nSchool type (ref.category: \nMoE): TVET\n-0.582***\n0.049\n-0.588***\n0.050\nSchool type (ref.category: \nMoE): Other\n-0.234***\n0.049\n-0.249***\n0.051\nAcademic competence (Mean \nPISA score)\n0.024*\n0.012\nObservations\n17,467\n17,467\n16,960\n16,941\n16,326\n16,941\n16,941 Standard errors (se); ***p < 0.01, **p < 0.05, *p < 0.1 3 J. 4.3 \u0007Conclusion Marquez et al. 820 Table 7 Output from linear regression models: socio-demographic differences in positive affects (standardized scale)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nSe\nConstant\n-0.034\n0.019\n-0.087*** 0.024\n-0.031\n0.026\n-0.077**\n0.028\n-0.053\n0.031\n-0.024\n0.025\n0.026***\n0.025\nEmirate (ref.category: Abu Dhabi): \nDubai\n0.013\n0.026\n0.015\n0.026\n0.021\n0.026\n0.007\n0.026\n0.033\n0.028\n0.050*\n0.025\n0.028\n0.026\nEmirate (ref.category: Abu Dhabi): \nSharjah\n0.041\n0.046\n0.036\n0.048\n0.049\n0.048\n0.058\n0.050\n0.065\n0.049\n0.063\n0.039\n0.055\n0.039\nEmirate (ref.category: Abu Dhabi): \nAjman\n0.162*** 0.044\n0.161***\n0.042\n0.151***\n0.043\n0.179***\n0.041\n0.193***\n0.042\n0.141***\n0.039\n0.147***\n0.038\nEmirate (ref.category: Abu Dhabi): \nUAQ\n0.123\n0.069\n0.120*\n0.058\n0.105\n0.058\n0.110*\n0.051\n0.098\n0.053\n0.091\n0.056\n0.089\n0.057\nEmirate (ref.category: Abu Dhabi): \nRAK\n0.166**\n0.054\n0.168**\n0.052\n0.149**\n0.051\n0.154**\n0.050\n0.139**\n0.052\n0.107*\n0.049\n0.102*\n0.048\nEmirate (ref.category: Abu Dhabi): \nFujairah\n0.217*** 0.057\n0.219***\n0.051\n0.202***\n0.051\n0.204***\n0.049\n0.189***\n0.050\n0.179***\n0.047\n0.175***\n0.045\nFemale\n0.098***\n0.025\n0.079**\n0.025\n0.071**\n0.025\n0.066*\n0.026\n0.071***\n0.020\n0.054*\n0.020\nExpatriate\n-0.067**\n0.022\n0.017\n0.025\n0.056*\n0.026\n0.063*\n0.028\n0.023\n0.029\nSocio-economic status\n0.119***\n0.012\n0.121***\n0.012\n0.148***\n0.012\n0.142***\n0.012\nPrivate school\n-0.082**\n0.028\nSchool type (ref.category: MoE): \nBritish\n-0.331*** 0.044\n-0.391***\n0.043\nSchool type (ref.category: MoE): \nAmerican\n-0.173*** 0.033\n-0.205***\n0.035\nSchool type (ref.category: MoE): \nIndian\n0.033\n0.040\n-0.018\n0.040\nSchool type (ref.category: MoE): \nTVET\n-0.308*** 0.068\n-0.326***\n0.063 Geographic, Socio‑Demographic and School Type Variation… 821 1\nTable 7 (continued)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nSe\nSchool type (ref.category: MoE): \nOther\n-0.124**\n0.042\n-0.173***\n0.046\nAcademic competence (Mean PISA \nscore)\n0.075***\n0.013\nObservations\n17,083\n16,593\n16,579\n15,980\n16,579\n16,579\n16,941\nStandard errors (se); ***p < 0.01, **p < 0.05, *p < 0.1 3 822 J. Marquez et al. 4.3 \u0007Conclusion Table 8 Output from linear regression models: socio-demographic differences in negative affects (standardized scale)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nB\nse\nConstant\n-0.006\n0.024\n-0.178***\n0.030\n-0.299***\n0.033\n-0.280***\n0.035\n-0.378***\n0.038\n-0.408***\n0.031\n0.439\n0.032\nEmirate (ref.category: Abu \nDhabi): Dubai\n0.158***\n0.038\n0.165***\n0.034\n0.115***\n0.032\n0.121***\n0.033\n0.047\n0.033\n-0.034\n0.027\n-0.021\n0.028\nEmirate (ref.category: Abu \nDhabi): Sharjah\n0.066\n0.081\n0.050\n0.068\n0.017\n0.065\n0.013\n0.066\n-0.014\n0.060\n-0.017\n0.049\n-0.012\n0.048\nEmirate (ref.category: Abu \nDhabi): Ajman\n-0.127**\n0.048\n-0.128**\n0.047\n-0.141**\n0.048\n-0.153**\n0.048\n-0.173***\n0.052\n-0.109**\n0.037\n-0.112**\n0.036\nEmirate (ref.category: Abu \nDhabi): UAQ\n-0.098\n0.083\n-0.109\n0.077\n-0.065\n0.078\n-0.066\n0.077\n-0.009\n0.081\n-0.020\n0.066\n-0.019\n0.065\nEmirate (ref.category: Abu \nDhabi): RAK\n-0.204***\n0.060\n-0.197**\n0.062\n-0.143*\n0.060\n-0.144*\n0.059\n-0.083\n0.062\n-0.061\n0.057\n-0.058\n0.057\nEmirate (ref.category: Abu \nDhabi): Fujairah\n-0.208**\n0.066\n-0.201***\n0.056\n-0.143**\n0.055\n-0.143**\n0.054\n-0.078\n0.054\n-0.087\n0.053\n-0.085\n0.053\nFemale\n0.324***\n0.036\n0.332***\n0.034\n0.335***\n0.033\n0.347***\n0.033\n0.348***\n0.023\n0.359***\n0.023\nExpatriate\n0.226***\n0.025\n0.191***\n0.028\n0.011\n0.029\n-0.015\n0.024\n0.010\n0.025\nSocio-economic status\n-0.051**\n0.016\n-0.052**\n0.016\n-0.081***\n0.014\n-0.077*** 0.014\nPrivate school\n0.354***\n0.036\nSchool type (ref.category: \nMoE): British\n0.606***\n0.057\n0.643***\n0.056\nSchool type (ref.category: \nMoE): American\n0.483***\n0.034\n0.503***\n0.035\nSchool type (ref.category: \nMoE): Indian\n0.519***\n0.035\n0.551***\n0.035\nSchool type (ref.category: \nMoE): TVET\n0.462***\n0.064\n0.472***\n0.062 Geographic, Socio‑Demographic and School Type Variation… 823 1\nTable 8 (continued)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nB\nse\nSchool type (ref.category: \nMoE): Other\n0.513***\n0.048\n0.544***\n0.051\nAcademic competence (Mean \nPISA score)\n-0.047*** 0.012\nObservations\n16,999\n16,999\n16,524\n16,512\n15,910\n16,512\n16,512\nStandard errors (se); ***p < 0.01, **p < 0.05, *p < 0.1 Table 8 (continued)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nB\nse\nSchool type (ref.category: \nMoE): Other\n0.513***\n0.048\n0.544***\n0.051\nAcademic competence (Mean \nPISA score)\n-0.047*** 0.012\nObservations\n16,999\n16,999\n16,524\n16,512\n15,910\n16,512\n16,512 Standard errors (se); ***p < 0.01, **p < 0.05, *p < 0.1 3 J. Marquez et al. 4.3 \u0007Conclusion 824 Table 9 Output from linear regression models: socio-demographic differences in meaning and purpose in life (standardized scale)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nConstant\n-0.023\n0.019\n-0.039\n0.025\n0.032\n0.030\n0.010\n0.032\n0.079*\n0.036\n0.123***\n0.027\n0.125\n0.027\nEmirate (ref.category: Abu \nDhabi): Dubai\n-0.100**\n0.033\n-0.099**\n0.033\n-0.083*\n0.033\n-0.090**\n0.034\n-0.021\n0.035\n0.032\n0.031\n0.032\n0.031\nEmirate (ref.category: Abu \nDhabi): Sharjah\n0.031\n0.065\n0.029\n0.065\n0.048\n0.064\n0.052\n0.065\n0.073\n0.061\n0.071\n0.043\n0.071\n0.043\nEmirate (ref.category: Abu \nDhabi): Ajman\n0.184***\n0.046\n0.184***\n0.045\n0.187***\n0.047\n0.200***\n0.046\n0.229***\n0.050\n0.141***\n0.040\n0.141*** 0.040\nEmirate (ref.category: Abu \nDhabi): UAQ\n0.165*\n0.069\n0.164*\n0.066\n0.140*\n0.064\n0.143*\n0.062\n0.103\n0.067\n0.096\n0.063\n0.096\n0.063\nEmirate (ref.category: Abu \nDhabi): RAK\n0.203***\n0.051\n0.204***\n0.050\n0.173***\n0.050\n0.176***\n0.049\n0.139**\n0.051\n0.093*\n0.047\n0.092*\n0.046\nEmirate (ref.category: Abu \nDhabi): Fujairah\n0.349***\n0.060\n0.350***\n0.059\n0.317***\n0.059\n0.318***\n0.058\n0.277***\n0.057\n0.263***\n0.050\n0.263*** 0.050\nFemale\n0.030\n0.034\n0.018\n0.034\n0.014\n0.034\n0.004\n0.034\n0.005\n0.022\n0.005\n0.022\nExpatriate\n-0.108***\n0.026\n-0.068*\n0.029\n0.065*\n0.029\n0.089***\n0.025\n0.088*** 0.025\nSocio-economic status\n0.057***\n0.015\n0.059***\n0.015\n0.100***\n0.013\n0.100*** 0.013\nPrivate school\n-0.259*** 0.034\nSchool type (ref.category: \nMoE): British\n-0.616***\n0.059\n-0.618*** 0.058\nSchool type (ref.category: \nMoE): American\n-0.407***\n0.036\n-0.408*** 0.036\nSchool type (ref.category: \nMoE): Indian\n-0.233***\n0.038\n-0.234*** 0.039\nSchool type (ref.category: \nMoE): TVET\n-0.477***\n0.058\n-0.478*** 0.058 Geographic, Socio‑Demographic and School Type Variation… 825 1\nTable 9 (continued)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nSchool type (ref.category: \nMoE): Other\n-0.473***\n0.048\n-0.474*** 0.050\nAcademic competence (Mean \nPISA score)\n0.002\n0.014\nObservations\n17,570\n17,570\n17,046\n17,034\n16,415\n17,034\n17,034\nStandard errors (se); ***p < 0.01, **p < 0.05, *p < 0.1 Table 9 (continued)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nSchool type (ref.category: \nMoE): Other\n-0.473***\n0.048\n-0.474*** 0.050\nAcademic competence (Mean \nPISA score)\n0.002\n0.014\nObservations\n17,570\n17,570\n17,046\n17,034\n16,415\n17,034\n17,034\nS\nd d\n(\n) ***\n0 01 **\n0 05 *\n0 1 Standard errors (se); ***p < 0.01, **p < 0.05, *p < 0.1 3 J. Declarations Conflict of Interest We confirm that there are no conflict of interests affecting the submission of this \nmanuscript. Informed Consent In this research, we exclusively used secondary data collected by the OECD (Programme \nfor International Student Assessment) and publically available here. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, \nwhich permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long \nas you give appropriate credit to the original author(s) and the source, provide a link to the Creative \nCommons licence, and indicate if changes were made. The images or other third party material in this \narticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit \nline to the material. If material is not included in the article’s Creative Commons licence and your \nintended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain \npermission directly from the copyright holder. To view a copy of this licence, visit http://creativeco\nmmons.org/licenses/by/4.0/. 4.3 \u0007Conclusion Marquez et al. 826 Table 10 Output from linear regression models: socio-demographic differences in internalizing difficulties (standardized scale)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nConstant\n0.075*\n0.032\n-0.135*** 0.031\n-0.031\n0.034\n-0.028\n0.034\n0.024\n0.038\n0.006\n0.035\n0.010\n0.034\nEmirate (ref.category: Abu Dhabi): \nDubai\n-0.139*** 0.041\n-0.130*** 0.037\n-0.087*\n0.035\n-0.086*\n0.035\n-0.055\n0.036\n-0.009\n0.031\n-0.003\n0.030\nEmirate (ref.category: Abu Dhabi): \nSharjah\n-0.028\n0.051\n-0.051\n0.043\n-0.019\n0.041\n-0.018\n0.041\n-0.008\n0.041\n0.003\n0.038\n0.006\n0.038\nEmirate (ref.category: Abu Dhabi): \nAjman\n-0.144**\n0.053\n-0.149**\n0.045\n-0.142***\n0.041\n-0.144***\n0.041\n-0.131**\n0.043\n-0.135*** 0.038\n-0.137*** 0.038\nEmirate (ref.category: Abu Dhabi): \nUAQ\n0.027\n0.076\n0.004\n0.039\n-0.038\n0.039\n-0.038\n0.039\n-0.072\n0.043\n-0.048\n0.038\n-0.047\n0.037\nEmirate (ref.category: Abu Dhabi): \nRAK\n-0.107\n0.062\n-0.099\n0.056\n-0.146**\n0.055\n-0.145**\n0.055\n-0.194*** 0.057\n-0.149**\n0.053\n-0.147**\n0.053\nEmirate (ref.category: Abu Dhabi): \nFujairah\n-0.100\n0.069\n-0.096\n0.060\n-0.155*\n0.062\n-0.153*\n0.062\n-0.188**\n0.061\n-0.159**\n0.058\n-0.158**\n0.058\nFemale\n0.395***\n0.030\n0.390***\n0.028\n0.390***\n0.028\n0.387***\n0.029\n0.383***\n0.023\n0.388***\n0.023\nExpatriate\n-0.196***\n0.027\n-0.202***\n0.026\n-0.111*** 0.029\n-0.073**\n0.028\n-0.061*\n0.027\nSocio-economic status\n-0.008\n0.013\n-0.010\n0.014\n-0.025\n0.013\n-0.022\n0.013\nPrivate school\n-0.177*** 0.032\nSchool type (ref.category: MoE): \nBritish\n-0.118**\n0.038\n-0.099*\n0.039\nSchool type (ref.category: MoE): \nAmerican\n-0.138*** 0.040\n-0.128**\n0.041\nSchool type (ref.category: MoE): \nIndian\n-0.497*** 0.042\n-0.481*** 0.043\nSchool type (ref.category: MoE): \nTVET\n-0.017\n0.050\n-0.012\n0.049 Geographic, Socio‑Demographic and School Type Variation… 827 1\nTable 10 (continued)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nSchool type (ref.category: MoE): \nOther\n-0.297*** 0.037\n-0.281*** 0.038\nAcademic competence (Mean PISA \nscore)\n0.024\n0.012\nObservations\n16,056\n16,056\n15,601\n15,589\n16,326\n15,021\n15,589\nStandard errors (se); ***p < 0.01, **p < 0.05, *p < 0.1 Table 10 (continued)\nVariable\nModel 1\nModel 2\nModel 3\nModel 4\nModel 5\nModel 6\nModel 7\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nb\nse\nSchool type (ref.category: MoE): \nOther\n-0.297*** 0.037\n-0.281*** 0.038\nAcademic competence (Mean PISA \nscore)\n0.024\n0.012\nObservations\n16,056\n16,056\n15,601\n15,589\n16,326\n15,021\n15,589 3 828 J. Marquez et al. References Children and Youth Services Review, 33(4), 548–556. https://doi.\n/10 1016/j hild\nth 2010 05 010 org/10.1016/j.childyouth.2010.05.010 org/10.1016/j.childyouth.2010.05.010\nBradshaw, J., Martorano, B., Natali, L., & de Neubourg, C. (2013). Children’s subjective well-\nbeing in rich countries. Child Indicators Research, 6(4), 619–635. https://doi.org/10.1007/\ns12187-013-9196-4 Buckner, E. (2018). The other gap: Examining low-income Emiratis’ educational achievement. Al Qasimi \nFoundation. https://doi.org/10.18502/aqf.0120f Buttazzoni, A., Doherty, S., & Minaker, L. (2022). How do urban environments affect young people’s \nmental health? A novel conceptual framework to bridge public health, planning, and neurourban-\nism. Public Health Reports, 137(1), 48–61. https://doi.org/10.1177/0033354920982088 Campbell, O. L. K., Bann, D., & Patalay, P. (2021). The gender gap in adolescent mental health: A cross-\nnational investigation of 566,829 adolescents across 73 countries. SSM - Population Health, 13, \n100742. https://doi.org/10.1016/j.ssmph.2021.100742 p\ng\nj\np\nCarmona, M. (2019). Place value: Place quality and its impact on health, social, economic and environmental \noutcomes. Journal of Urban Design, 41(1), 1–48. https://doi.org/10.1080/13574809.2018.1472523 Casas, F., Bello, A., Gonzalez, M., & Aligue, M. (2013). Children’s subjective well-being measured \nusing a composite index: What impacts Spanish first-year secondary education students’ subjective \nwell-being? Child Indicators Research, 6, 433–460. https://doi.org/10.1007/s12187-013-9182-x Casas, F., & González-Carrasco, M. (2019). Subjective well-being decreasing with age: New research o\nchildren over 8. Child Development, 90(2), 375–394. https://doi.org/10.1111/cdev.13133 Casas, F., & González-Carrasco, M. (2021). Analysing comparability of four multi-item well-being psy-\nchometric scales among 35 countries using Children’s Worlds 3rd wave 10 and 12-year-olds sam-\nples. Child Indicators Research, 14(5), 1829–1861. Cavioni, V., Grazzani, I., Ornaghi, V., Agliati, A., & Pepe, A. (2021). Adolescents’ mental health at \nschool: The mediating role of life satisfaction. Frontiers in Psychology, 12, 720628. https://doi.org/\n10.3389/fpsyg.2021.720628 Chan, M. F., Al Balushi, R., Al Falahi, M., Mahadevan, S., Al Saadoon, M., & Al-Adawi, S. (2021). Child and adolescent mental health disorders in the GCC: A systematic review and meta-analysis. International Journal of Pediatrics and Adolescent Medicine, 8(3), 134–145. https://doi.org/10.\n1016/j.ijpam.2021.04.002 Charara, R., El Bcheraoui, C., Khalil, I., Moradi-Lakeh, M., Afshin, A., Kassebaum, N. J., … & Mokdad, \nA. H. (2018). The burden of mental disorders in the Eastern Mediterranean region, 1990–2015: \nFindings from the global burden of disease 2015 study. International Journal of Public Health, 63, \n25–37. https://doi.org/10.1007/s00038-017-1006-1 Charara, R., Forouzanfar, M., Naghavi, M., Moradi-Lakeh, M., Afshin, A., Vos, T., … & Mokdad, A. H. (2017). The burden of mental disorders in the Eastern Mediterranean region, 1990–2013. PLoS \nONE, 12(1), e0169575. References Agnafors, S., Barmark, M., & Sydsjö, G. (2021). Mental health and academic performance: A study on \nselection and causation effects from childhood to early adulthood. Social Psychiatry and Psychiat-\nric Epidemiology, 56(5), 857–866. https://doi.org/10.1007/s00127-020-01934-5 Alivernini, F., Cavicchiolo, E., Manganelli, S., Chirico, A., & Lucidi, F. (2020). Students’ psychologi-\ncal well-being and its multilevel relationship with immigrant background, gender, socioeconomic \nstatus, achievement, and class size. School Effectiveness and School Improvement, 31(2), 172–191. https://doi.org/10.1080/09243453.2019.1642214 Al-Yateem, N., Bani Issa, W., Rossiter, R. C., Al-Shujairi, A., Radwan, H., Awad, M., Fakhry, R., & \nMahmoud, I. (2020). Anxiety related disorders in adolescents in the United Arab Emirates: A \npopulation based cross-sectional study. BMC Pediatrics, 20(1), 245. https://doi.org/10.1186/\ns12887-020-02155-0 Alzahrani, O. (2020). Depressive disorders in the Arabian Gulf Cooperation Council countries: A lit-\nerature review. The Journal of International Medical Research, 48(10), 300060520961917. https://\ndoi.org/10.1177/0300060520961917 Amone-P’Olak, K., Burger, H., Huisman, M., Oldehinkel, A. J., & Ormel, J. (2011). Parental psycho-\npathology and socioeconomic position predict adolescent offspring’s mental health independently \nand do not interact: The TRAILS study. Journal of Epidemiology and Community Health, 65(1), \n57–63. https://doi.org/10.1136/jech.2009.092569 Auerbach, R. P., Mortier, P., Bruffaerts, R., Alonso, J., Benjet, C., Cuijpers, P., Demyttenaere, K., … \n& Collaborators, W.H.O.W.M.H.-I.C.S. (2018). WHO World Mental Health Surveys International \nCollege Student Project: Prevalence and distribution of mental disorders. Journal of Abnormal \nPsychology, 127(7), 623–638. https://doi.org/10.1037/abn0000362 Badri, M. A., Alkhaili, M., Aldhaheri, H., Yang, G., Albahar, M., & Alrashdi, A. (2022). Exploring the \nreciprocal relationships between happiness and life satisfaction of working adults-evidence from \nAbu Dhabi. International Journal of Environmental Research and Public Health, 19(6), 3575. https://doi.org/10.3390/ijerph19063575 Ben-Arieh, A. (2007). The child indicators movement: Past, present, and future. Child Indicators \nResearch, 1(1), 3–16. https://doi.org/10.1007/s12187-007-9003-1 Berry, J. W., & Hou, F. (2017). Acculturation, discrimination and wellbeing among second generation of \nimmigrants in Canada. International Journal of Intercultural Relations, 61, 29–39. https://doi.org/\n10.1016/j.ijintrel.2017.08.003 Bohlmeijer, E., & Westerhof, G. (2021). The model for sustainable mental health: Future directions for \nintegrating positive psychology into mental health care. Frontiers in Psychology, 12, 747999. https://doi.org/10.3389/fpsyg.2021.747999 1 3 Geographic, Socio‑Demographic and School Type Variation… 829 Borgonovi, F., & Han, S. W. (2021). Gender disparities in fear of failure among 15-year-old students: The \nrole of gender inequality, the organisation of schooling and economic conditions. Journal of Ado-\nlescence, 86, 28–39. https://doi.org/10.1016/j.adolescence.2020.11.009 Bradshaw, J., Keung, A., Rees, G., & Goswami, H. (2011). Children’s subjective well-being: Interna-\ntional comparative perspectives. References https://doi.org/10.1371/journal.pone.0169575f Chen, X., Cai, Z., He, J., & Fan, X. (2019). Gender differences in life satisfaction among children and \nadolescents: A meta-analysis. Journal of Happiness Studies, 21, 2279–2307. https://doi.org/10.\n1007/s10902-019-00169-9 Cho, E.Y.-N., & Chan, T. M. S. (2020). Children’s wellbeing in a high-stakes testing environment: The \ncase of Hong Kong. Children and Youth Services Review, 109, 104694. https://doi.org/10.1016/j.\nchildyouth.2019.104694 y\nChzhen, Y., Bruckauf, Z., Kwok, N., Pavlova, D., Torsheim, T., & Gaspar de Matos, M. (2016). Inequalities \nin adolescent health and life satisfaction: Evidence from the health behaviour in school-aged children \nstudy. Innocenti Working Paper No. 2016–09, UNICEF Office of Research, United Nations Children’s fi\nFund (UNICEF). https://www.unicef-irc.org/publications/pdf/IWP_2016_09.pdf. Accessed 29 Oct 202 Colizzi, M., Lasalvia, A., & Ruggeri, M. (2020). Prevention and early intervention in youth mental \nhealth: Is it time for a multidisciplinary and trans-diagnostic model for care? International Journal \nof Mental Health Systems, 14(23). https://doi.org/10.1186/s13033-020-00356-9 Cosma, A., Stevens, G., Martin, G., Duinhof, E. L., Walsh, S. D., Garcia-Moya, I., Költő, A., Gobina, I., \nCanale, N., Catunda, C., Inchley, J., & de Looze, M. (2020). Cross-national time trends in adoles-\ncent mental well-being from 2002 to 2018 and the explanatory role of schoolwork pressure. The \nJournal of Adolescent Health, 66(6S), S50–S58. https://doi.org/10.1016/j.jadohealth.2020.02.010 1 3 830 J. Marquez et al. Cyranowski, J. M., Frank, E., Young, E., & Shear, M. K. (2000). Adolescent onset of the gender differ-\nence in lifetime rates of major depression: A theoretical model. Archives of General Psychiatry, \n57(1), 21–27. https://doi.org/10.1001/archpsyc.57.1.21 Deighton, J., Humphrey, N., Belsky, J., Boehnke, J., Vostanis, P., & Patalay, P. (2018). Longitudinal path-\nways between mental health difficulties and academic performance during middle childhood and \nearly adolescence. The British Journal of Developmental Psychology, 36(1), 110–126. https://doi.\norg/10.1111/bjdp.12218lf DeNeve, J. E., & Oswald, A. (2012). Estimating the influence of life satisfaction and positive affect on \nlater income using sibling fixed effects. Proceedings of the National Academy of Sciences of the \nUSA, 109(49), 19953–19958. https://doi.org/10.1073/pnas.1211437109 Diener, E., Lucas, R., & Oishi, S. (2002). Subjective well-being: The science of happiness and life sat-\nisfaction. In C. R. Snyder & S. J. Lopez (Eds.), The handbook of positive psychology (pp. 63–73). Oxford University Press. Diener, E., Oishi, S., & Tay, L. (2018). Advances in subjective well-being research. Nature Huma\nBehaviour, 2, 253–260. https://doi.org/10.1038/s41562-018-0307-6 Dryden-Peterson, S. (2018). References Family–school relationships in immigrant children’s well-being: The inter-\nsection of demographics and school culture in the experiences of Black African immigrants in the \nUnited States. Race Ethnicity and Education, 21(4), 486–502. https://doi.org/10.1080/13613324.\n2017.1294562 Ebbert, A. M., Infurna, F. J., & Luthar, S. S. (2019a). Mapping developmental changes in perceived par-\nent–adolescent relationship quality throughout middle school and high school. Development and \nPsychopathology, 31(4), 1541–1556. https://doi.org/10.1017/S0954579418001219 Ebbert, A. M., Kumar, N. L., & Luthar, S. S. (2019b). Complexities in adjustment patterns among the \n“best and the brightest”: Risk and resilience in the context of high achieving schools. Research in \nHuman Development, 16, 21–34. https://doi.org/10.1080/15427609.2018.1541376 Elgar, F. J., Gariépy, G., Torsheim, T., & Currie, C. (2017). Early-life income inequality and adolescent \nhealth and well-being. Social Science & Medicine, 174, 197–208. https://doi.org/10.1016/j.socsc\nimed.2016.10.014 Eloul, L., Ambusaidi, A., & Al-Adawi, S. (2009). Silent epidemic of depression in women in the Middle \nEast and North Africa Region: Emerging tribulation or fallacy? Sultan Qaboos University Medical \nJournal, 9(1), 5–15. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3074757/. ( )\np\ng\np\nEl-Sholkamyl, M., & Al-Saleh, Y. (2017, March). Paying for education in Dubai: Is it really worth it? Mohammed Bin Rashid School of Government. https://www.mbrsg.ae/home/research/education-\npolicy/paying-for-education-in-dubai-is-it-really-worth. Accessed 29 Oct 2022 Filion, N., Fenelon, A., & Boudreaux, M. (2018). Immigration, citizenship, and the mental health of ad\nlescents. PLoS ONE, 13(5), e0196859. https://doi.org/10.1371/journal.pone.0196859 Gavin, B., Lyne, J., & McNicholas, F. (2021). The global impact on mental health almost 2 years into the \nCOVID-19 pandemic. Irish Journal of Psychological Medicine, 38(4), 243–246. https://doi.org/10.\n1017/ipm.2021.75 p\nGiota, J., & Gustafsson, J.-E. (2017). Perceived demands of schooling, stress and mental health: Changes \nfrom grade 6 to grade 9 as a function of gender and cognitive ability. Stress and Health, 33(3), \n253–266. https://doi.org/10.1002/smi.2693 p\ng\nGoodman, A., Joshi, H., Nasi, B., & Tyler, C. (2015). Social and emotional skills in childhood and their \nlong-term effects on adult life. E. I. Foundation. https://www.eif.org.uk/public/report/social-and-\nemotional-skills-in-childhood-and-their-long-term-effects-on-adult-life. Accessed 29 Oct 2022 gf\nGorski-Steiner, I., O’Dell, S., Bandeen-Roche, K., Volk, H. E., Goes, F. S., & Schwartz, B. S. (2022). Geographic disparities in new onset of internalizing disorders in Pennsylvania adolescents using \nelectronic health records. Spatial and Spatio-Temporal Epidemiology, 41, 100439. https://doi.org/\n10.1016/j.sste.2021.100439 j\nGovorova, E., Benítez, I., & Muñiz, J. (2020). Predicting student well-being: Network analysis based on \nPISA 2018. International Journal of Environmental Research and Public Health, 17(11), 4014. https://doi.org/10.3390/ijerph17114014 Gross-Manos, D. (2017). References Material well-being and social exclusion association with children’s subjective \nwell-being: Cross-national analysis of 14 countries. Children and Youth Services Review, 80, 116–\n128. https://doi.org/10.1016/j.childyouth.2017.06.048 Gutman, L. M., & Vorhaus, J. (2012). The impact of pupil behaviour and wellbeing on educational \noutcomes. Childhood Wellbeing Research Centre, University of London. http://dera.ioe.ac.uk/id/\neprint/16093. 1 3 Geographic, Socio‑Demographic and School Type Variation… 831 Guzmán, J., Green, J. G., Oblath, R., & Holt, M. K. (2020). Life satisfaction mediates the association \nbetween mental health risk and perceptions of school functioning among children and adolescents. Contemporary School Psychology, 24, 389–399. https://doi.org/10.1007/s40688-019-00257-wf Hamama, L., & Hamama-Raz, Y. (2021). Meaning in life, self-control, positive and negative affect: \nExploring gender differences among adolescents. Youth & Society, 53(5), 699–722. https://doi.org/\n10.1177/0044118X19883736 Heller-Sahlgren, G. (2018). Smart but unhappy: Independent-school competition and the wellbeing-\nefficiency trade-off in education. Economics of Education Review, 62, 66–81. https://doi.org/10.\n1016/j.econedurev.2017.10.005 j\nHenderson, M., Anders, J., Green, F., & Henseke, G. (2020). Private schooling, subject choice, upper \nsecondary attainment and progression to university. Oxford Review of Education, 46(3), 295–312. https://doi.org/10.1080/03054985.2019.1669551 https://doi.org/10.1080/03054985.2019.166955 Högberg, B., Strandh, M., & Hagquist, C. (2020). Gender and secular trends in adolescent mental health \nover 24 years - The role of school-related stress. Social Science & Medicine, 250, 112890. https://\ndoi.org/10.1016/j.socscimed.2020.112890 Huang, L., Kern, M. L., & Oades, L. G. (2020). Strengthening university student wellbeing: Lan-\nguage and perceptions of Chinese international students. International Journal of Environmental \nResearch and Public Health, 17(15), 5538. https://doi.org/10.3390/ijerph17155538 Islam, M. I., Ormsby, G. M., Kabir, E., & Khanam, R. (2021). Estimating income-related and area-based \ninequalities in mental health among nationally representative adolescents in Australia: The concen-\ntration index approach. PLoS ONE, 16(9), e0257573. https://doi.org/10.1371/journal.pone.02575\n73 Johnson, D., Dupuis, G., Piche, J., Clayborne, Z., & Colman, I. (2018). Adult mental health outcomes of \nadolescent depression: A systematic review. Depression and Anxiety, 35(8), 700–716. https://doi.\norg/10.1002/da.22777 Joshanloo, M., Van de Vliert, E., & Jose, P.E. (2021). Four fundamental distinctions in conceptions of \nwellbeing across cultures. In M. L. Kern, & M. L. Wehmeyer (Eds.), The Palgrave handbook of \npositive education. Palgrave Macmillan. https://doi.org/10.1007/978-3-030-64537-3_26f Kansky, J., Allen, J. P., & Diener, E. (2016). Early adolescent affect predicts later life outcomes. Applie\nPsychology, Health and Well-Being, 8(2), 192–212. https://doi.org/10.1111/aphw.12068 y\ngy\ng\np\ng\np\naye-Tzadok, A., Kim, S. S., & Main, G. (2017). Children’s subjective well-being in relation to gender \n— What can we learn from dissatisfied children? References https://doi.org/10.1057/palcomms.2016.18 Lombardo, P., Jones, W., Wang, L., Shen, X., & Goldner, E. M. (2018). The fundamental association \nbetween mental health and life satisfaction: Results from successive waves of a Canadian national \nsurvey. BMC Public Health, 18(1), 342. https://doi.org/10.1186/s12889-018-5235-x g\nbetween mental health and life satisfaction: Results from successive waves of a Canadian national \nsurvey. BMC Public Health, 18(1), 342. https://doi.org/10.1186/s12889-018-5235-x Long, E., Zucca, C., & Sweeting, H. (2021). School climate, peer relationships, and adolescent men-\ntal health: A social ecological perspective. Youth & Society, 53(8), 1400–1415. https://doi.org/10.\n1177/0044118x20970232 Luthar, S. S., Kumar, N. L., & Zillmer, N. (2020). High-achieving schools connote risks for adolescents: \nProblems documented, processes implicated, and directions for interventions. American Psycholo-\ngist, 75(7), 983–995. https://doi.org/10.1037/amp0000556 g\n( )\np\ng\np\nMaalouf, F. T., Alamiri, B., Atweh, S., Becker, A. E., Cheour, M., Darwish, H., … & Akl, E. A. (2019). Mental health research in the Arab region: Challenges and call for action. The Lancet Psychiatry, \n6(11), 961–966. https://doi.org/10.1016/s2215-0366(19)30124-5 p\ng\nMain, G. (2014). Child poverty and children’s subjective well-being. Child Indicators Research, 7(3), \n451–472. https://doi.org/10.1007/s12187-014-9237-7ff Marquez, J. (2021). Does school impact adolescents’ life satisfaction differently for students of different \nsocio-economic status? A comparative study in 33 countries. Education Inquiry, 1–16,. https://doi.\norg/10.1080/20004508.2021.1930345 Marquez, J., & Long, E. (2020). A global decline in adolescents’ subjective well-being: A comparative \nstudy exploring patterns of change in the life satisfaction of 15-year-old students in 46 countries. Child Indicators Research, 14, 1251–1292. https://doi.org/10.1007/s12187-020-09788-8 Marquez, J., & Main, G. (2020). Can schools and education policy make children happier? A com-\nparative study in 33 countries. Child Indicators Research, 14, 283–339. https://doi.org/10.1007/\ns12187-020-09758-0 Marquez, J., Katsantonis, I., Sellers, R., & Knies, G. (2022a). Life satisfaction and mental health from \nage 17 to 21 years in a general population sample. Current Psychology. https://doi.org/10.1007/\ns12144-022-03685-9 Marquez, J., Lambert, L., Ridge, N., & Walker, S. (2022b). The PISA performance gap between national \nand expatriate students in the United Arab Emirates. Journal of Research in International Educa-\ntion, 21(1), 22–45. https://doi.org/10.1177/14752409221090440f Marquez, J., Inchley, J., & Long, E. (2022c). Cross-country and gender differences in factors associated \nwith the decline in adolescents’ life satisfaction. Child Indicators Research. https://doi.org/10.1007/\ns12187-022-09930-8 McManus, S., Gunnell, D., Cooper, C., Bebbington, P. E., Howard, L. M., Brugha, T., Jenkins, R., Hassio-\ntis, A., Weich, S., & Appleby, L. (2019). References Children and Youth Services Review, 80, 96–104. Kaye-Tzadok, A., Kim, S. S., & Main, G. (2017). Children’s subjective well-being in relation to gender \n— What can we learn from dissatisfied children? Children and Youth Services Review, 80, 96–104. https://doi.org/10.1016/j.childyouth.2017.06.058 p\ng\nj\ny\nKeyes, C. (2005). Mental illness and/or mental health? Investigating axioms of the complete state model \nof health. Journal of Consulting and Clinical Psychology, 73, 539–548. https://doi.org/10.1037/\n0022-006X.73.3.539 Khan, F., Aradi, W., Schwalje, W., Buckner, E., & Fernandez-Carag, M. (2018). Women’s participation in \ntechnical and vocational education and training in the Gulf States. International Journal of Train-\ning Research, 15(3), 229–244. https://doi.org/10.1080/14480220.2017.1374666 p\ng\nKillingsworth, M. A. (2021). Experienced well-being rises with income, even above $75,000 per year. PNAS, 118(4), e2016976118. https://doi.org/10.1073/pnas.2016976118 Kinderman, P., Tai, S., Pontin, E., Schwannauer, M., Jarman, I., & Lisboa, P. (2015). Causal and mediat-\ning factors for anxiety, depression and well-being. The British Journal of Psychiatry, 206(6), 456–\n460. https://doi.org/10.1192/bjp.bp.114.147553f Knies, G. (2022). Effects of income and material deprivation on children’s life satisfaction: Evidence \nfrom longitudinal data for England (2009–2018). Journal of Happiness Studies, 23, 1469–1492. https://doi.org/10.1007/s10902-021-00457-3 https://doi.org/10.1007/s10902-021-00457-3 p\ng\nnowledge and Human Development Authority (KHDA). (2021). 2020 Dubai Student Wellbeing Census \n– Fourth Year. https://www.khda.gov.ae/en/dswc2020. Accesed 29 Oct 2022 ansford, J. E., Ben Brik, A., & Al Fara, H. (2019). A framework for child well-being in the Gulf coun-\ntries. Child Indicators Research, 12, 1971–1987. https://doi.org/10.1007/s12187-019-9620-5 Lau, M., & Bradshaw, J. (2018). Material well-being, social relationships and children’s overall \nlife satisfaction in Hong Kong. Child Indicators Research, 11, 185. https://doi.org/10.1007/\ns12187-016-9426-7l Lee, S. (2019). Does identity matter for adaptation? The influence of acculturation identity on the well-\nbeing and delinquency of adolescent students in Europe. Journal of Youth Studies, 23(6), 797–817. https://doi.org/10.1080/13676261.2019.1636950 1 3 832 J. Marquez et al. Lindorff, A. (2021). The impact of promoting student wellbeing on student academic and non-academic \noutcomes: An analysis of the evidence. USA: Oxford University Press. https://oxfordimpact.oup.\ncom/wp-content/uploads/2020/10/Wellbeing-Impact-Study-Report.pdf. Liu, X., & Zhao, J. (2016). Chinese migrant adolescents’ perceived discrimination and psychologi-\ncal well-being: The moderating roles of group identity and the type of school. PLoS ONE, 11(1), \n0146559 h\n//d i\n/10 1371/j\nl\n0146559 g\ng\ng\np\ny\nyp\n(\ne0146559. https://doi.org/10.1371/journal.pone.0146559 g\ng\ng\np\ny\ne0146559. https://doi.org/10.1371/journal.pone.0146559 g\nj\nLomas, T. (2016). Flourishing as a dialectical balance: Emerging insights from second-wave positive psy-\nchology. Palgrave Communications, 2, 16018. References Prevalence of non-suicidal self-harm and service contact in \nEngland, 2000–2014: Repeated cross-sectional surveys of the general population. The Lancet Psy-\nchiatry, 6(7), 573–581. https://doi.org/10.1016/S2215-0366(19)30188-9 Merz, E. C., Tottenham, N., & Noble, K. G. (2018). Socioeconomic status, amygdala volume, and internal-\nizing symptoms in children and adolescents. Journal of Clinical Child and Adolescent Psychology, \n47(2), 312–323. https://doi.org/10.1080/15374416.2017.1326122 Mogilner, C., Whillans, A., & Norton, M. I. (2018). Time, money, and subjective wellbeing. In E. Diener, \nS. Oishi, & L. Tay (Eds.), Handbook of well-being. DEF Publishers. https://www.nobascholar.com/\nbooks/1. Mojtabai, R., Olfson, M., & Han, B. (2016). National trends in the prevalence and treatment of depres-\nsion in adolescents and young adults. Pediatrics, 138(6), e20161878. https://doi.org/10.1542/peds.\n2016-1878 Murphy, L. E., Jack, H. E., Concepcion, T. L., & Collins, P. Y. (2020). Integrating urban adolescent mental \nhealth into urban sustainability collective action: An application of Shiffman & Smith’s framework \nfor global health prioritization. Frontiers in Psychiatry, 11, 44. https://doi.org/10.3389/fpsyt.2020.\n00044 1 3 3 Geographic, Socio‑Demographic and School Type Variation… 833 Mutumba, M., & Schulenberg, J. (2020). Cross-national variation in the subjective wellbeing of youth in \nlow and middle income countries: The role of structural and micro-level factors. Journal of Youth \nStudies, 23(2), 252–268. https://doi.org/10.1080/13676261.2019.1597257 p\ng\nNHS Digital. (2018). Mental health of children and young people in England, 2017. https://digital.nhs.uk/\ndata-and-information/publications/statistical/mental-health-of-children-and-young-people-in-engla NHS Digital. (2018). Mental health of children and young people in England, 2017. https://digital.nhs.uk/\ndata-and-information/publications/statistical/mental-health-of-children-and-young-people-in-engla\nd/2017/2017 A\nd 29 O\n2022 nd/2017/2017. Accessed 29 Oct 2022 NHS Digital. (2021). Mental health of children and young people in England, 2021. NHS Digital. https://\ndigital.nhs.uk/data-and-information/publications/statistical/mental-health-of-children-and-young-\npeople-in-england/2021-follow-up-to-the-2017-survey p\np\ng\np\ny\nOrganization for Economic Cooperation and Development (OECD). (2009). PISA Data Analysis Manual: \nSPSS, Second Edition. PISA OECD Publishing. https://doi.org/10.1787/9789264056275-en Organization for Economic Cooperation and Development (OECD). (2017). PISA 2015 results (Volume \nIII): Students’ well-being. OECD Publishing. https://doi.org/10.1787/9789264273856-en Organization for Economic Cooperation and Development (OECD). (2018). PISA 2015 technical repor\nOECD Publishing. https://www.oecd.org/pisa/data/pisa2018technicalreport/. Accessed 29 Oct 202 Organization for Economic Cooperation and Development (OECD). (2019). United Arab Emirates - \nCountry note - PISA 2018 results. http://www.oecd.org/pisa/publications/PISA2018_CN_ARE.pdf. Accessed 29 Oct 2022 Organization for Economic Cooperation and Development (OECD). (2021). OECD review of well-being \npolicies and practices in Dubai’s private school sector. OECD Publishing. https://doi.org/10.1787/\n0e9aa172-en. Accessed 29 October 2022 Otto, C., Reiss, F., Voss, C., Wüstner, A., Meyrose, A.-K., Hölling, H., & Ravens-Sieberer, U. (2021). References Mental health and well-being from childhood to adulthood: Design, methods and results of the \n11-year follow-up of the BELLA study. European Child & Adolescent Psychiatry, 30, 1559–1577. https://doi.org/10.1007/s00787-020-01630-4 Pagerols, M., Prat, R., Rivas, C., Español-Martín, G., Puigbó, J., Pagespetit, E., Haro, J. P., Ramos-Qui-\nroga, J. A., Casas, M., & Bosch, R. (2022). The impact of psychopathology on academic perfor-\nmance in school-age children and adolescents. Scientific Reports, 12, 4291. https://doi.org/10.1038/\ns41598-022-08242-9 Paparusso, A. (2021). Immigrant integration in Europe: A subjective well-being perspective. Springer. https://doi.org/10.1007/978-3-030-78505-5 p\ng\nPatalay, P., & Fitzsimons, E. (2016). Correlates of mental illness and wellbeing in children: Are they the \nsame? Results from the UK Millennium Cohort Study. Journal of the American Academy of Child \nAdolescent Psychiatry, 55(9), 771–783. https://doi.org/10.1016/j.jaac.2016.05.019 Patalay, P., & Fitzsimons, E. (2018). Development and predictors of mental ill-health and wellbeing from \nchildhood to adolescence. Social Psychiatry & Psychiatric Epidemiology, 53, 1311–1323. https://\ndoi.org/10.1007/s00127-018-1604-0 g\nPatton, G. C., & Viner, R. (2007). Pubertal transitions in health. The Lancet, 369(9567), 1130–1139. https://doi.org/10.1016/s0140-6736(07)60366-3 Petersen, I. T., Lindhiem, O., LeBeau, B., Bates, J. E., Pettit, G. S., Lansford, J. E., & Dodge, K. A. (2018). Development of internalizing problems from adolescence to emerging adulthood: Accounting for \nheterotypic continuity with vertical scaling. Developmental Psychology, 54(3), 586–599. https://doi.\norg/10.1037/dev0000449 g\nPeverill, M., Dirks, M. A., Narvaja, T., Herts, K. L., Comer, J. S., & McLaughlin, K. A. (2021). Socioeco-\nnomic status and child psychopathology in the United States: A meta-analysis of population-based \nstudies. Clinical Psychology Review, 83, 101933. https://doi.org/10.1016/j.cpr.2020.101933 Racine, N., McArthur, B. A., Cooke, J. E., Eirich, R., Zhu, J., & Madigan, S. (2021). Global prevalence of \ndepressive and anxiety symptoms in children and adolescents during COVID-19: A meta-analysis. JAMA Pediatrics, 175(11), 1142–1150. https://doi.org/10.1001/jamapediatrics.2021.2482 Rees, P. L., Main, G., & Bradshaw, J. R. (2015). The good childhood report 2015. The Children’s Socie\nhttp://www.york.ac.uk/inst/spru/research/pdf/GCReport2015.pdf. Accessed 29 October 2022 Rees, G., Pople, L., & Goswami, H. (2011). Links between family economic factors and children’s \nsubjective well-being: Initial findings from wave 2 and wave 3 quarterly surveys. The Children’s \nSociety. Reiss, F. (2013). Socioeconomic inequalities and mental health problems in children and adolescents: A \nsystematic review. Social Science & Medicine, 90, 24–31. https://doi.org/10.1016/j.socscimed.2013.\n04.026 1 3 834 J. Marquez et al. Reiss, F., Meyrose, A. K., Otto, C., Lampert, T., Klasen, F., & Ravens-Sieberer, U. (2019). References Socioeconomic \nstatus, stressful life situations and mental health problems in children and adolescents: Results of the \nGerman BELLA cohort-study. PLoS ONE, 14(3), e0213700. https://doi.org/10.1371/journal.pone.\n0213700 Rentfrow, P. J. (2018). Geographical variation in subjective well-being. In E. Diener, S. Oishi, & L. Tay \n(Eds.), Handbook of well-being. USA: DEF Publishers. https://www.nobascholar.com/books/1. Richards, M., & Huppert, F. A. (2011). Do positive children become positive adults? Evidence from a \nlongitudinal birth cohort study. The Journal of Positive Psychology, 6(1), 75–87. https://doi.org/10.\n1080/17439760.2011.536655 Riglin, L., Petrides, K. V., Frederickson, N., & Rice, F. (2014). The relationship between emotional prob-\nlems and subsequent school attainment: A meta-analysis. Journal of Adolescence, 37(4), 335–346. https://doi.org/10.1016/j.adolescence.2014.02.010 https://doi.org/10.1016/j.adolescence.2014.02.0 p\ng\nj\nRodríguez, S., Valle, A., Gironelli, L. M., Guerrero, E., Regueiro, B., & Estévez, I. (2020). Performance \nand well-being of native and immigrant students. Comparative analysis based on PISA 2018. Jour-\nnal of Adolescence, 85, 96–105. https://doi.org/10.1016/j.adolescence.2020.10.001f Ryff, C. D., Boylan, J. M., & Kirsch, J. A. (2021). Eudaimonic and hedonic well-being: An integrative \nperspective with linkages to sociodemographic factors and health. In M. T. Lee, L. D. Kubzansky, & \nT. J. VanderWeele (Eds.), Measuring well-being: Interdisciplinary perspectives from the social sci-\nences and the humanities (pp. 92–135). Oxford University Press. https://doi.org/10.1093/oso/97801\n97512531.003.0005 Sarriera, J. C., Casas, F., Bedin, L., Abs, D., Strelhow, M. R., Gross-Manos, D., & Giger, J. (2014). Mate-\nrial resources and children’s subjective well-being in eight countries. Child Indicators Research, \n8(1), 199–209. https://doi.org/10.1007/s12187-014-9284-0 Schirripa, F., Salvati, N., D’Agostino, A., & Nicaise, I. (2020). The use of sampling weights in the M-quan-\ntile random-effects regression: An application to PISA mathematics scores. of the Royal Statistical \nSociety Series C 69(4), 991–1012. https://doi.org/10.1111/rssc.12418 Schoon, I., & Henseke, G. (2022). Social inequalities in young people’s mental distress during the COVID-\n19 pandemic: Do psychosocial resource factors matter? Frontiers in Public Health, 10, 820270. https://doi.org/10.3389/fpubh.2022.820270 Schwartz, S. J., Waterman, A. S., Umaña-Taylor, A. J., Lee, R. M., Kim, S. Y., Vazsonyi, A. T., Huynh, Q., \nWhitbourne, S. K., Park, I. J. K., Hudson, M., Zamboanga, B. L., Bersamin, M. M., & Williams, M. K. (2013). Acculturation and well-being among college students from immigrant families. Journal \nof Clinical Psychology, 69(4), 298–318. https://doi.org/10.1002/jclp.21847 Shah, S. M., Al Dhaheri, F., Albanna, A., Al Jaberi, N., Al Eissaee, S., Alshehhi, N. A., … & Betancourt, \nT. S. (2020). Self-esteem and other risk factors for depressive symptoms among adolescents in \nUnited Arab Emirates. StataCorp. (2017). Stata statistical software: Release (p. 15). StataCorp LLC. References https://doi.org/10.1016/S2215-0366(20)30481-8 Tomyn, A. J., Mellor, D., Fuller-Tyszkiewicz, M., Cummins, R. A., & Tanton, R. (2017). Geographic dif-\nferences in subjective well-being among Indigenous and non-Indigenous Australian adolescents and \nadults. Journal of Community Psychology, 45(1), 81–99. https://doi.org/10.1002/jcop.21835fi Trotta, A., Arseneault, L., Caspi, A., Moffitt, T. E., Danese, A., Pariante, C., & Fisher, H. L. (2020). Mental \nhealth and functional outcomes in young adulthood of children with psychotic symptoms: A longi-\ntudinal cohort study. Schizophrenia Bulletin, 46(2), 261–271. https://doi.org/10.1093/schbul/sbz069 Tsai, S. L., Smith, M. L., & Hauser, R. M. (2018). Gender gaps in student academic achievement and ine-\nquality. Research in the Sociology of Education, 20, 181–218. https://doi.org/10.1108/S1479-35392\n0180000020008 NICEF. (2021). The state of the world’s children 2021: On my mind – Promoting, protecting and caring \nfor children’s Mental health. https://www.unicef.org/reports/state-worlds-children-2021 United Nations (1989). The United Nations Convention on the Rights of th United Nations (1989). The United Nations Convention on the Rights of the Child. https://www.unicef.org.\nuk/wp-content/uploads/2010/05/UNCRC PRESS200910web.pdf United Nations (1989). The United Nations Convention on the Rights of the Child. https://www.unicef.or\nk/\nt\nt/\nl\nd /2010/05/UNCRC PRESS200910\nb df uk/wp-content/uploads/2010/05/UNCRC_PRESS200910web.pdff van Hek, M., Kraaykamp, G., & Pelzer, B. (2018). Do schools affect girls’ and boys’ reading performance \ndifferently? A multilevel study on the gendered effects of school resources and school practices. School Effectiveness and School Improvement. Routledge, 29(1), 1–21. https://doi.org/10.1080/\n09243453.2017.1382540 Vanaken, G. J., & Danckaerts, M. (2018). Impact of green space exposure on children’s and adolescents’ \nmental health: A systematic review. International Journal of Environmental Research and Public \nHealth, 15(12), 2668. https://doi.org/10.3390/ijerph15122668 (\n)\np\ng\nj p\nVan Phan, P., & O’Brien, M. (2019). Multidimensional wellbeing inequality in a developing coun-\ntry: A case study of Vietnam. Social Indicators Research, 145, 157–183. https://doi.org/10.1007/\ns11205-019-02104-0 Wang, Z. (2021). Mind the gap: Examining migrant-native disparities in reading performance and subjec-\ntive wellbeing among 15-year-old students in different education systems. International Journal of \nEducation Research Open, 2, 100087. https://doi.org/10.1016/j.ijedro.2021.100087 Ward, J. S., Duncan, J. S., Jarden, A., & Stewart, T. (2016). The impact of children’s exposure to greens-\npace on physical activity, cognitive development, emotional wellbeing, and ability to appraise risk. Health & Place, 40, 44–50. https://doi.org/10.1016/j.healthplace.2016.04.015 p\ng\nj\np\nWesterhof, G. J., & Keyes, C. L. M. (2010). Mental illness and mental health: The two continua \nmodel across the lifespan. Journal of Adult Development, 17, 110–119. References PLoS ONE, 15(1), e0227483. https://doi.org/10.1371/journal.pone.0227483 Sharpe, H., Patalay, P., Fink, E., Vostanis, P., Deighton, J., & Wolpert, M. (2016). Exploring the relation-\nship between quality of life and mental health problems in children: Implications for measurement \nand practice. European Child and Adolescent Psychiatry, 25(6), 659–667. https://doi.org/10.1007/\ns00787-015-0774-5 Sheikh Saud bin Saqr Al Qasimi Foundation for Policy Research (2020). Education in the United Ara\nEmirates and Ras Al Khaimah. Fact Sheet. https://doi.org/10.18502/aqf.0154.f Shoshani, A., Steinmetz, S., & Kanat-Maymon, Y. (2016). Effects of the Maytiv positive psychology \nschool program on early adolescents’ well-being, engagement, and achievement. Journal of School \nPsychology, 57, 73–92. https://doi.org/10.1016/j.jsp.2016.05.003 Silva, M., Loureiro, A., & Cardoso, G. (2016). Social determinants of mental health: A review of the evi-\ndence. European Journal of Psychiatry, 30, 259–292. Solmi, M., Radua, J., Olivola, M., Croce, E., Soardo, L., Salazar de Pablo, G., Il Shin, J., Kirkbride, J. B., \nJones, P., Kim, J. H., Kim, J. Y., Carvalho, A. F., Seeman, M. V., Correll, C. U., & Fusar-Poli, P. (2021). Age at onset of mental disorders worldwide: Large-scale meta-analysis of 192 epidemiologi-\ncal studies. Molecular Psychiatry, 27, 281–295. https://doi.org/10.1038/s41380-021-01161-7 Jones, P., Kim, J. H., Kim, J. Y., Carvalho, A. F., Seeman, M. V., Correll, C. U., & Fusar-Poli, P. (2021). Age at onset of mental disorders worldwide: Large-scale meta-analysis of 192 epidemiologi-\ncal studies Molecular Psychiatry 27 281–295 https://doi org/10 1038/s41380-021-01161-7 Sonmark, K., Godeau, E., Augustine, L., Bygren, M., & Modin, B. (2016). Individual and contextual \nexpressions of school demands and their relation to psychosomatic health a comparative study of \nstudents in France and Sweden. Child Indicators Research, 9(1), 93–109. https://doi.org/10.1007/\ns12187-015-9299-1 StataCorp. (2017). Stata statistical software: Release (p. 15). StataCorp LLC. 1 3 Geographic, Socio‑Demographic and School Type Variation… 835 Sullivan, A., Parsons, S., Ploubidis, G., Wiggins, R. D., & Green, F. (2021). Education and psychologi-\ncal distress in adolescence and mid-life: Do private schools make a difference? British Educational \nResearch Journal, 47(3), 599–615. https://doi.org/10.1002/berj.3674 Tang, Y. (2019). Immigration status and adolescent life satisfaction: An international comparative anal-\nysis based on PISA 2015. Journal of Happiness Studies, 20, 1499–1518. https://doi.org/10.1007/\ns10902-018-0010-3 Taylor, L. J., DeNeve, J.-E., DeBrost, L., & Khanna, D. (2022). Wellbeing in education in childhood and \nadolescence (Report No. 1). International Baccalaureate Organisation. https://ibo.org/research/pol-\nicy-research/well-being-in-education-in-childhood-and-adolescence-2022/. y\ng\nThapar, A., Stewart-Brown, S., & Harold, G. T. (2021). What has happened to children’s wellbeing in the \nUK? Lancet Psychiatry, 8(1), 5–6. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps \nand institutional affiliations. References https://doi.org/10.1007/\ns10804-009-9082-y Zaborskis, A., Grincaite, M., Lenzi, M., Tesler, R., Moreno-Maldonado, C., & Mazur, J. (2019a). Social \ninequality in adolescent life satisfaction: Comparison of measure approaches and correlation with \nmacro-level indices in 41 countries. Social Indicators Research, 141, 1055–1079. https://doi.org/10.\n1007/s11205-018-1860-0 Zaborskis, A., Ilionsky, G., Tesler, R., & Heinz, A. (2019b). The association between cyberbullying, \nschool bullying, and suicidality among adolescents: Findings from the cross-national study HBSC \nin Israel, Lithuania, and Luxembourg. Crisis, 40(2), 100–114. https://doi.org/10.1027/0227-5910/\na000536 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps \nand institutional affiliations. 1 3 J. Marquez et al. 836 Jose Marquez1 · Louise Lambert2 · Megan Cutts1 Louise Lambert \nltlamber@yahoo.com\nMegan Cutts \nmegan.cutts@manchester.ac.uk Louise Lambert \nltlamber@yahoo.com\nMegan Cutts \nmegan.cutts@manchester.ac.uk Louise Lambert \nltlamber@yahoo.com 1\t\nManchester Institute of Education, University of Manchester, Oxford Road, \nManchester M13 9PL, UK 2\t\nCanadian University Dubai, Dubai, UAE 1 3"
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Horizontal Plasmid Transfer by Transformation in Escherichia coli: Environmental Factors and Possible Mechanisms
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*Correspondence: *Correspondence:
Sumio Maeda
smaeda@cc.nara-wu.ac.jp Horizontal Plasmid Transfer by
Transformation in Escherichia coli:
Environmental Factors and Possible
Mechanisms Haruka Hasegawa, Erika Suzuki and Sumio Maeda*
Graduate School of Humanities and Sciences, Nara Women’s University, Nara, Japan Transformation is one mode of horizontal gene transfer (HGT) in bacteria, wherein
extracellular naked DNA is taken up by cells that have developed genetic competence. Sensitivity to DNase, which degrades naked DNA, is the key to distinguishing
transformation from the DNase-resistant HGT mechanisms. In general, Escherichia
coli is not believed to be naturally transformable; it develops high competence only
under artificial conditions, including exposure to high Ca2+ concentrations. However,
E. coli can reportedly express modest competence under certain conditions that are
feasible in natural environments outside laboratory. In addition, recent data suggest
that environmental factors influence multiple routes of transformation. In this mini
review, we (1) summarize our studies on transformation-based HGT using E. coli
experimental systems and (2) discuss the possible occurrence of transformation via
multiple mechanisms in the environment and its possible impact on the spread of
antibiotic resistance genes. MINI REVIEW MINI REVIEW
published: 04 October 2018
doi: 10.3389/fmicb.2018.02365 INTRODUCTION Specialty section:
This article was submitted to
Antimicrobials, Resistance
and Chemotherapy,
a section of the journal
Frontiers in Microbiology Horizontal gene transfer (HGT) between bacterial cells contributes to bacterial adaptation to
various environments and, in the long term, to bacterial evolution (Lorenz and Wackernagel,
1994; Bushman, 2002; Thomas and Nielsen, 2005). However, in human environments, it
causes undesirable spread of pathogenicity, antibiotic resistance, or artificially engineered genes
(Bushman, 2002; Keese, 2008; Kelly et al., 2009a,b). Three mechanisms of HGT in bacteria
are generally accepted: conjugation, transduction, and transformation (Bushman, 2002; von
Wintersdorffet al., 2016). Conjugation and transduction involve specific apparatus for DNA
transfer from donor to recipient cells; these are conjugative pili and phage virions, respectively. Transformation is primarily a function of recipient cells that express competence to take up
extracellular naked DNA. Received: 21 June 2018
Accepted: 14 September 2018
Published: 04 October 2018 Edited by:
Dongchang Sun,
Zhejiang University of Technology,
China Reviewed by:
Nathalie J. A. Campo,
UMR5100 Laboratoire
de Microbiologie et Génétique
Moléculaires (LMGM), France
Rosemary Redfield,
The University of British Columbia,
Canada
Radoslaw Pluta,
Institute for Research in Biomedicine,
Spain Keywords: plasmid transformation, horizontal plasmid transfer, Escherichia coli, antibiotic resistance, solid-air
biofilm Keywords: plasmid transformation, horizontal plasmid transfer, Escherichia coli, antibiotic resistance, solid-air
biofilm Citation: Hasegawa H, Suzuki E and
Maeda S (2018) Horizontal Plasmid
Transfer by Transformation
in Escherichia coli: Environmental
Factors and Possible Mechanisms. Front. Microbiol. 9:2365. doi: 10.3389/fmicb.2018.02365 Transformation competence can be naturally or artificially induced, but not all bacterial species
develop natural competence (Lorenz and Wackernagel, 1994; Johnston et al., 2014). In naturally
transformable bacteria, competence is usually transient and induced by alterations in the growth
state of organism (Johnston et al., 2014). A group of “competence genes” has been identified,
and general mechanistic models have been proposed (Chen and Dubnau, 2004), although precise October 2018 | Volume 9 | Article 2365 Frontiers in Microbiology | www.frontiersin.org 1 Multiplicity of E. coli Transformation Hasegawa et al. mechanisms for individual bacterial species have not been
sufficiently elucidated (Cameron and Redfield, 2006, 2008;
Sinha et al., 2009; Seitz and Blokesch, 2013; Johnston et al.,
2014; Jaskólska and Gerdes, 2015). Because transformation
requires extracellular naked DNA as the substrate, sensitivity
to DNase, which degrades naked DNA, is key in distinguishing
transformation from other DNase-resistant HGT mechanisms
(Lorenz and Wackernagel, 1994; Giovanetti et al., 2005; Marshall
et al., 2010; Rohrer et al., 2012; Blesa and Berenguer, 2015). mechanisms for individual bacterial species have not been
sufficiently elucidated (Cameron and Redfield, 2006, 2008;
Sinha et al., 2009; Seitz and Blokesch, 2013; Johnston et al.,
2014; Jaskólska and Gerdes, 2015). Because transformation
requires extracellular naked DNA as the substrate, sensitivity
to DNase, which degrades naked DNA, is key in distinguishing
transformation from other DNase-resistant HGT mechanisms
(Lorenz and Wackernagel, 1994; Giovanetti et al., 2005; Marshall
et al., 2010; Rohrer et al., 2012; Blesa and Berenguer, 2015). and inner membrane proteins are involved. This mechanism is
regulated by internal transcriptional regulators, RpoS and CRP,
therefore it was suggested that this mechanism is also a genetically
controlled natural process. In this mini review, we summarize our studies on HGT
using E. coli experimental systems and discuss the possible
occurrence of transformation by multiple mechanisms in natural
environments and its possible impact on the spread of antibiotic
resistance genes. g
In general, Escherichia coli is not believed to be naturally
transformable; it develops high genetic competence only
under artificial conditions, including exposure to high Ca2+
concentrations and temperature shock (Mandel and Higa, 1970;
Hanahan, 1983; Sambrook et al., 1989), polyethylene glycol
treatment (Chung et al., 1989; Sambrook et al., 1989), or electrical
shock (Sambrook and Russell, 2006). However, reportedly, E. Citation: coli
can express modest competence under certain conditions that
are feasible in its natural environments (Baur et al., 1996, Bauer
et al., 1999; Tsen et al., 2002; Woegerbauer et al., 2002). In the
following, we define transformation wherein plasmid was added
externally as plasmid transformation (PT) and transformation
wherein plasmid DNA comes from dead bacterial cells (from the
environment) as horizontal plasmid transfer by transformation
(HPTT). PT in Food Extracts Human foods are excellent culture media for many bacteria. However, little attention has been paid to the effects of foods
on bacterial physiology other than growth and survival. We
investigated the possibility that foods act as media for bacterial
transformation. Foods often contain millimolar concentrations
of divalent metal ions (Ca2+ and Mg2+) and are often stored
in a refrigerator or freezer followed by rapid warming (i.e., heat
shocked). These conditions are conducive to the development
of competency in E. coli (Mandel and Higa, 1970; Huang and
Reusch, 1995; Baur et al., 1996); because E. coli is a common
food contaminant, it is interesting to determine whether it can
be transformed in foods. Certain foods can indeed act as media
that induce competency in E. coli (Maeda et al., 2003). Of 42 food
samples tested, >10 exhibited an ability to induce competency
at a frequency of 10−7−10−9. Among these, the supernatant
from tofu (a cheese-like food made of curdled soybean milk)
exhibited the highest activity (one in 10−7−10−8 recipient
cells), corresponding to approximately one-half of the efficiency
obtained with 100 mM CaCl2. However, there were no clear
correlations between transformation frequencies and chemical
characteristics of the foods (Ca2+ or Mg2+ concentrations and
pH), suggesting that complex factors within the foods affect
competency development. Similar effects of foods in inducing
transformation have been reported in E. coli (Bauer et al., 1999)
and Bacillus subtilis (Brautigam et al., 1997; Zenz et al., 1998). Escherichia coli seems to possess multiple DNA-uptake
mechanisms, including two popular ones: one that is dependent
on the “competence genes,” which commonly work in many
gram-negative and -positive bacteria (Finkel and Kolter, 2001;
Palchevskiy and Finkel, 2006; Sinha et al., 2009; Sinha and
Redfield, 2012; Seitz and Blokesch, 2013; Johnston et al.,
2014; Jaskólska and Gerdes, 2015). This mechanism is mainly
conducted by the specific molecular apparatus formed around
the cell surface structure, which pass through the cell membranes
only linear single-stranded DNA produced using a specific
periplasmic nuclease. In E. coli, these genes are not considered to
contribute to PT because PT requires the uptake of intact double-
stranded circular DNA (Sinha and Redfield, 2012; Johnston
et al., 2014). Therefore, it is unlikely that this mechanism
contributes to PT in the environment. PT in Food Extracts The second mechanism
is that dependent on external environmental factors, such as
divalent metal ions, heat shock, and physical stresses (Mandel
and Higa, 1970; Hanahan, 1983; Yoshida, 2007; Rodríguez-
Beltrán et al., 2013). These stimuli are commonly considered to
induce the formation of pore-like structures in cell surface for
the passing of intact double-stranded DNA, including circular
plasmids, although the details remain unclear (Reusch et al.,
1986; Reusch and Sadoff, 1988; Huang and Reusch, 1995;
Sun et al., 2013; Asif et al., 2017). Ca2+ and Mg2+ ions are
the most typical competence-inducing factors. Environmental
habitats often contain several millimolar of these ions, whose
concentrations are sufficient to induce weak but detectable
competence in E. coli (Baur et al., 1996, Bauer et al., 1999;
Maeda et al., 2003). Therefore, this mechanism is possible in the
environment outside laboratories. In addition to the above two
mechanisms, another mechanism has been proposed by Sun et al. (2006, 2009), Zhang et al. (2012), Guo et al. (2015), and Sun
(2016), in which an ABC transporter and specific periplasmic High Frequency of HPTT Induced by P1
Phage g
By assessing combinations of several strains and plasmids for
horizontal plasmid transfer, the E. coli strain CAG18439 was
found to act as both a plasmid donor and a plasmid recipient
in combination with the plasmid pHSG299 and could frequently
transfer the plasmid in a mixed cell culture even in a liquid
medium (Etchuuya et al., 2011). This HGT was demonstrated
to be a type of transformation because the high frequency
plasmid transfer (10−5−10−8) was DNase-sensitive. Further
studies revealed that this phenomenon exhibits some specific
characteristics: (1) promotion by proteinaceous factor released
from CAG18439 (Etchuuya et al., 2011); (2) promotion by an
88-bp sequence on pHSG299 (Sobue et al., 2011); (3) high
transfer frequency (Etchuuya et al., 2011; Sobue et al., 2011); and
(4) dependence on specific genes (Kurono et al., 2012; Matsuda
et al., 2012). With respect to (1), a later study revealed that
these proteinaceous factors include a P1vir phage particle (or a
derivative thereof) and that externally added P1vir phage can
reproduce horizontal plasmid transfer between E. coli cells and
the three other major features of CAG18439-dependent HPTT
(Sugiura et al., 2017). This phenomenon was also largely DNase-
sensitive, suggesting that a large part of this plasmid transfer
is due to transformation despite the involvement of P1 phage. The transformation mechanism of P1vir phage-induced plasmid
transfer may be due to phage infection or spontaneous awakening
of lysogenized phage in plasmid-harboring cells, leading to cell
lysis and subsequent intracellular plasmid DNA release in a
usable form for transformation. Although such a mechanism
is generally feasible, there have been few clear demonstrations
of it in E. coli. A recent study by Keen et al. (2017) using
other phage system also demonstrated a similar phage-induced
transformation mechanism in E. coli. However, HPTT by P1vir
or CAG18439 cannot be adequately explained only by enhanced
DNA supply from phage-induced cell lysis, and it differs from
simple transformation in E. coli (Hanahan, 1983) in terms of its PT of Wild E. coli Strains in Water Our and others’ results suggest that environmental E. coli can
potentially acquire foreign DNA via transformation. However,
there are few previous reports of investigations into the
transformability of natural E. coli strains (Woegerbauer et al.,
2002; Sinha and Redfield, 2012). Therefore, we examined the
potential of natural E. coli strains to develop competence under
environmental conditions. We used a standard E. coli collection
of reference (ECOR) strains as our model of natural E. coli
(Ochman and Selander, 1984) because these ECOR strains have
been widely used in various studies on the physiology, behavior,
and genotypic variation of natural E. coli (Tenaillon et al.,
2010). We found that some ECOR strains exhibited detectable
transformability (10−10−10−11) in natural water (commercially
available bottled natural pure water) at constant and varying
temperatures between 5 and 35◦C and at winter temperatures in
a field experiment, suggesting that natural E. coli can potentially
develop competence under certain conditions that could feasibly
occur in the environment (Matsumoto et al., 2016b). PT in Solid-Air Biofilm Many bacteria exist as biofilms in natural and artificial
environments (Davey and O’Toole, 2000). Biofilms are aggregates
of microbes that form at solid-liquid or solid-air (SA) interfaces
(Anderl et al., 2000; Carmen et al., 2004). Cells in these high-
density cultures interact with one another and express distinctive
physiological functions compared with their free planktonic
forms. Previous studies on E. coli transformation exclusively
focused on planktonic cells (Mandel and Higa, 1970; Hanahan,
1983), but we showed that E. coli cells within SA biofilms develop
competence at a frequency of 10−6−10−8 on various solid media,
including LB and H2O agar and various moist foods (Maeda et al.,
2004). Living cells generally coexist with dead cells in biofilms,
and the latter can release their DNA and certain divalent metal October 2018 | Volume 9 | Article 2365 Frontiers in Microbiology | www.frontiersin.org 2 Multiplicity of E. coli Transformation Hasegawa et al. (Maeda et al., 2004), both these factors contribute to HPTT
in biofilms. By simply co-culturing a plasmid-free strain with
one harboring a non-conjugative plasmid in a SA biofilm on
antibiotic-free agar media, transformed cells were produced at
low frequency (10−9−10−10) within 24–48 h (Maeda et al.,
2006). Liquid cultures of the same strains in LB broth produced
no or few transformants, suggesting the importance of SA
biofilm formation for plasmid transfer. Essentially, the same
phenomenon occurred in SA biofilms on food-based media
(Ando et al., 2009). This phenomenon also occurred between
popular laboratory strains such as DH5, HB101, and MG1655
(Etchuuya et al., 2011), which are lysogenic phage-free and
conjugative apparatus-free, suggesting that the low frequency of
horizontal plasmid transfer in SA biofilms can occur without the
aid of phage or conjugation machinery and, therefore, that this
DNA transfer is due to a kind of transformation. However, since
rpoS−mutation did not affect this HPTT (Maeda et al., 2006), the
RpoS-dependent mechanism (Zhang et al., 2012) is unlikely to be
involved. ions, including Ca2+ and Mn2+, into the local microenvironment
of the biofilm (Davey and O’Toole, 2000; Whitchurch et al.,
2002). These conditions may be conducive for the development
of transformation and may not be exclusive to SA biofilms since
a similar enhancement in E. coli air-liquid biofilms has also been
reported (Król et al., 2011). Freeze–Thaw-Induced HPTT in Natural
Waters and Food Extracts In the environment, naked DNA can be naturally supplied
from dead cells to neighboring cells within the same habitat
or microenvironment. Therefore, it is worth investigating the
possibility of HPTT in a closed system under some feasible
conditions. Freeze–thaw is a common process in the handling
of foodstuffs and also occurs in nature. Freeze–thaw treatment
of E. coli cells may promote DNA leakage from dead cells and
subsequent uptake by surviving cells because they respond to
heat shock, resulting in in situ transformation (Li et al., 1992;
Takahashi et al., 1992). This treatment of condensed suspensions
of mixed E. coli strains in natural waters and food extracts caused
in situ lateral transfer of non-conjugative plasmids at a frequency
of 10−8−10−10 (Ishimoto et al., 2008). This phenomenon also
occurred even after 1–2 months of storage at −20◦C, and its
sensitivity to DNase demonstrated that it was mediated via a
transformation mechanism. Low Frequency of HPTT in SA Biofilms Low Frequency of HPTT in SA Biofilms
Biofilms are thought to be suitable environments for in situ
transformation because living and dead cells coexist in close
proximity, and DNA released from dead cells often accumulates
around living cells. In addition, as described above, because
E. coli cells can develop modest competence in SA biofilms October 2018 | Volume 9 | Article 2365 Frontiers in Microbiology | www.frontiersin.org 3 Hasegawa et al. Multiplicity of E. coli Transformation POSSIBLE MECHANISMS AND
FEASIBILITY OF PT AND HPTT IN E. coli
IN THE ENVIRONMENT With respect to
(3), this high-frequency transfer cannot be explained by the
simple PT ability of CAG18439 and other strains used because
simple PT in those strains under the equivalent culture condition
was 105–102 times less frequent (Etchuuya et al., 2011). It was,
therefore, suggested that a CAG18439-derived proteinaceous
factor, with size estimated between 9 and 30 kDa (Etchuuya et al.,
2011) could also be involved in promoting HPTT. This factor
presumably assists in DNA uptake by recipient cells, probably
in combination with the 88-bp sequence on the transforming
DNA. Lastly, with respect to (4), later genome-wide screening
studies for recipient genes involved in HPTT suggested that
multiple genes participate in the mechanism (Kurono et al.,
2012; Matsuda et al., 2012; Shibata et al., 2014a). These include
those that have not been reported to be involved in natural
or artificial transformation in E. coli (such as rodZ) and a few
known competence gene homologs, such as ybaV and yhiR
(Finkel and Kolter, 2001; Palchevskiy and Finkel, 2006), but do
not include rpoS and other genes related to the RpoS-dependent
mechanism (Zhang et al., 2012). Overall, these results point
toward an unknown, complex mechanism of phage-induced,
high-frequency HPTT that may partly share the pathway of
natural transformation. Examples of PT and freeze–thaw-induced and low-frequency
HPTT introduced in this mini-review are probably more
related to the pore-forming mechanism than the competence
gene-dependent mechanism because foods and natural waters
often contain mM levels of Ca2+ and Mg2+ ions (Baur
et al., 1996, Bauer et al., 1999; Maeda et al., 2003), and the
biofilm environment supply living cells with the content of
dead cells, including divalent metal ions and transformable
plasmid DNA. As we described previously (Maeda et al.,
2006),
an
SA
biofilm
(diameter,
10–12
mm;
thickness,
0.5–0.8 mm) contains approximately 2–5 × 109 cells. In
addition, gut bacteria in mammals generally amount to
approximately 1011 cells/g (Zoetendal et al., 2004; Sekirov et al.,
2010). Considering the enormous scale of the environment,
even transformation frequencies of 10−9−10−10 cannot be
underestimated as they will have an impact on the bacteria
populations. High-frequency HPTT described in this article may involve
not only the pore-forming mechanism but also a part of the
competence gene functions and possibly another unknown
mechanism, as mentioned above. POSSIBLE MECHANISMS AND
FEASIBILITY OF PT AND HPTT IN E. coli
IN THE ENVIRONMENT Because bacteriophages are one
of the most abundant organisms in the biosphere and ubiquitous
in the environment (Clokie et al., 2011), phage-induced HPTT
is also considered to be feasible in the environment, as well as
ordinary transduction and other phage-derived ways of HGT,
e.g., gene transfer agents (Lang et al., 2012). POSSIBLE MECHANISMS AND
FEASIBILITY OF PT AND HPTT IN E. coli
IN THE ENVIRONMENT distinctive characteristics (2–4). With respect to (2), the 88-bp
sequence on pHSG299 is not homologous to the part of the
P1 phage genome sequence. This sequence is often found in
databases among general cloning vector sequences but not in
any natural source. By tracing back the construction process
of pHSG299 (Hashimoto-Gotoh et al., 1981; Brady et al., 1984;
Takeshita et al., 1987), however, we suspect that the 88-bp
sequence originates from R6-5, a conjugative R plasmid. This
sequence, and similar DNA elements, may contribute to HPTT
of R and other plasmids in the environment. With respect to
(3), this high-frequency transfer cannot be explained by the
simple PT ability of CAG18439 and other strains used because
simple PT in those strains under the equivalent culture condition
was 105–102 times less frequent (Etchuuya et al., 2011). It was,
therefore, suggested that a CAG18439-derived proteinaceous
factor, with size estimated between 9 and 30 kDa (Etchuuya et al.,
2011) could also be involved in promoting HPTT. This factor
presumably assists in DNA uptake by recipient cells, probably
in combination with the 88-bp sequence on the transforming
DNA. Lastly, with respect to (4), later genome-wide screening
studies for recipient genes involved in HPTT suggested that
multiple genes participate in the mechanism (Kurono et al.,
2012; Matsuda et al., 2012; Shibata et al., 2014a). These include
those that have not been reported to be involved in natural
or artificial transformation in E. coli (such as rodZ) and a few
known competence gene homologs, such as ybaV and yhiR
(Finkel and Kolter, 2001; Palchevskiy and Finkel, 2006), but do
not include rpoS and other genes related to the RpoS-dependent
mechanism (Zhang et al., 2012). Overall, these results point
toward an unknown, complex mechanism of phage-induced,
high-frequency HPTT that may partly share the pathway of
natural transformation. distinctive characteristics (2–4). With respect to (2), the 88-bp
sequence on pHSG299 is not homologous to the part of the
P1 phage genome sequence. This sequence is often found in
databases among general cloning vector sequences but not in
any natural source. By tracing back the construction process
of pHSG299 (Hashimoto-Gotoh et al., 1981; Brady et al., 1984;
Takeshita et al., 1987), however, we suspect that the 88-bp
sequence originates from R6-5, a conjugative R plasmid. This
sequence, and similar DNA elements, may contribute to HPTT
of R and other plasmids in the environment. CONCLUSION AND PERSPECTIVE HPTT Between Natural E. coli Strains
To further assess the generality and variety of HPTT in E. coli
strains, natural strains (the aforementioned ECOR strains) were
used in a study of HPTT. Several combinations of ECOR
strains were co-cultured in liquid media, resulting in DNase-
sensitive horizontal transfer of natural antibiotic resistance
genes (Matsumoto et al., 2016a,b). Plasmid isolation from these
new transformants demonstrated horizontal plasmid transfer
between ECOR strains (Matsumoto et al., 2016a,b). Simple
PT experiments using the same ECOR strains revealed that
HPTT occurs much more frequently (10−6−10−8) than simple
PT (below 10−10) under the same culture conditions, which
suggested that HPTT is unique and effective. Moreover, we
discovered that 6 of 12 combinations of the ECOR strains,
some of which produce no plaque-forming phages (Shibata
et al., 2014b), exhibited DNase-sensitive gene transfer, leading
us to suspect that HPTT is rather common in natural E. coli
strains. Overall, these data suggest that some phage- and
conjugation-free transformation mechanism(s) also naturally
exist in some E. coli strains and that HPTT of antibiotic-
resistant natural plasmids (such as plasmids of the ECOR24
strain: Accession Nos. AB905284 and AB905285) can be
a pathway for producing multidrug-resistant natural E. coli
cells. Overall, our results and related previous data indicate that
multiple mechanisms induce transformation-type HGT in E. coli
based on various environmental and cellular circumstances
such as the nature of the media (e.g., water and food),
variable temperature from sub-zero to ∼40◦C, high cell
density in biofilms, and varying genetic backgrounds of
the strains involved. The contribution of transformation-
type HGT to genetic dynamics in the environment may be
underestimated (Bushman, 2002; Thomas and Nielsen, 2005),
and our studies indicate that HPTT in E. coli occurs at substantial
transfer frequencies (10−5−10−10) under the conditions that
can be feasibly encountered in the environment. Therefore,
transformation-type HGT can contribute to the spread of
antibiotic resistance genes and emergence of multidrug-resistant
bacteria in the real environment outside laboratories. Further
studies are required to understand the precise role and
contribution of transformation-type HGT in spreading antibiotic
resistance. AUTHOR CONTRIBUTIONS
HH, ES, and SM wrote the paper. REFERENCES doi: 10.1016/0378-1119(84)90143-4 Johnston, C., Martin, B., Fichant, G., Polard, P., and Claverys, J. P. (2014). Bacterial
transformation: distribution, shared mechanisms and divergent control. Nat. Rev. Microbiol. 12, 181–196. doi: 10.1038/nrmicro3199 Brautigam, M., Hertel, C., and Hammes, W. P. (1997). Evidence for natural
transformation of Bacillus subtilis in foodstuffs. FEMS Microbiol. Lett. 155,
93–98. doi: 10.1016/S0378-1097(97)00372-8 Keen, E. C., Bliskovsky, V. V., Malagon, F., Baker, J. D., Prince, J. S., Klaus, J. S.,
et al. (2017). Novel “superspreader” bacteriophages promote horizontal gene
transfer by transformation. mBio 8:e02115-16. doi: 10.1128/mBio.02115-16 Bushman, F. (2002). Lateral DNA Transfer. Cold Spring Harbor. New York, NY:
Laboratory Press, Cold Spring Harbor. Keese, P. (2008). Risks from GMOs due to horizontal gene transfer. Environ. Biosafety Res. 7, 123–149. doi: 10.1051/ebr:2008014 Cameron, A. D. S., and Redfield, R. J. (2006). Non-canonical CRP sites control
competence regulons in Escherichia coli and many other g-proteobacteria. Nucleic Acids Res. 34, 6001–6014. doi: 10.1093/nar/gkl734 Kelly, B. G., Vespermann, A., and Bolton, D. J. (2009a). Gene transfer events and
their occurrence in selected environments. Food Chem. Toxicol. 47, 978–983. doi: 10.1016/j.fct.2008.06.012 Cameron, A. D. S., and Redfield, R. J. (2008). CRP binding and transcription
activation at CRP-S sites. J. Mol. Biol. 383, 313–323. doi: 10.1016/j.jmb.2008. 08.027 Kelly, B. G., Vespermann, A., and Bolton, D. J. (2009b). Horizontal gene transfer of
virulence determinants in selected bacterial foodborne pathogens. Food Chem. Toxicol. 47, 969–977. doi: 10.1016/j.fct.2008.02.007 Carmen, J. C., Nelson, J. L., Beckstead, B. L., Runyan, C. M., Robinson, R. A.,
Schaalje, G. B., et al. (2004). Ultrasonic-enhanced gentamicin transport through
colony biofilms of Pseudomonas aeruginosa and Escherichia coli. J. Infect. Chemother. 10, 193–199. doi: 10.1007/s10156-004-0319-1 Król, J. E., Nguyen, H. D., Rogers, L. M., Beyenal, H., Krone, S. M., and Top, E. M. (2011). Increased transfer of a multidrug resistance plasmid in Escherichia coli
biofilms at the air-liquid interface. Appl. Environ. Microbiol. 77, 5079–5088. doi: 10.1128/AEM.00090-11 Chen, I., and Dubnau, D. (2004). DNA uptake during bacterial transformation. Nat. Rev. Microbiol. 3, 241–249. doi: 10.1038/nrmicro844 Kurono, N., Matsuda, A., Etchuya, R., Sobue, R., Sakaki, Y., Ito, M., et al. (2012). Genome-wide screening of Escherichia coli genes involved in execution and
promotion of cell-to-cell transfer of non-conjugative plasmids: rodZ (yfgA)
is essential for plasmid acceptance in recipient cells. Biochem. Biophys. Res. Commun. 421, 119–123. doi: 10.1016/j.bbrc.2012.03.127 Chung, C. T., Niemera, S. L., and Miller, R. H. (1989). REFERENCES erythromycin resistance in Streptococcus pyogenes. J. Antimicrob. Chemother. 55, 445–451. doi: 10.1093/jac/dki049 erythromycin resistance in Streptococcus pyogenes. J. Antimicrob. Chemother. 55, 445–451. doi: 10.1093/jac/dki049 Anderl, J. N., Franklin, M. J., and Stewart, P. S. (2000). Role of antibiotic
penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin
and ciprofloxacin. Antimicrob. Agents Chemother. 44, 1818–1824. doi: 10.1128/
AAC.44.7.1818-1824.2000 Guo, M., Wang, H., Xie, N., and Xie, Z. (2015). Positive Effect of carbon sources on
natural transformation in Escherichia coli: role of low-level cyclic AMP (cAMP)-
cAMP receptor protein in the derepression of rpoS. J. Bacteriol. 197, 3317–3328. doi: 10.1128/JB.00291-15 Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–580. doi: 10.1016/S0022-2836(83)80284-8 Ando, T., Itakura, S., Uchii, K., Sobue, R., and Maeda, S. (2009). Horizontal transfer
of non-conjugative plasmid in colony biofilm of Escherichia coli on food-based
media. World J. Microbiol. Biotechnol. 25, 1865–1869. doi: 10.1007/s11274-009-
0070-y Hashimoto-Gotoh, T., Franklin, F. C., Nordheim, A., and Timmis, K. N. (1981). Specific-purpose plasmid cloning vectors. I. Low copy number, temperature-
sensitive, mobilization-defective pSC101-derived containment vectors. Gene
16, 227–235. doi: 10.1016/0378-1119(81)90079-2 Asif, A., Mohsin, H., Tanvir, R., and Rehman, Y. (2017). Revisiting the mechanisms
involved in calcium chloride induced bacterial transformation. Front. Microbiol. 8:2169. doi: 10.3389/fmicb.2017.02169 Huang, R., and Reusch, R. N. (1995). Genetic competence in Escherichia
coli requires poly-beta-hydroxybutyrate/calcium polyphosphate membrane
complexes and certain divalent cations. J. Bacteriol. 177, 486–490. doi: 10.1128/
jb.177.2.486-490.1995 Bauer, F., Hertel, C., and Hammes, W. P. (1999). Transformation of Escherichia coli
in foodstuffs. Syst. Appl. Microbiol. 22, 161–168. doi: 10.1016/S0723-2020(99)
80061-7 Baur, B., Hanselmann, K., Schlimme, W., and Jenni, B. (1996). Genetic
transformation in freshwater: Escherichia coli is able to develop natural
competence. Appl. Environ. Microbiol. 62, 3673–3678. Ishimoto, Y., Kato, S., and Maeda, S. (2008). Freeze-thaw-induced lateral transfer
of non-conjugative plasmids by in situ transformation in Escherichia coli in
natural waters and food extracts. World J. Microbiol. Biotechnol. 24, 2731–2735. doi: 10.1007/s11274-008-9761-z Blesa, A., and Berenguer, J. (2015). Contribution of vesicle-protected extracellular
DNA to horizontal gene transfer in Thermus spp. Int. Microbiol. 18, 177–187. doi: 10.2436/20.1501.01.248 Jaskólska, M., and Gerdes, K. (2015). CRP-dependent positive autoregulation and
proteolytic degradation regulate competence activator Sxy of. Escherichia coli. Mol. Microbiol. 95, 833–845. doi: 10.1111/mmi.12901 Brady, G., Jantzen, H. M., Bernard, H. U., Brown, R., Schütz, G., and Hashimoto-
Gotoh, T. (1984). New cosmid vectors developed for eukaryotic DNA cloning. Gene 27, 223–232. AUTHOR CONTRIBUTIONS HH, ES, and SM wrote the paper. October 2018 | Volume 9 | Article 2365 Frontiers in Microbiology | www.frontiersin.org 4 Multiplicity of E. coli Transformation Hasegawa et al. FUNDING This
work
was
supported
by
JSPS
KAKENHI
(Grant #25292051). We are grateful to Enago (www.enago.jp) for English editing and
proofreading services. We are grateful to Enago (www.enago.jp) for English editing and
proofreading services. REFERENCES One-step preparation of
competent Escherichia coli: transformation and storage of bacterial cells in the
same solution. Proc. Natl. Acad. Sci. U.S.A. 86, 2172–2175. doi: 10.1073/pnas. 86.7.2172 Clokie, M. R., Millard, A. D., Letarov, A. V., and Heaphy, S. (2011). Phages in
nature. Bacteriophage 1, 31–45. doi: 10.4161/bact.1.1.14942 Lang, A. S., Zhaxybayeva, O., and Beatty, J. T. (2012). Gene transfer agents:
phage-like elements of genetic exchange. Nat. Rev. Microbiol. 10, 472–482. doi: 10.1038/nrmicro2802 Davey, M. E., and O’Toole, G. A. (2000). Microbial biofilms: from ecology to
molecular genetics. Microbiol. Mol. Biol. Rev. 64, 847–867. doi: 10.1128/MMBR. 64.4.847-867.2000 Li, M., Stern, B., and Kamp, D. (1992). Ultrafast plasmid DNA preparation for rapid
transformation. Biotechniques 13, 692–696. Lorenz, M. G., and Wackernagel, W. (1994). Bacterial gene transfer by natural
genetic transformation in the environment. Microbiol. Rev. 58, 563–602. Etchuuya, R., Ito, M., Kitano, S., Shigi, F., Sobue, R., and Maeda, S. (2011). Cell-to-
cell transformation in Escherichia coli: a novel type of natural transformation
involving cell-derived DNA and a putative promoting pheromone. PLoS One
6:e16355. doi: 10.1371/journal.pone.0016355 Maeda, S., Ito, M., Ando, T., Ishimoto, Y., Fujisawa, Y., Takahashi, H., et al. (2006). Horizontal transfer of nonconjugative plasmids in a colony biofilm of
Escherichia coli. FEMS Microbiol. Lett. 255, 115–120. doi: 10.1111/j.1574-6968. 2005.00072.x Finkel, S. E., and Kolter, R. (2001). DNA as a nutrient: novel role for bacterial
competence gene homologs. J. Bacteriol. 183, 6288–6293. doi: 10.1128/JB.183. 21.6288-6293.2001 Maeda, S., Kakihara, N., and Koishi, Y. (2003). Competency development of
Escherichia coli in foodstuffs. Microbes Environ. 18, 100–103. doi: 10.1264/
jsme2.18.100 Giovanetti, E., Brenciani, A., Vecchi, M., Manzin, A., and Varaldo, P. E. (2005). Prophage association of mef(A) elements encoding efflux-mediated October 2018 | Volume 9 | Article 2365 Frontiers in Microbiology | www.frontiersin.org 5 Hasegawa et al. Multiplicity of E. coli Transformation Maeda, S., Sawamura, A., and Matsuda, A. (2004). Transformation of colonial
Escherichia coli on solid media. FEMS Microbiol. Lett. 236, 61–64. doi: 10.1016/
j.femsle.2004.05.023 Sugiura, C., Miyaue, S., Shibata, Y., Matsumoto, A., and Maeda, S. (2017). Bacteriophage P1vir-induced cell-to-cell plasmid transformation in Escherichia
coli. AIMS Microbiol. 3, 784–797. doi: 10.3934/microbiol.2017.4.784 Sun, D. (2016). Two different routes for double-stranded DNA transfer in
natural and artificial transformation of Escherichia coli. Biochem. Biophys. Res. Commun. 471, 213–218. doi: 10.1016/j.bbrc.2016.01.137 Mandel, M., and Higa, A. (1970). Calcium-dependent bacteriophage DNA
infection. J. Mol. Biol. 53, 159–162. doi: 10.1016/0022-2836(70)90051-3 Marshall, K. M., Bradshaw, M., and Johnson, E. A. REFERENCES (2010). Conjugative botulinum
neurotoxin-encoding plasmids in Clostridium botulinum. PLoS One 5:e11087. doi: 10.1371/journal.pone.0011087 Sun, D., Wang, B., Zhu, L., Chen, M., and Zhan, L. (2013). Block and boost DNA
transfer: opposite roles of OmpA in natural and artificial transformation of
Escherichia coli. PLoS One 8:e59019. doi: 10.1371/journal.pone.0059019 Matsuda, A., Kurono, N., Kawano, C., Shirota, K., Hirabayashi, A., Horino, M.,
et al. (2012). Genome-wide screen for Escherichia coli genes involved in
repressing cell-to-cell transfer of non-conjugative plasmids. Biochem. Biophys. Res. Commun. 428, 445–450. doi: 10.1016/j.bbrc.2012.10.098 Sun, D., Zhang, X., Wang, L., Prudhomme, M., Xie, Z., Martin, B., et al. (2009). Transforming DNA uptake gene orthologs do not mediate spontaneous
plasmid transformation in Escherichia coli. J. Bacteriol. 191, 713–719. doi: 10.1128/JB.01130-08 Matsumoto,
A.,
Sekoguchi,
A.,
Imai,
J.,
Kondo,
K.,
Shibata,
Y.,
and
Maeda, S. (2016a). Natural Escherichia coli strains undergo cell-to-cell
plasmid transformation. Biochem. Biophys. Res. Commun. 481, 59–62. doi: 10.1016/j.bbrc.2016.11.018 Sun, D., Zhang, Y., Mei, Y., Jiang, H., Xie, Z., and Liu, H. (2006). Escherichia coli
is naturally transformable in a novel transformation system. FEMS Microbiol. Lett. 265, 249–255. doi: 10.1111/j.1574-6968.2006.00503.x j
Matsumoto, A., Sekoguchi, A., Murakami, Y., Imai, J., Kondo, K., Shibata, Y.,
et al. (2016b). “Competence development and horizontal plasmid transfer in
natural Escherichia coli strain,” in Microbes in the Spotlight: Recent Progress in
the Understanding of Beneficial and Harmful Microorganisms, ed. M. A. Vilas
(Florida, FL: Brown Walker Press), 468–473. Takahashi, R., Valeika, S. R., and Glass, K. W. (1992). A simple method of plasmid
transformation of E. coli by rapid freezing. Biotechniques 13, 711–715. Takeshita, S., Sato, M., Toba, M., Masahashi, W., and Hashimoto-Gotoh, T. (1987). High-copy-number
and
low-copy-number
plasmid
vectors
for
lacZα-complementation
and
chloramphenicol-
or
kanamycin-resistance
selection. Gene 61, 63–74. doi: 10.1016/0378-1119(87)90365-9 Ochman, H., and Selander, R. K. (1984). Standard reference strains of Escherichia
coli from natural populations. J. Bacteriol. 157, 690–693. Tenaillon, O., Skurnik, D., Picard, B., and Denamur, E. (2010). The population
genetics of commensal Escherichia coli. Nat. Rev. Microbiol. 8, 207–217. doi: 10.1038/nrmicro2298 Palchevskiy, V., and Finkel, S. E. (2006). Escherichia coli competence gene
homologs are essential for competitive fitness and the use of DNA as a nutrient. J. Bacteriol. 188, 3902–3910. doi: 10.1128/JB.01974-05 Thomas, C. M., and Nielsen, K. M. (2005). Mechanisms of, and barriers to,
horizontal gene transfer between bacteria. Nat. Rev. Microbiol. 3, 711–721. doi: 10.1038/nrmicro1234 Reusch, R. N., Hiske, T. W., and Sadoff, H. L. (1986). REFERENCES Poly-β-hydroxybutyrate
membrane structure and its relationship to genetic transformability in
Escherichia coli. J. Bacteriol. 168, 553–562. doi: 10.1128/jb.168.2.553-562.1986 Tsen, S. D., Fang, S. S., Chen, M. J., Chien, J. Y., Lee, C. C., and Tsen, D. H. (2002). Natural plasmid transformation in Escherichia coli. J. Biomed. Sci. 9, 246–252. doi: 10.1159/000059425 Reusch, R. N., and Sadoff, H. L. (1988). Putative structure and functions of a
poly-β-hydroxybutyrate/calcium polyphosphate channel in bacterial plasma
membranes. Proc. Natl. Acad. Sci. U.S.A. 85, 4176–4180. doi: 10.1073/pnas.85. 12.4176 von Wintersdorff, C. J., Penders, J., van Niekerk, J. M., Mills, N. D., Majumder, S.,
van Alphen, L. B., et al. (2016). Dissemination of antimicrobial resistance in
microbial ecosystems through horizontal gene transfer. Front. Microbiol. 7, 173. doi: 10.3389/fmicb.2016.00173 Rodríguez-Beltrán, J., Rodríguez-Rojas, A., Yubero, E., and Blázquez, J. (2013). The animal food supplement sepiolite promotes a direct horizontal transfer
of antibiotic resistance plasmids between bacterial species. Antimicrob. Agents
Chemother. 57, 2651–2653. doi: 10.1128/AAC.02363-12 Whitchurch, C. B., Tolker-Nielsen, T., Ragas, P. C., and Mattick, J. S. (2002). Extracellular DNA required for bacterial biofilm formation. Science 295, 1487. doi: 10.1126/science.295.5559.1487 Rohrer, S., Holsten, L., Weiss, E., Banghezal, M., Fischer, W., and Haas, R. (2012). Multiple pathways of plasmid DNA transfer in Helicobacter pylori. PLoS One
7:e45623. doi: 10.1371/journal.pone.0045623 Woegerbauer, M., Jenni, B., Thalhammer, F., Graninger, W., and Burgmann, M. (2002). Natural genetic transformation of clinical isolates of Escherichia coli in
urine and water. Appl. Environ. Microbiol. 68, 440–443. doi: 10.1128/AEM.68.1. 440-443.2002 Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A
Laboratory Manual, 2nd Edn. New York, NY: Cold Spring Harbor Laboratories. Yoshida, N. (2007). Discovery and application of the Yoshida effect: nano-sized
acicular materials enable penetration of bacterial cells by sliding friction force. Recent Pat. Biotechnol. 1, 194–201. doi: 10.2174/187220807782330147 Sambrook, J., and Russell, D. R. (2006). Transformation of E. coli by
electroporation. Cold Spring Harb. Protoc. 21, doi: 10.1101/pdb.prot3933 Seitz, P., and Blokesch, M. (2013). Cues and regulatory pathways involved in
natural competence and transformation in pathogenic and environmental
Gram-negative bacteria. FEMS Microbiol. Rev. 37, 336–363. doi: 10.1111/j.1574-
6976.2012.00353.x Zenz, K. I., Neve, H., Geis, A., and Heller, K. J. (1998). Bacillus subtilis develops
competence for uptake of plasmid DNA when growing in milk products. Syst. Appl. Microbiol. 21, 28–32. doi: 10.1016/S0723-2020(98)80005-2 Sekirov, I., Russell, S. L., Antunes, L. C., and Finlay, B. B. (2010). Gut microbiota in
health and disease. Physiol. Rev. 90, 859–904. REFERENCES doi: 10.1152/physrev.00045.2009 Zhang, Y., Shi, C., Yu, J., Ren, J., and Sun, D. (2012). RpoS regulates a
novel type of plasmid DNA transfer in Escherichia coli. PLoS One 7:e33514. doi: 10.1371/journal.pone.0033514 Shibata, Y., Matsumoto, A., Horino, M., Hirabayashi, A., Shirota, K., Kawano, C.,
et al. (2014a). Genome-wide screen for Escherichia coli genes involved in
repressing cell-to-cell transfer of a nonconjugative pSC101-derived plasmid. Am. J. Life Sci. 2, 345–350. doi: 10.11648/j.ajls.20140206.13 Zoetendal, E. G., Collier, C. T., Koike, S., Mackie, R. I., and Gaskins, H. R. (2004). Molecular ecological analysis of the gastrointestinal microbiota. J. Nutr. 134,
465–472. doi: 10.1093/jn/134.2.465 Shibata, Y., Ugumori, C., Takahashi, A., Sekoguchi, A., and Maeda, S. (2014b). Survey of lysogenic phages in the 72 strains of Escherichia coli collection of
reference (ECOR) and identification of a phage derived from the ECOR52
strain. Am. J. BioSci. 2, 32–37. doi: 10.11648/j.ajbio.20140202.12 Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Sinha, S., Cameron, A. D. S., and Redfield, R. J. (2009). Sxy Induces a CRP-
S Regulon in Escherichia coli. J. Bacteriol. 191, 5180–5195. doi: 10.1128/JB. 00476-09 Copyright © 2018 Hasegawa, Suzuki and Maeda. 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. Sinha, S., and Redfield, R. J. (2012). Natural DNA Uptake by Escherichia coli. PLoS
One 7:e35620. doi: 10.1371/journal.pone.0035620 Sobue, R., Kurono, N., Etchuya, R., and Maeda, S. (2011). Identification of a
novel DNA element that promotes cell-to-cell transformation in Escherichia
coli. FEBS Lett. 585, 2223–2228. doi: 10.1016/j.febslet.2011.05.040 October 2018 | Volume 9 | Article 2365 Frontiers in Microbiology | www.frontiersin.org 6
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Th1/Th17 Cell Induction and Corresponding Reduction in ATP Consumption following Vaccination with the Novel Mycobacterium tuberculosis Vaccine MVA85A
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Abstract Vaccination with Bacille Calmette-Gue´rin (BCG) has traditionally been used for protection against disease caused by the
bacterium Mycobacterium tuberculosis (M.tb). The efficacy of BCG, especially against pulmonary tuberculosis (TB) is variable. The best protection is conferred in temperate climates and there is close to zero protection in many tropical areas with a
high prevalence of both tuberculous and non-tuberculous mycobacterial species. Although interferon (IFN)-c is known to
be important in protection against TB disease, data is emerging on a possible role for interleukin (IL)-17 as a key cytokine in
both murine and bovine TB vaccine studies, as well as in humans. Modified Vaccinia virus Ankara expressing Antigen 85A
(MVA85A) is a novel TB vaccine designed to enhance responses induced by BCG. Antigen-specific IFN-c production has
already been shown to peak one week post-MVA85A vaccination, and an inverse relationship between IL-17-producing cells
and regulatory T cells expressing the ectonucleosidease CD39, which metabolises pro-inflammatory extracellular ATP has
previously been described. This paper explores this relationship and finds that consumption of extracellular ATP by
peripheral blood mononuclear cells from MVA85A-vaccinated subjects drops two weeks post-vaccination, corresponding to
a drop in the percentage of a regulatory T cell subset expressing the ectonucleosidase CD39. Also at this time point, we
report a peak in co-production of IL-17 and IFN-c by CD4+ T cells. These results suggest a relationship between extracellular
ATP and effector responses and unveil a possible pathway that could be targeted during vaccine design. Citation: Griffiths KL, Pathan AA, Minassian AM, Sander CR, Beveridge NER, et al. (2011) Th1/Th17 Cell Induction and Corresponding Reduction in ATP
Consumption following Vaccination with the Novel Mycobacterium tuberculosis Vaccine MVA85A. PLoS ONE 6(8): e23463. doi:10.1371/journal.pone.0023463 Editor: Ludovic Tailleux, Institut Pasteur, France Copyright: 2011 Griffiths 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: Funding was provided by KLG’s Wellcome Trust studentship (www.wellcome.ac.uk; WT086338RP) and HM’s Wellcome Trust Senior Fellowship (www. wellcome.ac.uk; WT076943MA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract Competing Interests: The authors have read the journal’s policy and have the following conflicts: AVSH, AAP, and HM are named inventors in a patent filing
related to MVA85A and are shareholders in a joint venture, OETC, formed for the future development of this vaccine. AVSH and HM are named as co-inventors on
patents related to heterologous prime-boost immunization. There are no other conflicts of interest. These conflicts of interest will not in any way interfere with
the authors’ adherence to the journal’s policies on sharing data and materials. * E-mail: Helen.Mcshane@ndm.ox.ac.uk ¤ Current address: Centre for Infection, Immunity and Disease Mechanisms, Biosciences, School of Health Sciences and Social Care, B
West London, United Kingdom e for Infection, Immunity and Disease Mechanisms, Biosciences, School of Health Sciences and Social Care, Brunel University, Uxbridge
d Th1/Th17 Cell Induction and Corresponding Reduction in
ATP Consumption following Vaccination with the Novel
Mycobacterium tuberculosis Vaccine MVA85A Kristin L. Griffiths, Ansar A. Pathan¤, Angela M. Minassian, Clare R. Sander, Natalie E. R. Beveridge,
Adrian V. S. Hill, Helen A. Fletcher, Helen McShane*
The Jenner Institute Oxford University Oxford United Kingdom Kristin L. Griffiths, Ansar A. Pathan¤, Angela M. Minassian, Clare R. Sander, Natalie E. R. Beveridge,
Adrian V. S. Hill, Helen A. Fletcher, Helen McShane* The Jenner Institute, Oxford University, Oxford, United Kingdom The Jenner Institute, Oxford University, Oxford, United Kingdom PLoS ONE | www.plosone.org ation: Griffiths KL, Pathan AA, Minassian AM, Sander CR, Beveridge NER, et al. (2011) Th1/Th17 Cell Induction and Correspond
nsumption following Vaccination with the Novel Mycobacterium tuberculosis Vaccine MVA85A. PLoS ONE 6(8): e23463. doi:10.1371/jou ATP consumption following MVA85A vaccination follows
a distinct pattern and can be inhibited using an apyrase
inhibitor ATP consumption following MVA85A vaccination follows
a distinct pattern and can be inhibited using an apyrase
inhibitor Consumption of extracellular ATP was measured in PBMC
from vaccinated subjects at 0, 1, 2, 4 and 24 weeks post-
vaccination using the CellTiter-Glo cell viability assay and plotting
against a standard curve. There was a significant difference in
ATP consumption 2 weeks post-vaccination compared to baseline
(p = 0.008) (Fig. 1A). Paired analysis between 0 and 2 weeks is
shown in figure 2B. In order to verify that ATP consumption was
attributable to the action of an apyrase, cells were treated with
ARL67156 at the time of ATP addition, which reduced ATP
consumption (Fig. 1C). Regulatory T cells, defined here as CD4+CD25+CD39hi, have
previously been shown to decrease in number post-vaccination
with MVA85A [10]. Since CD39 is an eNDTPase metabolising
ATP, percentages of these cells in PBMC of vaccinated subjects
were compared to levels of ATP consumption. There was a small
dip in percentages of CD39+ Treg cells between 1 and 2 weeks
post-vaccination and followed the pattern of ATP consumption
(Fig. 1D). Here we show that CD39+ Treg percentages drop 2 weeks post-
MVA85A vaccination, coincident with a drop in ATP consump-
tion by PBMC from MVA85A-vaccinated subjects. This also
coincides with an increase in percentages IFN-c and IL-17 double-
producing CD4+ T cells. p
g
Clearance and control of M.tb infection is at least partly
dependent upon interferon (IFN)-c production by CD4+ T
helper 1 (Th1) cells [20], however IL-17 has recently been
identified as being induced by M.tb in murine lungs following
vaccination with adjuvanted peptides derived from ESAT-6, an
immunodominant
secreted
protein
specific
to
M.tb
[21]. Vaccination of mice lacking IL-23 subunits, the cytokine
essential for Th17 expansion, resulted in the loss of accelerated
vaccine-induced recruitment of Th1 cells to the lungs following
M.tb infection, suggesting that IL-17-producing cells (Th17)
contribute to vaccine-induced protection against M.tb challenge
through recruitment of Th1 cells to the lung. These hypotheses
are supported by a different study in which mice were
vaccinated with either BCG or BCG followed by a construct
designed to produce anti-IL-12 antibodies within the animal, or
with the anti-IL-12-inducing construct alone [22]. IFN-c and IL-17 double positive cells peak 2 weeks post-
vaccination In PBMC from healthy MVA85A-vaccinated subjects, T cells
producing only IL-17 were not detectable by intracellular cytokine
staining, using a peptide pool of 66 Ag85A peptides as the
stimulant (data not shown). In contrast, CD4+ T cells producing
both IFN-c and IL-17 simultaneously in response to stimulation
with Ag85A peptides were readily detected in response to the same
stimulant. IFN-c+IL-17+ cells (gating shown in Fig. 2A) peaked 2
weeks post-vaccination (Fig. 2B). Since there appeared to be CD39
activity in the PBMC samples (Fig. 1C), the effect of ARL67156
treatment on antigen-specific cytokine production was examined. Addition of ARL67156 enhanced production of both cytokines
both 1 and 2 weeks post-vaccination and the change in cytokine
production compared to baseline in this experiment was significant
(p = 0.047 at 1 week and 0.02 at 2 weeks post-vaccination)
(Fig. 2C). ATP consumption following MVA85A vaccination follows
a distinct pattern and can be inhibited using an apyrase
inhibitor Following an
M.tb challenge, results showed higher bacterial load (cfu) in
lungs
and
spleen
from
mice
with
anti-IL-12
antibodies
compared to no treatment, but no difference in cfu between
BCG or BCG+anti-IL-12 groups, which both had significantly
lower cfu than unvaccinated mice. Interestingly, higher IL-17
and IL-6 levels were detected in the vaccinated compared to the
unvaccinated groups, suggesting that control during primary
intravenous infection depends on a Th1 response, but on an IL-
17-driven response following vaccination. Introduction The combination of these events drives the
cleavage of pro-IL-1, induced by Toll-like receptor (TLR)
activation, to IL-1b by Caspase-1 [16]. IL-1b, in synergy with
IL-6, has in turn been shown to induce IL-17 production by
Th17 cells [17]. Reduced percentages of Foxp3+CD39+ Treg
have been described in PBMC of patients with multiple sclerosis
compared to healthy donors [18,19], and the CD39+ cells that
were present had impaired ATP-hydrolysing capacity [19],
providing evidence for a link between CD39 expression on Treg
cells
and
a
function
in
regulating
inflammation
through
controlling extracellular ATP levels. environment as a natural process during inflammation [13] as
well as being released by dead cells. It has been identified as a
proinflammatory agent and is known to activate the NALP3
inflammasome through binding to the P2X7 receptor and
inducing a K+ efflux, as well as activating the Pannexin-1
channel [14,15]. The combination of these events drives the
cleavage of pro-IL-1, induced by Toll-like receptor (TLR)
activation, to IL-1b by Caspase-1 [16]. IL-1b, in synergy with
IL-6, has in turn been shown to induce IL-17 production by
Th17 cells [17]. Reduced percentages of Foxp3+CD39+ Treg
have been described in PBMC of patients with multiple sclerosis
compared to healthy donors [18,19], and the CD39+ cells that
were present had impaired ATP-hydrolysing capacity [19],
providing evidence for a link between CD39 expression on Treg
cells
and
a
function
in
regulating
inflammation
through
controlling extracellular ATP levels. Results ATP consumption following MVA85A vaccination follows
a distinct pattern and can be inhibited using an apyrase
inhibitor Introduction to both BCG and M.tb, is a vaccine designed to enhance the low
level of T cell responses induced by BCG through expansion of
antigen
85A-specific
T
cells [4,5]. The
MVA85A-induced
interferon (IFN)-c response has been well-characterised across
several groups of vaccinated individuals and peaks 1–2 weeks
post-vaccination [4,5,6,7,8]. Recently, however, other immuno-
logical parameters have been investigated, in particular those
pertaining to immune regulation. MVA85A has been shown to
induce a reduction in transforming growth factor (TGF)-b in
the serum [9] as well as a reduction in percentages of
CD25+Foxp3+CD39+
Treg
in
peripheral
blood
monocytes
(PBMC) from vaccinated subjects [10]. CD39, an ectonucleosi-
dase
triphosphate
diphosphohydrolase
(eNDTPase;
apyrase)
hydrolysing
extracellular
adenosine
triphosphate
(ATP)
to
adenosine monophosphate (AMP) [11], is expressed on leuko-
cytes, including neutrophils and T cells. In humans, CD4+ T cells
can be divided according to their expression of CD25, CD39 and
Foxp3, with CD4+CD25+CD39+Foxp3+ cells representing a
regulatory population [12]. ATP is released into the extracellular Mycobacterium tuberculosis (M.tb), the causative agent of tubercu-
losis (TB), infects ,30% of the World’s population and is endemic
in Asia and sub-Saharan Africa. M.tb is transmitted by aerosol and
infects macrophages in the lung. Infection with M.tb can result in
acute (primary) disease, or, more commonly, remain latent. In
10% of cases, this latent infection reactivates later in life causing
disease, which is usually pulmonary but can occur in other organs
including spleen, stomach, bowel or brain. Co-infection with HIV,
also endemic in TB-endemic areas, results in a significant increase
in the risk of reactivation of this latent infection [1,2,3]. Bacille
Calmette-Gue´rin (BCG), the current TB vaccine, is an attenuated
form of M.bovis and offers varying degrees of protection. Notably,
the level of protection is lowest in areas endemic for M.tb and the
development of a novel, more effective vaccine against TB is
urgently needed. Modified Vaccinia virus Ankara expressing antigen 85A
(MVA85A), a secreted and highly immunogenic protein common August 2011 | Volume 6 | Issue 8 | e23463 1 August 2011 | Volume 6 | Issue 8 | e23463 MVA85A-Induced Th1/Th17 Cells and ATP Consumption environment as a natural process during inflammation [13] as
well as being released by dead cells. It has been identified as a
proinflammatory agent and is known to activate the NALP3
inflammasome through binding to the P2X7 receptor and
inducing a K+ efflux, as well as activating the Pannexin-1
channel [14,15]. IL-17+ and IFN-c+IL-17+ cells are present at a higher
frequency in whole blood compared to PBMC of
vaccinated subjects PBMC from MVA85A-vaccin
subjects were plated out at 56104 cells/well in 50 mL. Cells were incubated with either 50 mM ATP or 50 mM+100 mM ARL67156 before addition o
luciferase reagent. A standard curve starting at 50 mM ATP was set up and negative controls were cells with no ATP added. (A) Shows change in
consumption over time post-vaccination. (B) Paired representation of change in ATP consumption between 0 and 2 weeks post-MVA85A. Effe
addition of ARL67156 is show in (C). (n = 10–12). Note that the observation of a greater effect of the inhibitor is potentially due to saturatio
binding sites for ATP by the inhibitor at this timepoint, whereas the greater percentage of CD39+ cells present at other timepoints mean
concentration of ARL67156 was not high enough to completely block all available binding sites. Percentages of CD25+CD39+ Treg in MVA
vaccinated subjects were calculated as a percentage of CD4+ T cells and shown in (D), plotted over ATP consumption. doi:10.1371/journal.pone.0023463.g001
MVA85A-Induced Th1/Th17 Cells and ATP Consum MVA85A-Induced Th1/Th17 Cells and ATP Consumption Figure 1. ATP consumption by PBMC and CD39+ Treg percentages dip 2 weeks post-vaccination. PBMC from MVA85A-vaccinated
subjects were plated out at 56104 cells/well in 50 mL. Cells were incubated with either 50 mM ATP or 50 mM+100 mM ARL67156 before addition of the
luciferase reagent. A standard curve starting at 50 mM ATP was set up and negative controls were cells with no ATP added. (A) Shows change in ATP
consumption over time post-vaccination. (B) Paired representation of change in ATP consumption between 0 and 2 weeks post-MVA85A. Effect of
addition of ARL67156 is show in (C). (n = 10–12). Note that the observation of a greater effect of the inhibitor is potentially due to saturation of
binding sites for ATP by the inhibitor at this timepoint, whereas the greater percentage of CD39+ cells present at other timepoints meant the
concentration of ARL67156 was not high enough to completely block all available binding sites. Percentages of CD25+CD39+ Treg in MVA85A-
vaccinated subjects were calculated as a percentage of CD4+ T cells and shown in (D), plotted over ATP consumption. doi:10.1371/journal.pone.0023463.g001 difference in peak response comparing whole blood and PBMC
following Ag85A peptide pool stimulation (Fig. 3C) (mean 6 SD
for WB (1 week post-vaccination): 0.0548% (60.0355); PBMC (2
weeks post-vaccination): 0.0096% (60.0083), p,0.001). IL-17+ and IFN-c+IL-17+ cells are present at a higher
frequency in whole blood compared to PBMC of
vaccinated subjects j
Other
trials
involving
vaccination
with
MVA85A
have
investigated cytokine responses in whole blood as opposed to
PBMC. One marked difference between these two compartments
is that IFN-c-producing CD8+ T cells are readily detectable in
whole blood but not in PBMC following vaccination ([5,26] and
Satti, I. unpublished data). To directly compare CD4+ T cell
cytokine production in these two compartments, we investigated
IL-17 and IFN-c production following stimulation with Ag85A
peptides in whole blood and PBMC from the same BCG-
vaccinated healthy subjects. Whole blood was stimulated with
peptide pools of 85A. Both IL-17+ and IFN-c+IL-17+ cells peaked
1 week post-vaccination (Figs. 3A & B), in contrast to the
undetectable IL-17 response and the peak of IFN-c+IL-17+ cells
observed 2 weeks post-vaccination in PBMC. The magnitude of
response was also significantly higher one week post-vaccination in
whole blood, with a mean of 0.0548% (60.0355) compared to
0.0065% (60.0063) for PBMC, p = 0.002 following stimulation
with the Ag85A peptide pool. Furthermore, there was a significant Further support for the involvement of IL-17 in control of M.tb
infection comes from a recent study comparing cytokine levels in
tuberculin skin test (TST) negative and TST positive (considered
latently infected) individuals in a TB endemic area. These results
showed that IL-17, IL-23 and RORct, the transcription factor
implicated in Th17 development, were downregulated in TST+
individuals [23] suggesting that higher IL-17 production favours
clearance or control of M.tb. MVA85A has previously been shown to increase interleukin
(IL)-17 production in both humans and cattle [10,24]. Further-
more, in cattle, vaccine-induced IL-17 production both pre- and
post- M.bovis challenge has been correlated with vaccine-induced
protection against TB disease [24]. IL-17 has also been detected in
whole blood of MVA85A-vaccinated adolescents and children,
where the IL-17+ cells were also found to produce IFN-c, tumour
necrosis factor (TNF)-a and IL-2 [25]. Here we suggest a possible
link
between CD39+
Treg cells
and
potentially
protective
MVA85A-induced IFN-c and IL-17 production. August 2011 | Volume 6 | Issue 8 | e23463 August 2011 | Volume 6 | Issue 8 | e23463 PLoS ONE | www.plosone.org 2 difference in peak response comparing whole blood and PBMC
following Ag85A peptide pool stimulation (Fig. 3C) (mean 6 SD
for WB (1 week post-vaccination): 0.0548% (60.0355); PBMC (2
Discussion
M.tb is a resilient intracellular pathogen that has evolve
Figure 1. ATP consumption by PBMC and CD39+ Treg percentages dip 2 weeks post-vaccination. Discussion M.tb is a resilient intracellular pathogen that has evolved to
survive successfully inside human macrophages in a delicate August 2011 | Volume 6 | Issue 8 | e23463 August 2011 | Volume 6 | Issue 8 | e23463 PLoS ONE | www.plosone.org 3 MVA85A-Induced Th1/Th17 Cells and ATP Consumption Figure 2. IL-17 and IFN-c production in PBMC peaks 2 weeks post-vaccination. PBMC from vaccinated subjects were stimulated with
Ag85A peptide pools with or without 100 uM ARL67156. No stimulation and phorbol 12-myristate 13-acetate with ionomycin were used as negative
and positive controls. Percentages shown are unstimulated subtracted from Ag85A stimulation. Following staining, cells were gated as shown in (A):
Lymphocytes were gated for on FSC vs. SSC. Singlets were then gated and dead cells, B cells and monocytes were gated out. CD3+ cells were selected
for CD4+CD82 cells. Antigen-specific cytokine expression from these cells was evaluated. Cells expressing both IL-17 and IFN-c were quantified and
shown in (B). The effect of ARL67156 on cytokine expression was investigated by addition during ICS stimulation (C). doi:10.1371/journal.pone.0023463.g002 Figure 2. IL-17 and IFN-c production in PBMC peaks 2 weeks post-vaccination. PBMC from vaccinated subjects were stimulated with
Ag85A peptide pools with or without 100 uM ARL67156. No stimulation and phorbol 12-myristate 13-acetate with ionomycin were used as negative
and positive controls. Percentages shown are unstimulated subtracted from Ag85A stimulation. Following staining, cells were gated as shown in (A):
Lymphocytes were gated for on FSC vs. SSC. Singlets were then gated and dead cells, B cells and monocytes were gated out. CD3+ cells were selected
for CD4+CD82 cells. Antigen-specific cytokine expression from these cells was evaluated. Cells expressing both IL-17 and IFN-c were quantified and
shown in (B). The effect of ARL67156 on cytokine expression was investigated by addition during ICS stimulation (C). doi:10.1371/journal.pone.0023463.g002 ulcerative colitis [27] as well as in response to mycobacterial
antigens [28]. Our data support the hypothesis that extracellular
ATP may help to drive the development of these cells. This
hypothesis is supported by the fact that IL-17+ and IFN-c+IL-17+
cells are more readily detectable in whole blood compared to
PBMC; the extracellular environment of whole blood is far more
complex than that of PBMC and our experiments have shown a
higher concentration of ATP in whole blood compared to PBMC
following stimulation. Furthermore, ATP has previously been
shown to induce IL-17 production by T cells in mice [29]. Discussion Phytohaemagglutinin (PHA)-treated and untreated cells were used as positive and negative
controls respectively. Lymphocytes were gated for and cytokine-expressing cells quantified as described above. Percentages of IL-17+ and IFN-c+IL-
17+ cells responding to the Ag85A peptide pool (with the percentages from unstimulated cells subtracted) are shown in (A) and (B). Differences in
percentages of cells at the relevant peak time point in PBMC vs. WB is shown in (C). n = 7. doi:10.1371/journal.pone.0023463.g003 Figure 3. IL-17 and IFN-c production in whole blood peaks 1 week post-vaccination. Whole blood (WB) from vaccinated subjects was
stimulated for 6 h with a pool of 66 Ag85A peptides. Phytohaemagglutinin (PHA)-treated and untreated cells were used as positive and negative
controls respectively. Lymphocytes were gated for and cytokine-expressing cells quantified as described above. Percentages of IL-17+ and IFN-c+IL-
17+ cells responding to the Ag85A peptide pool (with the percentages from unstimulated cells subtracted) are shown in (A) and (B). Differences in
percentages of cells at the relevant peak time point in PBMC vs. WB is shown in (C). n = 7. doi:10.1371/journal.pone.0023463.g003 Figure 3. IL-17 and IFN-c production in whole blood peaks 1 week post-vaccination. Whole blood (WB) from vaccinated subjects was
stimulated for 6 h with a pool of 66 Ag85A peptides. Phytohaemagglutinin (PHA)-treated and untreated cells were used as positive and negative
controls respectively. Lymphocytes were gated for and cytokine-expressing cells quantified as described above. Percentages of IL-17+ and IFN-c+IL-
17+ cells responding to the Ag85A peptide pool (with the percentages from unstimulated cells subtracted) are shown in (A) and (B). Differences in
percentages of cells at the relevant peak time point in PBMC vs. WB is shown in (C). n = 7. doi:10.1371/journal.pone.0023463.g003 control of TB disease, it needs to be under tight regulation by
other aspects of the immune response in order to avoid induction
of immunopathology. A further contributing factor to inflammasome activation and
IL-17 induction might be the viral vector MVA, which has been
found to activate the NALP3 inflammasome in THP-1 cells
following its endocytosis [32]. These findings demonstrate vaccine-mediated induction of a
subset of IFN-c+IL-17+ cells, whose peak corresponds with a
reduction in the ability of these cells to hydrolyse ATP in a CD39-
mediated manner. Should this cell subset prove to be protective in
vaccine-induce protection against TB disease, this pathway
represents a potential target for manipulation for their enhance-
ment. Discussion balance with the host’s immune system. Should this balance be
tipped in favour of the pathogen, the result is potentially fatal
active disease. Vaccination is therefore essential in order to either
prevent initial infection or, failing that, to prevent development of
active TB disease. Consequently, it is important to understand the
types of immune cells that should optimally be induced by
vaccination and the mechanisms through which these cells can be
induced. As well as measuring effector responses in terms of Th1
immunity, it is also important to measure other types of effector
responses as well as the corresponding regulatory response induced
by vaccination as protection induced by any vaccine will be the
outcome of the balance of all these responses. The link between these observations following MVA85A
vaccination is still under investigation. One mechanism that could
contribute to increased cytokine production is activation of the
NALP3 inflammasome. This leads to IL-1b production, which in
turn acts on induction of CD4+ T cells to produce IL-17 [17]. Since CD39 metabolises ATP, a vaccine-induced reduction in
circulating CD39+ Treg may result in increased concentrations of
pro-inflammatory extracellular ATP, known to act through the
P2X7 receptor to activate the inflammasome [14,30]. Adding to
this effect, a decrease in circulating CD39+ Treg would also reduce
the concentration of breakdown products of ATP, such as
adenosine, which is known to act in an inhibitory fashion through
the A2A receptor on activated T cells [31]. Here we show that there is a relationship between ATP
consumption, and IFN-c and IL-17 expression by CD4+ T cells
following vaccination with MVA85A, which also relates to
percentages of CD39+ Treg in the PBMC. In healthy BCG and
MVA85A-vaccinated subjects, CD39+ Treg numbers decrease post-
vaccination, with a maximum decrease 2 weeks post-vaccination. This is associated with a decrease in ATP consumption, suggesting
that ATP consumption is driven at least in part by CD39+ Treg. Strikingly, at 2 weeks post-vaccination, we also show an increase in
the percentage of IFN-c+IL-17+ cells. IFN-c+IL-17+ cells have been
previously been described in autoimmune diseases [19] and PLoS ONE | www.plosone.org August 2011 | Volume 6 | Issue 8 | e23463 4 MVA85A-Induced Th1/Th17 Cells and ATP Consumption Figure 3. IL-17 and IFN-c production in whole blood peaks 1 week post-vaccination. Whole blood (WB) from vaccinated subjects was
stimulated for 6 h with a pool of 66 Ag85A peptides. August 2011 | Volume 6 | Issue 8 | e23463 1. Churchyard GJ, Kleinschmidt I, Corbett EL, Mulder D, De Cock KM (1999)
Mycobacterial disease in South African gold miners in the era of HIV infection.
Int J Tuberc Lung Dis 3: 791–798. Statistical analysis Given the non-parametric nature of the data, Wilcoxon Sign
Rank
Tests
and
Mann-Whitney
Tests
were
performed
as
appropriate tests for statistical analysis. MVA85A-Induced Th1/Th17 Cells and ATP Consumption MVA85A-Induced Th1/Th17 Cells and ATP Consumption 0.2 ug/mL phorbol 12-myristate 13-acetate (PMA) and 2 ug/mL
ionomycin, 2 ug/mL Ag85A peptide pool, 2 ug/mL Ag85A
peptide pool +100 uM ATP, 2 ug/mL Ag85A peptide pool
+100 uM ARL67156. After 2 hours, 5 ug/mL Brefeldin A and
0.7 uL GolgiStop (BD Biosciences) were added. Vaccine study participants PBMC were from subjects recruited for a trial approved by the
review committees indicated below. Subjects (aged 18–50) were
recruited on the basis of prior BCG vaccination (maximum
Mantoux test 15 mm, ,10 sfc/million ELISpot counts in response
to ESAT-6 and CFP10 peptide pools) and were seronegative for
HIV and hepatitis B and C viruses. The trial was registered on the
clinical trials database (ClinicalTrials.gov ID: NCT00465465). Subjects from whom samples were taken received a dose of
MVA85A at 16108 pfu as two intradermal injections, administered
simultaneously, one in each arm. The MVA85A vaccine was
manufactured to Good Manufacturing Practice (GMP) by Im-
pfstoffwerk Dessau-Tornau (IDT) Biologika GmbH in Germany. Samples from weeks 0, 1, 2, 4 and 24 were investigated. After stimulation, cells were sedimented at 1300 rpm for 5 min
at 4uC and transferred to a flexible 96 well plate for staining. Cells were stained with ViViD Live/Dead cell stain prior to
surface staining with eFluor450 anti-CD19, eFluor450 anti-CD14,
APC anti-CD25 and PE-Cy7 anti-CD39. Cytofix/Cytoperm was
used to permeabilise cells prior to staining intracellularly with PE-
Cy5 anti-CD3, Qdot655 anti-CD4, APC-AlexaFluor780 anti-
CD8, FITC anti-IFN-c and PE anti-IL-17. Following permeabi-
lisation, washes between stains included sedimentation for 5 min
at 1800 rpm, 4uC. Staining was analysed using an LSR II flow cytometer (BD
Biosciences). Staining was analysed using an LSR II flow cytometer (BD
Biosciences). The efficacy and safety data from this trial is in the process of
being written up but has yet to be published; immunological data
from the same trial has been published in Beveridge et al. 2008 [26]. Ethics statement All clincal trials are fully approved by the ethical and regulatory
agencies (Centre of Research: Ethical Campaign, and Medicines
and Healthcare Products Regulatory Agency), and also local
GMO and NHS committees as required (the Gene Therapy
Advisory Committee), and full written consent was obtained from
each subject prior to enrolment in the trial. Storage of samples for
exploratory immunological analyses is fully ethically approved. Antibodies and reagents Anti-human antibodies (Pacific blue anti-CD19, eFluor450 anti-
CD19, Pacific blue anti-CD14, eFluor450 anti-CD14, PE-Cy5
anti-CD3, APC-AlexaFluor780 anti-CD8, APC anti-CD25, PE-
Cy7 anti-CD39, FITC anti-IFN-c, PE anti-IL-17) were obtained
from eBioscience, and Qdot655 anti-CD4 and the ViViD Live/
Dead cell stain were from Invitrogen. The Cytofix/Cytoperm
intracellular staining kit was from BD Biosciences. ATP was
purchased from Millipore, and ARL67156 from Tocris Biosci-
ence. The CellTiter-Glo cell viability kit was from Promega. Brefeldin A was supplied by Sigma Aldrich and GolgiStop by BD. Author Contributions Cells were resuspended at 16106 cells/mL and each sample
divided into seven 1 mL aliquots in 5 mL polystyrene round
bottom tubes (BD Falcon). Cells were stimulated for 18 hours at
37uC in 5% CO2 with one of the following: no stimulation, no
stimulation +100 uM ATP, no stimulation +100 uM ARL67156, Conceived and designed the experiments: KLG HAF HM. Performed the
experiments: KLG. Analyzed the data: KLG HAF. Contributed reagents/
materials/analysis tools: AAP AMM CRS NERB HAF. Wrote the paper:
KLG. Head of the laboratory: AVSH. ATP consumption assay PBMC from vaccinated subjects were cryopreserved in liquid
nitrogen at time of acquisition in aliquots of 56106 cells in 50%
fetal bovine serum (FBS; Biosera Ltd.), 40% RPMI 1640, 10%
dimethylsulphoxide (DMSO; both from Sigma Aldrich). Prior to
use, cells were thawed in 9 mL R10 (10% FBS, 2 mM L-
glutamine, 100 U/mL penicillin, 100 ug/mL streptomycin in
RPMI 1640). Cells were treated with 67.2 U/mL Benzonase
(Novagen) for at least 2 hours at 37uC in 5% CO2. Cells were
washed and resuspended to ,16106 cells/mL and counted using
a CASY cell counter (Scha¨rfe System, GmbH). ATP consumption was measured as described Borsellino et al. [18]. Briefly, PBMC from subjects were resuspended at 16106
cells/mL and aliquoted into 6 wells/sample of a white 96 well
plate (Nunc) at 56104 cells/well. Cells were treated with either
nothing, 50 uM ATP or 50 uM ATP +100 uM ARL67156 for
10 min at room temperature. One volume CellTiter-Glo solution
was added to each well and cells were incubated for a further
10 min at room temperature in the dark. Luminescence was
recorded using a Varioskan Flash spectral scanning multimode
reader (Thermo Scientific). ATP consumption was calculated
using a standard curve of known ATP concentrations and
expressed in nmol. Acknowledgments The authors thank the volunteers who took part in the clinical trials
reported here, and Ian Poulton and Alison Lawrie for assistance with the
clinical trials administration. Whole blood ICS Freshly collected heparinised whole blood from MVA85A-
vaccinated volunteers was stimulated for 10–11 hours at 37uC
with either recombinant Ag85A, a pool of 66 peptides of Ag85A,
PPD or BCG. No stimulation and Phytohaemagglutinin were used
as negative and positive controls, respectively. After 5–6 hours,
Brefeldin A was added for the final 5 hours. Cells were harvested
by adding 1 mL FACS Lysing Solution (BD Bioscience) and
sedimenting. Cells were frozen in 10% DMSO in FCS and stored
at 280uC. 2. WHO (2010) Global Tuberculosis Control. World Health Organisation.
3. Dye C, Williams BG (2010) The population dynamics and control of
tuberculosis. Science 328: 856–861. Discussion The role of IFN-c+IL-17+ cells in protection against mycobac-
terial infection is not clear, however both cytokines individually are
known to be important in vaccine-induced protection. So far in
mice, it has been shown that ablation of an IL-17 response
following both vaccination (antigen-specific production) [21] and
high-dose M.tb challenge (production by innate cells) [33] leads to
reduced protection against M.tb infection. However, it would also
appear that pathology induced as a result of prolonged exposure to
mycobacterial antigens is IL-17-dependent [34]. Measurement of
IL-17 production in lungs following either vaccination or M.tb
infection is not possible in human studies so it is difficult to draw
comparisons and to predict the role of IL-17, especially since the
mouse model of M.tb disease is not an ideal representation of
human disease. As discussed above, it has been found that IL-17
levels in blood in both humans and cattle correlate with protection
against mycobacterial exposure [23] and mycobacterial infection
[24] respectively, so it may be, as proposed by Torrado and
Cooper [35], that while IL-17 may be essential in vaccine-induced Knowledge of mechanisms through which IL-17-, and IFN-c
and IL-17-producing cells can be induced is important with regard
to vaccine development, with the next step being to determine
their role in vaccine-induced protection or pathology. Furthermore, it is important to investigate regulatory aspects of
cellular immunity, as the total outcome of any vaccine-induced
immunity will be the result of a balance between both effector and
regulatory responses. The work here helps to dissect this
interaction between regulatory and effector responses following
vaccination and provides potential avenues for manipulation of
immune responses in order to provide improved vaccine-induced
protection. PLoS ONE | www.plosone.org August 2011 | Volume 6 | Issue 8 | e23463 August 2011 | Volume 6 | Issue 8 | e23463 5 . WHO (2010) Global Tuberculosis Control. World Health Organisa MVA85A-Induced Th1/Th17 Cells and ATP Consumption MVA85A-Induced Th1/Th17 Cells and ATP Consumption MVA85A-Induced Th1/Th17 Cells and ATP Consumption 4. McShane H, Pathan AA, Sander CR, Goonetilleke NP, Fletcher HA, et al. (2005) Boosting BCG with MVA85A: the first candidate subunit vaccine for
tuberculosis in clinical trials. Tuberculosis (Edinb) 85: 47–52. 20. Altare F, Durandy A, Lammas D, Emile JF, Lamhamedi S, et al. (1998)
Impairment of mycobacterial immunity in human interleukin-12 receptor
deficiency. Science 280: 1432–1435. (
)
5. McShane H, Pathan AA, Sander CR, Keating SM, Gilbert SC, et al. (2004)
Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts
BCG-primed and naturally acquired antimycobacterial immunity in humans. Nat Med 10: 1240–1244. 21. Khader SA, Bell GK, Pearl JE, Fountain JJ, Rangel-Moreno J, et al. (2007) IL-
23 and IL-17 in the establishment of protective pulmonary CD4+ T cell
responses after vaccination and during Mycobacterium tuberculosis challenge. Nat Immunol 8: 369–377. 6. Sander CR, Pathan AA, Beveridge NE, Poulton I, Minassian A, et al. (2009)
Safety and immunogenicity of a new tuberculosis vaccine, MVA85A, in
Mycobacterium tuberculosis-infected individuals. Am J Respir Crit Care Med
179: 724–733. 22. Freches D, Romano M, Korf H, Renauld JC, Van Snick J, et al. (2011)
Increased pulmonary TNF-{alpha}, IL-6 and IL-17A responses compensate for
decreased IFN-{gamma} production in anti-IL-12 auto-vaccine treated, BCG
vaccinated mice. Clin Vaccine Immunol 18: 95–104. 7. Minassian A, Rowland R, Beveridge NE, Poulton I, Satti I, et al. (Under
submission) MVA85A, a candidate TB vaccine, is safe and immunogenic in
HIV-infected adults. 23. Babu S, Bhat SQ, Kumar NP, Kumaraswami V, Nutman TB (2010) Regulatory
T cells modulate Th17 responses in patients with positive tuberculin skin test
results. J Infect Dis 201: 20–31. 8. Brookes RH, Hill PC, Owiafe PK, Ibanga HB, Jeffries DJ, et al. (2008) Safety
and immunogenicity of the candidate tuberculosis vaccine MVA85A in West
Africa. PLoS One 3: e2921. 24. Vordermeier HM, Villarreal-Ramos B, Cockle PJ, McAulay M, Rhodes SG,
et al. (2009) Viral booster vaccines improve Mycobacterium bovis BCG-induced
protection against bovine tuberculosis. Infect Immun 77: 3364–3373. 9. Fletcher HA, Pathan AA, Berthoud TK, Dunachie SJ, Whelan KT, et al. (2008)
Boosting BCG vaccination with MVA85A down-regulates the immunoregula-
tory cytokine TGF-beta1. Vaccine 26: 5269–5275. 25. Scriba TJ, Tameris M, Mansoor N, Smit E, van der Merwe L, et al. MVA85A-Induced Th1/Th17 Cells and ATP Consumption (2010)
Modified vaccinia Ankara-expressing Ag85A, a novel tuberculosis vaccine, is
safe in adolescents and children, and induces polyfunctional CD4+ T cells. Eur J Immunol 40: 279–290. 10. De Cassan S, Pathan A, Sander C, Minassian A, Hill AV, et al. (2010)
Investigating the induction of vaccine induced Th17 and regulatory T cells in
healthy, BCG immunised adults vaccinated with a new tuberculosis vaccine,
MVA85A. Clin Vaccine Immunol 17: 1066–1073. 26. Beveridge NE, Fletcher HA, Hughes J, Pathan AA, Scriba TJ, et al. (2008) A
comparison of IFNgamma detection methods used in tuberculosis vaccine trials. Tuberculosis (Edinb) 88: 631–640. 11. Robson SC, Sevigny J, Zimmermann H (2006) The E-NTPDase family of
ectonucleotidases: Structure function relationships and pathophysiological
significance. Purinergic Signal 2: 409–430. 27. Rovedatti L, Kudo T, Biancheri P, Sarra M, Knowles CH, et al. (2009)
Differential regulation of interleukin 17 and interferon gamma production in
inflammatory bowel disease. Gut 58: 1629–1636. 12. Dwyer KM, Hanidziar D, Putheti P, Hill PA, Pommey S, et al. (2010)
Expression of CD39 by human peripheral blood CD4+ CD25+ T cells denotes a
regulatory memory phenotype. Am J Transplant 10: 2410–2420. 28. Acosta-Rodriguez EV, Rivino L, Geginat J, Jarrossay D, Gattorno M, et al. (2007) Surface phenotype and antigenic specificity of human interleukin 17-
producing T helper memory cells. Nat Immunol 8: 639–646. 13. Corriden R, Chen Y, Inoue Y, Beldi G, Robson SC, et al. (2008) Ecto-
nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1/CD39) regulates
neutrophil chemotaxis by hydrolyzing released ATP to adenosine. J Biol Chem
283: 28480–28486. 29. Atarashi K, Nishimura J, Shima T, Umesaki Y, Yamamoto M, et al. (2008) ATP
drives lamina propria T(H)17 cell differentiation. Nature 455: 808–812. 30. Pelegrin P, Barroso-Gutierrez C, Surprenant A (2008) P2X7 receptor
differentially couples to distinct release pathways for IL-1beta in mouse
macrophage. J Immunol 180: 7147–7157. 14. Pelegrin P, Surprenant A (2006) Pannexin-1 mediates large pore formation and
interleukin-1beta release by the ATP-gated P2X7 receptor. EMBO J 25:
5071–5082. 31. Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, et al. (2007) Adenosine
generation catalyzed by CD39 and CD73 expressed on regulatory T cells
mediates immune suppression. J Exp Med 204: 1257–1265. 15. Pelegrin P, Surprenant A (2009) The P2X(7) receptor-pannexin connection to
dye uptake and IL-1beta release. Purinergic Signal 5: 129–137. 32. Delaloye J, Roger T, Steiner-Tardivel QG, Le Roy D, Knaup Reymond M,
et al. References PLoS ONE | www.plosone.org August 2011 | Volume 6 | Issue 8 | e23463 6 MVA85A-Induced Th1/Th17 Cells and ATP Consumption (2009) Innate immune sensing of modified vaccinia virus Ankara (MVA) is
mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome. PLoS
Pathog 5: e1000480. 16. Petrilli V, Papin S, Dostert C, Mayor A, Martinon F, et al. (2007) Activation of
the NALP3 inflammasome is triggered by low intracellular potassium
concentration. Cell Death Differ 14: 1583–1589. 17. Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F (2007)
Interleukins 1beta and 6 but not transforming growth factor-beta are essential
for the differentiation of interleukin 17-producing human T helper cells. Nat
Immunol 8: 942–949. 33. Okamoto Yoshida Y, Umemura M, Yahagi A, O’Brien RL, Ikuta K, et al. (2010) Essential role of IL-17A in the formation of a mycobacterial infection-
induced granuloma in the lung. J Immunol 184: 4414–4422. 34. Cruz A, Fraga AG, Fountain JJ, Rangel-Moreno J, Torrado E, et al. (2010)
Pathological role of interleukin 17 in mice subjected to repeated BCG
vaccination after infection with Mycobacterium tuberculosis. J Exp Med 207:
1609–1616. 18. Borsellino G, Kleinewietfeld M, Di Mitri D, Sternjak A, Diamantini A, et al. (2007) Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of
extracellular ATP and immune suppression. Blood 110: 1225–1232. 19. Fletcher JM, Lonergan R, Costelloe L, Kinsella K, Moran B, et al. (2009)
CD39+Foxp3+ regulatory T Cells suppress pathogenic Th17 cells and are
impaired in multiple sclerosis. J Immunol 183: 7602–7610. 35. Torrado E, Cooper AM (2010) IL-17 and Th17 cells in tuberculosis. Cytokine
Growth Factor Rev 21: 455–462. PLoS ONE | www.plosone.org August 2011 | Volume 6 | Issue 8 | e23463 7
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English
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Successful en bloc venous resection with reconstruction and subsequent radiotherapy for 2 consecutive recurrences of intravenous leiomyoma—a case report
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BMC cancer
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cc-by
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© 2016 Zhang 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. * Correspondence: shengxiugui@163.com; czhou@med.unc.edu
1Department of Gynecologic Oncology, Shandong Cancer Hospital and
Institute, 440 Jiyan Road, Jinan 250017, China
3Division of Gynecologic Oncology, University of North Carolina at Chapel
Hill, Chapel Hill, NC, USA
Full list of author information is available at the end of the article Abstract Background: Intravenous leiomyomas are a rare variant of uterine leiomyoma. Although histologically benign,
these tumors are associated with a poor prognosis due to propensity for metastasis, high recurrence rate, difficulty
of obtaining complete resection, and frequent extension into and along major veins. Case presentation: We describe a 43-year-old patient initially presenting with lower abdominal pain. Clinical
examination revealed a large right pelvic mass that was shown by computed tomography (CT) to surround the
right external iliac vein, right common iliac vein and distal inferior vena cava. The patient had a history of total
abdominal hysterectomy with bilateral ovarian cystectomies for uterine leiomyoma approximately 3 years prior
to her presentation. Her past surgical history also included removal of an ovarian endometriosis cyst and right
hydrosalpinx. The patient underwent an exploratory laparotomy. Operative findings included complete occlusion of
the right iliac vessels and distal vena cava by a large tumor that filled the pelvis and extended to the level of the
right kidney. The mass was resected en bloc with the involved veins and synthetic vascular grafts were placed. This
highly technical procedure was complicated by hemorrhage requiring a total of 32 units of red blood cells and
2.0 L of plasma. Pathologic examination confirmed intravenous leiomyoma. On Immunohistochemical staining, the
tumor cells were positive for CD32, CD34, Vimentin and smooth muscle actin. Eight months after this procedure,
the patient again presented with an abdominal mass. She was diagnosed with a pelvic recurrence and noted to
have intravascular extension into the left iliac vein and inferior vena cava. For this tumor she underwent radiation
treatment with three-dimensional conformal radiation therapy (total dose 4500 cGy). The tumor gradually
decreased in size during follow-up and became undetectable by CT. Conclusions: Surgical excision is the mainstay of treatment of intravenous leiomyoma. Radiation therapy may be
an effective alternative in patients with unresectable disease or poor surgical candidates. Keywords: Intravenous leiomyomas, Iliac vessel reconstruction, Radiotherapy tissue. IVL was first described in 1896 by Birch-Hirschfeld. Although it is a benign tumor, IVL has malignant biologic
behavior making its management difficult. Because IVL is
often diagnosed after it has metastasized to other locations,
it generally has a poor prognosis [1]. The tumor can
progress to involve the iliac veins, the inferior vena cava
(IVC), and even the right atrium [2]. Successful en bloc venous resection
with reconstruction and subsequent
radiotherapy for 2 consecutive recurrences
of intravenous leiomyoma—a case report Ying Zhang1,2, Leslie H. Clark3, Xiugui Sheng1* and Chunxiao Zhou3,4* Ying Zhang1,2, Leslie H. Clark3, Xiugui Sheng1* and Chunxiao Zhou3,4* * Correspondence: shengxiugui@163.com; czhou@med.unc.edu
1Department of Gynecologic Oncology, Shandong Cancer Hospital and
Institute, 440 Jiyan Road, Jinan 250017, China
3Division of Gynecologic Oncology, University of North Carolina at Chapel
Hill, Chapel Hill, NC, USA
Full list of author information is available at the end of the article Zhang et al. BMC Cancer (2016) 16:6
DOI 10.1186/s12885-015-2045-8 Zhang et al. BMC Cancer (2016) 16:6
DOI 10.1186/s12885-015-2045-8 Case presentation The patient is a 43-year-old woman who presented with
a large pelvic mass and was admitted to Shandong
Cancer Hospital and Institute in June 2006. Her only
complaint was lower abdominal pain. Pertinent past
medical and surgical history include: benign hypertension
for 2 years, prior cesarean delivery, prior hysterectomy,
and inferior vena cava filter placement approximately
three years prior for history of venous thrombus. Surgery was performed under general anesthesia on
July 5, 2006. Upon abdominal entry, two large masses
were noted in the right retroperitoneum. One mass was
abdominal and measured approximately 20 cm × l8 cm ×
10 cm. This mass encased the distal IVC,the right com-
mon iliac vein and right external and internal iliac veins. It was closely adherent to the right internal iliac artery. The other mass measured approximately 10 cm × 10 cm ×
8 cm and was adherent to the bladder wall, pelvic floor,
and right iliac veins. Upon further exploration, the two
masses were connected as a single tumor in a dumb-bell
shape. The right external and internal iliac veins, right
common iliac vein and IVC were completely occluded by
the tumor. The right internal iliac and common iliac arter-
ies were dissected off the tumor. The right internal iliac
vein was ligated distal to the tumor. The tumor was dis-
sected off the pelvic sidewall. Next the distal IVC and right
external iliac vein were ligated. Finally, the left common
iliac vein was ligated to free all venous attachments from
the tumor and obtain a surgical margin. The tumor had a
predominant blood supply from a single pedicle which
was divided and the tumor removed en bloc. The en bloc
resection included the distal IVC with proximal left com-
mon iliac vein, entire right common iliac vein, and prox-
imal portion of the right internal and external iliac veins. An 8 mm expanded polytetrafluoroethylene vascular graft
(by Bard Medical) was placed to reconnect the distal IVC
to the left common iliac vein with 4-0 Prolene suture to
restore venous return to the left lower extremity and pel-
vis. Given that the patient had developed significant The patient reported undergoing a total hysterectomy
and bilateral ovarian cystectomies for uterine fibroids
3 years prior to this presentation at a local hospital. Background Leiomyomas are the most common type of uterine neo-
plasm; however, there are several less common variants of
leiomyomas. Angioleiomyoma or intravenous leiomyoma
(IVL) are a rare variant that originates from mesenchymal Here we present a rare case of uterine IVL with two
recurrences. Our patient was treated with multiple opera-
tions, including an extensive resection with grafting of the Page 2 of 6 Zhang et al. BMC Cancer (2016) 16:6 Page 2 of 6 Page 2 of 6 tumors blood supply was likely originating from the iliac
artery. iliac veins, as well as radiation therapy in order to obtain
tumor control. We are the first authors to present the use
of radiation therapy for this indication. Based on the physical exam and radiological findings,
combined with the medical history, a preliminary diagno-
sis of intravascular leiomyoma was made with extension
into the right iliac vein and IVC. The recommended treat-
ment was surgical resection. The patient was counseled
that surgery would include en bloc resection of the mass
with vascular grafting of the distal IVC and common iliac
vessels as needed. Case presentation The tissue was rich in blood vessels, and tumor cells were arranged around the blood vessels (hematoxylin and eosin, 100× Fig. 2 Pathologic diagnosis. The tissue was rich in blood vessels, and tumor cells were arranged around the blood vessels (hem March 2007. Clinical examination revealed a mass
measuring approximately 6 cm × 6 cm in the left pelvis. CT examination revealed a soft tissue mass close to the
previously resected tumor involving the right internal iliac
vessels, measuring 3.5 cm × 4 cm × 4 cm. A separate mass
was also seen posterior to the bladder on the left, measur-
ing 3 cm × 5 cm × 6.5 cm (Fig. 3). This was felt to repre-
sent a pelvic recurrence of IVL. The patient was deemed
to not be an operative candidate given deconditioning
from the prior surgery. Three-dimensional conformal ra-
diation therapy was performed for tumor control. External
beam radiation was planned using an ADAC Treatment
Planning System, delivered with 15MV X-ray from a
Varian 21EX (Palo Alto, CA) and designed from CT scans. External irradiation was 2.0 Gy daily fraction, 4 fractions
per week, the total dose of radiation was 4500 cGy. collateral flow with no right extremity edema or venous
stasis, the right iliac veins were not reconstructed. Re-
moval of the tumor was challenging with an estimated in-
traoperative blood loss of 5.0 L. In total the patient
was transfused 32 units of packed red blood cells and
2.0
L
of
plasma. Postoperatively
the
patient
was
closely observed for distal pulses, evidence of edema,
and lower extremity compartment syndrome. Postoperative pathology confirmed this mass to be an
intravenous leiomyoma arising from the right internal
iliac vein (Fig. 2). Immunohistochemistry shows the endo-
thelial cells to be positive for CD31, CD34, Vimentin and
smooth muscle actin. The cells stain negative for cytokera-
tin with low expression of p53 protein and high expres-
sion of progesterone receptor and Bcl-2. The patient was managed postoperatively on anticoa-
gulation and given nutritional support with intravenous
nutrition until able to tolerate oral intake. She ultimately
recovered from the operation and was discharged in a
stable condition on postoperative day 20. Following radiation therapy, the patient was followed
closely. A subsequent CT scan in October 2010 demon-
strated a pelvic mass on the right of the bladder and
rectum. Case presentation Patho-
logic review of this specimen shows uterine IVL based on
the Immunohistochemical staining profile and <5 mitotic
figures per 10 high-power fields. Additionally, in April
2006, three months prior to her current presentation, the
patient was diagnosed with a pelvic mass with complaints
of lower abdominal pain and underwent a resection of an
ovarian cyst and right salpingectomy. The histopathological
assessment of the specimen was an ovarian endometriosis
cyst and right hydrosalpinx. At the time of presentation, gynecologic bimanual exam
revealed a palpable mass in the right pelvis, measuring
approximately 10 cm × 10 cm × 8 cm. It was firm and
non-tender to palpation. The mass appeared to invade the
rectovaginal space and extend beyond the pelvis into the
abdomen. Contrast-enhanced computed tomography (Fig. 1) of
the abdomen and pelvis demonstrated a large soft tissue
mass with partial cystic degeneration, measuring 25 cm ×
13.5 cm × 7 cm. The mass was noted to be surrounding
the right external and internal iliac veins, right common
iliac vein and distal IVC. It was in close proximity to the
right common iliac artery and abdominal aorta up to the
level of the right kidney. Doppler ultrasound showed that Fig. 1 June 16, 2006. A large soft tissue mass with dumb-bell shape and partial cystic degeneration, measuring 25 cm × 13.5 cm × 7 cm. The mass
was enlarged since the prior operation 2 months previously. It surrounded the right external and internal iliac veins, right common iliac vein and
distal inferior vena cava. It was close in proximity to the right common and internal iliac arteries and aorta up to the level of the right kidney Fig. 1 June 16, 2006. A large soft tissue mass with dumb-bell shape and partial cystic degeneration, measuring 25 cm × 13.5 cm × 7 cm. The mass
was enlarged since the prior operation 2 months previously. It surrounded the right external and internal iliac veins, right common iliac vein and
distal inferior vena cava. It was close in proximity to the right common and internal iliac arteries and aorta up to the level of the right kidney Zhang et al. BMC Cancer (2016) 16:6 Page 3 of 6 Fig. 2 Pathologic diagnosis. The tissue was rich in blood vessels, and tumor cells were arranged around the blood vessels (hematoxylin and eosin, 100×) nosis. Case presentation It was again felt to be surrounding the distal
IVC and iliac blood vessels. The largest cross-sectional
area was 4.0 cm × 2.0 cm (Fig. 4). This lesion was not
further treated, but followed with serial imaging and Eight months following her surgical resection, the pa-
tient represented complaining of an enlarging abdomen
and frequent urination. She was readmitted to hospital in Fig. 3 March 26, 2007. There was a soft tissue mass close to the site of the prior tumor near the right internal iliac vessels, measuring 3.5 cm ×
4.0 cm × 4.0 cm. Additionally, a left sided mass behind the bladder, measured 3.0 cm × 5.0 cm × 6.5 cm Fig. 3 March 26, 2007. There was a soft tissue mass close to the site of the prior tumor near the right internal iliac vessels, measuring 3.5 cm ×
4.0 cm × 4.0 cm. Additionally, a left sided mass behind the bladder, measured 3.0 cm × 5.0 cm × 6.5 cm Zhang et al. BMC Cancer (2016) 16:6 Page 4 of 6 Fig. 4 Oct 7, 2010. A pelvic mass to the right of the bladder and rectum measuring 4 .0 cm × 2.0 cm Fig. 4 Oct 7, 2010. A pelvic mass to the right of the bladder and rectum measuring 4 .0 cm × 2.0 cm It was necessary to determine the size and shape of
the mass as well as the scope and extent of the involved
adjacent organs, especially in relationship to the iliac veins
and IVC. This evaluation can be performed using contrast-
enhanced
CT,
magnetic
resonance
imaging,
nuclear
magnetic resonance angiography, and digital subtraction
angiography [5, 9]. In the presented case, the complex in-
volvement of the mass with critical veins made complete
resection of the tumor unlikely, without damaging the as-
sociated veins. Therefore, the decision was made to remove
the tumor en bloc with the affected veins using vascular
grafting. By performing en bloc resection with reconstruc-
tion of the iliac vessels, there was an improved chance for
optimal cytoreduction, and therefore reduced risk of recur-
rence with associated improvement in survival [7]. resolved. At most recent follow-up 8 years after radi-
ation therapy in July 2015 the patient remains without
evidence of recurrence on physical examination and CT
images (Fig. 5). Consent Written informed consent was obtained from the patient
for publication of this case report and any accompanying
images (Additional file 1). A copy of the written consent
is available for review by the editor of this journal. References 1. Sahu L, Tempe A, Agrawal A. Angioleiomyoma of uterus. J Obstet Gynaecol. 2012;32(7):713–4. 1. Sahu L, Tempe A, Agrawal A. Angioleiomyoma of uterus. J Obstet Gynaecol. 2012;32(7):713–4. 1. Sahu L, Tempe A, Agrawal A. Angioleiomyoma of uterus. J Obstet Gynaecol. 2012;32(7):713–4. 2. Zizi-Sermpetzoglou A, Myoteri D, Arkoumani E, Koulia K, Tsavari A, Alamanou E,
et al. Angioleiomyoma of the uterus: report of a distinctive benign leiomyoma
variant. Eur J Gynaecol Oncol. 2015;36(2):210–2. 3. Hsu TL, Changchien CC, Huang CC, Lin H. Angioleiomyoma originating from
the ovary of an eleven-year-old premenarchal girl. Gynecol Obstet Invest. 2008;65(4):262–5. 4. Peng HJ, Zhao B, Yao QW, Qi HT, Xu ZD, Liu C. Intravenous leiomyomatosis:
CT findings. Abdom Imaging. 2012;37(4):628–31. 5. Dalainas I. Vascular smooth muscle tumors: review of the literature. Int J
Surg. 2008;6(2):157–63. Competing interests Competing interests
The authors declare that they have no competing interests. Authors’ contributions
d
l
d YZ and XS were involved in the clinical management of the patient. YZ and
XS wrote the main structure of the manuscript. LC and CZ revised the
manuscript. All authors read and approved the final manuscript. It is well-documented that radiation therapy is a
commonly used means of treating malignant tumors. There are also case reports of selectively using radi-
ation treatments for benign tumors, especially those
with biologic activity mimicking malignancy [15, 16]. While malignant tumors usually display immediate
response to radiation therapy, our patient displayed a
delayed response to radiation therapy with appreciable
decrease in the size of the tumor beginning 3 months
after completion of treatment. No residual tumor was
seen on imagining 6 months following treatment. We
suspect that the overall slow response rate to radi-
ation observed was a result of the relatively slow
growth rate of IVL compared to malignancy. Given
that radiation relies on DNA damage leading to the
inability
to
replicate,
this
relatively
slow
growing
tumor took longer to respond to radiation than would
be expected for a malignancy. When radiation therapy
is applied to benign tumors, it can be used at lower
doses. This offers the advantage of fewer side effects,
which may be more acceptable to patients, especially
in those who are not surgical candidates. Discussion Some
studies suggest that IVL is hormone-dependent making
anti-hormonal therapy a possible treatment strategy [12]. Anti-estrogens such as GnRH agonists like Leuprorelin
Acetate have been used for treatment of IVL [13]. But the
role of anti-estrogens therapy for IVL in general remains
unclear [11, 14]. diagnosis and treatment of any recurrences [11]. Some
studies suggest that IVL is hormone-dependent making
anti-hormonal therapy a possible treatment strategy [12]. Anti-estrogens such as GnRH agonists like Leuprorelin
Acetate have been used for treatment of IVL [13]. But the
role of anti-estrogens therapy for IVL in general remains
unclear [11, 14]. Additional file Determining the best curative treatment option for
a patient who is not suitable for surgery is challenging. In
the present case there were concerns surrounding the
use of hormonal therapy in a patient with prior
venous thromboembolism. Given that the patient was
deemed not to be a surgical candidate due to decon-
ditioning and concerns surrounding use of hormonal
therapy, she was counseled by her team and opted
for radiation therapy. Ultimately, this strategy was
shown to be successful with effective control of the
tumor, and its gradual reduction to no visible tumor
on her most recent CT (Fig. 5). Additional file 1: CARE Checklist (2013) of information to include
when writing a case report. (DOCX 1527 kb) Acknowledgements The authors wish to acknowledge the appreciated contribution of Dr. Xiaoling Zhang for the patient’s continuing care and follow up. We also
thank Dr. Dapeng Li, Dr. Yajie Yin and Dr. Nan Gao for manuscript
preparation. We gratefully acknowledge the patient and her relatives for
collaboration. Author details
1 1Department of Gynecologic Oncology, Shandong Cancer Hospital and
Institute, 440 Jiyan Road, Jinan 250017, China. 2School of Medicine and Life
Sciences, Shandong Academy of Medical Sciences, University of Jinan, Jinan,
China. 3Division of Gynecologic Oncology, University of North Carolina at
Chapel Hill, Chapel Hill, NC, USA. 4Lineberger Comprehensive Cancer Center,
University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Received: 23 August 2015 Accepted: 30 December 2015 5.
Dalainas I. Vascular smooth muscle tumors: review of the literature. Int J
Surg. 2008;6(2):157–63. Discussion IVL is a rare benign neoplasm arising from the vascular
smooth muscle [1]. It presents as a painful mass in ~60 %
of cases [3]. IVL is the benign hyperplasia of smooth
muscle cells, as is seen in uterine fibroids, but it has pro-
pensity to grow and metastasize like a malignant tumor. It
has been reported to involve the IVC, right atrium, and
lungs and may be potentially life-threatening [2]. A patient
presenting following a hysterectomy for leiomyoma with a
solid abdominopelvic mass on clinical examination or im-
aging, should prompt providers to consider a recurrence
of IVL [4, 5]. Undertaking
such
an
extensive
operation
carries
significant risks for the patients and surgeons. Careful
preparations preoperatively, correct judgment intraopera-
tively, and close postoperative observation is essential. Success also requires close cooperation among multiple
teams
ranging
from
vascular
surgeons
to
general
surgeons to anesthesiologists and intensive care unit
(ICU) physicians. In complex cases of IVL, whether a tumor can be
completely resected depends primarily on the extent of
involvement of associated vessels [6]. Forced detachment
can easily damage vessels, especially veins with thin,
fragile walls, and result in severe hemorrhage. An in-
complete resection can result in the residual tumor
cells on the vascular surface recurring in the future. The
controversy in these types of cases includes
whether to resect and reconstruct the iliac veins, as
well as how this should be done. As a result of the
development of advanced vascular surgical techniques
and vascular grafts, we were able to completely resect
the tumor en bloc with the involved vessels, and
implant vascular grafts [7, 8]. The mainstay of treatment for IVL is surgical removal,
including cases of recurrence and metastasis. Prognosis
of IVL depends on whether the tumor is completely
resected [10]. As a result, patients not desiring future
fertility should be advised to undergo total hysterectomy. However, up to 30 % of patients can develop recurrence
and metastasis after initial tumor resection [10]. There-
fore, whether or not the tumor is completely resected,
routine postoperative follow-up is necessary for prompt Fig. 5 July 3, 2015. No signs of recurrence were detected at follow up Fig. 5 July 3, 2015. No signs of recurrence were detected at follow up Zhang et al. BMC Cancer (2016) 16:6 Page 5 of 6 Zhang et al. BMC Cancer (2016) 16:6 diagnosis and treatment of any recurrences [11]. 7.
Schneider JR, Sener SF, Barrera Jr E. Combined replacement of infrarenal
aorta and inferior vena cava after en bloc resection of retroperitoneal
extraosseous osteosarcoma. J Vasc Surg. 2008;48(2):478–9. Abbreviations CT: computed tomography; IVL: intravenous leiomyoma; IVC: inferior vena
cava; ICU: intensive care unit. Competing interests
The authors declare that they have no competing interests. 10.
Lin J, Song X, Liu C. Pelvic intravascular leiomyomatosis associated with
benign pulmonary metastasizing leiomyoma: clinicopathologic, clonality,
and copy number variance analysis. Intl J Gynecol Pathol. 2014;33(2):140–5.
11.
Fasih N, Prasad Shanbhogue AK, Macdonald DB, Fraser-Hill MA, Papadatos D,
Kielar AZ, et al. Leiomyomas beyond the uterus: unusual locations, rare
manifestations. Radiographics. 2008;28(7):1931–48.
12.
Valdes Devesa V, Conley CR, Stone WM, Collins JM, Magrina JF. Update on
intravenous leiomyomatosis: report of five patients and literature review.
Eur J Obstet Gynecol Reprod Biol. 2013;171(2):209–13.
13.
Morice P, Chapelier A, Dartevelle P, Castaigne D, Lhomme C. Late
intracaval and intracardiac leiomyomatosis following hysterectomy for
benign myomas treated by surgery and GnRH agonist. Gynecol Oncol.
2001;83(2):422–3.
14.
Liu B, Liu C, Guan H, Li Y, Song X, Shen K, et al. Intravenous leiomyomatosis
with inferior vena cava and heart extension. J Vasc Surg. 2009;50(4):897–902.
15.
Yoneoka Y, Tsumanuma I, Fukuda M, Tamura T, Morii K, Tanaka R, et al.
Cranial base chordoma–long term outcome and review of the literature.
Acta Neurochir. 2008;150(8):773–8. discussion 778.
16.
Schulz-Ertner D, Nikoghosyan A, Thilmann C, Haberer T, Jakel O, Karger C,
et al. Carbon ion radiotherapy for chordomas and low-grade chondrosarcomas
of the skull base. Results in 67 patients. Strahlenther Onkol. 2003;179(9):598–605. 15.
Yoneoka Y, Tsumanuma I, Fukuda M, Tamura T, Morii K, Tanaka R, et al.
Cranial base chordoma–long term outcome and review of the literature.
Acta Neurochir. 2008;150(8):773–8. discussion 778. 16.
Schulz-Ertner D, Nikoghosyan A, Thilmann C, Haberer T, Jakel O, Karger C,
et al. Carbon ion radiotherapy for chordomas and low-grade chondrosarcomas
of the skull base. Results in 67 patients. Strahlenther Onkol. 2003;179(9):598–605. 14.
Liu B, Liu C, Guan H, Li Y, Song X, Shen K, et al. Intravenous leiomyomatosis
with inferior vena cava and heart extension. J Vasc Surg. 2009;50(4):897–902. Zhang et al. BMC Cancer (2016) 16:6 13.
Morice P, Chapelier A, Dartevelle P, Castaigne D, Lhomme C. Late
intracaval and intracardiac leiomyomatosis following hysterectomy for
benign myomas treated by surgery and GnRH agonist. Gynecol Oncol.
2001;83(2):422–3. Conclusion For IVL, radical surgical excision remains the mainstay
of treatment. In cases where dissection of the tumor off
involved vessels appears to be challenging or not feas-
ible, consideration should be given to vascular resection
en bloc with graft placement in order to obtain a
complete tumor resection. In the event of a recurrence
or tumor in a patient deemed a poor surgical candidate,
radiation therapy is feasible and effective alternative to
surgical or medical management for prevention or treat-
ment of recurrence. 6. Anaya-Ayala JE, Cheema ZF, Davies MG, Lumsden AB, Reardon MJ. Concomitant reconstruction of infrarenal aorta and inferior vena cava after
en bloc resection of retroperitoneal rhabdomyosarcoma. Vasc Endovascular
Surg. 2011;45(8):769–72. 7. Schneider JR, Sener SF, Barrera Jr E. Combined replacement of infrarenal
aorta and inferior vena cava after en bloc resection of retroperitoneal
extraosseous osteosarcoma. J Vasc Surg. 2008;48(2):478–9. 8. Schwarzbach MH, Hormann Y, Hinz U, Leowardi C, Bockler D, Mechtersheimer G,
et al. Clinical results of surgery for retroperitoneal sarcoma with major blood
vessel involvement. J Vasc Surg. 2006;44(1):46–55. 9. An JY, Heo JS, Noh JH, Sohn TS, Nam SJ, Choi SH, et al. Primary malignant
retroperitoneal tumors: analysis of a single institutional experience. Eur J Surg Oncol. 2007;33(3):376–82. Page 6 of 6 Zhang et al. BMC Cancer (2016) 16:6 10. Lin J, Song X, Liu C. Pelvic intravascular leiomyomatosis associated with
benign pulmonary metastasizing leiomyoma: clinicopathologic, clonality,
and copy number variance analysis. Intl J Gynecol Pathol. 2014;33(2):140–5. 10. Lin J, Song X, Liu C. Pelvic intravascular leiomyomatosis associated with
benign pulmonary metastasizing leiomyoma: clinicopathologic, clonality,
and copy number variance analysis. Intl J Gynecol Pathol. 2014;33(2):140–5. 11. Fasih N, Prasad Shanbhogue AK, Macdonald DB, Fraser-Hill MA, Papadatos D,
Kielar AZ, et al. Leiomyomas beyond the uterus: unusual locations, rare
manifestations. Radiographics. 2008;28(7):1931–48. 12. Valdes Devesa V, Conley CR, Stone WM, Collins JM, Magrina JF. Update on
intravenous leiomyomatosis: report of five patients and literature review. Eur J Obstet Gynecol Reprod Biol. 2013;171(2):209–13. 13. Morice P, Chapelier A, Dartevelle P, Castaigne D, Lhomme C. Late
intracaval and intracardiac leiomyomatosis following hysterectomy for
benign myomas treated by surgery and GnRH agonist. Gynecol Oncol. 2001;83(2):422–3. 14. Liu B, Liu C, Guan H, Li Y, Song X, Shen K, et al. Intravenous leiomyomatosis
with inferior vena cava and heart extension. J Vasc Surg. 2009;50(4):897–902. 15. Yoneoka Y, Tsumanuma I, Fukuda M, Tamura T, Morii K, Tanaka R, et al. 12.
Valdes Devesa V, Conley CR, Stone WM, Collins JM, Magrina JF. Update on
intravenous leiomyomatosis: report of five patients and literature review.
Eur J Obstet Gynecol Reprod Biol. 2013;171(2):209–13. 10.
Lin J, Song X, Liu C. Pelvic intravascular leiomyomatosis associated with
benign pulmonary metastasizing leiomyoma: clinicopathologic, clonality,
and copy number variance analysis. Intl J Gynecol Pathol. 2014;33(2):140–5 Conclusion Cranial base chordoma–long term outcome and review of the literature. Acta Neurochir. 2008;150(8):773–8. discussion 778. 16. Schulz-Ertner D, Nikoghosyan A, Thilmann C, Haberer T, Jakel O, Karger C,
et al. Carbon ion radiotherapy for chordomas and low-grade chondrosarcomas
of the skull base. Results in 67 patients. Strahlenther Onkol. 2003;179(9):598–605. 11. Fasih N, Prasad Shanbhogue AK, Macdonald DB, Fraser-Hill MA, Papadatos D,
Kielar AZ, et al. Leiomyomas beyond the uterus: unusual locations, rare
manifestations. Radiographics. 2008;28(7):1931–48. 12. Valdes Devesa V, Conley CR, Stone WM, Collins JM, Magrina JF. Update on
intravenous leiomyomatosis: report of five patients and literature review. Eur J Obstet Gynecol Reprod Biol. 2013;171(2):209–13. 16. Schulz-Ertner D, Nikoghosyan A, Thilmann C, Haberer T, Jakel O, Karger C,
et al. Carbon ion radiotherapy for chordomas and low-grade chondrosarcomas
of the skull base. Results in 67 patients. Strahlenther Onkol. 2003;179(9):598–605. • We accept pre-submission inquiries
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https://openalex.org/W1967133088
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https://figshare.com/articles/journal_contribution/Finite_Element_Prediction_of_Surface_Strain_and_Fracture_Strength_at_the_Distal_Radius/10754372/1/files/19265591.pdf
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Finite element prediction of surface strain and fracture strength at the distal radius
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Medical engineering & physics
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cc-by
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*Manuscript *Manuscript Original Manuscript 2
FINITE ELEMENT PREDICTION OF SURFACE STRAIN AND FRACTURE
3
STRENGTH AT THE DISTAL RADIUS
4
5
W. Brent Edwards a, Karen L. Troy ab
6
7
a Department of Kinesiology and Nutrition
8
b Department of Bioengineering
9
University of Illinois at Chicago,
10
Chicago, IL 60612, USA
11
12
Corresponding Author:
13
W. Brent Edwards
14
University of Illinois at Chicago
15
Department of Kinesiology and Nutrition
16
1919 W. Taylor Street
17
650 AHSB, M/C 517
18
Chicago, IL 60612
19
Phone: 312-996-1582
20
E-mail: edwardsb@uic.edu
21
22 FINITE ELEMENT PREDICTION OF SURFACE STRAIN AND FRACTURE
3
STRENGTH AT THE DISTAL RADIUS
4
5
W. Brent Edwards a, Karen L. Troy ab
6
7
a Department of Kinesiology and Nutrition
8
b Department of Bioengineering
9
University of Illinois at Chicago,
10
Chicago, IL 60612, USA
11
12
Corresponding Author:
13
W. Brent Edwards
14
University of Illinois at Chicago
15
Department of Kinesiology and Nutrition
16
1919 W. Taylor Street
17
650 AHSB, M/C 517
18
Chicago, IL 60612
19
Phone: 312-996-1582
20
E-mail: edwardsb@uic.edu
21
22 STRENGTH AT THE DISTAL RADIUS ABSTRACT
23
To better understand the mechanisms underlying distal radius fracture we have developed finite
24
element models to predict radius bone strain and fracture strength under loading conditions
25
simulating a fall. This study compares experimental surface strains and fracture loads of the
26
distal radius with specimen-specific finite element models to validate our model-generating
27
algorithm. Five cadaveric forearms were instrumented with strain gage rosettes, loaded non-
28
destructively to 300 N, and subsequently loaded until failure. Finite element models were created
29
from computed tomography data; three separate density-elasticity relationships were examined. 30
Fracture strength was predicted for three specimens that failed at the distal radius using six
31
different failure theories. The density-elasticity relationship providing the strongest agreement
32
between measured and predicted strains had a correlation of r=0.90 and a root mean squared
33
error 13% of the highest measured strain. Mean absolute percent error (11.6%) between
34
measured and predicted fracture loads was minimized with Coulomb-Mohr failure theory and a
35
tensile-compressive strength ratio of 0.5. These results suggest that our modeling method is a
36
suitable candidate for the in vivo assessment of distal radius bone strain and fracture strength
37
under fall type loading configurations. 38 ABSTRACT
23
To better understand the mechanisms underlying distal radius fracture we have developed finite
24
element models to predict radius bone strain and fracture strength under loading conditions
25
simulating a fall. This study compares experimental surface strains and fracture loads of the
26
distal radius with specimen-specific finite element models to validate our model-generating
27
algorithm. Five cadaveric forearms were instrumented with strain gage rosettes, loaded non-
28
destructively to 300 N, and subsequently loaded until failure. Finite element models were created
29
from computed tomography data; three separate density-elasticity relationships were examined. 30
Fracture strength was predicted for three specimens that failed at the distal radius using six
31
different failure theories. The density-elasticity relationship providing the strongest agreement
32
between measured and predicted strains had a correlation of r=0.90 and a root mean squared
33
error 13% of the highest measured strain. Mean absolute percent error (11.6%) between
34
measured and predicted fracture loads was minimized with Coulomb-Mohr failure theory and a
35
tensile-compressive strength ratio of 0.5. Keywords: Finite element model; Density-elasticity relationship; Failure criteria; Experimental
40
validation; Falls
41 STRENGTH AT THE DISTAL RADIUS These results suggest that our modeling method is a
36
suitable candidate for the in vivo assessment of distal radius bone strain and fracture strength
37
under fall type loading configurations. 38 Keywords: Finite element model; Density-elasticity relationship; Failure criteria; Experimental
40
validation; Falls
41 42 2 INTRODUCTION
43 44
Distal radius fractures are the most common upper extremity fractures in adults 65 years
45
and older [1]. Nearly 80% of distal radius fractures result from a fall [2]. Because many of these
46
falls occur from standing height or lower, fractures of the distal radius are considered low-energy
47
fractures and are associated with age-related declines in bone quality. Distal radius fractures are
48
a source of considerable morbidity; approximately 40% of all dollars spent on physical therapy
49
following osteoporotic fractures go to treat the distal forearm, 50% of patients are dissatisfied
50
with their functional outcome six months post-fracture, and more than one third experience pain
51
or weakness [3]. 52 The propensity for skeletal fracture to occur as a result of a fall is dependent on the
53
loading intensity during impact (i.e., stress and strain within the bone itself). Previous research
54
has focused on preventive strategies, such as surface compliance [4] and fall arrest manipulations
55
[5], to reduce peak bone loading. These studies have used the external reaction force as a
56
surrogate measure of bone loading because the direct measurement of bone strain in vivo requires
57
invasive surgical procedures [6]. Unfortunately, the relationship between external force and
58
internal bone loading is often complex and nonintuitive. This is because external forces generate
59
location-specific triaxial stress-strain states that are dependent on bone size, shape, and material
60
properties. Thus, the ability to quantify radius bone strain non-invasively, and ultimately define
61
fracture strength, would greatly aid in the development and evaluation of preventive measures to
62
minimize the occurrence of distal radius fracture. 63 The propensity for skeletal fracture to occur as a result of a fall is dependent on the
53
loading intensity during impact (i.e., stress and strain within the bone itself). Previous research
54
has focused on preventive strategies, such as surface compliance [4] and fall arrest manipulations
55
[5], to reduce peak bone loading. These studies have used the external reaction force as a
56 Subject-specific finite element models have been an effective tool for both bone strain
64
and fracture strength assessment. The accuracy of these models is heavily dependent on the
65
chosen constitutive equation that relates stress to strain [7, 8], and the chosen failure criterion
66 3 that defines fracture threshold [9]. INTRODUCTION
43 The purpose of this study was to compare experimental
67
surface strains and fracture loads of the distal radius with specimen-specific finite element
68
models for the purpose of validating our model-generating algorithm. Our immediate use for the
69
model is to gain a better understanding of the mechanisms underlying distal radius fracture. For
70
this reason, the model was validated under loading conditions simulating a fall. 71 that defines fracture threshold [9]. The purpose of this study was to compare experimental
67
surface strains and fracture loads of the distal radius with specimen-specific finite element
68
models for the purpose of validating our model-generating algorithm. Our immediate use for the
69
model is to gain a better understanding of the mechanisms underlying distal radius fracture. For
70
this reason, the model was validated under loading conditions simulating a fall. 71
METHODS
72
Specimens
73
Five female cadaveric right forearms with hand intact (mean age 78 yrs, range 59-93 yrs)
74
were obtained through anatomical gift. Specimens were freshly-frozen and stored at -20 °C, but
75
thawed to room temperature for: 1) computed tomography (CT) data acquisition 2) specimen
76
dissection and potting and 3) strain gage application and mechanical testing. In these instances, a
77
saline solution spray was used periodically to keep the specimens moist. The distal most 12 cm
78
of the forearms were imaged with a clinical CT scanner (BrightSpeed; GE Medical Systems,
79
Milwaukee, WI, 120 kV, 180 mA, voxel size: 234 x 234 x 625 µm). Images were reconstructed
80
with GE’s high spatial frequency (bone) algorithm. A subsequent identical scanning session of a
81
calibration phantom (QRM, Moehrendorf, Germany) with calcium hydroxyapatite equivalent
82
concentrations of 0, 400, and 800 mg/cm3 was used to establish the following linear relationship
83
between CT Hounsfield units (Hu) and calcium hydroxyapatite equivalent density (ρha) in g/cm3:
84
(r2 = 0.9993). 85
Experimentation
86
Hu
*
0007
.0
0069
.0
ha
Five female cadaveric right forearms with hand intact (mean age 78 yrs, range 59-93 yrs)
74
were obtained through anatomical gift. Specimens were freshly-frozen and stored at -20 °C, but
75
thawed to room temperature for: 1) computed tomography (CT) data acquisition 2) specimen
76
dissection and potting and 3) strain gage application and mechanical testing. In these instances, a
77
saline solution spray was used periodically to keep the specimens moist. INTRODUCTION
43 The distal most 12 cm
78
of the forearms were imaged with a clinical CT scanner (BrightSpeed; GE Medical Systems,
79
Milwaukee, WI, 120 kV, 180 mA, voxel size: 234 x 234 x 625 µm). Images were reconstructed
80
with GE’s high spatial frequency (bone) algorithm. A subsequent identical scanning session of a
81
calibration phantom (QRM, Moehrendorf, Germany) with calcium hydroxyapatite equivalent
82
concentrations of 0, 400, and 800 mg/cm3 was used to establish the following linear relationship
83
between CT Hounsfield units (Hu) and calcium hydroxyapatite equivalent density (ρha) in g/cm3:
84
(r2 = 0.9993). 85
Hu
*
0007
.0
0069
.0
ha
All soft tissue proximal to the wrist joint capsule was removed and radial/ulnar
87
osteotomy was performed 14 cm proximal to Lister’s Tubercle. The proximal most 8 cm of the
88
forearms were embedded in polymethylmethacrylate (PMMA), leaving 6 cm exposed below
89 4 4 Lister’s Tubercle (Figure 1). Six rectangular strain gage rosettes (TS1N-K120M-PK06-LE,
90
Micro-Flextronics Ltd, Coleraine, N. Ireland) were adhered circumferentially to the periosteal
91
surface of the radius. The active gage length for each individual grid within the rosette was 1.5
92
mm, which corresponded to an overall gage length of 4.2 mm in a stacked rosette configuration. 93
Three rosettes were mounted distally, immediately proximal to Lister’s Tubercle, and three were
94
mounted 3 cm proximal to the distal gage locations (Figure 1). These two locations were chosen
95
to elicit a large range in periosteal surface strains – whereas the distal location was comprised
96
primarily of trabecular bone, the proximal location was comprised primarily of cortical bone. 97
Prior to strain gage attachment the periosteum was removed, the surface was cleaned with
98
isopropyl alcohol, sanded, and recleaned with isopropyl alcohol. Gages were adhered with
99
cyanoacrylate glue and covered with a polyurethane coating. 100
For strain assessment, specimens were loaded in compression using a uniaxial-driven
101
materials testing machine (MiniBionix 858, MTS Systems, Eden Prairie, MN). Force was
102
applied to the palm of the hand with a custom made fixture mounted to the load actuator (Figure
103
2). The fixture consisted of a flat aluminum plate with an angular adjustment to mimic ground
104
contact during a fall onto an outstretched hand. The aluminum plate was angled 60° from vertical
105
such that the wrist was extended 60° to simulate falling conditions [10]. INTRODUCTION
43 The amount of wrist
106
extension was confirmed by a goniometer and an additional aluminum plate was brought into
107
contact with the dorsal surface of the hand to prevent further extension. The bottom surface of
108
the PMMA was placed on a smooth, flat, unconstrained aluminum surface mounted to the MTS
109
load cell. The PMMA was sanded and coated with lubricant to reduce frictional shear forces
110
during testing [11]. The actuator was driven at a fixed displacement rate of 0.1 mm/s until a load
111
of 300 N was reached. Force and displacement data were collected concurrently at 100 Hz, and
112 Lister’s Tubercle (Figure 1). Six rectangular strain gage rosettes (TS1N-K120M-PK06-LE,
90
Micro-Flextronics Ltd, Coleraine, N. Ireland) were adhered circumferentially to the periosteal
91
surface of the radius. The active gage length for each individual grid within the rosette was 1.5
92
mm, which corresponded to an overall gage length of 4.2 mm in a stacked rosette configuration. 93
Three rosettes were mounted distally, immediately proximal to Lister’s Tubercle, and three were
94
mounted 3 cm proximal to the distal gage locations (Figure 1). These two locations were chosen
95
to elicit a large range in periosteal surface strains – whereas the distal location was comprised
96
primarily of trabecular bone, the proximal location was comprised primarily of cortical bone. 97
Prior to strain gage attachment the periosteum was removed, the surface was cleaned with
98
isopropyl alcohol, sanded, and recleaned with isopropyl alcohol. Gages were adhered with
99
cyanoacrylate glue and covered with a polyurethane coating. 100 Lister’s Tubercle (Figure 1). Six rectangular strain gage rosettes (TS1N-K120M-PK06-LE,
90
Micro-Flextronics Ltd, Coleraine, N. Ireland) were adhered circumferentially to the periosteal
91
surface of the radius. The active gage length for each individual grid within the rosette was 1.5
92
mm, which corresponded to an overall gage length of 4.2 mm in a stacked rosette configuration. 93
Three rosettes were mounted distally, immediately proximal to Lister’s Tubercle, and three were
94
mounted 3 cm proximal to the distal gage locations (Figure 1). These two locations were chosen
95
to elicit a large range in periosteal surface strains – whereas the distal location was comprised
96
primarily of trabecular bone, the proximal location was comprised primarily of cortical bone. INTRODUCTION
43 97
Prior to strain gage attachment the periosteum was removed, the surface was cleaned with
98
isopropyl alcohol, sanded, and recleaned with isopropyl alcohol. Gages were adhered with
99
cyanoacrylate glue and covered with a polyurethane coating. 100 For strain assessment, specimens were loaded in compression using a uniaxial-driven
101
materials testing machine (MiniBionix 858, MTS Systems, Eden Prairie, MN). Force was
102
applied to the palm of the hand with a custom made fixture mounted to the load actuator (Figure
103
2). The fixture consisted of a flat aluminum plate with an angular adjustment to mimic ground
104
contact during a fall onto an outstretched hand. The aluminum plate was angled 60° from vertical
105
such that the wrist was extended 60° to simulate falling conditions [10]. The amount of wrist
106
extension was confirmed by a goniometer and an additional aluminum plate was brought into
107
contact with the dorsal surface of the hand to prevent further extension. The bottom surface of
108
the PMMA was placed on a smooth, flat, unconstrained aluminum surface mounted to the MTS
109
load cell. The PMMA was sanded and coated with lubricant to reduce frictional shear forces
110
during testing [11]. The actuator was driven at a fixed displacement rate of 0.1 mm/s until a load
111
of 300 N was reached. Force and displacement data were collected concurrently at 100 Hz, and
112 For strain assessment, specimens were loaded in compression using a uniaxial-driven
101
materials testing machine (MiniBionix 858, MTS Systems, Eden Prairie, MN). Force was
102
applied to the palm of the hand with a custom made fixture mounted to the load actuator (Figure
103
2). The fixture consisted of a flat aluminum plate with an angular adjustment to mimic ground
104
contact during a fall onto an outstretched hand. The aluminum plate was angled 60° from vertical
105
such that the wrist was extended 60° to simulate falling conditions [10]. The amount of wrist
106
extension was confirmed by a goniometer and an additional aluminum plate was brought into
107
contact with the dorsal surface of the hand to prevent further extension. The bottom surface of
108
the PMMA was placed on a smooth, flat, unconstrained aluminum surface mounted to the MTS
109
load cell. The PMMA was sanded and coated with lubricant to reduce frictional shear forces
110
during testing [11]. INTRODUCTION
43 Fifteen total tests were
114
performed allowing for five repeat trials to be collected for each gage. Load repetitions were
115
separated by approximately two minutes. Following strain assessment, specimens were loaded in
116
an identical fashion until failure. The fracture load was identified by a rapid decrease in the slope
117
of the force/displacement curve. Strain gage locations within the CT imaging coordinate system
118
were determined following mechanical testing, by overlaying dissected cross sections of the
119
specimens with their respective CT images. 120 an additional six synchronized analog channels were available for strain information. Therefore,
113
only data from two rosettes could be collected during each test. Fifteen total tests were
114
performed allowing for five repeat trials to be collected for each gage. Load repetitions were
115
separated by approximately two minutes. Following strain assessment, specimens were loaded in
116
an identical fashion until failure. The fracture load was identified by a rapid decrease in the slope
117
of the force/displacement curve. Strain gage locations within the CT imaging coordinate system
118
were determined following mechanical testing, by overlaying dissected cross sections of the
119
specimens with their respective CT images. 120 p
p
g
Modeling
121
Stereolithographic models of the radius, scaphoid, and lunate based on segmented CT
122
data from Mimics (Materialise, Leuven, Belgium) were imported into IA-FEMesh (University of
123
Iowa, Iowa City, IA) for finite element model creation (Figure 3). IA-FEMesh allows the user to
124
define a series of blocks around the surface of interest. Each block is composed of a mesh
125
seeding with a user-specified refinement that is projected onto the surface, laying the foundation
126
for the finite element geometry [12]. The models consisted of 18,231 ± 2,402 8-node hexahedral
127
elements with 21,406 ± 2,600 degrees of freedom depending on specimen size. A nominal
128
element size of 1 mm3 was chosen in accordance with a preliminary mesh convergence analysis. 129
The scaphoid and lunate were modeled as non-deformable rigid bodies. Articular cartilage was
130
included in the model by extruding elements of the radial-carpal bone articular surface 1 mm
131
[13] in a local-normal direction (producing a tissue thickness “just touching” the carpal bones). 132
The cartilage was modeled as a neo-Hookean hyperelastic material with a modulus of 10 MPa
133
[14]; near-incompressibility was assumed [15], thus Poisson’s ratio was set to 0.49. INTRODUCTION
43 The actuator was driven at a fixed displacement rate of 0.1 mm/s until a load
111
of 300 N was reached. Force and displacement data were collected concurrently at 100 Hz, and
112 5 5 an additional six synchronized analog channels were available for strain information. Therefore,
113
only data from two rosettes could be collected during each test. Fifteen total tests were
114
performed allowing for five repeat trials to be collected for each gage. Load repetitions were
115
separated by approximately two minutes. Following strain assessment, specimens were loaded in
116
an identical fashion until failure. The fracture load was identified by a rapid decrease in the slope
117
of the force/displacement curve. Strain gage locations within the CT imaging coordinate system
118
were determined following mechanical testing, by overlaying dissected cross sections of the
119
specimens with their respective CT images. 120
Modeling
121
Stereolithographic models of the radius, scaphoid, and lunate based on segmented CT
122
data from Mimics (Materialise, Leuven, Belgium) were imported into IA-FEMesh (University of
123
Iowa, Iowa City, IA) for finite element model creation (Figure 3). IA-FEMesh allows the user to
124
define a series of blocks around the surface of interest. Each block is composed of a mesh
125
seeding with a user-specified refinement that is projected onto the surface, laying the foundation
126
for the finite element geometry [12]. The models consisted of 18,231 ± 2,402 8-node hexahedral
127
elements with 21,406 ± 2,600 degrees of freedom depending on specimen size. A nominal
128
element size of 1 mm3 was chosen in accordance with a preliminary mesh convergence analysis. 129
The scaphoid and lunate were modeled as non-deformable rigid bodies. Articular cartilage was
130
included in the model by extruding elements of the radial-carpal bone articular surface 1 mm
131
[13] in a local-normal direction (producing a tissue thickness “just touching” the carpal bones). 132
The cartilage was modeled as a neo-Hookean hyperelastic material with a modulus of 10 MPa
133
[14]; near-incompressibility was assumed [15], thus Poisson’s ratio was set to 0.49. For the
134
radius internal elements were assigned the median ρh of the comprising voxels; surface
135 an additional six synchronized analog channels were available for strain information. Therefore,
113
only data from two rosettes could be collected during each test. INTRODUCTION
43 For the
134
radius, internal elements were assigned the median ρha of the comprising voxels; surface
135 6 6 elements were assigned the maximum ρha to avoid partial volume effects. Three previously
136
established density-elasticity relationships (Eqs. i – iii) were investigated that allowed for
137
inhomogeneous linearly-isotropic material properties to be assigned to the finite element models
138
(Figure 3):
139 elements were assigned the maximum ρha to avoid partial volume effects. Three previously
136
established density-elasticity relationships (Eqs. i – iii) were investigated that allowed for
137
inhomogeneous linearly-isotropic material properties to be assigned to the finite element models
138
(Figure 3):
139 elements were assigned the maximum ρha to avoid partial volume effects. Three previously
136
established density-elasticity relationships (Eqs. i – iii) were investigated that allowed for
137
inhomogeneous linearly-isotropic material properties to be assigned to the finite element models
138
(Figure 3):
139 (i)
[16],
140
(ii)
[17],
141
(iii)
[18],
142
29
.2
ash
10500
E
49
.1
app
6950
E
3
app
2875
E
where E is expressed in MPa, and ρapp (apparent density) and ρash (ash density) are expressed in
143
g/cm3. For Eq. (i), calcium hydroxyapatite equivalent density was converted to ρash using:
144
[19]. 145
ha
839
.0
0698
.0
ash [19]. 145
ha
839
.0
0698
.0
ash For Eqs. (ii) and (iii), ρash was divided by 0.6 to obtain ρapp [8]. Moduli lower than 0.01 MPa
146
were assigned a new value of 0.01 MPa [20]. We determined that sufficient model accuracy (%
147
change in principal strains less than 1%) could be obtained by binning moduli in increments
148
corresponding to 20 Hu, or ρha = 0.014 g/cm3. However, moduli were binned in increments
149
corresponding to 10 Hu, or ρha = 0.007 g/cm3, because the increased computational time was
150
negligible. This resulted in 239 ± 13 bins ranging from 0.01 to 21,547 ± 2,728 MPa for Eq. (i),
151
23,714 ± 1,997 MPa for Eq. (ii), and 34,218 ± 5,565 MPa for Eq. (iii), depending on ρha range. 152
Each bin was assigned a Poisson’s ratio of 0.4 [11, 21]. 153 For Eqs. (ii) and (iii), ρash was divided by 0.6 to obtain ρapp [8]. Moduli lower than 0.01 MPa
146
were assigned a new value of 0.01 MPa [20]. INTRODUCTION
43 We determined that sufficient model accuracy (%
147
change in principal strains less than 1%) could be obtained by binning moduli in increments
148
corresponding to 20 Hu, or ρha = 0.014 g/cm3. However, moduli were binned in increments
149
corresponding to 10 Hu, or ρha = 0.007 g/cm3, because the increased computational time was
150
negligible. This resulted in 239 ± 13 bins ranging from 0.01 to 21,547 ± 2,728 MPa for Eq. (i),
151
23,714 ± 1,997 MPa for Eq. (ii), and 34,218 ± 5,565 MPa for Eq. (iii), depending on ρha range. 152
Each bin was assigned a Poisson’s ratio of 0.4 [11, 21]. 153 Finite element analyses were performed using FEBio software (Musculoskeletal
154
Research Laboratories, Salt Lake City, UT). The proximal end of the radius was fully
155
constrained at the location of potting. To simulate the boundary conditions imparted by 60°
156
wrist extension, the scaphoid and lunate were rotated about the flexion-extension axis 50° and
157
35°, respectively. These rotations were based on average values from in vivo and in vitro
158 Finite element analyses were performed using FEBio software (Musculoskeletal
154
Research Laboratories, Salt Lake City, UT). The proximal end of the radius was fully
155
constrained at the location of potting. To simulate the boundary conditions imparted by 60°
156
wrist extension, the scaphoid and lunate were rotated about the flexion-extension axis 50° and
157
35°, respectively. These rotations were based on average values from in vivo and in vitro
158 7 7 examinations of carpal bone kinematics as a function of wrist angle [22, 23]. Contact was
159
modeled between the surfaces of the radius and scaphoid, and the radius and lunate. We assumed
160
that during load application, the radial-carpal ligaments and wrist joint capsule kept the carpal
161
bones seated within the articular cartilage. Therefore, a “tied” interface contact model was
162
utilized in which the carpal bones were not free to slide once initial contact was made. The
163
contact constraints were enforced using the augmented Lagrangian method [24]. A ramped
164
quasi-static load of 300 N was applied to the centroids of the scaphoid (180 N) and lunate (120
165
N) based on the assumption that the scaphoid bears 60% of the load transmitted through the wrist
166
[25, 26]. INTRODUCTION
43 These criteria, which are summarized in Ta
173
will occur when the factor of safety is less than or equal to 1
174
Hoffman (Hσ), Hoffman Strain Analog (Hε), and Maximum P
175
for different tensile (σyt, εyt) and compressive (σyc, εyc) failure
176
εyt=k εyc, we examined four different values of k to investigat
177
0.75, 0.5, and 0.25. In general, a material’s behavior become
178
compressive strength ratio, k, approaches zero [28]. Cortical
179
[29] and cancellous bone an εyc of 0.011 [30]; σyc was determ
180
respective element’s E. For both cortical and cancellous bone
181 examinations of carpal bone kinematics as a function of wrist angle [22, 23]. Contact was
159
modeled between the surfaces of the radius and scaphoid, and the radius and lunate. We assumed
160
that during load application, the radial-carpal ligaments and wrist joint capsule kept the carpal
161
bones seated within the articular cartilage. Therefore, a “tied” interface contact model was
162
utilized in which the carpal bones were not free to slide once initial contact was made. The
163
contact constraints were enforced using the augmented Lagrangian method [24]. A ramped
164
quasi-static load of 300 N was applied to the centroids of the scaphoid (180 N) and lunate (120
165
N) based on the assumption that the scaphoid bears 60% of the load transmitted through the wrist
166
[25, 26]. The line of action of the resultant force vector was determined for each specimen using
167
an unsymmetrical beam theory analysis based on proximal strain gage and CT information
168
(Figure 4; See Appendix for specific details). 169 examinations of carpal bone kinematics as a function of wrist angle [22, 23]. Contact was
159
modeled between the surfaces of the radius and scaphoid, and the radius and lunate. We assumed
160
that during load application, the radial-carpal ligaments and wrist joint capsule kept the carpal
161
bones seated within the articular cartilage. Therefore, a “tied” interface contact model was
162
utilized in which the carpal bones were not free to slide once initial contact was made. The
163
contact constraints were enforced using the augmented Lagrangian method [24]. INTRODUCTION
43 The line of action of the resultant force vector was determined for each specimen using
167
an unsymmetrical beam theory analysis based on proximal strain gage and CT information
168
(Figure 4; See Appendix for specific details). 169
Bone failure was simulated with the finite element method by applying a ramped load up
170
to 3000 N in increments of 120 N. Six different stress- and strain-based failure criteria were
171
evaluated based on previous successful predictions of distal radius fracture [27] and femoral
172
fracture load [9]. These criteria, which are summarized in Table 1, assume that element failure
173
will occur when the factor of safety is less than or equal to 1. The Coulomb-Mohr (CM),
174
Hoffman (Hσ), Hoffman Strain Analog (Hε), and Maximum Principal Strain (εmax) theories allow
175
for different tensile (σyt, εyt) and compressive (σyc, εyc) failure strengths. Assuming, σyt=k σyc and
176
εyt=k εyc, we examined four different values of k to investigate a range of material behaviors: 1,
177
0.75, 0.5, and 0.25. In general, a material’s behavior becomes more brittle as the tensile-
178
compressive strength ratio, k, approaches zero [28]. Cortical bone was assigned an εyc of 0.0154
179
[29] and cancellous bone an εyc of 0.011 [30]; σyc was determined by multiplying εyc by the
180
respective element’s E. For both cortical and cancellous bone, γy was assigned a value 0.0146
181 examinations of carpal bone kinematics as a function of wris
159
modeled between the surfaces of the radius and scaphoid, an
160
that during load application, the radial-carpal ligaments and w
161
bones seated within the articular cartilage. Therefore, a “tied
162
utilized in which the carpal bones were not free to slide once
163
contact constraints were enforced using the augmented Lagra
164
quasi-static load of 300 N was applied to the centroids of the
165
N) based on the assumption that the scaphoid bears 60% of t
166
[25, 26]. The line of action of the resultant force vector was d
167
an unsymmetrical beam theory analysis based on proximal st
168
(Figure 4; See Appendix for specific details). 169
Bone failure was simulated with the finite element m
170
to 3000 N in increments of 120 N. Six different stress- and s
171
evaluated based on previous successful predictions of distal r
172
fracture load [9]. INTRODUCTION
43 The criterion alpha level was set to
198
0.05 for ICC, Pearson’s r, and linear regression analyses. Scatter was assessed using Bland-
199
Altman plots. These illustrate the difference between predicted and measured strains, expressed
200
as a percentage of the mean, versus the mean of the predicted and measured strains. The density-
201
elasticity relationship resulting in the highest correlation and least amount of error was used for
202
failure simulations. Discrepancies between modeled and experimental fracture loads were
203 [31]. Failure was determined for each element at each load increment. Bone fracture (i.e., crack
182
propagation) was assumed to occur when a cluster of contiguous failed elements exceeded a
183
predefined volume. A failed volume of approximately 150 mm3 has been proposed for micro-
184
finite element models of the distal radius [27], while 405 mm3 has been proposed for continuum
185
models of the proximal femur [11]. Thus, we examined a range of failed volumes from 150 to
186
450 mm3, in increments of 100 mm3. 187 [31]. Failure was determined for each element at each load increment. Bone fracture (i.e., crack
182
propagation) was assumed to occur when a cluster of contiguous failed elements exceeded a
183
predefined volume. A failed volume of approximately 150 mm3 has been proposed for micro-
184
finite element models of the distal radius [27], while 405 mm3 has been proposed for continuum
185
models of the proximal femur [11]. Thus, we examined a range of failed volumes from 150 to
186
450 mm3, in increments of 100 mm3. 187 Experimental strain readings from each rosette were used to calculate maximum and
189
minimum principal strains at the instant the target load of 300 N was reached. The between-trial
190
reliability of principal strains at 300 N was examined using interclass correlations (ICC) and
191
variability was assessed using standard error of measurement (SEM), where SEM = standard
192
deviation*(1-ICC)½ [32]. Model predicted strains for nodes corresponding to each rosette
193
location were transformed into a local coordinate system with a unit normal to the model exterior
194
surface. Maximum and minimum principal strains in the surface plane were calculated and nodal
195
values were averaged at each rosette location. Model predicted and experimentally measured
196
principal strains at 300 N were compared using Pearson’s r correlation, linear regression, root
197
mean squared error (RMSE), and maximum error (Max err). INTRODUCTION
43 A ramped
164
quasi-static load of 300 N was applied to the centroids of the scaphoid (180 N) and lunate (120
165
N) based on the assumption that the scaphoid bears 60% of the load transmitted through the wrist
166
[25, 26]. The line of action of the resultant force vector was determined for each specimen using
167
an unsymmetrical beam theory analysis based on proximal strain gage and CT information
168
(Figure 4; See Appendix for specific details). 169 Bone failure was simulated with the finite element method by applying a ramped load up
170
to 3000 N in increments of 120 N. Six different stress- and strain-based failure criteria were
171
evaluated based on previous successful predictions of distal radius fracture [27] and femoral
172
fracture load [9]. These criteria, which are summarized in Table 1, assume that element failure
173
will occur when the factor of safety is less than or equal to 1. The Coulomb-Mohr (CM),
174 8 8 [31]. Failure was determined for each element at each load increment. Bone fracture (i.e., crack
182
propagation) was assumed to occur when a cluster of contiguous failed elements exceeded a
183
predefined volume. A failed volume of approximately 150 mm3 has been proposed for micro-
184
finite element models of the distal radius [27], while 405 mm3 has been proposed for continuum
185
models of the proximal femur [11]. Thus, we examined a range of failed volumes from 150 to
186
450 mm3, in increments of 100 mm3. 187
Data Analysis
188
Experimental strain readings from each rosette were used to calculate maximum and
189
minimum principal strains at the instant the target load of 300 N was reached. The between-trial
190
reliability of principal strains at 300 N was examined using interclass correlations (ICC) and
191
variability was assessed using standard error of measurement (SEM), where SEM = standard
192
deviation*(1-ICC)½ [32]. Model predicted strains for nodes corresponding to each rosette
193
location were transformed into a local coordinate system with a unit normal to the model exterior
194
surface. Maximum and minimum principal strains in the surface plane were calculated and nodal
195
values were averaged at each rosette location. Model predicted and experimentally measured
196
principal strains at 300 N were compared using Pearson’s r correlation, linear regression, root
197
mean squared error (RMSE), and maximum error (Max err). INTRODUCTION
43 The criterion alpha level was set to
198
0.05 for ICC, Pearson’s r, and linear regression analyses. Scatter was assessed using Bland-
199
Altman plots. These illustrate the difference between predicted and measured strains, expressed
200
as a percentage of the mean, versus the mean of the predicted and measured strains. The density-
201
elasticity relationship resulting in the highest correlation and least amount of error was used for
202
failure simulations. Discrepancies between modeled and experimental fracture loads were
203 9 9 expressed as a percent error, and the correspondence in fracture location was examined
204
qualitatively. 205
RESULTS
206
Experimentation
207
Experimentally measured principal strains were highly reliable (gage dehiscence
208
occurred for the distal strain gages of a single specimen during mechanical testing, so these data
209
were not included). For example, the ICC for maximum principal strains at 300 N measured
210
across 5 trials was 0.997 (p<0.001); ICC was 0.994 (p<0.001) for minimum principal strains. 211
The SEM was 10 με for maximum principal strains and 18 με for minimum principal strains. 212
These SEM values corresponded to approximately 1.6% and 1.8% of the largest measured
213
maximum (640 με) and minimum (-977 με) principal strain, respectively. 214
Of the five specimens loaded until failure, three fractured at the distal radius, one
215
fractured at the scaphoid, and one wrist dislocated. Interestingly, the line of action of the
216
resultant force vector fell outside the bone cross section for the dislocated specimen, indicating
217
poor alignment of the specimen within the testing fixture (Figure 4). Only the three specimens
218
with distal radius fracture were used for failure analysis. Distal radius fracture occurred at
219
loading magnitudes of 813, 971, and 1,214 N. 220
Comparison between predicted and measured strains
221
The finite element predicted strains varied as a function of Eqs. (i-iii) (Figure 5). 222
Correlation coefficients for experimentally measured strains versus predicted strains ranged from
223 expressed as a percent error, and the correspondence in fracture location was examined
204
qualitatively. 205
RESULTS
206
Experimentation
207
Experimentally measured principal strains were highly reliable (gage dehiscence
208
occurred for the distal strain gages of a single specimen during mechanical testing, so these data
209
were not included). For example, the ICC for maximum principal strains at 300 N measured
210
across 5 trials was 0.997 (p<0.001); ICC was 0.994 (p<0.001) for minimum principal strains. INTRODUCTION
43 229 not different from zero (p=0.056), the regression slope for Eq. (ii) was different from unity
227
(p<0.001). The RMSE among density-elasticity Eqs. ranged from 13% to 14% of the highest
228
measured strain. Max error was smallest for Eq. (i) and largest for Eq. (ii). 229 Bland-Altman plots illustrated a randomly distributed scatter across strain magnitudes for
230
Eqs. (i) and (iii) (Figure 5). In contrast, Eq. (ii) illustrated systematic scatter in which strains
231
were under-predicted at high strain magnitudes and over-predicted at low strain magnitudes. 232
Overall, predicted strains using the Eq. (i) were most closely matched to measured strains in
233
terms of regression coefficients, error, and scatter. Therefore, the finite element models created
234
using Eq. (i) were used for failure analyses. 235 Bland-Altman plots illustrated a randomly distributed scatter across strain magnitudes for
230
Eqs. (i) and (iii) (Figure 5). In contrast, Eq. (ii) illustrated systematic scatter in which strains
231
were under-predicted at high strain magnitudes and over-predicted at low strain magnitudes. 232
Overall, predicted strains using the Eq. (i) were most closely matched to measured strains in
233
terms of regression coefficients, error, and scatter. Therefore, the finite element models created
234
using Eq. (i) were used for failure analyses. 235 Comparison between predicted and measured fracture loads
236 Comparison between predicted and measured fracture loads
236 The predicted fracture loads varied among failure theories, tensile-compressive strength
237
ratio k, and contiguous volume assumptions. For a given failure theory and volume, changing k
238
from 0.25 to 0.5, from 0.5 to 0.75, and from 0.75 to 1 increased fracture loads an average of
239
26%, 9%, and 4%, respectively., For a given failure theory and k, changing volume from 150 to
240
250 mm3, from 250 to 350 mm3, and from 350 to 450 mm3 increased fracture loads an average of
241
7%, 5%, and 4%, respectively. For a contiguous volume of 150 mm3, mean absolute percent
242
error was minimized with Hσ theory and k=0.5 (Figure 6). For contiguous volumes of 250, 350,
243
and 450 mm3, mean absolute percent error was minimized with CM theory and k=0.5. In all of
244
these instances, mean absolute percent error varied from 11.6% (range 2.2-25.4% for 350 mm3)
245
to 12.9% (range 3.90-18.45% for 150 mm3). INTRODUCTION
43 211
The SEM was 10 με for maximum principal strains and 18 με for minimum principal strains. 212
These SEM values corresponded to approximately 1.6% and 1.8% of the largest measured
213
maximum (640 με) and minimum (-977 με) principal strain, respectively. 214
Of the five specimens loaded until failure, three fractured at the distal radius, one
215
fractured at the scaphoid, and one wrist dislocated. Interestingly, the line of action of the
216 expressed as a percent error, and the correspondence in fracture location was examined
204
qualitatively. 205 Of the five specimens loaded until failure, three fractured at the distal radius, one
215
fractured at the scaphoid, and one wrist dislocated. Interestingly, the line of action of the
216
resultant force vector fell outside the bone cross section for the dislocated specimen, indicating
217
poor alignment of the specimen within the testing fixture (Figure 4). Only the three specimens
218
with distal radius fracture were used for failure analysis. Distal radius fracture occurred at
219
loading magnitudes of 813, 971, and 1,214 N. 220 Of the five specimens loaded until failure, three fractured at the distal radius, one
215
fractured at the scaphoid, and one wrist dislocated. Interestingly, the line of action of the
216
resultant force vector fell outside the bone cross section for the dislocated specimen, indicating
217
poor alignment of the specimen within the testing fixture (Figure 4). Only the three specimens
218
with distal radius fracture were used for failure analysis. Distal radius fracture occurred at
219
loading magnitudes of 813, 971, and 1,214 N. 220 Comparison between predicted and measured strains
221 The finite element predicted strains varied as a function of Eqs. (i-iii) (Figure 5). 222 Correlation coefficients for experimentally measured strains versus predicted strains ranged from
223
r=0.90 (p<0.001) for Eq (i) to r=0.86 (p<0.001) for Eq (ii) (Table 2). For Eqs. (i) and (iii)
224 regression slopes were not different from unity (p≥0.270) and intercepts were not different from
225
zero (p≥0.178). Despite having a relatively high correlation coefficient and an intercept that was
226 10 not different from zero (p=0.056), the regression slope for Eq. (ii) was different from unity
227
(p<0.001). The RMSE among density-elasticity Eqs. ranged from 13% to 14% of the highest
228
measured strain. Max error was smallest for Eq. (i) and largest for Eq. (ii). INTRODUCTION
43 246 The centroids of failed contiguous volumes were located within the distal radius
247
cancellous region for all failure theories. Crack propagation was not explicitly simulated and as
248
such, failed elements (failure criterion value ≥ 1) were not observed at the external surface of the
249 11 models. However, the CM failure contours (k=0.5) illustrated higher values at locations where
250
experimental fracture manifested at the surface (Figure 7). 251 models. However, the CM failure contours (k=0.5) illustrated higher values at locations where
250
experimental fracture manifested at the surface (Figure 7). 251 models. However, the CM failure contours (k=0.5) illustrated higher values at locations where
250
experimental fracture manifested at the surface (Figure 7). 251
DISCUSSION
252
Non-invasive methods to quantify bone strain and fracture strength on a subject-specific
253
basis are needed so that preventive measures to reduce the incidence of distal radius fracture can
254
be evaluated. The purpose of this study was to compare experimental surface strains and fracture
255
loads at the distal radius with specimen-specific finite element models to validate our model
256
generating algorithm. Of the three density-elasticity relationships investigated, the models
257
developed using Eq. (i) [16] predicted principal strains that most closely matched the
258
experimentally measured strains. Average percent error between experimentally measured and
259
model predicted fracture loads was minimized with the use of CM failure theory, a tensile-
260
compressive strength ratio k=0.5, and a contiguous volume assumption of 350 mm3. In addition,
261
surface elements illustrating the largest magnitudes of CM failure qualitatively agreed with the
262
locations where experimental fracture was observed at the surface. 263 Non-invasive methods to quantify bone strain and fracture strength on a subject-specific
253
basis are needed so that preventive measures to reduce the incidence of distal radius fracture can
254
be evaluated. The purpose of this study was to compare experimental surface strains and fracture
255
loads at the distal radius with specimen-specific finite element models to validate our model
256
generating algorithm. Of the three density-elasticity relationships investigated, the models
257
developed using Eq. (i) [16] predicted principal strains that most closely matched the
258
experimentally measured strains. Average percent error between experimentally measured and
259
model predicted fracture loads was minimized with the use of CM failure theory, a tensile-
260
compressive strength ratio k=0.5, and a contiguous volume assumption of 350 mm3. INTRODUCTION
43 In addition,
261
surface elements illustrating the largest magnitudes of CM failure qualitatively agreed with the
262
locations where experimental fracture was observed at the surface. 263 Very few studies have investigated the influence of density-elasticity relationships on the
264
accuracy of specimen-specific finite element predicted strains [7, 8, 33]. Schileo et al. [8]
265
compared three density-elasticity relationships for the human femur under several loading
266
scenarios and concluded that the relationship described by Morgan et al. [17], Eq. (ii) in the
267
present study, produced the closest agreement between numerical and experimental results. 268
Austman et al. [7] compared six density-elasticity relationships for the human ulna under a
269
simplified cantilever bending scenario and observed the most accurate results using the Carter
270
and Hayes relationship [18], corresponding to Eq. (iii) here, as well as a pooled bone site
271
relationship described by Morgan et al. [17]. The discrepancy in density-elasticity relationship
272 12 accuracy between the current and aforementioned studies is not surprising. Density-elasticity
273
relationships depend on variables such as anatomical site [17] and strain rate [18]. Eq. (ii), which
274
provided the poorest agreement between experimental and predicted surface strains in the present
275
study, was developed for femoral trabecular bone. Similar to Austman et al. [7], we found
276
reasonable agreement with Eq. (iii), which is logical given the anatomic similarity of the radius
277
and ulna. Unfortunately, we are unaware of a density-elasticity relationship specific to the distal
278
radius. We hesitate to implicate strain rate as a discriminating factor in the present study because
279
all three density-elasticity relationships investigated were determined using strain rates of 0.01 to
280
1 s-1 [16-18]. Our strain rates were substantially lower than this with maximum measured values
281
of 2.5 x 10-4 to 4.5 x 10-4 s-1. 282 accuracy between the current and aforementioned studies is not surprising. Density-elasticity
273
relationships depend on variables such as anatomical site [17] and strain rate [18]. Eq. (ii), which
274
provided the poorest agreement between experimental and predicted surface strains in the present
275
study, was developed for femoral trabecular bone. Similar to Austman et al. [7], we found
276
reasonable agreement with Eq. (iii), which is logical given the anatomic similarity of the radius
277
and ulna. Unfortunately, we are unaware of a density-elasticity relationship specific to the distal
278
radius. INTRODUCTION
43 In contrast, the finite element model involves some assumptions about how density
300
relates to modulus of elasticity, and how to best simulate element failure. Our interpretation is
301
that these last two assumptions are the true subject of the finite element model validation. 302
Although we adopted a method to approximate the line of action of the resulting force vector
303
based on unsymmetrical beam theory, there is still some uncertainty in simulating this
304
“physiological” contact scenario including: the exact load share distribution between the
305
scaphoid and lunate, the exact carpal bone translations/rotations that occur relative to the radius
306
with wrist extension, and the possibility of shear forces at the lubricated PMMA/aluminum
307
interface. Changes in these parameters can influence load transition through the radius [39] and
308
thus periosteal surface strain, and may have contributed to our observed error. 309 predicted strain is dependent on how well our finite element model represents both the structural
296
characteristics of and boundary conditions applied to the radius. The boundary conditions that
297
were applied to the scaphoid and lunate were estimated based on measured surface strains and
298
radius geometry. Both of these quantities are direct and repeatable measures derived from the
299
bone itself. In contrast, the finite element model involves some assumptions about how density
300
relates to modulus of elasticity, and how to best simulate element failure. Our interpretation is
301
that these last two assumptions are the true subject of the finite element model validation. 302 Although we adopted a method to approximate the line of action of the resulting force vector
303
based on unsymmetrical beam theory, there is still some uncertainty in simulating this
304
“physiological” contact scenario including: the exact load share distribution between the
305
scaphoid and lunate, the exact carpal bone translations/rotations that occur relative to the radius
306
with wrist extension, and the possibility of shear forces at the lubricated PMMA/aluminum
307
interface. Changes in these parameters can influence load transition through the radius [39] and
308
thus periosteal surface strain, and may have contributed to our observed error. 309 The volume of failed contiguous elements chosen to represent bone fracture in this study
310
was a topic of uncertainty. INTRODUCTION
43 We hesitate to implicate strain rate as a discriminating factor in the present study because
279
all three density-elasticity relationships investigated were determined using strain rates of 0.01 to
280
1 s-1 [16-18]. Our strain rates were substantially lower than this with maximum measured values
281
of 2.5 x 10-4 to 4.5 x 10-4 s-1. 282 Here, we observed a best-fit correlation of 0.90 between experimental and predicted
283
strains using Eq. (i). Similar in vitro validation studies have reported various levels of accuracy
284
ranging from r=0.679 to 0.955 [8, 34-38]. Several factors can explain this relatively large range
285
in model accuracy including: the number of specimens used, constitutive law applied, loading
286
scenario(s) investigated, as well as the incorporated model meshing technique (voxel vs. 287
geometry based). These studies focused on the femur, pelvis, and scapula. In all cases but one
288
[36], complex bone articulations were not incorporated into in vitro testing and modeling. This
289
approach is sufficient for bones like the femur, for which the boundary conditions in a fall-type
290
load configuration are relatively straightforward (e.g. side impact to the greater trochanter). For
291
the wrist however, load is transferred to the distal radius through its articulating carpal bones. 292
Finite element models developed to examine the mechanisms underlying distal radius
293
fracture should be validated with the wrist joint fully intact, allowing the model’s behavior under
294
physiological loading conditions to be investigated. It is important to note that our accuracy in
295 Here, we observed a best-fit correlation of 0.90 between experimental and predicted
283
strains using Eq. (i). Similar in vitro validation studies have reported various levels of accuracy
284
ranging from r=0.679 to 0.955 [8, 34-38]. Several factors can explain this relatively large range
285
in model accuracy including: the number of specimens used, constitutive law applied, loading
286
scenario(s) investigated, as well as the incorporated model meshing technique (voxel vs. 287 13 predicted strain is dependent on how well our finite element model represents both the structural
296
characteristics of and boundary conditions applied to the radius. The boundary conditions that
297
were applied to the scaphoid and lunate were estimated based on measured surface strains and
298
radius geometry. Both of these quantities are direct and repeatable measures derived from the
299
bone itself. INTRODUCTION
43 This approach, which has been used by others to predict the fracture
311
strength of the distal radius [27, 40] and proximal femur [11, 20], assumes that a given amount of
312
tissue must fail in order for a crack to propagate. This approach also reduces the potential error
313
caused by CT scanning and finite element modeling artifacts that may underestimate the failure
314
strength of individual elements. Although, changing this volume influenced the predicted
315
fracture load, the mean absolute percent errors were not substantially altered by volume
316
assumption (See Figure 6). This is because the most accurate volume for fracture strength
317
prediction varied amongst specimens. This specific response may be related to differences in the
318 14 age of the specimens tested to failure (59, 71, and 93 yrs). The bone of younger adults can
319
undergo more plastic deformation before failure [41], which would require an increased
320
contiguous volume assumption to replicate in our linear elastic models. Further study with an
321
increased sample size and a thorough statistical analysis would be necessary to verify this
322
assumption. 323 assumption. 323
Here we observed a fracture strength prediction accuracy of 11.6 to 12.9%, depending on
324
the chosen contiguous volume. This is comparable to the 13% accuracy reported for microCT
325
finite element models of the distal radius [27]. Our most accurate predictions were obtained
326
using CM and Hσ theories with k=0.5. Investigations of bovine trabecular bone have reported
327
tensile-compressive strength ratios ranging from 0.3 to 0.7 [42, 43]. Both CM and Hσ theories are
328
stress-based criteria intended to be applicable across a range of material types (i.e., ability to
329
account for different tensile and compressive strengths). In their simplest form where k=1, CM
330
and Hσ theories are equivalent to Tresca (max shear stress) and von Mises (max distortion
331
energy) criteria, respectively. These findings suggest that shear or distortion modes of failure
332
play an important role in bone fracture, at least at the continuum level. At the microstuctural
333
level, bone fracture is indeed strain controlled [44]. Thus from a theoretical standpoint the
334
appropriate failure criterion should be strain-based as well. Unfortunately, our continuum and
335
linearly isotropic assumptions do not allow us to properly model the microstructural properties of
336
bone. INTRODUCTION
43 For present purposes it is more important to determine a robust failure theory that
337
phenomenologically describes fracture load and location given the various simplifications and
338
limitations of the modeling procedure. 339
This study is limited by the relatively small sample size of five specimens for strain
340 This study is limited by the relatively small sample size of five specimens for strain
340
assessment and three specimens for failure analysis. However, most specimen specific finite
341 15 element model validation studies have relied on sample sizes of three or less [34-38, 45, 46],
342
with only a few having reported sample sizes greater than this [8, 11]. Here, we dealt with partial
343
volume effects by assigning surface elements the maximum density of the comprising voxels,
344
which could be considered a less refined method than other published techniques [47], and may
345
have contributed to the observed scatter between measured and predicted strains. However, given
346
the homogeneity of cortical bone, variation in Hu within elements at the bone surface would
347
largely be explained by partial volume artifacts, providing rationale for the assignment of
348
maximum density. 349 element model validation studies have relied on sample sizes of three or less [34-38, 45, 46],
342
with only a few having reported sample sizes greater than this [8, 11]. Here, we dealt with partial
343
volume effects by assigning surface elements the maximum density of the comprising voxels,
344
which could be considered a less refined method than other published techniques [47], and may
345
have contributed to the observed scatter between measured and predicted strains. However, given
346
the homogeneity of cortical bone, variation in Hu within elements at the bone surface would
347
largely be explained by partial volume artifacts, providing rationale for the assignment of
348
maximum density. 349 For this initial validation, a slow rate of loading (0.1 mm/s) was used for fracture
350
analysis corresponding to approximately 10-20 N/s. Actual loading rates during a fall can
351
approach 90 to 180 kN/s [48]. Our future work will focus on validating similar models able to
352
predict bone strain and fracture load at rates of loading consistent with a fall. Presumably, this
353
would require us to incorporate strain-rate dependent behavior into our models, which could be
354
done for Young’s modulus by including a second power-term in the density-elasticity
355
relationship [18]. INTRODUCTION
43 373
CONFLICT OF INTEREST
374
The authors have no conflict of interest
375
APPENDIX A
376
The axial force, P, and bending moments, Mx and My, acting at the cross section
377
corresponding to the proximal gage locations were resolved using unsymmetrical beam theory a
378
described by Rybicki et al., [51]. Assuming the origin of the reference system is at the cross
379
section centoid, the axial strain εzz at any point (x, y) can be determined as:
380
y
κ
x
κ
ε
ε
x
y
0
zz
381
where ε0 is the strain created by the axial force, and κy and κx are the radii of curvature about the
382
x and y-axis, respectivity. Using the measured strain from the axial gage at each of the three
383
rosette locations the unknown parameters ε0, κy, and κx can be determined. The axial force, P, an
384
bending moments, Mx and My, acting at the cross section are then:
385
0
ε
A
E
P
386
I
κ
I
κ
E
M
387 In summary, the present study has shown that our model generating algorithm provides
365
realistic measures of radius bone strain and fracture strength under a physiological loading
366
scenario simulating a fall. Given our model’s level of accuracy for strain (r=0.90, RMSE=13% of
367
the highest measured strain) and fracture prediction (mean absolute percent error of 11.6%), we
368
consider it a suitable candidate for in vivo examinations of preventive strategies to minimize the
369
occurrence of distal radius fracture. 370 365 Partial funding for this study was provided by the Department of Kinesiology and Nutrition at
372
the University of Illinois at Chicago. 373 The axial force, P, and bending moments, Mx and My, acting at the cross section
377
corresponding to the proximal gage locations were resolved using unsymmetrical beam theory as
378
described by Rybicki et al., [51]. Assuming the origin of the reference system is at the cross
379
section centoid, the axial strain εzz at any point (x, y) can be determined as:
380 where ε0 is the strain created by the axial force, and κy and κx are the radii of curvature about the
382
x and y-axis, respectivity. INTRODUCTION
43 Additionally, bone elicits a ductile-to-brittle transition with increases in strain
356
rate, which influences post-yield behavior [49]. This would likely require smaller contiguous
357
volume assumptions [46] and different ultimate failure strengths. Alternatively, an elastic-plastic
358
material model could be incorporated with strain-rate dependent post-yield behavior. Such a
359
material model would also likely improve our overall prediction accuracy [50].Unfortunately we
360
were unable to compare experimental and predicted fracture location in a quantitative manner. 361
This stems from our inability to identify the location of fracture onset during experimentation. 362
Future studies could incorporate high-speed video to approximate the location of fracture onset
363
[46], provided that crack nucleation occurred at the periosteal surface. 364 For this initial validation, a slow rate of loading (0.1 mm/s) was used for fracture
350
analysis corresponding to approximately 10-20 N/s. Actual loading rates during a fall can
351
approach 90 to 180 kN/s [48]. Our future work will focus on validating similar models able to
352
predict bone strain and fracture load at rates of loading consistent with a fall. Presumably, this
353
would require us to incorporate strain-rate dependent behavior into our models, which could be
354
done for Young’s modulus by including a second power-term in the density-elasticity
355 16 In summary, the present study has shown that our model generating algorithm provides
365
realistic measures of radius bone strain and fracture strength under a physiological loading
366
scenario simulating a fall. Given our model’s level of accuracy for strain (r=0.90, RMSE=13% of
367
the highest measured strain) and fracture prediction (mean absolute percent error of 11.6%), we
368
consider it a suitable candidate for in vivo examinations of preventive strategies to minimize the
369
occurrence of distal radius fracture. 370 In summary, the present study has shown that our model generating algorithm provides
365
realistic measures of radius bone strain and fracture strength under a physiological loading
366
scenario simulating a fall. Given our model’s level of accuracy for strain (r=0.90, RMSE=13% o
367
the highest measured strain) and fracture prediction (mean absolute percent error of 11.6%), we
368
consider it a suitable candidate for in vivo examinations of preventive strategies to minimize the
369
occurrence of distal radius fracture. 370
ACKNOWLEDGEMENTS
371
Partial funding for this study was provided by the Department of Kinesiology and Nutrition at
372
the University of Illinois at Chicago. INTRODUCTION
43 Using the measured strain from the axial gage at each of the three
383
rosette locations the unknown parameters ε0, κy, and κx can be determined. The axial force, P, and
384
bending moments, Mx and My, acting at the cross section are then:
385
0
ε
A
E
P
386
0
ε
A
E
P
386
xy
y
xx
x
x
I
κ
I
κ
E
M
387 17
yy
y
xy
x
y
I
κ
I
κ
E
M
yy
y
xy
x
y
I
κ
I
κ
E
M
388 388 where E is the elastic modulus, A is the cross sectional area, and Ixx, Iyy, and Ixy are the cross
389
sectional moments of inertia defined as:
390 where E is the elastic modulus, A is the cross sectional area, and Ixx, Iyy, and Ixy are the cross
389 where E is the elastic modulus, A is the cross sectional area, and Ixx, Iyy, and Ixy are the cross
389 where E is the elastic modulus, A is the cross sectional area, and Ixx, Iyy, and Ixy are the cross
389 sectional moments of inertia defined as:
390 sectional moments of inertia defined as:
390 sectional moments of inertia defined as:
390
n
i
i
d
1
A
A
391
i
n
i
i
dA
y
I
1
2
xx
392
i
n
i
i
dA
x
I
1
2
yy
393
i
n
i
i dA
(xy)
I
1
xy
394 were n is the number of bone pixels and dAi is the ith bone pixel area. The line of action of the
395
applied force was then calculated by assuming that it was directed from the scaphoid and lunate
396
centroids through location (xact, yact), using the following formulae:
397 were n is the number of bone pixels and dAi is the ith bone pixel area. The line of action of the
395
applied force was then calculated by assuming that it was directed from the scaphoid and lunate
396
centroids through location (xact, yact), using the following formulae:
397 P
My
x act
and
P
Mx
yact
. INTRODUCTION
43 398 P
My
x act
and
P
Mx
yact
. It can be seen that the calculation of xact and yact is independent of the chosen E. 399 It can be seen that the calculation of xact and yact is independent of the chosen E. 399 It can be seen that the calculation of xact and yact is independent of the chosen E. 399 18 REFERENCES
400
[1] Vogt MT, Cauley JA, Tomaino MM, Stone K, William JR, Herndon JH. Distal Radius
401
Fractures in Older Women: A 10-Year Follow-Up Study of Descriptive Characteristics and Risk
402
Factors. the Study of Osteoporotic Fractures. Journal of the American Geriatrics Society 2002;
403
50(1): 97-103. 404
405
[2] Oskam J, Kingma J, Klasen HJ. Fracture of the Distal Forearm: Epidemiological
406
Developments in the Period 1971-1995. Injury 1998; 29(5): 353-355. 407
408
[3] Melton LJ. Adverse Outcomes of Osteoporotic Fractures in the General Population. Journal
409
of Bone and Mineral Research 2003; 18(6): 1139-1141. 410
411
[4] Robinovitch SN, Chiu J. Surface Stiffness Affects Impact Force during a Fall on the
412
Outstretched Hand. Journal of Orthopaedic Research 1998; 16(3): 309-313. 413
414
[5] Kim KJ, Ashton-Miller JA. Biomechanics of Fall Arrest using the Upper Extremity: Age
415
Differences. Clinical Biomechanics 2003; 18(4): 311-318. 416
417
[6] Foldhazy Z, Arndt A, Milgrom C, Finestone A, Ekenman I. Exercise-Induced Strain and
418
Strain Rate in the Distal Radius. The Journal of Bone and Joint Surgery 2005; 87(2): 261-266. 419
420 REFERENCES
400 420 19 [7] Austman RL, Milner JS, Holdsworth DW, Dunning CE. The Effect of the Density-Modulus
421
Relationship Selected to Apply Material Properties in a Finite Element Model of Long Bone. 422
Journal of Biomechanics 2008; 41(15): 3171-3176. 423 [7] Austman RL, Milner JS, Holdsworth DW, Dunning CE. The Effect of the Density-Modulus
421
Relationship Selected to Apply Material Properties in a Finite Element Model of Long Bone. 422
Journal of Biomechanics 2008; 41(15): 3171-3176. 423
424
[8] Schileo E, Taddei F, Malandrino A, Cristofolini L., Viceconti M. Subject-Specific Finite
425
Element Models can Accurately Predict Strain Levels in Long Bones. Journal of Biomechanics
426
2007; 40(13): 2982-2989. 427
428
[9] Keyak JH, Rossi SA. Prediction of Femoral Fracture Load using Finite Element Models: An
429
Examination of Stress- and Strain-Based Failure Theories. Journal of Biomechanics 2000; 33(2):
430
209-214. 431
432
[10] Troy KL, Grabiner MD. Asymmetrical Ground Impact of the Hands After a Trip-Induced
433
Fall: Experimental Kinematics and Kinetics. Clinical Biomechanics 2007; 22(10): 1088-1095. 434
435
[11] Keyak JH, Rossi SA, Jones KA, Skinner HB. Prediction of Femoral Fracture Load using
436
Automated Finite Element Modeling. Journal of Biomechanics 1998; 31(2): 125-133. 437
438
[12] Grosland NM, Shivanna KH, Magnotta VA, Kellemeyn NA, DeVries NA, Tadepalli SC,
439
Lisle C. IA-FEMesh: An Open-Source, Interactive, Multiblock Approach to Anatomic Finite
440
Element Model Development. Computer Methods and Programs in Biomedicine 2009; 94(1):
441
96-107. 442 443 20 [13] Pillai RR, Thoomukuntla B, Ateshian GA, Fischer KJ. MRI-Based Modeling for
444
Evaluation of in Vivo Contact Mechanics in the Human Wrist during Active Light Grasp. 445
Journal of Biomechanics 2007; 40(12): 2781-2787. 446
447
[14] Anderson DD, Deshpande BR, Daniel TE, Baratz ME. A Three-Dimensional Finite Ele
448
Model of the Radiocarpal Joint: Distal Radius Fracture Step-Off and Stress Transfer. The Iow
449
Orthopaedic Journal 2005; 25: 108-117. 450
451
[15] Armstrong CG, Lai WM, Mow VC. An Analysis of the Unconfined Compression of
452
Articular Cartilage. Journal of Biomechanical Engineering 1984; 106(2): 165-173. 453
454
[16] Keller TS. Predicting the Compressive Mechanical Behavior of Bone. Journal of
455
Biomechanics 1994; 27(9): 1159-1168. 456
457
[17] Morgan EF, Bayraktar HH, Keaveny TM. Trabecular Bone Modulus-Density Relations
458
Depend on Anatomic Site. Journal of Biomechanics 2003; 36(7): 897-904. 459
460
[18] Carter DR, Hayes WC. The Compressive Behavior of Bone as a Two-Phase Porous
461
Structure. [19] Kaneko TS, Bell JS, Pejcic MR, Tehranzaden J, Keyak JH. Mechanical Properties Density
464
and Quantitative CT Scan Data of Trabecular Bone with and without Metastases. Journal of
465
Biomechanics 2004; 37(4): 523-530.
466 [20] Keyak JH, Rossi SA, Jones KA, Les CM, Skinner HB. Prediction of Fracture Location in
468
the Proximal Femur using Finite Element Models. Medical Engineering & Physics 2001; 23(9):
469
657-664.
470 REFERENCES
400 The Journal of Bone and Joint Surgery 1977; 59(7): 954-962. 462
463
[19] Kaneko TS, Bell JS, Pejcic MR, Tehranzaden J, Keyak JH. Mechanical Properties Dens
464
and Quantitative CT Scan Data of Trabecular Bone with and without Metastases. Journal of
465
Biomechanics 2004; 37(4): 523-530. 466 [13] Pillai RR, Thoomukuntla B, Ateshian GA, Fischer KJ. MRI-Based Modeling for
444
Evaluation of in Vivo Contact Mechanics in the Human Wrist during Active Light Grasp. 445
Journal of Biomechanics 2007; 40(12): 2781-2787. 446 [13] Pillai RR, Thoomukuntla B, Ateshian GA, Fischer KJ. MRI-Based Modeling for
444
Evaluation of in Vivo Contact Mechanics in the Human Wrist during Active Light Grasp. 445
Journal of Biomechanics 2007; 40(12): 2781-2787. 446 [18] Carter DR, Hayes WC. The Compressive Behavior of Bone as a Two-Phase Porous
461
Structure. The Journal of Bone and Joint Surgery 1977; 59(7): 954-962. 462 21 467 [20] Keyak JH, Rossi SA, Jones KA, Les CM, Skinner HB. Prediction of Fracture Location in
468
the Proximal Femur using Finite Element Models. Medical Engineering & Physics 2001; 23(9):
469
657-664. 470
471
[21] Reilly DT, Burstein AH. The Elastic and Ultimate Properties of Compact Bone Tissue. 472
Journal of Biomechanics 1975; 8(6): 393-405. 473
474
[22] Moojen TM, Snel JG, Ritt MJ, Kauer JM, Verema HW, Bos KE. Three-Dimensional Carpal
475
Kinematics in Vivo. Clinical Biomechanics 2002; 17(7): 506-514. 476
477
[23] Kobayashi M, Berger RA, Nagy L, Linscheid RL, Uchiyama S, Ritt M, An KN. Normal
478
Kinematics of Carpal Bones: A Three-Dimensional Analysis of Carpal Bone Motion Relative to
479
the Radius. Journal of Biomechanics 1997; 30(8): 787-793. 480
481
[24] Mass S, Rawlins D, Weiss J, Ateshian G FEBio Theory Manual Version 1.2 URL:
482
http://mrl.sci.utah.edu/software/febio
483
484
[25] Majima M, Horii E, Matsuki H, Hirata H, Genda E. Load Transmission through the Wrist in
485
the Extended Position. The Journal of Hand Surgery 2008; 33(2): 182-188. 486
487 [20] Keyak JH, Rossi SA, Jones KA, Les CM, Skinner HB. Prediction of Fracture Location in
468
the Proximal Femur using Finite Element Models. Medical Engineering & Physics 2001; 23(9):
469
657-664. 470 471
[21] Reilly DT, Burstein AH. The Elastic and Ultimate Properties of Compact Bone Tissue. 472
Journal of Biomechanics 1975; 8(6): 393-405. 473
474
[22] Moojen TM, Snel JG, Ritt MJ, Kauer JM, Verema HW, Bos KE. Three-Dimensional Carpal
475
Kinematics in Vivo. REFERENCES
400 Clinical Biomechanics 2002; 17(7): 506-514. 476
477
[23] Kobayashi M, Berger RA, Nagy L, Linscheid RL, Uchiyama S, Ritt M, An KN. Normal
478
Kinematics of Carpal Bones: A Three-Dimensional Analysis of Carpal Bone Motion Relative to
479
the Radius. Journal of Biomechanics 1997; 30(8): 787-793. 480
481
[24] Mass S, Rawlins D, Weiss J, Ateshian G FEBio Theory Manual Version 1.2 URL:
482 [21] Reilly DT, Burstein AH. The Elastic and Ultimate Properties of Compact Bone Tissue. 472 Journal of Biomechanics 1975; 8(6): 393-405. 473 [22] Moojen TM, Snel JG, Ritt MJ, Kauer JM, Verema HW, Bos KE. Three-Dimensional Carpal
475
Kinematics in Vivo. Clinical Biomechanics 2002; 17(7): 506-514. 476 487 22 [26] Schuind F, Cooney WP, Linscheid RL, An KN, Chao EY. Force and Pressure Transmission
488
through the Normal Wrist. A Theoretical Two-Dimensional Study in the Posteroanterior Plane. 489
Journal of Biomechanics 1995; 28(5): 587-601. 490
491
[27] Pistoia W, van Rietbergen B, Lochmuller EM, Lill CA, Eckstein F, Ruegsegger P. 492
Estimation of Distal Radius Failure Load with Micro-Finite Element Analysis Models Based on
493
Three-Dimensional Peripheral Quantitative Computed Tomography Images. Bone 2002; 30(6):
494
842-848. 495
496
[28] Christensen, RM. A Two-Property Yield, Failure (Fracture) Criterion for Homogeneous,
497
Isotropic Materials. Journal of Engineering Materials and Technology 2004; 126: 45-52. 498
499
[29] Steinhauser E, Diehl P, Hadaller M Schauwecker J, Busch R, Gradinger R, Mittelmeier W. 500
Biomechanical Investigation of the Effect of High Hydrostatic Pressure Treatment on the
501
Mechanical Properties of Human Bone. Journal of Biomedical Materials Research. Part B
502
Applied Biomaterials 2006; 76(1): 130-135. 503
504
[30] Rohl L, Larsen E, Linde F, Odgaard A. Tensile and Compressive Properties of Cancellous
505
Bone. Journal of Biomechanics 1991; 24(12): 1143-1149. 506
507 [26] Schuind F, Cooney WP, Linscheid RL, An KN, Chao EY. Force and Pressure Transmission
488
through the Normal Wrist. A Theoretical Two-Dimensional Study in the Posteroanterior Plane. 489
Journal of Biomechanics 1995; 28(5): 587-601. 490 [26] Schuind F, Cooney WP, Linscheid RL, An KN, Chao EY. Force and Pressure Transmission
488
through the Normal Wrist. A Theoretical Two-Dimensional Study in the Posteroanterior Plane. 489
Journal of Biomechanics 1995; 28(5): 587-601. 490 [30] Rohl L, Larsen E, Linde F, Odgaard A. Tensile and Compressive Properties of Cancellous
505
Bone. Journal of Biomechanics 1991; 24(12): 1143-1149. 506 23 511
[32] Harvill LM. Standard Error of Measurement. [37] Keyak JH, Fourkas MG, Meagher JM, Skinner HB. Validation of an Automated Method of
531
Three-Dimensional Finite Element Modelling of Bone. Journal of Biomedical Engineering 1993;
532
15(6): 505-509.
533 [32] Harvill LM. Standard Error of Measurement. Educational Measurement: Issues and Practice
512
1991; 10: 33-41.
513 [43] Stone JL, Beaupre GS, Hayes W C. Multiaxial Strength Characteristics of Trabecular Bone.
554
Journal of Biomechanics 1983; 16(9): 743-752.
555 REFERENCES
400 Educational Measurement: Issues and Practice
512
1991; 10: 33-41. 513
514
[33] Barker DS, Netherway DJ, Krishnan J, Hearn TC. Validation of a Finite Element Model of
515
the Human Metacarpal. Medical Engineering & Physics 2005; 27(2): 103-113. 516
517
[34] Ota T, Yamamoto I, Morita R. Fracture Simulation of the Femoral Bone using the Finite-
518
Element Method: How a Fracture Initiates and Proceeds. Journal of Bone and Mineral
519
Metabolism 1999; 17(2): 108-112. 520
521
[35] Gupta S, van der Helm FC, Sterk JC, van Keulen F, Kaptien BL. Development and
522
Experimental Validation of a Three-Dimensional Finite Element Model of the Human Scapula. 523
Proceedings of the Institution of Mechanical Engineers. Part H Journal of Engineering in
524
Medicine 2004; 218(2): 127-142. 525
526
[36] Anderson AE, Peters CL, Tuttle BD, Weiss JA. Subject-Specific Finite Element Model of
527
the Pelvis: Development Validation and Sensitivity Studies. Journal of Biomechanical
528
Engineering 2005; 127(3): 364-373. 529
530
[37] Keyak JH, Fourkas MG, Meagher JM, Skinner HB. Validation of an Automated Method of
531
Three-Dimensional Finite Element Modelling of Bone. Journal of Biomedical Engineering 1993;
532
15(6): 505-509. 533 [34] Ota T, Yamamoto I, Morita R. Fracture Simulation of the Femoral Bone using the Finite-
518
Element Method: How a Fracture Initiates and Proceeds. Journal of Bone and Mineral
519
Metabolism 1999; 17(2): 108-112. 520 521
[35] Gupta S, van der Helm FC, Sterk JC, van Keulen F, Kaptien BL. Development and
522
Experimental Validation of a Three-Dimensional Finite Element Model of the Human Scapula. 523
Proceedings of the Institution of Mechanical Engineers. Part H Journal of Engineering in
524
Medicine 2004; 218(2): 127-142. 525 24 534 [38] Taddei F, Cristofolini L, Martelli S, Gill HS, Viceconti M. Subject-Specific Finite Element
535
Models of Long Bones: An in Vitro Evaluation of the overall Accuracy. Journal of
536
Biomechanics 2006; 39(13): 2457-2467. 537 Models of Long Bones: An in Vitro Evaluation of the overall Accuracy. Journal of
536
Biomechanics 2006; 39(13): 2457-2467. 537
538
[39] Ulrich D, van Rietbergen B, Laib A, Ruegsegger P. Load Transfer Analysis of the Distal
539
Radius from in-Vivo High-Resolution CT-Imaging. Journal of Biomechanics 1999; 32(8): 821-
540
828. 541
542
[40] Troy KL, Grabiner MD. Off-Axis Loads Cause Failure of the Distal Radius at Lower
543
Magnitudes than Axial Loads: A Finite Element Analysis. Journal of Biomechanics 2007; 40(8):
544
1670-1675. rnal of Biomechanics 1983; 16(9): 743-752. [44] Nalla RK, Kinney JH, Ritchie RO. Mechanistic Fracture Criteria for the Failure of Human
557
Cortical Bone. Nature Materials 2003; 2(3): 164-168.
558 REFERENCES
400 545
546
[41] Bernstein AH, Reilly D T, Martens M. Aging of Bone Tissue: Mechanical Properties. 547
Journal of Bone and Joint Surgery 1976; 58: 82-86. 548
549
[42] Keaveny TM, Wachtel EF, Ford C M, Hayes WC. Differences between the Tensile and
550
Compressive Strengths of Bovine Tibial Trabecular Bone Depend on Modulus. Journal of
551
Biomechanics 1994; 27(9): 1137-1146. 552
553
[43] Stone JL, Beaupre GS, Hayes W C. Multiaxial Strength Characteristics of Trabecular Bone. 554
Journal of Biomechanics 1983; 16(9): 743-752. 555
556 [39] Ulrich D, van Rietbergen B, Laib A, Ruegsegger P. Load Transfer Analysis of the Distal
539
Radius from in-Vivo High-Resolution CT-Imaging. Journal of Biomechanics 1999; 32(8): 821-
540
828. 541 556 25 [44] Nalla RK, Kinney JH, Ritchie RO. Mechanistic Fracture Criteria for the Failure of Human
557
Cortical Bone. Nature Materials 2003; 2(3): 164-168. 558
559
[45] Gray HA, Taddei F, Zavatsky AB, Cristofolini L, Gill HS. Experimental Validation of a
560
Finite Element Model of a Human Cadaveric Tibia. Journal of Biomechanical Engineering 2008;
561
130(3): 031016-1-9. 562
563
[46] Schileo E, Taddei F, Cristofolini L, Viceconti M. Subject-Specific Finite Element Models
564
Implementing a Maximum Principal Strain Criterion are Able to Estimate Failure Risk and
565
Fracture Location on Human Femurs Tested in Vitro. Journal of Biomechanics 2008; 41(2): 356-
566
367. 567
568
[47] Helgason B, Taddei F, Palsson H Schileo E, Cristofolini L, Viceconti M, Brynjolfsson S. A
569
Modified Method for Assigning Material Properties to FE Models of Bones. Medical
570
Engineering & Physics 2008; 30(4): 444-453. 571
572
[48] DeGoede KM, Ashton-Miller JA, Schultz AB. Fall-Related Upper Body Injuries in the
573
Older Adult: A Review of the Biomechanical Issues. Journal of Biomechanics 2003; 36(7):
574
1043 1053 [44] Nalla RK, Kinney JH, Ritchie RO. Mechanistic Fracture Criteria for the Failure of Human
557
Cortical Bone. Nature Materials 2003; 2(3): 164-168. 558 559
[45] Gray HA, Taddei F, Zavatsky AB, Cristofolini L, Gill HS. Experimental Validation of a
560
Finite Element Model of a Human Cadaveric Tibia. Journal of Biomechanical Engineering 2008;
561
130(3): 031016-1-9. 562
563
[46] Schileo E, Taddei F, Cristofolini L, Viceconti M. Subject-Specific Finite Element Models
564
Implementing a Maximum Principal Strain Criterion are Able to Estimate Failure Risk and
565
Fracture Location on Human Femurs Tested in Vitro. Journal of Biomechanics 2008; 41(2): 356-
566
367. [51] Rybicki EF, Mills EJ. In Vivo and Analytical Studies of Forces and Moments in Equine
584
Long Bones. Journal of Biomechanics 1977; 10(11/12): 701-705.
585 [50] Keyak JH. Improved Prediction of Proximal Femoral Fracture Load using Nonlinear Finite
581
Element Models. Medical Engineering & Physics 2001; 23(3): 165-173.
582 REFERENCES
400 567
568
[47] Helgason B, Taddei F, Palsson H Schileo E, Cristofolini L, Viceconti M, Brynjolfsson S. A
569
Modified Method for Assigning Material Properties to FE Models of Bones. Medical
570
Engineering & Physics 2008; 30(4): 444-453. 571
572
[48] DeGoede KM, Ashton-Miller JA, Schultz AB. Fall-Related Upper Body Injuries in the
573
Older Adult: A Review of the Biomechanical Issues. Journal of Biomechanics 2003; 36(7):
574
1043-1053. 575
576
[49] Hansen U, Zioupos P, Simpson R, Currey JD, Hynd D. The Effect of Strain Rate on the
577
Mechanical Properties of Human Cortical Bone. Journal of Biomechanical Engineering 2008;
578
130(1): 011011. 579 26 580 [50] Keyak JH. Improved Prediction of Proximal Femoral Fracture Load using Nonlinear Finite
581
Element Models. Medical Engineering & Physics 2001; 23(3): 165-173. 582
583
[51] Rybicki EF, Mills EJ. In Vivo and Analytical Studies of Forces and Moments in Equine
584
Long Bones. Journal of Biomechanics 1977; 10(11/12): 701-705. 585 [50] Keyak JH. Improved Prediction of Proximal Femoral Fracture Load using Nonlinear Finite
581
Element Models. Medical Engineering & Physics 2001; 23(3): 165-173. 582 Element Models. Medical Engineering & Physics 2001; 23(3): 165-173. 582 27 Tables TABLES TABLES
Table 1. The six failure criterion investigated with their respective equations. Criterion
Equation
CM (Coulomb-Mohr)
(σ1/σyt)-(σ3/σyc) ≥ 1
Hσ (Hoffman)
(1/2σytσyc)[(σ1-σ2)2+(σ1-σ3)2(σ2-σ3)2]+…
[(1/σyt)-(1/σyc)](σ1+σ2+σ3) ≥ 1
Hε (Hoffman Strain Analog)
(1/2εytεyc)[(ε1-ε2)2+(ε1-ε3)2(ε2-ε3)2]+…
[(1/εyt)-(1/εyc)](ε1+ε2+ε3) ≥ 1
εmax (Maximum Principal Strain)
(ε1/εyt) ≥ 1 or (ε3/εyc) ≤ -1
εeff (Effective Strain)
(1/εy)(2U/E)½ ≥ 1
γmax (Maximum Shear Strain)
(γmax/γy) ≥ 1
σ1, σ2, and σ3 are the principal stresses for a given element (σ1 > σ2> σ3), ε1 , ε 2, and ε 3 are the Table 1. The six failure criterion investigated with their respective equations. Table 1. The six failure criterion investigated with their respective equations. Criterion
Equation
CM (Coulomb-Mohr)
(σ1/σyt)-(σ3/σyc) ≥ 1
Hσ (Hoffman)
(1/2σytσyc)[(σ1-σ2)2+(σ1-σ3)2(σ2-σ3)2]+…
[(1/σyt)-(1/σyc)](σ1+σ2+σ3) ≥ 1
Hε (Hoffman Strain Analog)
(1/2εytεyc)[(ε1-ε2)2+(ε1-ε3)2(ε2-ε3)2]+…
[(1/εyt)-(1/εyc)](ε1+ε2+ε3) ≥ 1
εmax (Maximum Principal Strain)
(ε1/εyt) ≥ 1 or (ε3/εyc) ≤ -1
εeff (Effective Strain)
(1/εy)(2U/E)½ ≥ 1
γmax (Maximum Shear Strain)
(γmax/γy) ≥ 1
σ1, σ2, and σ3 are the principal stresses for a given element (σ1 > σ2> σ3), ε1 , ε 2, and ε 3 are the σ1, σ2, and σ3 are the principal stresses for a given element (σ1 > σ2> σ3), ε1 , ε 2, and ε 3 are the principal strains for a given element (ε 1 > ε 2> ε 3), γmax is the maximum shear strain, U is the
strain energy density, and σy, εy, and γy are the normal failure stress, normal failure strain, and
shear failure strain, respectively. CM, Hσ, Hε, and εmax allow for different tensile (σyt, εyt) and
compressive (σyc, εyc) failure strengths (σyc and εyc >0). Table 2. Validation parameters as a function of Eqs. (i-iii). Eq. (i)
Eq. (ii)
Eq. (iii)
r
0.90
0.86
0.88
Slope
0.94 (CI: 0.82-1.07) ns
0.51 (CI: 0.42-0.59) a
0.92 (CI: 0.78-1.06) ns
Intercept (με) -31.54 (CI: -77.87-14.79) ns -28.83 (CI: -58.48-0.81) ns -18.32 (CI: -68.74-32.09) ns
RMSE (με)
128.59
138.51
130.15
RMSE% b
13.17
14.18
13.33
Max err (με)
476.78
750.87
642.82
Max err% b
48.82
76.88
65.82
ns Not significantly different from 1(slope) or 0 (intercept). a Significantly different from 1 (slope) or 0 (intercept). b Percentage of the maximum absolute measured strain. b Percentage of the maximum absolute measured strain. b Percentage of the maximum absolute measured strain. Figures Figures FIGURE CAPTIONS Figure 1. Dorsal, sagittal, and planar views of strain gage rosettes. Three rosettes were mounted
distally, immediately proximal to Lister’s Tubercle, and three were mounted 3 cm proximal to
distal rosettes. Figure 2. Left – three dimensional illustration of experimental setup. A flat aluminum plate was
positioned 60° from vertical (120° as shown here) and brought into contact with the palm of the
hand. A second flat aluminum plate was then brought into contact with the dorsal surface of the
hand to maintain 60° wrist extension. Right – sagittal view of typical experimental setup. Figure 3. Left – representative finite element model illustrating surface ρash distribution. Top-
right – transverse cross sections illustrating internal ρash distributions. Bottom-right – Plot of
Young’s modulus as a function of ρash for the three density-elasticity relationships investigated
(Eqs. i-iii). Figure 4. Representative proximal cross sections for two specimens illustrating location of
centroid ( ), strain gage rosettes (▬), and line of action (●). The wrist joint dislocated during
fracture testing for specimen on the right. Note the line of action fell outside the bone cross
section for this specimen. The line of action was determined using an unsymmetrical beam
theory analysis (See Appendix). Figure 5. Top – predicted versus measured principal strains at 300 N for Eqs. (i-iii). Bottom –
Bland-Altman plots for Eqs. (i-iii). Solid line is the mean difference between predicted and
measured strain. Dashed lines are the 95% limits of agreement. Figure 6. The specimen-mean absolute percent error between experimentally measured and finite
element predicted fracture strength as a function of failure theory, tensile-compressive strength
ratio k, and contiguous volume assumption. Figure 7. Surface fracture locations of the distal radius vs. finite element failure contours for CM
theory, k = 0.5, and volume = 350mm3. Note that surface elements did not fail (CM failure ≤ 1),
but displayed higher values at locations where experimental surface fracture was observed. FIGURES FIGURES Figure 1. Doral View
Palmer View
Sagittal View Figure 1. Figure 1. Figure 2. Figure 2. Figure 2. Figure 3. 0
0.2
0.4
0.6
0.8
1
1.2
5
10
15
20
ρash(g/cm3)
E (GPa)
Eq. (i)
Eq. (ii)
Eq. (iii)
1.20
0.96
0.72
0.48
0.24
ρash(g/cm3)
0
a
a
b
b
c
c
a. b. c. 0 b. c. Figure 3. Figure 4. X
Y
X
Y
PALMAR
DORSAL
(x1,y1)
(x2,y2)
(x3,y3)
(x1,y1)
(x3,y3)
(x2,y2)
DORSAL
PALMAR
(0,0)
(0,0)
(xact,yact)
(xact,yact) X
Y
(x1,y1)
(x3,y3)
(x2,y2)
DORSAL
PALMAR
(0,0)
(xact,yact) Figure 4. X
Y
PALMAR
DORSAL
(x1,y1)
(x2,y2)
(x3,y3)
(0,0)
(xact,yact) DORSAL (x2,y2) (x2,y2) (x2,y2) Figure 5. -1000
0
1000
-1000
-500
0
500
1000
Eq. (i)
Predicted (
)
Measured (
)
-1000
0
1000
-1000
-500
0
500
1000
Eq. (ii)
Measured (
)
-1000
0
1000
-1000
-500
0
500
1000
Eq. (iii)
Measured (
)
-1000
0
1000
-200
-100
0
100
200
Mean Predicted-Measured (
)
Diff Predicted-Measured/
Mean Predicted-Measured (%)
-1000
0
1000
-200
-100
0
100
200
Mean Predicted-Measured (
)
-1000
0
1000
-200
-100
0
100
200
Mean Predicted-Measured (
) 0
-1000
0
1000
-1000
Measured (
)
0
-1000
0
1000
-200
-100
0
100
200
Mean Predicted-Measured (
) Figure 5. Figure 6. 0
10
20
30
40
50
60
0
0.25
0.5
0.75
1
1.25
Absolute Percent Error
k
350 mm3
0
10
20
30
40
50
60
0
0.25
0.5
0.75
1
1.25
k
450 mm3
0
10
20
30
40
50
60
0
0.25
0.5
0.75
1
1.25
Absolute Percent Error
150 mm3
0
10
20
30
40
50
60
0
0.25
0.5
0.75
1
1.25
250 mm3
CM
Hσ
Hε
εmax
εeff
γmax Figure 6. 0
10
20
30
40
50
60
0
0.25
0.5
0.75
1
1.25
Absolute Percent Error
k
350 mm3
0
10
20
30
40
50
60
0
0.25
0.5
0.75
1
1.25
k
450 mm3
0
10
20
30
40
50
60
0
0.25
0.5
0.75
1
1.25
Absolute Percent Error
150 mm3
0
10
20
30
40
50
60
0
0.25
0.5
0.75
1
1.25
250 mm3
CM
Hσ
Hε
εmax
εeff
γmax CM
Hσ
Hε
εmax
εeff
γmax Figure 6. k
k
CM
Hσ
Hε
εmax
εeff
γmax Fi
7
Experimental
Fracture
CM Failure
0
0.2 0.4 0.6 0.8 1.0 Experimental
Fracture
CM Failure
0
0.2 0.4 0.6 0.8 1.0 Figure 7. Figure 7.
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Reactivity of bromoselenophenes in palladium-catalyzed direct arylations
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Beilstein journal of organic chemistry
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To cite this version: Aymen Skhiri, Ridha Ben Salem, Jean-François Soulé, Henri Doucet. Reactivity of bromoselenophenes
in palladium-catalyzed direct arylations. Beilstein Journal of Organic Chemistry, 2017, 13, pp.2862-
2868. 10.3762/bjoc.13.278. hal-01709524 Distributed under a Creative Commons Attribution 4.0 International License Abstract The reactivity of 2-bromo- and 2,5-dibromoselenophenes in Pd-catalyzed direct heteroarylation was investigated. From 2-bromo-
selenophene, only the most reactive heteroarenes could be employed to prepare 2-heteroarylated selenophenes; whereas, 2,5-
dibromoselenophene generally gave 2,5-di(heteroarylated) selenophenes in high yields using both thiazole and thiophene deriva-
tives. Moreover, sequential catalytic C2 heteroarylation, bromination, catalytic C5 arylation reactions allowed the synthesis of un-
symmetrical 2,5-di(hetero)arylated selenophene derivatives in three steps from selenophene. Email: * Corresponding author HAL Id: hal-01709524
https://univ-rennes.hal.science/hal-01709524v1
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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 Full Research Paper Beilstein J. Org. Chem. 2017, 13, 2862–2868. doi:10.3762/bjoc.13.278 Address:
1Institut des Sciences Chimiques de Rennes, UMR 6226
CNRS-Université de Rennes 1, "Organométalliques: Matériaux et
Catalyse", Campus de Beaulieu, 35042 Rennes, France and
2Laboratoire de Laboratoire de Chimie Organique LR 17ES08,
Université de Sfax, Faculté des Sciences de Sfax, Route de la Soukra
km 4, 3038 Sfax, Tunisia Received: 24 August 2017
Accepted: 04 December 2017
Published: 22 December 2017
Associate Editor: M. Rueping
© 2017 Skhiri et al.; licensee Beilstein-Institut. License and terms: see end of document. Reactivity of bromoselenophenes in palladium-catalyzed
direct arylations
Aymen Skhiri1,2, Ridha Ben Salem*2, Jean-François Soulé*1 and Henri Doucet*1 Full Research Paper
Open Access
Address:
1Institut des Sciences Chimiques de Rennes, UMR 6226
CNRS-Université de Rennes 1, "Organométalliques: Matériaux et
Catalyse", Campus de Beaulieu, 35042 Rennes, France and
2Laboratoire de Laboratoire de Chimie Organique LR 17ES08,
Université de Sfax, Faculté des Sciences de Sfax, Route de la Soukra
km 4, 3038 Sfax, Tunisia
Email:
Ridha Ben Salem* - ridhabensalem@yahoo.fr; Jean-François Soulé* -
jean-francois.soule@univ-rennes1.fr; Henri Doucet* -
henri.doucet@univ-rennes1.fr
* Corresponding author
Keywords:
C–H bond activation; catalysis; heteroarenes; palladium; selenophene
Beilstein J. Org. Chem. 2017, 13, 2862–2868. doi:10.3762/bjoc.13.278
Received: 24 August 2017
Accepted: 04 December 2017
Published: 22 December 2017
Associate Editor: M. Rueping
© 2017 Skhiri et al.; licensee Beilstein-Institut. License and terms: see end of document. Full Research Paper
Open Access
Address:
1Institut des Sciences Chimiques de Rennes, UMR 6226
CNRS-Université de Rennes 1, "Organométalliques: Matériaux et
Catalyse", Campus de Beaulieu, 35042 Rennes, France and
2Laboratoire de Laboratoire de Chimie Organique LR 17ES08,
Université de Sfax, Faculté des Sciences de Sfax, Route de la Soukra
km 4, 3038 Sfax, Tunisia
Email:
Ridha Ben Salem* - ridhabensalem@yahoo.fr; Jean-François Soulé* -
jean-francois.soule@univ-rennes1.fr; Henri Doucet* -
henri.doucet@univ-rennes1.fr
* Corresponding author
Keywords:
C–H bond activation; catalysis; heteroarenes; palladium; selenophene
Beilstein J. Org. Chem. 2017, 13, 2862–2868. doi:10.3762/bjoc.13.278
Received: 24 August 2017
Accepted: 04 December 2017
Published: 22 December 2017
Associate Editor: M. Rueping
© 2017 Skhiri et al.; licensee Beilstein-Institut. License and terms: see end of document. Introduction Table 1: Influence of the reaction conditions for the coupling of 2-ethyl-
4-methylthiazole with 2-bromoselenophene.a
Entry
Catalyst
Solvent
Base
Yield (%)
1
Pd(OAc)2
DMA
KOAc
55b
2
Pd(OAc)2
DMA
KOAc
64c
3
Pd(OAc)2
DMA
KOAc
80
4
PdCl2
DMA
KOAc
45
5
PdCl(C3H5)(dppb)
DMA
KOAc
48
6
Pd(OAc)2
DMA
K2CO3
41
7
Pd(OAc)2
DMA
Cs2CO3
9
8
Pd(OAc)2
DMA
CsOAc
52
9
Pd(OAc)2
DMA
NaOAc
17
10
Pd(OAc)2
DMF
KOAc
53
11
Pd(OAc)2
xylene
KOAc
48
aConditions: catalyst (0.02 equiv), 2-bromoselenophene (1 equiv),
2-ethyl-4-methylthiazole (1.5 equiv), base (2 equiv), 24 h, 90 °C, isolat-
ed yields of 1. b130 °C, c110 °C. Table 1: Influence of the reaction conditions for the coupling of 2-ethyl-
4-methylthiazole with 2-bromoselenophene.a
Entry
Catalyst
Solvent
Base
Yield (%)
1
Pd(OAc)2
DMA
KOAc
55b
2
Pd(OAc)2
DMA
KOAc
64c
3
Pd(OAc)2
DMA
KOAc
80
4
PdCl2
DMA
KOAc
45
5
PdCl(C3H5)(dppb)
DMA
KOAc
48
6
Pd(OAc)2
DMA
K2CO3
41
7
Pd(OAc)2
DMA
Cs2CO3
9
8
Pd(OAc)2
DMA
CsOAc
52
9
Pd(OAc)2
DMA
NaOAc
17
10
Pd(OAc)2
DMF
KOAc
53
11
Pd(OAc)2
xylene
KOAc
48
aConditions: catalyst (0.02 equiv), 2-bromoselenophene (1 equiv),
2-ethyl-4-methylthiazole (1.5 equiv), base (2 equiv), 24 h, 90 °C, isolat-
ed yields of 1. b130 °C, c110 °C. Table 1: Influence of the reaction conditions for the coupling of 2-ethyl-
4-methylthiazole with 2-bromoselenophene.a Scheme 1: Reported Pd-catalyzed heteroarylations of bromose-
lenophenes. in 2014 that using ethyl oxazole-4-carboxylate as reaction
partner, the corresponding 2,5-bis(oxazol-2-yl)selenophene de-
rivative was formed in 45% yield (Scheme 1c) [17]. Moreover,
to our knowledge the Pd-catalyzed direct heteroarylation of
2-bromoselenophene has not yet been described. Therefore, the
reactivity of 2-bromo- and 2,5-dibromoselenophenes in Pd-cata-
lyzed direct couplings with heteroarenes needed to be investi-
gated. aConditions: catalyst (0.02 equiv), 2-bromoselenophene (1 equiv),
2-ethyl-4-methylthiazole (1.5 equiv), base (2 equiv), 24 h, 90 °C, isolat-
ed yields of 1. b130 °C, c110 °C. Herein, we wish to report on the reactivity of 2-bromoseleno-
phene, 2,5-dibromoselenophene and 2-aryl-5-bromose-
lenophenes in palladium-catalyzed direct heteroarylations with
a variety of heteroarenes using a phosphine-free palladium cata-
lyst. Then, we investigated the scope of the coupling of 2-bromo-
selenophene with a set of heteroarenes in the presence of
2 mol % Pd(OAc)2, KOAc as the base in DMA at 90 °C
(Scheme 2). The reaction of 2-isopropyl-4-methylthiazole gave
the desired product 2 in 82% yield. Introduction Conversely, low yields in
the target products 3–5 were obtained for the reactions with
thiophene-2-carbonitrile, 2-chlorothiophene and 2-pentylthio-
phene, although complete conversions of 2-bromoselenophene Introduction (MX) as waste. In recent years, the Pd-catalyzed arylation, via a
C–H bond activation, of a broad range of heteroaromatics using
aryl halides as reaction partners was demonstrated to be particu-
larly effective for the preparation of bi(hetero)aryls [22-31]. Among the reported results, a few examples of Pd-catalyzed
direct arylations via the C–H bond activation of selenophenes
using aryl halides as coupling partners have been reported [32-
35]. Conversely, C–H bond activation methodology was em-
ployed in only in one case for the preparation of a heteroary-
lated selenophene from a haloselenophene. Wipf et al. reported (Hetero)aryl-substituted selenophenes represent a class of mole-
cules which exhibit useful physical properties, especially for the
preparation of artificial photosynthetic systems for solar energy
conversion or for thin film transistor applications [1-3]. In most
cases, these (hetero)arylated selenophenes are currently pre-
pared through the use of transition-metal-mediated reactions
such as Stille [3-9], Suzuki [10-19], or Kumada [20] couplings
[21] (Scheme 1a and b). However, all these procedures require
the preparation of an organometallic or a boron derivative of
one of the coupling partners, and provide an organometallic salt 2862
2862 Beilstein J. Org. Chem. 2017, 13, 2862–2868. Scheme 1: Reported Pd-catalyzed heteroarylations of bromose-
lenophenes. matics such as thiazole, pyrrole, furan or imidazole derivatives
with aryl bromides [36]. 2-Bromoselenophene, which was
easily prepared by reaction of selenophene with N-bromosuc-
cinimide [37], and 2-ethyl-4-methylthiazole were employed as
model substrates for our study. Reactions performed at 130 °C
or 110 °C gave the expected arylated selenophene 1 in 55%
and 64% yields, respectively, with complete conversions of
2-bromoselenophene; whereas 1 was obtained in a higher
yield of 80% when the reaction was conducted at 90 °C
(Table 1, entries 1–3). At elevated temperature (110–130 °C),
2-bromoselenophene seems to afford larger amounts of
selenophene oligomers as side-products. The use of PdCl2 or
PdCl(C3H5)(dppb) as catalysts instead of Pd(OAc)2 or other
bases such as K2CO3, Cs2CO3, CsOAc or NaOAc afforded 1 in
lower yields (Table 1, entries 4–9). The influence of two other
solvents in this cross-coupling reaction was also examined. We
observed that both DMF and xylene in the presence of 2 mol %
Pd(OAc)2 catalyst with KOAc gave 1 in moderate yields
(Table 1, entries 10 and 11). Results and Discussion First, we examined the influence of the reaction temperature,
using DMA as solvent, KOAc as base and 2 mol % Pd(OAc)2
as catalyst (Table 1). We had previously observed that these
reaction conditions allowed the coupling of several heteroaro- 2863 Beilstein J. Org. Chem. 2017, 13, 2862–2868. Scheme 2: Palladium-catalyzed heteroarylations of 2-bromoseleno-
phene. *: 110 °C at C5-position of thiophene or pyrrole derivatives, for reaction
which proceed via concerted metallation–deprotonation [38-40],
are higher than that of thiazoles (see bottom of Scheme 2) [42]. We assume that, due to these higher energies of activation for
reactions with thiophene or pyrrole, larger amounts of
selenophene oligomers were formed in the presence of these
less reactive heteroarenes. Then, the reactivity of imidazo[1,2-
a]pyridine, which contains a very reactive C–H bond at C3-po-
sition was examined. The expected product 7 was obtained in a
high yield of 81%. Thus, for Pd-catalyzed direct heteroaryla-
tions of 2-bromoselenophene, only the heteroarenes containing
C–H bonds with low Gibbs free energies of activation [41]
should be employed. By contrast, the direct arylation reactions with 2,5-dibromo-
selenophene were found to tolerate both thiazole and thiophene
derivatives (Scheme 3). The coupling of 3 equiv of thiazole de-
rivatives with 2,5-dibromoselenophene in the presence of
2 mol % Pd(OAc)2 and KOAc as base gave the corresponding
2,5-diheteroarylated selenophenes 8 and 9 in 78% and 80%
yields, respectively. The use of 2-pentyl- and 2-chlorothio-
phenes also gave the desired products 10 and 11 in high yields. In general, the Pd-catalyzed direct arylation of 3-substituted
thiophenes with aryl halides afforded quite regioselectively the
C2-arylated thiophenes [30]. A similar regioselectivity was
oberved for the coupling of thiophene derivatives containing
methyl- or chloro-substituents at the C3-position with 2,5-
dibromoselenophene. In both cases, regioselective arylations at
the C2-positions were observed, affording the 2,5-diarylated
selenophenes 12 and 13 in 69% and 72% yields, respectively. From 2,5-dibromoselenophene and an excess of 1-methyl-
pyrrole, the expected 2,5-diarylated selenophene 14 was ob-
tained in 81% yield. Scheme 2: Palladium-catalyzed heteroarylations of 2-bromoseleno-
phene. *: 110 °C were observed. A similar result was obtained for the reaction
with 1-phenylpyrrole. Reactions performed at a higher tempera-
ture with thiophene-2-carbonitrile, 2-chlorothiophene afforded 3
and 4 in slightly lower yields. Gorelsky calculated that the
Gibbs free energies of activation for the cleavage of C–H bonds Scheme 3: Palladium-catalyzed 2,5-diheteroarylation of 2,5-dibromoselenophene. Scheme 3: Palladium-catalyzed 2,5-diheteroarylation of 2,5-dibromoselenophene. Scheme 3: Palladium-catalyzed 2,5-diheteroarylation of 2,5-dibromoselenophene. Results and Discussion 2864 Beilstein J. Org. Chem. 2017, 13, 2862–2868. Conclusion Finally, we show a sequential transformation leading to 2-aryl-
5-(heteroaryl)selenophenes in three steps from commercially
available compounds (Scheme 4). Bromination at the C5-posi-
tion of 2-arylselenophenes containing nitrile, acetyl or chloro
substituents on the aryl moiety, which could be easily obtained
in good yields from selenophene and aryl bromides via a
Pd-catalyzed direct arylation using a reported procedure [33],
afforded the 2-aryl-5-bromoselenophenes 15–17 in 84–90%
yields. Then, a second Pd-catalyzed direct arylation using
heteroarenes and 15–17 as reaction partners, provided the target
compounds 18–26 in high yields. Both thiazole and thiophene
derivatives were successfully employed in this transformation. The reaction tolerates useful functional groups on both cou-
pling partners such as nitrile, acetyl or chloro. It should be
mentioned that again a regioselective arylation at the C2-posi-
tion of 3-chlorothiophene was observed affording 26 in 72%
yield. Finally, we show a sequential transformation leading to 2-aryl-
5-(heteroaryl)selenophenes in three steps from commercially
available compounds (Scheme 4). Bromination at the C5-posi-
tion of 2-arylselenophenes containing nitrile, acetyl or chloro
substituents on the aryl moiety, which could be easily obtained
in good yields from selenophene and aryl bromides via a
Pd-catalyzed direct arylation using a reported procedure [33],
afforded the 2-aryl-5-bromoselenophenes 15–17 in 84–90%
yields. Then, a second Pd-catalyzed direct arylation using
heteroarenes and 15–17 as reaction partners, provided the target
compounds 18–26 in high yields. Both thiazole and thiophene
derivatives were successfully employed in this transformation. The reaction tolerates useful functional groups on both cou-
pling partners such as nitrile, acetyl or chloro. It should be
mentioned that again a regioselective arylation at the C2-posi-
tion of 3-chlorothiophene was observed affording 26 in 72%
yield. In summary, the reactivity of 2-bromoselenophenes was investi-
gated and revealed that the C2-heteroarylation of 2-bromo-
selenophene in high yields is only possible with specific
heteroarenes such as thiazoles and imidazopyridines, whereas
thiophene or pyrroles gave the desired products in low yields. Conversely, 2,5-dibromoselenophene was successfully coupled
with both thiazoles and thiophenes in the presence of phos-
phine-free Pd(OAc)2 catalyst precursor and KOAc as inexpen-
sive base, affording the desired 2,5-diheteroarylated seleno-
phenes in high yields. We also described that the sequential cat-
alytic C2-arylation, bromination, and catalytic C5-arylation of
selenophene provides the controled double (hetero)arylation at
the C2 and C5 positions of selenophene in good yields. General procedure for palladium-catalyzed
direct mono-heteroarylations of 2-bromo-5-
arylselenophenes EtOAc/pentane 2:98 for compounds 1 and 6. EtOAc/pentane
10:90 for compound 2 and 5 and EtOAc/pentane 40:60 for com-
pound 7. The reaction of the heteroarene (1.2 mmol), 2-bromo-5-arylse-
lenophene 15–17 (1 mmol) and KOAc (0.392 g, 4 mmol) at
150 °C during 16 h in DMA (4 mL) in the presence of
Pd(OAc)2 (4.5 mg, 0.02 mmol), under argon affords the cou-
pling products 18–26 after evaporation of the solvent and purifi-
cation on silica gel. Eluents: Pentane for compounds 21 and 22. EtOAc/pentane 5:95 for compounds 18 and 19. EtOAc/pentane
10:90 for compounds 20, 23, 24 and 26. EtOAc/pentane 20:80
for compound 25. General procedure for palladium-catalyzed
direct mono-heteroarylations of 2-bromo-
selenophene Although the mechanism of these reactions was not elucidated,
the catalytic cycle shown on Scheme 5 can be proposed. The
first step is probably the oxidative addition of the 2-bromo-
selenophene to Pd(0) to afford the Pd(II) intermediate A. Then, after elimination of KBr with KOAc, a concerted metala-
tion–deprotonation pathway involving an heteroarene gives B. Reductive elimination affords the 2-heteroarylated selenophene
with regeneration of the Pd(0) species. The reaction of the heteroarene (1.5 mmol), 2-bromoseleno-
phene (0.210 g, 1 mmol) and KOAc (0.196 g, 2 mmol) at 90 °C
during 24 h in DMA (4 mL) in the presence of Pd(OAc)2
(4.5 mg, 0.02 mmol), under argon affords the coupling
products 1–7 after evaporation of the solvent and purification
on silica gel. Eluents: Pentane for compounds 3 and 4. Scheme 4: Synthesis of 2-aryl-5-(heteroaryl)selenophenes. Scheme 4: Synthesis of 2-aryl-5-(heteroaryl)selenophenes. Scheme 4: Synthesis of 2-aryl-5-(heteroaryl)selenophenes. 2865 Beilstein J. Org. Chem. 2017, 13, 2862–2868. Scheme 5: Proposed catalytic cycle. Scheme 5: Proposed catalytic cycle. General procedure for the synthesis of
5-bromo-2-arylselenophenes 15–17 Supporting Information File 1
Additional experimental and analytical data and copies of
NMR spectra. To a mixture of the 2-arylselenophene [2] (2 mmol) in DMF
(5 mL) at 0 °C, N-bromosuccinimide (0.392 g, 2.2 mmol) was
slowly added. Then, the mixture was allowed to increase to
room temperature and stirred during 20 h. After addition of
water, the extraction was carried out with diethyl ether. Then,
the organic phase was dried over magnesium sulphate. Finally, evaporation of the solvent and purification on silica gel
afforded the 5-bromo-2-arylselenophenes 15–17. Eluents:
Pentane for compounds 15 and 16. EtOAc:pentane 5:95 for
compound 17. [http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-13-278-S1.pdf] [http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-13-278-S1.pdf] General procedure for palladium-catalyzed
direct diheteroarylations The reaction of the heteroarene (3 mmol), 2,5-dibromoseleno-
phene (0.289 g, 1 mmol) and KOAc (0.392 g, 4 mmol) at 90 °C
during 40 h in DMA (4 mL) in the presence of Pd(OAc)2
(4.5 mg, 0.02 mmol), under argon affords the coupling prod-
ucts 8–14 after evaporation of the solvent and purification on
silica gel. Eluents: Pentane for compounds 10–13. EtOAc/
pentane 5:95 for compounds 8, 9 and 14. Acknowledgements We are grateful the CNRS, Rennes Metropole and Scientific
Ministry of Higher Education and Research of Tunisia for pro-
viding financial support. 2866 Beilstein J. Org. Chem. 2017, 13, 2862–2868. ORCID® iDs 20.Shabana, R.; Galal, A.; Mark, H. B., Jr.; Zimmer, H.; Gronowitz, S.;
Hörnfeldt, A. B. J. Chem. Soc., Chem. Commun. 1988, 988–989. doi:10.1039/C39880000988
See for the synthesis of heteroarylated selenophenes via Kumada
coupling. Ridha Ben Salem - https://orcid.org/0000-0002-5179-351X
Jean-François Soulé - https://orcid.org/0000-0002-6593-1995
Henri Doucet - https://orcid.org/0000-0002-1410-3663 Henri Doucet - https://orcid.org/0000-0002-1410-3663 21.Skhiri, A.; Ben Salem, R.; Soulé, J.-F.; Doucet, H. ChemCatChem
2017, 9, 2895–2913. doi:10.1002/cctc.201700256
See for a review on the arylation of selenophenes via Pd-catalyzed
reactions. References 1. Hollinger, J.; Jahnke, A. A.; Coombs, N.; Seferos, D. S. J. Am. Chem. Soc. 2010, 132, 8546–8547. doi:10.1021/ja103191u 1. Hollinger, J.; Jahnke, A. A.; Coombs, N.; Seferos, D. S. 1. Hollinger, J.; Jahnke, A. A.; Coombs, N.; Seferos, D. S. J. Am. Chem. Soc. 2010, 132, 8546–8547. doi:10.1021/ja103191u 22.Akita, Y.; Inoue, A.; Yamamoto, K.; Ohta, A.; Kurihara, T.; Shimizu, M. Heterocycles 1985, 23, 2327–2333. doi:10.3987/R-1985-09-2327 2. Yen, Y.-S.; Lee, C.-T.; Hsu, C.-Y.; Chou, H.-H.; Chen, Y.-C.; Lin, J. T. Chem. – Asian J. 2013, 8, 809–816. doi:10.1002/asia.201201173 2. Yen, Y.-S.; Lee, C.-T.; Hsu, C.-Y.; Chou, H.-H.; Chen, Y.-C.; Lin, J. T. Chem. – Asian J. 2013, 8, 809–816. doi:10.1002/asia.201201173 23.Alberico, D.; Scott, M. E.; Lautens, M. Chem. Rev. 2007, 107,
174–238. doi:10.1021/cr0509760 3. Liu, Y.; Yang, Y.; Chen, C.-C.; Chen, Q.; Dou, L.; Hong, Z.; Li, G.;
Yang, Y. Adv. Mater. 2013, 25, 4657–4662. doi:10.1002/adma.201301716 24.Satoh, T.; Miura, M. Chem. Lett. 2007, 36, 200–205. doi:10.1246/cl.2007.200 4. Li, R.; Lv, X.; Shi, D.; Zhou, D.; Cheng, Y.; Zhang, G.; Wang, P. J. Phys. Chem. C 2009, 113, 7469–7479. doi:10.1021/jp900972v 25.Li, B.-J.; Yang, S.-D.; Shi, Z.-J. Synlett 2008, 949–957. doi:10.1055/s-2008-1042907 25.Li, B.-J.; Yang, S.-D.; Shi, Z.-J. Synlett 2008, 949–957. doi:10.1055/s-2008-1042907 5. Haid, S.; Mishra, A.; Weil, M.; Uhrich, C.; Pfeiffer, M.; Bäuerle, P. Adv. Funct. Mater. 2012, 22, 4322–4333. doi:10.1002/adfm.2012010 26.Ackermann, L. Chem. Rev. 2011, 111, 1315–1345. doi:10.1021/cr100412j 26.Ackermann, L. Chem. Rev. 2011, 111, 1315–1345. 6. Khim, D.; Lee, W.-H.; Baeg, K.-J.; Kim, D.-Y.; Kang, I.-N.; Noh, Y.-Y. J. Mater. Chem. 2012, 22, 12774–12783. doi:10.1039/c2jm16546e 27.Wencel-Delord, J.; Glorius, F. Nat. Chem. 2013, 5, 369–375. doi:10.1038/nchem.1607 7. Lee, W.-H.; Son, S. K.; Kim, K.; Lee, S. K.; Shin, W. S.; Moon, S.-J.;
Kang, I.-N. Macromolecules 2012, 45, 1303–1312. doi:10.1021/ma2020112 28.Rossi, R.; Bellina, F.; Lessi, M.; Manzini, C. Adv. Synth. Catal. 2014,
356, 17–117. doi:10.1002/adsc.201300922 29.Yuan, K.; Soulé, J.-F.; Doucet, H. ACS Catal. 2015, 5, 978–991. doi:10.1021/cs501686d 8. Lee, W.-H.; Lee, S. K.; Son, S. K.; Choi, J.-E.; Shin, W. S.; Kim, K.;
Lee, S.-H.; Moon, S.-J.; Kang, I.-N. J. Polym. Sci., Part A: Polym. Chem. 2012, 50, 551–561. doi:10.1002/pola.25064 30.Bheeter, C. B.; Chen, L.; Soulé, J.-F.; Doucet, H. Catal. Sci. Technol. 30.Bheeter, C. B.; Chen, L.; Soulé, J.-F.; Doucet, H. Catal. Sci. Technol. 2016 6 2005 2049 doi:10 1039/C5CY02095F 30.Bheeter, C. B.; Chen, L.; Soulé, J.-F.; Doucet, H 2016, 6, 2005–2049. doi:10.1039/C5CY02095F 31.Rossi, R.; Lessi, M.; Manzini, C.; Marianetti, G.; Bellina, F. doi:10.3987/COM-89-S90 38.Davies, D. L.; Donald, S. M. A.; Macgregor, S. A. J. Am. Chem. Soc. 38.Davies, D. L.; Donald, S. M. A.; Macgregor, S. A. J. Am. Chem. Soc. 2005, 127, 13754–13755. doi:10.1021/ja052047w 38.Davies, D. L.; Donald, S. M. A.; Macgregor, S. A. J 2005, 127, 13754–13755. doi:10.1021/ja052047w 14.Mishra, S. P.; Javier, A. E.; Zhang, R.; Liu, J.; Belot, J. A.; Osaka, I.;
McCullough, R. D. J. Mater. Chem. 2011, 21, 1551–1561. doi:10.1039/C0JM00602E 39.Lafance, M.; Fagnou, K. J. Am. Chem. Soc. 2006, 128, 16496–16497. doi:10.1021/ja067144j 40, 922–924. doi:10.1246/cl.2011.922 40, 922–924. doi:10.1246/cl.2011.922 10.Shabana, R.; Galal, A.; Mark, H. B., Jr.; Zimmer, H.; Gronowitz, S.;
Hörnfeldt, A.-B. Phosphorus, Sulfur Silicon Relat. Elem. 1990, 48,
239–244. doi:10.1080/10426509008045904 33.Rampon, D. S.; Wessjohann, L. A.; Schneider, P. H. J. Org. Che 33.Rampon, D. S.; Wessjohann, L. A.; Schneide 2014, 79, 5987–5992. doi:10.1021/jo500094t 34.Lu, T.-J.; Lin, P.-H.; Lee, K.-M.; Liu, C.-Y. Eur. J. Org. Chem. 2017, 34.Lu, T.-J.; Lin, P.-H.; Lee, K.-M.; Liu, C.-Y. Eur. J. Org. Chem. 2017,
111 123 doi:10 1002/ejoc 201601257 11.Prediger, P.; Moro, A. V.; Nogueira, C. W.; Savegnago, L.;
Menezes, P. H.; Rocha, J. B. T.; Zeni, G. J. Org. Chem. 2006, 71,
3786–3792. doi:10.1021/jo0601056 111–123. doi:10.1002/ejoc.201601257 35.Skhiri, A.; Ben Salem, R.; Soulé, J.-F.; Doucet, H. Chem. – Eur. 35.Skhiri, A.; Ben Salem, R.; Soulé, J.-F.; Doucet, H. Chem. 2017, 23, 2788–2791. doi:10.1002/chem.201700202 2017, 23, 2788–2791. doi:10.1002/chem.201700202 12.Kong, H.; Jung, Y. K.; Cho, N. S.; Kang, I.-N.; Park, J.-H.; Cho, S.;
Shim, H.-K. Chem. Mater. 2009, 21, 2650–2660. doi:10.1021/cm803409q 36.Roger, J.; Doucet, H. Tetrahedron 2009, 65, 9772–9781 36.Roger, J.; Doucet, H. Tetrahedron 2009, 65, 9772–9781. doi:10.1016/j.tet.2009.09.084 doi:10.1016/j.tet.2009.09.084 37.Gronowitz, S.; Peters, D. Heterocycles 1990, 30, 645–658. doi:10.3987/COM-89-S90 13.Kong, H.; Chung, D. S.; Kang, I.-N.; Park, J.-H.; Park, M.-J.; Jung, I. H. Park, C. E.; Shim, H.-K. J. Mater. Chem. 2009, 19, 3490–3499. doi:10.1039/b823082j doi:10.1039/C0JM00602E 40.Lapointe, D.; Fagnou, K. Chem. Lett. 2010, 39, 1118–1126. d i 10 1246/ l 2010 1118 40.Lapointe, D.; Fagnou, K. Chem. Lett. 2010, 39, 1118–1126. doi:10.1246/cl.2010.1118 40.Lapointe, D.; Fagnou, K. Chem. Lett. 2010, 39, 1118–1126. 15.Haid, S.; Mishra, A.; Uhrich, C.; Pfeiffer, M.; Bäuerle, P. Chem. Mater. 2011, 23, 4435–4444. doi:10.1021/cm201392c 15.Haid, S.; Mishra, A.; Uhrich, C.; Pfeiffer, M.; Bäuerle, P. Chem. Mater. 40.Lapointe, D.; Fagnou, K. Chem. Lett. 2010, 39, 1118–1126. doi:10.1246/cl.2010.1118 doi:10.1246/cl.2010.1118 2011, 23, 4435–4444. doi:10.1021/cm201392c 41.Gorelsky, S. I. Coord. Chem. Rev. 2013, 257, 153–164. 16.Klingstedt, T.; Shirani, H.; Åslund, K. O. A.; Cairns, N. J.;
Sigurdson, C. J.; Goedert, M.; Nilsson, K. P. R. Chem. – Eur. J. 2013,
19, 10179–10192. doi:10.1002/chem.201301463 16.Klingstedt, T.; Shirani, H.; Åslund, K. O. A.; Cairns, N. J.; References Tetrahedron 31.Rossi, R.; Lessi, M.; Manzini, C.; Marianetti, G.; Be 9. Agrawal, V.; Shahjad; Bhardwaj, D.; Bhargav, R.; Sharma, G. D 9. Agrawal, V.; Shahjad; Bhardwaj, D.; Bhargav, R.; Sharma, G. D.;
Bhardwaj, R. K.; Patra, A.; Chand, S. Electrochim. Acta 2016, 192,
52–60. doi:10.1016/j.electacta.2016.01.157 2016, 72, 1795–1837. doi:10.1016/j.tet.2016.02.037 32.Tamba, S.; Fujii, R.; Mori, A.; Hara, K.; Koumura, N. Chem. Lett. 2011, 32.Tamba, S.; Fujii, R.; Mori, A.; Hara, K.; Koumura, N. Che doi:10.1016/j.ccr.2012.06.016 doi:10.1016/j.ccr.2012.06.016 Sigurdson, C. J.; Goedert, M.; Nilsson, K. P. R. Chem. – Eur. J. 2013,
19, 10179–10192. doi:10.1002/chem.201301463 42.Zimmer, H.; Shabana, R.; Galal, A.; Mark, H. B., Jr.; Gronowitz, 42.Zimmer, H.; Shabana, R.; Galal, A.; Mark, H. B., Jr.; Gronowitz, S.;
Hörnfeldt, A. B. Phosphorus, Sulfur Silicon Relat. Elem. 1989, 42,
171–176. doi:10.1080/10426508908054892 Hörnfeldt, A. B. Phosphorus, Sulfur Silicon Relat. Elem. 1989, 4 7.Salamoun, J.; Anderson, S.; Burnett, J. C.; Gussio, R.; Wipf, P. 17.Salamoun, J.; Anderson, S.; Burnett, J. C.; Gussio, R.; Wipf, P. Org. Lett. 2014, 16, 2034–2037. doi:10.1021/ol500620m p
Org. Lett. 2014, 16, 2034–2037. doi:10.1021/ol500620m 171–176. doi:10.1080/10426508908054892 18.Shirani, H.; Linares, M.; Sigurdson, C. J.; Lindgren, M.; Norman, P.;
Nilsson, K. P. R. Chem. – Eur. J. 2015, 21, 15133–15137. doi:10.1002/chem.201502999 18.Shirani, H.; Linares, M.; Sigurdson, C. J.; Lindgren, M.; Norman, P.; Nilsson, K. P. R. Chem. – Eur. J. 2015, 21, 15133–15137. 19.Ho, P.-Y.; Siu, C.-H.; Yu, W.-H.; Zhou, P.; Chen, T.; Ho, C.-L.; 19.Ho, P.-Y.; Siu, C.-H.; Yu, W.-H.; Zhou, P.; Chen, T.; Ho, C.-L.;
Lee, L. T. L.; Feng, Y.-H.; Liu, J.; Han, K.; Lo, Y. H.; Wong, W.-Y. J. Mater. Chem. C 2016, 4, 713–726. doi:10.1039/C5TC03308J Lee, L. T. L.; Feng, Y.-H.; Liu, J.; Han, K.; Lo, Y. H.; Wong, W.-Y J. Mater. Chem. C 2016, 4, 713–726. doi:10.1039/C5TC03308J 2867 Beilstein J. Org. Chem. 2017, 13, 2862–2868. License and Terms
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The definitive version of this article is the electronic one
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doi:10.3762/bjoc.13.278 License and Terms
This is an Open Access article under the terms of the
Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
(http://www.beilstein-journals.org/bjoc)
The definitive version of this article is the electronic one
which can be found at:
doi:10.3762/bjoc.13.278 Beilstein J. Org. Chem. 2017, 13, 2862–2868. License and Terms doi:10.3762/bjoc.13.278 2868
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Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 eISSN: 2317-2096
DOI: 10.35699/2317-2096.2021.26194 1 “Robinson Crusoe [...] is the dream of being the first man in the world [...]. It is the
dream of being suddenly, just as one is, in unquestionable control of the physical world
[...]. It is the dream of total power”. “Combinação possível de palavras”:
linguagem e dominação em Robinson Crusoé e em Foe
“Energy of Words”: Language and Dominance
in Robinson Crusoe and in Foe Daniel Lago Monteiro
Universidade Estadual de Campinas (UNICAMP), Campinas, São Paulo / Brasil
daniellagomonteiro1980@gmail.com
daniel.l.monteiro@hotmail.com
https://orcid.org/0000-0001-6520-087X Daniel Lago Monteiro
Universidade Estadual de Campinas (UNICAMP), Campinas, São Paulo / Brasil
daniellagomonteiro1980@gmail.com
daniel.l.monteiro@hotmail.com
https://orcid.org/0000-0001-6520-087X Resumo: O objetivo deste artigo é investigar o papel da linguagem em Robinson Crusoé
e em Foe. Neste, as relações de dominação são minadas pela quase completa ausência
de comunicação verbal entre Cruso e Sexta-Feira, que é mudo, aliado ao fracasso
da personagem e narradora, Susan Barton, em tramar um enredo para sua história;
naquele, o personagem e narrador habilmente manipula os signos verbais de modo a
atribuir um novo significado à ilha deserta, assumindo controle sobre ela, semelhante
ao domínio que irá exercer sobre os demais habitantes, sobretudo Sexta-Feira. Assim,
neste artigo, procuraremos ligar alguns pontos entre o uso descritivo e denotativo da
linguagem, próprio do realismo formal de Robinson Crusoé, e da missão colonizadora. Do mesmo modo, ao colocar a própria linguagem referencial em jogo, Foe oferece
uma alternativa original e desafiadora sobre as possibilidades de se contrapor à herança
colonial e suas formas narrativas. Palavras-chave: Robinson Crusoé; Foe; Linguagem; Realismo Formal; Colonialismo. Abstract: This paper aims at investigating the role of language in Robinson Crusoe
and in Foe. In the latter, relations of dominance are undermined by the near absence
of verbal communication between Cruso and Friday, who is speechless, allied with
the failure of the character and narrator, Susan Barton, in weaving a plot to her story;
in the former, the character and narrator skillfully manipulates verbal signs so as to eISSN: 2317-2096
DOI: 10.35699/2317-2096.2021.26194 eISSN: 2317-2096
DOI: 10.35699/2317-2096.2021.26194 Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 68 attribute a new meaning to the desert island, thus taking control over it, similarly to the
dominion he will exercise over its inhabitants, mainly Friday. Therefore, in this paper,
I aim at connecting some dots between the descriptive and denotative use of language,
proper to the formal realism of Robinson Crusoe, and the colonial mission. Likewise,
in putting referential language itself at stake, Foe offers an original and challenging
alternative to the possibilities of opposing colonial heritage and its narrative forms. Keywords: Robinson Crusoe; Foe; Language; Formal Realism; Colonialism. Keywords: Robinson Crusoe; Foe; Language; Formal Realism; Colonialism Que a peste o pegue,
Por me ensinar sua língua! (SHAKESPEARE, 2011) Que a peste o pegue,
Por me ensinar sua língua! (SHAKESPEARE, 2011) Em um breve ensaio de 1967, “Columbus and Crusoe”, o aclamado
escritor caribenho V. S. Naipaul propõe uma aproximação entre o
explorador genovês e o aventureiro inglês a partir de um ponto que se
tornaria crucial para os temas de resistência pós-coloniais: as aventuras
marítimas como instituidoras de um mito de controle e dominação. Robinson Crusoé, diz Naipaul (1991, p. 659), é essencialmente fabricador
de mito [myth-making]. No que consiste esse mito? Robinson Crusoé [...] é o sonho de ser o primeiro homem
no mundo [...]; é o sonho de subitamente estar, como
alguém poderia estar, em controle inquestionável do mundo
físico [...]. É o sonho do poder total (NAIPAUL, 1991,
p. 659, tradução nossa).1 A materialização do sonho é um componente indispensável para
a construção de qualquer mito; e Robinson Crusoé, para fazer valer a
expressão de Ian Watt (1996), tem o estatuto de mito moderno. De fato,
um dos alcances mais surpreendentes do mito consiste em uma espécie
de ofuscamento metonímico do texto: a história que conhecemos, aquela
do isolamento de Crusoé na ilha deserta, está tão impregnada em nosso
imaginário cultural que alguém que leia a obra pela primeira vez talvez
se surpreenda em descobrir que o livro contém outros episódios, que 1 “Robinson Crusoe [...] is the dream of being the first man in the world [...]. It is the
dream of being suddenly, just as one is, in unquestionable control of the physical world
[...]. It is the dream of total power”. Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 69 aborda uma grande variedade de temas e que explora diferentes técnicas
narrativas. 2 “The novel rewards analysis as many things – an exotic adventure story; a study of
solitary consciousness; a parable of sin, atonement, and redemption; a myth of economic
individualism; a displayed or encoded autobiography; an allegory of political defeat; a
prophesy of imperial expansion – yet none of these explanations exhausts it”. Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 A mera tentativa de definir Robinson Crusoé corre o risco
de circunscrevê-lo em uma categoria que passa ao largo de sua ampla
extensão, que é, ao certo, um dos charmes de seu estatuto mitológico. Nas
palavras de Thomas Keymer (2008, p. vii, tradução nossa): O romance gratifica análises de todos os tipos: uma história exótica
de aventura; um estudo da consciência solitária; uma parábola do
pecado, da expiação e da redenção; um mito do individualismo
econômico; uma autobiografia explícita ou codificada; uma
alegoria da derrota política; uma profecia da expansão imperial –
contudo, nenhuma dessas explicações esgota a obra.2 Os últimos trezentos anos dão prova da natureza inesgotável de
Robinson Crusoé, que vem estimulando uma variedade tão ampla de
leituras, adaptações e recriações jamais alcançadas por qualquer outro
romance da literatura ocidental. Portanto, meu esforço interpretativo aqui
não será outro senão modesto, a saber, o de destacar um aspecto que, a
meu ver, é essencial para a construção de qualquer mito: a manipulação
dos signos verbais ou, nas palavras de Crusoé, a combinação possível de
palavras (no original: the energy of words). Esse aspecto, como se verá
adiante, está no cerne da intenção do narrador de estabelecer uma relação
de dominação com as pessoas com quem ele se relaciona e com o meio no
qual se insere. Para uma leitura em contraponto, recorrerei a uma das mais
bem elaboradas interações dialéticas com o discurso colonial encarnado
em Robinson Crusoé: Foe, do escritor sul-africano, John Maxwell Coetzee
(2016), e a sua sustentação de que a ilha de Crusoé (ou Cruso, variação
que dá ao nome do herói) era tão árida e silenciosa que a comunicação
verbal beirava o desaparecimento. Assim, aproveitando-se da sugestão de
Naipaul (1991), falarei neste artigo de três aspirações ou sonhos próprios
ao mito de Crusoé – o de ser o primeiro homem no mundo; o de estar em
controle do mundo físico; e o do poder total –, bem como do fracasso
dessas aspirações quando se esgota a combinação possível de palavras. 2 “The novel rewards analysis as many things – an exotic adventure story; a study of
solitary consciousness; a parable of sin, atonement, and redemption; a myth of economic
individualism; a displayed or encoded autobiography; an allegory of political defeat; a
prophesy of imperial expansion – yet none of these explanations exhausts it”. Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 70 1. O sonho de ser o primeiro homem no mundo Robinson Crusoé é geralmente descrito como o primeiro romance
moderno, isto é, o primeiro romance que plenamente concretiza as
inovações do “realismo formal”, definido por Ian Watt (2007, p. 31) nos
seguintes termos: A premissa, ou a convenção básica, de que o romance constitui
um relato completo e autêntico da experiência humana e, portanto,
tem a obrigação de fornecer ao leitor detalhes da história como a
individualidade dos agentes envolvidos, os particulares das épocas
e locais de suas ações – detalhes que são apresentados através de
um emprego da linguagem muito mais referencial do que é comum
em outras formas literárias. Watt (2007) fundamenta o seu argumento ao comparar a tradição
estilística anterior, a ornamentação linguística e o decoro adequado ao
assunto, ao ponto de vista realístico na linguagem e na estrutura da
prosa de Robinson Crusoé. Essa mudança teve origem em um problema
semântico sobre a correspondência entre as palavras e as coisas,
substituindo, assim, as belezas intrínsecas “que o uso da retórica podia
conferir à descrição e ação” (WATT, 2007, p. 28). O emprego descritivo
e denotativo da linguagem ajudou a promover um movimento em direção
a uma prosa mais clara e simples, em direção a uma utilização mais
referencial da linguagem, que, segundo Watt (2007), é uma marca do
romance mais do que qualquer outro gênero literário. q
q
q
g
Apresentei aqui um sumário dos argumentos de Watt (2007) do
quanto a alteração semântica, da linguagem figurativa à referencial,
estabeleceu uma nova relação entre as palavras e as coisas e possibilitou
a ascensão do estilo claro e fácil na literatura. Ainda, segundo Watt
(2007, p. 27), “Os capítulos finais do terceiro livro do Essay concerning
human understanding [Ensaio sobre o entendimento humano], de Locke,
constituem provavelmente a evidência mais importante dessa corrente no
século XVII”. Defoe, lembra John Richetti (2012, p. 11), estava bastante
familiarizado com a obra e a estudou quando foi aluno na Academia de
Charles Morton. Esta Academia, continua Richetti (2012, p. 11), era um
dos principais centros da cultura protestante dissidente e nela seus alunos
“aprendiam línguas modernas, ciência moderna e filosofia, inclusive a
obra de Locke, Ensaio sobre o entendimento humano (1690), à época Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 71 banida em Oxford”. Portanto, farei uma breve discussão epistemológica
da natureza das palavras segundo Locke.3 Nesse mesmo capítulo de A ascensão do romance, Watt (2007,
p. 3 Parte da exposição de Locke que se segue foi desenvolvida a partir do livro de Daniel
Lago Monteiro (2018), No limiar da visão: a poética do sublime em Edmund Burke. 1. O sonho de ser o primeiro homem no mundo 27) se detém àquilo que chamou de “ceticismo nominalista com
relação à linguagem”, isto é, aos capítulos finais do terceiro livro de
Locke, quando ele confronta o principal objetivo da linguagem, a
compreensão, com o prazer enganoso da eloquência. Contudo, creio
ser necessário dar um passo para trás e analisar a relação que Locke
propõe entre as palavras e as coisas, o seu argumento de que as palavras
não representam os objetos, mas as nossas ideias acerca deles e que
se encontram, primordialmente, em nós e não no mundo exterior. Ao
confiar à experiência o precedente último de todo conhecimento, o autor
do Ensaio sobre o entendimento humano foi conduzido a rejeitar as
definições mais amplamente aceitas na tradição filosófica do termo ideia. Intimamente associado ao entendimento e à própria mente, a ideia para
Locke é despida de sua antiga roupagem ontológica – o mundo das ideias
de Platão, por exemplo – e passa a corresponder a um certo uso para o
conhecimento. Para Locke, não há nada que não possa ser tomado por
ideia. As próprias faculdades da mente, das quais dependem as ideais para
sua origem, quando se ocupam de si mesmas, são classificadas de ideia
de reflexão. Em suma, diz Locke (2012, p. 131): “Chamo de ideia tudo
aquilo que a mente percebe em si mesma, ou que é objeto imediato da
percepção, seja de pensamento ou de entendimento”. Imbuído do espírito
moderno, Locke (2012) buscou nesse sentido frouxo de ideia um modo
de se eximir dos embaraços metafísicos que envolvem o tratamento da
coisa ela mesma. Antes, o Ensaio sobre o entendimento humano ocupa-
se das ideias, das representações das coisas. Locke (2012) divide as ideias em simples e complexas. Se as ideias
são representações das qualidades sensíveis, as ideias simples são aquelas
que representam uma qualidade individual, separada das demais, como
dor e prazer, a cor, o volume ou a extensão de uma única impressão na
mente. As ideias complexas, como o próprio nome sugere, representam
combinações de ideias simples. Pertencem as elas os termos complexos,
os conceitos estéticos ou morais, assim como as substâncias, os modos
e as relações. Atribuir um termo a uma classe de ideais é um processo Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 72 que envolve um ajuste entre as palavras e as coisas. De todas as classes
de ideias, diz Locke (2012, p. 4 “The instability of generic categories registers an epistemological crisis”. 1. O sonho de ser o primeiro homem no mundo 388), as dos modos mistos são “as mais
suscetíveis à confusão”, as que mais contribuem para aquilo que ele
chama de imperfeição da linguagem, isto é, quando uma palavra “não
excita no ouvinte a mesma ideia que representa na mente do falante”
(LOCKE, 2012, p. 519). Portanto, a nomeação dos modos mistos merece
uma atenção maior. A peculiaridade do nome dos modos mistos se mostra mais
facilmente quando o contrastamos com o nome de outras espécies de
composição: as substâncias. A estas a mente adquire o hábito de recolher
na natureza seus componentes essenciais – cores, formas etc. –, os quais,
em seguida, são reunidos e emoldurados pelo nome. A palavra homem,
por exemplo, corresponde a uma coleção de ideias simples e complexas:
movimento, sensação, faculdade de raciocinar, vida etc. Desse modo, por
mais que o número e a espécie de ideias que entram em sua composição
não sejam exatamente as mesmas para todas as pessoas – segundo o
conhecimento maior ou menor que cada um tem do objeto –, caso se
queira estabelecer o veredito e decidir a quais ideias a palavra homem
corresponde: “devemos seguir a natureza, de tal maneira que nossas ideias
complexas convenham a existências reais e a significação de seus nomes
se regulem pelas coisas mesmas” (LOCKE, 2012, p. 524). Por contraste,
o percurso a ser percorrido na criação dos modos mistos é justamente
o inverso: são as coisas que devem ser ajustadas pelos nomes. Estes
funcionam como uma espécie de armação (frame) que reúne e sustenta
“a imensa [...] variedade de diferentes ideias” (LOCKE, 2012, p. 470). Portanto, o ato de emoldurar a ideia é, nesse caso, arbitrária e seu poder
de comunicação depende principalmente do sentimento que ela evoca. Ora, os sentimentos, os juízos estéticos e morais variam entre as pessoas,
é então que a regra do decoro (propriety), isto é, o uso comum, ajuda a
estabelecer o significado dos nomes e não a autoridade de um indivíduo. Estamos aqui lidando com uma transição de atitude em relação
ao problema da linguagem, em outras palavras, com aquilo que
Michael McKeon (1987, p. 20, tradução nossa) chamou de “questões
de verdade”: “a instabilidade de categorias genéricas registra uma
crise epistemológica”.4 Essa transição terá um impacto significativo na
afirmação da verdade em uma narrativa. Em seu cerne está a reivindicação Aletria, Belo Horizonte, v. 31, n. 2, p. 1. O sonho de ser o primeiro homem no mundo 67-89, 2021 73 da historicidade, que o suposto editor de Robinson Crusoé apresenta
no Prefácio: “O editor julga que o relato seja uma história fiel de fatos”
(DEFOE, 2012, p. 43). Mas esse princípio também vale para a natureza
fabricada da linguagem de que o narrador se utiliza para descrever seu
espaço. Sozinho na ilha, como se fosse o primeiro homem no mundo,
Crusoé não possui a mesma regra do decoro (propriety) para ajustar o uso
dos signos verbais e, com isso, ele estende a arbitrariedade na nomeação
dos modos mistos às substâncias. Há inúmeros exemplos do uso arbitrário na nomeação das coisas
em Robinson Crusoé, que se observa, por exemplo, quando o narrador
intencionalmente renomeia a ilha ou o resultado do seu trabalho e,
desse modo, molda a sua própria condição. Assim, ao longo dos anos de
confinamento na ilha deserta, os leitores assistem à gradual transformação
e ascensão do protagonista: de náufrago a soberano. Em um primeiro
momento, o lugar onde Crusoé se encontra não passava de “uma ilha
deserta e infeliz, a que dei o nome de ‘Ilha do Desespero’” (DEFOE,
2012, p. 126). Foi isso o que ele primeiro registrou em seu Diário, o qual
manteve durante os cinco primeiros anos de isolamento, e a linguagem
que ele ali se utiliza é ainda mais clara, simples e referencial do que aquela
que desenvolve à medida que a narrativa progride; uma linguagem que
tem por objetivo registrar seus meios de subsistência, como as peças que
recupera do navio naufragado. Quanto mais o local de Crusoé se torna
familiar, mais ele se torna consciente do quanto a linguagem o determina. Michael Seidel (2008, p. 191, tradução nossa) denomina esse
processo de “duplicação verbal” e oferece uma lista de exemplos que
condiz com o argumento que apresento aqui: Os mundos que Crusoé constrói em sua ilha se estendem para
a natureza fabricada da linguagem que usa para descrever seus
locais. É crucial que Crusoé nomeie as coisas que pertencem
à sua condição de tal modo que crie um universo replicado no
isolamento [...]. Ele tem plena consciência do quanto a linguagem
o determina e frequentemente se refere às coisas em duplos verbais
que se afinam ao estatuto real e mental de seu ambiente. 5 “The worlds Crusoe builds on his island extend to the fabricated nature of the language
he uses to describe his places. It is crucial that Crusoe names things that pertain to his
condition in such a way that he builds a replicate universe in isolation [...]. He is very
conscious about how his language determines him, often referring to things in verbal
doublets that key the actual and mental status of his environment. So when he digs out
the rock behind his tent to place his powder for shot he says, ‘I plac’d it in my new
Cave, which in my fancy I call’d my Kitchen’, just as he soon calls additions to his
cave his ‘Dining-room’ and his ‘Cellar’. When he plans to collect citrus fruit from the
lush side of the island he spends three days away from ‘Home, so I must call my Tent
and my Cave’. When he is frightened by the footprint he runs home to ‘my Castle, for
so I think I call’d it ever after this’”. Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 passa três dias fora de “casa, pois assim eu chamei minha tenda
ou caverna”. Quando se assusta com a vista da pegada, corre para
o “meu castelo, que foi como passei a chamar minha casa desde
então”.5 Se, diz Locke (2012), é o decoro ou o uso comum que garante o
fim último da comunicação, e não a autoridade de um indivíduo, o que
autoriza a duplicação verbal de Crusoé? Precisamente, o sonho de ser o
primeiro homem no mundo, uma espécie de qualidade adâmica da qual ele
se investe. Nos primeiros vinte e cinco anos de confinamento, o narrador
não tem ninguém com quem possa regular o significado das palavras
senão seus leitores e, sobretudo, sua consciência. Ainda comprometido
com o uso referencial da linguagem, a renomeação dos lugares adquire
uma função alegórica que promove adaptação e aprimoramento. É assim
que ele adquire controle sobre a ilha, que dentro em pouco, de ilha do
desespero se torna “minha querida ilha” (DEFOE, 2012, p. 207). 1. O sonho de ser o primeiro homem no mundo Assim,
quando ele extrai uma rocha por detrás da tenda para guardar a
pólvora de sua munição, diz ele, “Coloquei-a na minha caverna,
que na minha fantasia chamei de minha cozinha”, assim como, em
breve, ele a chamará de “sala de jantar” ou de “adega”. Quando
ele planeja colher frutas cítricas do lado mais exuberante da ilha, 74 2. O sonho de estar em controle do mundo físico Sabe-se que uma das principais inspirações para que Defoe
escrevesse Robinson Crusoé foi o relato que leu em The Englishman
(1713), de Richard Steele, sobre a história real de um marinheiro,
Alexander Selkirk, que por quatro anos permaneceu na ilha deserta Juan
Fernández, do Pacífico Sul. Segundo Richetti (2008, p. 123, tradução
nossa), não só Defoe cuidadosamente se apropriou do relato de Steele
como o fascínio que seu romance exerceu sobre os primeiros leitores se
deve, em grande parte, a “ostensivas reivindicações de que suas narrativas Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 75 seriam literalmente verdadeiras”.6 Na abertura do ensaio de Steele (2013,
tradução nossa), diz ele: “A pessoa de quem falo é Alexander Selkirk,
cujo nome é familiar aos homens curiosos, por sua fama de ter morado
por quatro anos e quatro meses sozinho na ilha de Juan Fernandez”.7
A página título de Robinson Crusoé – o único trecho do livro, junto ao
Prefácio, escrito em terceira pessoa – tem todo o sabor de uma notícia
de jornal: A VIDA e as Estranhas e Surpreendentes AVENTURAS de
Robinson Crusoé, de York, Marujo: que viveu vinte e oito
anos sozinho numa ilha deserta na costa da América, perto da
embocadura do grande rio Orinoco; tendo sido lançado à costa
por um naufrágio, no qual morreram todos os homens, menos
ele. Com um relato de como foi, afinal, estranhamente salvo por
piratas. Escrita pelo próprio (DEFOE, 2012, p. 41). A VIDA e as Estranhas e Surpreendentes AVENTURAS de
Robinson Crusoé, de York, Marujo: que viveu vinte e oito
anos sozinho numa ilha deserta na costa da América, perto da
embocadura do grande rio Orinoco; tendo sido lançado à costa
por um naufrágio, no qual morreram todos os homens, menos
ele. Com um relato de como foi, afinal, estranhamente salvo por
piratas. Escrita pelo próprio (DEFOE, 2012, p. 41). Ainda que o título não transmita uma descrição precisa dos
acontecimentos – pois, lembra Watt (1996, p. 146), não foram piratas,
mas amotinados que o resgataram –, a linguagem direta e denotativa de
sua prosa é uma de suas maiores inovações. Outra inovação importante
de Robinson Crusoé, que também contribui para sua reivindicação de
veracidade, é o modo como o personagem e narrador mergulha nos
meandros de sua mente, registrando suas esperanças e temores. Ainda
segundo Watt (1996, p. 6 “[...] Defoe’s narrative claim aggressively to be literally true”.
7 “The Person I speak of is Alexander Selkirk, whose Name is familiar to Men of
Curiosity, from the Fame of his having lived four years and four Months alone in the
Island of Juan Fernandez”.
8 “Nor is his life purely a mechanical series of laboring operations”.
9 Sobre o tema do tédio ver o artigo de Daniel Bonomo (2018), “Experimentum in
Insula: Robinson Crusoé nas origens do aborrecimento”. 9 Sobre o tema do tédio ver o artigo de Daniel Bonomo (2018), “Experimentum in
Insula: Robinson Crusoé nas origens do aborrecimento”. 2. O sonho de estar em controle do mundo físico 153, tradução nossa),8 “tampouco é sua vida [de
Crusoé] puramente uma série mecânica de operações laboriosas”: Crusoé
trabalha não só para lhe prover dos meios de subsistência ou para fugir
do tédio, mas para “usufruir [enjoy] dos poucos confortos que tinha no
mundo” (DEFOE, 2012, p. 123).9 Assim, por se tratar de alguém que por
vinte e cinco anos viveu em solidão, os sentimentos oriundos da falta
de convívio humano e, sobretudo, da voz humana são cuidadosamente
registrados. Também aqui a inspiração veio do relato de Steele (2013),
como se nota nesta passagem: “Era seu costume usar horas e lugares 6 “[...] Defoe’s narrative claim aggressively to be literally true”. 7 “The Person I speak of is Alexander Selkirk, whose Name is familiar to Men of
Curiosity, from the Fame of his having lived four years and four Months alone in the
Island of Juan Fernandez”. 7 “The Person I speak of is Alexander Selkirk, whose Name is familiar to Men of
Curiosity, from the Fame of his having lived four years and four Months alone in the
Island of Juan Fernandez”. “Nor is his life purely a mechanical series of laboring operations”. 9 Sobre o tema do tédio ver o artigo de Daniel Bonomo (2018), “Experimentum in
Insula: Robinson Crusoé nas origens do aborrecimento”. 9 Sobre o tema do tédio ver o artigo de Daniel Bonomo (2018), “Experimentum in
Insula: Robinson Crusoé nas origens do aborrecimento”. Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 76 certos para seus exercícios de devoção, que praticava em voz alta, a
fim de preservar a faculdade da fala e manifestar-se com mais energia”
(STEELE, 2013, tradução nossa).10 Um anseio semelhante se observa
de uma ponta a outra em Robinson Crusoé. Um dos maiores desesperos
de Crusoé era não ter “vivalma com quem possa falar” (DEFOE, 2012,
p. 121). Vez ou outra, ele temia que a falta de comunicação verbal levaria
à sua perdição. Isso fica evidente quando fala sobre seu cachorro: “só
me faltava que pudesse falar comigo, mas isso ele jamais conseguiu”
(DEFOE, 2012, p. 119); ou quando, ao domesticar o papagaio, Poll,
distraia-se ensinando-o a falar e se satisfazia ao ouvir o som: “‘Poll’, a
primeira palavra que ouvi pronunciada na ilha por uma boca que não a
minha” (DEFOE, 2012, p. 183). Mas nada poderia compensar a falta de
um semelhante. 10 “It was his Manner to use stated Hours and Places for Exercises of Devotion, which
he performed aloud, in order to keep up the Faculties of Speech, and to utter himself
with greater Energy”. 2. O sonho de estar em controle do mundo físico No vigésimo quarto ano de isolamento, quando sua mente se viu
perturbada por um sonho assustador, Crusoé acordou com raios e trovões
e foi “surpreendido por um barulho vindo do mar que entendi como um
disparo de canhão” (DEFOE, 2012, p. 261). Imediatamente, correu para
o local e percebeu se tratar de um “navio em dificuldade” (DEFOE, 2012,
p. 261), que naufragou de encontro a uma rocha no lado sul da ilha, o
mesmo lugar onde o navio de Crusoé naufragara. No dia seguinte, Crusoé
foi ao local, na esperança de encontrar um sobrevivente. Mas, para o seu
desespero, todos morreram. Assim, diz ele: Não sei explicar, com qualquer combinação possível de palavras
[energy of words], que estranha ânsia ou tumulto de desejos senti
em minha alma diante disso, e que me fazia dizer às vezes assim:
“Ah, se tivessem sobrado apenas um ou dois; na verdade, uma
única alma salva daquele navio, que viesse parar comigo, para
que me coubesse ao menos um companheiro, um semelhante que
falasse comigo, com quem eu pudesse conversar!” (DEFOE,
2012, p. 264, grifo nosso). É claro que a ânsia de Crusoé por ter alguém com quem pudesse
conversar corrobora a afirmação de Defoe de que sua narrativa fosse
uma verdade literal. Esta surgiu, nas palavras de Richetti (2008, p. 124, Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 77 tradução nossa), a partir “de um apetite crescente na Grã-Bretanha de fins
do século XVII e início do XVIII por ‘notícias’”.11 Mas o trecho faz mais
do que isso. No capítulo de Watt referido acima sobre Robinson Crusoé,
em Myths of Modern Individualism (1996, p. 141-171), ele afirma que
o mito possui três aspectos: retorno à natureza, dignidade do trabalho e
o homem econômico. A ilha oferece uma oportunidade de exploração
colonial para Crusoé e é pelo seu progresso tecnológico que ele reclama
propriedade sobre a ilha: “Eu era rei e senhor indisputável daquelas
terras, às quais tinha direito de posse” (DEFOE, 2012, p. 160). Mas sua
soberania, ou seu controle do mundo físico, nunca seria plenamente
realizável sem a “combinação possível de palavras” – mesmo quando
essas lhe faltavam, como no trecho acima – ou a manipulação dos signos
verbais; quarto aspecto que julgamos central à construção do mito. 11 “[...] grow out of an increasing appetite in the late seventeenth and early eighteenth
century in Britain for ‘News’”. 12 “By renaming this handsome, twenty-six-year-old savage, Crusoe assumes possession
of him in the same way that Columbus assumed possession of the land by his namings”.
13 “[...] reflects the heathen contexts we might associate with Anglo-Saxon mythology
and superstition”. 2. O sonho de estar em controle do mundo físico q
p
q
j g
A mistura de uma ânsia pela comunicação verbal e a linguagem
como meio de dominação é em nenhum outro momento mais explícita do
que no episódio do primeiro encontro com Sexta-Feira. Este fora salvo
por Crusoé, que derrubou um selvagem que o perseguia. Mas, ao se dar
conta de que o selvagem não morrera, Crusoé apontou para Sexta-Feira: a isso ele [Sexta-Feira] respondeu me dizendo algumas palavras
que, embora eu nada tenha entendido, achei muito agradáveis de
escutar, pois eram os primeiros sons de voz humana que eu ouvia,
tirando a minha própria, em mais de vinte e cinco anos (DEFOE,
2012, p. 282). “O homem é uma criatura sociável”, diz Locke (2012, p. 433),
“com a necessidade de relações com seus congêneres, e a inclinação
para tal [por meio] da linguagem”. Crusoé imediatamente reconheceu o
selvagem como um congênere ou um semelhante: ele tinha voz humana. Mas, linhas abaixo, pelo uso da linguagem referencial, Sexta-Feira se
torna “o meu selvagem, pois assim eu o chamava agora” (DEFOE, 2012,
p. 282). O uso do pronome possessivo marca a distinção não apenas
entre Sexta-Feira e o selvagem que derrubara, mas entre Sexta-Feira e
Crusoé. Aquele se torna sua propriedade, seu servo, assim como a ilha
era sua por direito de posse. 11 “[...] grow out of an increasing appetite in the late seventeenth and early eighteenth
century in Britain for ‘News’”. 78 Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 Que direito de domínio tem Crusoé sobre Sexta-Feira? Esse direito
foi adquirido do mesmo modo do “direito de posse” que exercia sobre a
ilha? Foi apenas depois de anos de intenso trabalho que Crusoé dominou
sua condição e seu ambiente, e esse domínio veio acompanhado, como
vimos, da renomeação do lugar. O controle do espaço físico e a regulação
do significado de seu nome caminhavam juntos. Contudo, no caso de
Sexta-Feira, a subjugação se deu primeiro no âmbito da linguagem, não
muito diferente do princípio lockiano quanto à nomeação dos modos
mistos, os juízos morais, em que as coisas devem se ajustar aos nomes: Primeiro, dei-lhe a saber que seu nome seria Sexta-Feira, o dia
em que eu tinha salvo a sua vida; dei-lhe este nome em memória
da data. Ensinei-lhe também a me chamar de “amo”, dando a
entender que era este meu nome (DEFOE, 2012, p. 285). Sobre essa passagem, o biógrafo de Defoe, Maximillian Novak
(1997, p. 117), escreveu: “Ao renomear esse selvagem belo, de vinte e
cinco anos, Crusoé assumia posse sobre ele do mesmo modo que Colombo
assumiu posse sobre a terra ao nomeá-la”.12 A subordinação sociopolítica
pela linguagem faz parte de um mito agressivo de supremacia, a lógica
da expansão colonial. Esse grande continente no qual nos encontramos,
América, tem seu nome a partir do explorador italiano, Américo Vespúcio,
que foi o primeiro a demonstrar que Colombo não havia chegado à Ásia,
mas ao Novo Mundo. Contudo, os nativos desta terra foram e ainda são
denominados de índios, mesmo após ter se tornado claro de que aqui não
era a Índia. Igualmente, “Crusoé não pergunta [o nome de Sexta-Feira],
mas lhe dá um” (Watt, 2007, p. 63). Em um artigo sobre o tema, Sandro Jung se pergunta o porquê
de Defoe ter escolhido o nome de Sexta-Feira para o companheiro de
Crusoé. Segundo Jung (2003, p. 275, tradução nossa), de um lado, o nome
Sexta-Feira – em inglês, Friday – “reflete o contexto pagão com o qual
associamos a mitologia e a superstição anglo-saxãs” (JUNG, 2003, p. 275, tradução nossa),13 reafirmando a natureza indômita do selvagem; do 12 “By renaming this handsome, twenty-six-year-old savage, Crusoe assumes possession
of him in the same way that Columbus assumed possession of the land by his namings”. 14 “Friday, the savage, is tamed and embedded into a civilised context [...]. In the
context of the protagonist’s conferring a name to Friday, we might consider the act of
naming as an attempt to create an independent religion, or in other words, a fusion of
the heathen culture, the primitive environment Crusoe encounters on the island and
the Christian, and more strongly Puritan, faith”. Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 Era notável, também, que, entre apenas três súditos, houvesse
três religiões diferentes. Meu Sexta-Feira era protestante, seu pai
era pagão e canibal, e o Espanhol era papista. Ainda assim, eu
consentia a liberdade de consciência em meus domínios (DEFOE,
2012, p. 326). Isso não significa, claro, que Crusoé, ou o protótipo do colono
britânico, exerce um poder menos imperioso sobre seu domínio do que
os de qualquer outro colonizador. Contudo, o modelo de subjugação
colonial para Crusoé está impregnado de problemas que concernem
a interpretação verbal e textual; problemas esses que não se resolvem
na solidão. Isso se associa perfeitamente ao puritanismo. Se, de um
lado, a religiosidade protestante é orientada pela ascese intramundana
(WEBER, 2012 p. 87-139); do outro, como assinalou Christopher Hill
(2002, p. 376-395), há um senso prático de comunhão, de apoio mútuo
e de conhecimento por intermédio das palavras. Sexta-Feira se torna não
apenas o servo de Crusoé, mas também um receptáculo, por meio do
qual ele pode exercer sua autoridade por meio de questões de linguagem
e de crença religiosa. Ao instruir Sexta-Feira no conhecimento da língua
inglesa e das Sagradas Escrituras, Crusoé dá testemunho de seu próprio
chamado. Dentro em pouco, observa McKeon (1987, p. 332, tradução
nossa): “[Sexta-Feira] rapidamente aprende a renunciar seu canibalismo,
e quando o senhor confronta novamente os selvagens, ele solicita a ajuda
de seu escravo civilizado”.15 15 “[Friday] quickly learns to renounce his cannibalism, and when the master next
confronts the savages, he enlists the aid of his civilized slave”. Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 13 “[...] reflects the heathen contexts we might associate with Anglo-Saxon mythology
and superstition”. Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 79 outro, o nome se refere ao contexto do calendário, que introduz ordem
aos dias da semana. Nas palavras de Jung (2003, p. 275, tradução nossa): Sexta-Feira, o selvagem, é domesticado e integrado a um contexto
civilizado [...]. No contexto de o protagonista conferir um nome
a Sexta-Feira, podemos considerar o ato de nomeação como
uma tentativa de criar uma religião independente, ou, em outras
palavras, uma fusão da cultura pagã, o ambiente primitivo que
Crusoé encontra na ilha, e a fé cristã ou ainda mais fortemente
puritana.14 Mas há outro aspecto do nome Sexta-Feira (Friday) que o artigo
de Jung (2003) não leva em consideração. De terça-feira à sexta-feira
(Tuesday a Friday), os dias da semana em inglês recebem o nome de
um deus da mitologia nórdica. Por que, então, Friday e não Tuesday,
Wednesday ou Thursday? O nome Friday vem de Frigg, a deusa que,
como Vênus, é associada ao amor e à beleza. O perfil que Crusoé traça de
Sexta-Feira é significativo nesse aspecto: “Tinha um semblante bondoso,
não um aspecto arrogante e feroz, mas parecia ter algo de muito másculo
no rosto, ao mesmo tempo que transmitia a doçura e a suavidade de um
Europeu também na expressão” (DEFOE, 2012, p. 284). Nas conhecidas
palavras de James Joyce (1912, apud RICHETTI, 2012, p. 39), o herói de
Defoe é “o verdadeiro protótipo do colono britânico, assim como Sexta-
Feira (o selvagem fiel que surge num dia de pouca sorte) é o símbolo das
raças submetidas”. Com isso em mente, meu argumento é que o nome
Friday sinaliza um tipo específico de dominação que Crusoé deseja
imprimir sobre sua colônia. Ao contrário das “barbaridades”, da “simples
carnificina” e da “crueldade sangrenta” dos espanhóis, o empreendimento
colonial de Crusoé é regido por Vênus, guiado, em suas palavras, por
“princípios ternos” e por uma “inclinação generosa do espírito” (DEFOE,
2012, p. 245). Quando a ilha de Crusoé se encontrava povoada, ele se
satisfez com a seguinte reflexão: 80 Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 3. Foe e o fracasso do sonho do poder total Estudos sobre a literatura colonial e pós-colonial discutem a
relação entre territórios coloniais e modelos de autoridade narrativa e, com
isso, apresentam uma revisão da teoria do romance. Este é precisamente
o caso de Cultura e Imperialismo, de Edward Said (2017). Said (2017, p. 129) argumenta que se importantes estudos sobre o romance, de autores
como Watt (1996; 2007), Richetti (1996; 2008) e McKeon (1987),
“tenham dedicado atenção especial à relação entre o romance e o espaço
social, a perspectiva imperialista foi negligenciada”. “Robinson Crusoé”,
continua Said (2017, p. 120), “é praticamente impensável sem a missão 15 “[Friday] quickly learns to renounce his cannibalism, and when the master next
confronts the savages, he enlists the aid of his civilized slave”. Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 81 colonizadora”. Ainda que a maioria dos romances realistas não aborde
explicitamente o domínio colonial, “sem império, não existe o romance
europeu tal como o conhecemos” (SAID, 2017, p. 128). Said não quer
dizer com isso que o romance causou o imperialismo, mas porque o
romance está fundamentalmente ligado à sociedade burguesa, e, em
razão de seu modelo narrativo de autoridade social, o imperialismo e o
romance “se fortaleceram reciprocamente a um tal grau que é impossível
[...] ler um sem estar lidando de alguma maneira com o outro” (SAID,
2017, p. 129). De modo semelhante, da década de 1960 em diante, escritores
frequentemente empregam um repertório consistente de temas coloniais
e pós-coloniais. Em geral, o que se critica é a ubiquidade de estereótipos;
ao mesmo tempo, almeja-se dar voz aos membros mais oprimidos e à
margem da comunidade global. Contudo, no caso específico da obra
Foe, de Coetzee (2016), de que falaremos aqui, a narrativa se afasta do
comum e, sob o aspecto linguístico, como veremos, afirma a posição
marginalizada do nativo.16 Ao reelaborar o cânone, ao invés de se voltar
para questões de conteúdo temático, o autor se volta, sobretudo, para
questões relativas à forma. Isso se observa, por exemplo, no modo como
o enredo não é articulado logicamente e o clímax é empurrado para as
margens do discurso. Para uma visão mais clara de como isso se dá
na obra, e para aqueles que não estão familiarizados com sua história,
apresentarei um breve sumário do enredo. 16 Sobre o tema, ver o artigo de Gayatri Chakravorty Spivak (1990), The Theory on the
Margin: Coetzee’s Foe Reading Defoe’s Crusoe/Roxana. 3. Foe e o fracasso do sonho do poder total Susan Barton, a personagem principal e narradora, está em
uma missão para encontrar sua filha sequestrada que ela sabe ter sido
levada ao Novo Mundo. Seu navio fica à deriva durante um motim em
Lisboa, quando ela chega em uma ilha deserta e encontra com Sexta-
Feira e Cruso, este em estado complacente, contentado a esquecer o
passado e viver na ilha com a assistência daquele. Ao fim do período de
confinamento – Barton fica na ilha por um ano até que o trio é resgatado
–, Cruso não sobrevive a viagem de volta à Inglaterra. Na Inglaterra com
Sexta-Feira, Barton resolve colocar a sua aventura no papel, mas sente
que sua história carece de apelo popular. Ela tenta convencer o romancista
Daniel Foe a ajudá-la com seu manuscrito, mas ele não concorda quanto
ao que há de mais interessante na aventura dela. Foe prefere que a história Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 82 na ilha deserta seja apenas um episódio de uma história formulada sobre
uma mãe à procura de sua filha, e quando escreve a história que ela
propõe, inventa as aventuras de Cruso, ao invés de narrar os fatos tal qual
aconteceram. Ao frustrar os esforços de Barton, Foe, que se torna seu
amante, está mais preocupado em pagar as dívidas e tem pouco tempo
e energia para escrever sobre qualquer coisa. A história de Barton passa
por uma reviravolta com o aparecimento de uma garota que afirma ser
sua filha. Barton não a reconhece e, por fim, substitui essa missão por
outra, a de escrever sua própria história. Coetzee (2016), desse modo, direciona a atenção do leitor para
a sedução e a tirania da fabulação. Barton não admite escrever uma
narrativa que “não [tenha] consideração pela verdade” (COETZEE,
2016, p. 86). Se, diz Barton a Sexta-Feira, “não enfrentamos perigos [...],
nenhum pirata aportou em nossas praias, nenhum flibusteiro, nenhum
canibal” (COETZEE, 2016, p. 75), qual aventura ela poderia narrar? Em
suma, Foe é sobre o fracasso de encontrar um enredo consistente para a
história de Barton e para a vida de Cruso na ilha; é sobre o fracasso do
sonho do poder total.l p
Isso se reflete no uso da linguagem na ilha, pois era “um lugar
árido e silencioso” (COETZEE, 2016, p. 55), assim como os utensílios
na ilha eram escassos ou resultado de um fabrico rústico. 3. Foe e o fracasso do sonho do poder total “Faltam-nos
muitas coisas nesta ilha” (COETZEE, 2016, p. 18), diz Susan; “Cruso não
mantinha um diário, talvez porque não tivesse papel e tinta, porém mais
provavelmente [...] porque não tinha o pendor de mantê-lo” (COETZEE,
2016, p. 17). Além disso, Susan fica intrigada com a aridez da linguagem,
seu uso quase exclusivo para transmitir instruções simples, e questiona
Cruso sobre a comunicação que estabelece com Sexta-Feira: “Quantas palavras em inglês Sexta-Feira conhece?”, perguntei. “Quantas palavras em inglês Sexta-Feira conhece?”, perguntei. “As necessárias”, Cruso respondeu. “Aqui não é a Inglaterra, não
precisamos de muitas palavras”. “O senhor fala como se a língua fosse uma das perdições da vida,
como o dinheiro ou a varíola”, eu disse. “No entanto, não teria
aliviado sua solidão se Sexta-Feira dominasse o inglês? O senhor
e ele podiam ter experimentado, todos esses anos, os prazeres
da conversação; o senhor poderia ter passado a ele algumas
das bênçãos da civilização e feito dele um homem melhor. Que
benefício existe numa vida de silêncio”. “O senhor fala como se a língua fosse uma das perdições da vida,
como o dinheiro ou a varíola”, eu disse. “No entanto, não teria
aliviado sua solidão se Sexta-Feira dominasse o inglês? O senhor
e ele podiam ter experimentado, todos esses anos, os prazeres
da conversação; o senhor poderia ter passado a ele algumas
das bênçãos da civilização e feito dele um homem melhor. Que
benefício existe numa vida de silêncio”. Cruso nada respondeu [...]. Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 83 Cruso puxou Sexta-Feira para perto de si. “Abra a boca”, disse, e
abriu a sua própria. Sexta-Feira abriu a boca. “Olhe”, disse Cruso. Eu olhei, mas não vi nada no escuro além do reluzir de dentes
brancos de marfim. “La-la-la”, disse Cruso, e gesticulou para Sexta-
Feira repetir. “Ha-ha-ha”, disse Sexta-Feira do fundo da garganta. “Ele não tem língua”, disse Cruso (COETZEE, 2016, p. 23). Cruso puxou Sexta-Feira para perto de si. “Abra a boca”, disse, e
abriu a sua própria. Sexta-Feira abriu a boca. “Olhe”, disse Cruso. Eu olhei, mas não vi nada no escuro além do reluzir de dentes
brancos de marfim. “La-la-la”, disse Cruso, e gesticulou para Sexta-
Feira repetir. “Ha-ha-ha”, disse Sexta-Feira do fundo da garganta. “Ele não tem língua”, disse Cruso (COETZEE, 2016, p. 23). 17 “Friday is in the margin as such, the wholly other”. 3. Foe e o fracasso do sonho do poder total Se Sexta-Feira perdeu a língua pelos traficantes de escravos da
África, segundo a história contada por Cruso, ou se foi mutilado pelo seu
próprio senhor, não sabemos. “Muitas histórias podem ser contadas sobre
a língua de Sexta-Feira”, diz Susan, “mas a verdadeira está sepultada
dentro de Sexta-Feira, que é mudo” (COETZEE, 2016, p. 106). Seja
como for, Sexta-Feira está impedido de contar sua própria história; nas
palavras de Susan: “A verdadeira história não será ouvida até que por
nosso próprio engenho encontremos um meio de dar voz a Sexta-Feira”
(COETZEE, 2016, p. 106). (
p
)
Qual engenho seria capaz de dar voz ao mudo Sexta-Feira? A
escolha de Coetzee, nesse sentido, parece minar a combinação possível
de palavras. Assim, ele retrata o nativo de modo diferente do que se
convencionou nos romances pós-coloniais do chamado Terceiro Mundo,
nos quais, nas palavras de Said (2017, p. 332), “o nativo outrora silenciado
fala e age em território tomado do colonizador, como parte de um
movimento geral de resistência”. Esse motivo é percebido por George
Lamming (1984, apud SAID, 2017, p. 333) como um modo de explodir
o velho mito do colono; mas isso só pode ocorrer, diz ele, quando: Mostrarmos a língua como produto do empenho humano;
quando colocarmos à disposição de todos o resultado de certos
empreendimentos levados a cabo por homens ainda vistos como
infelizes descendentes de escravos sem linguagem e deformados. O mudo Sexta-Feira e sua completa inabilidade em adquirir a
linguagem verbal, a despeito de todos os esforços de Barton de revelar
a ele a “magia das palavras” (COETZEE, 2016, p. 54), afina-se com o
fracasso de Barton ou mesmo de Foe em tecer um enredo. Segundo o
célebre artigo de Gayatri Chakravorty Spivak (1990, p. 4, tradução nossa),
a ausência de comunicação verbal do Sexta-Feira de Coetzee coloca-o
“à margem enquanto tal”; ele é “o outro por completo”.17 84 Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 Para se ter uma clareza maior da alternativa original e desafiadora
de Coetzee sobre as possibilidades de se contrapor à herança colonial
e suas formas narrativas, comparemos a representação do nativo, em
Foe, com os exemplos oferecidos por Said (2017, p. 328-344) em
“Temas da Cultura de Resistência”. 3. Foe e o fracasso do sonho do poder total São três os livros analisados ali –
The River Between (1965), de Ngũgĩ wa Thiong’o; Season of Migration
to the North (1966), de Tayeb Salih; e Une tempête (1969), de Aimé
Césaire –, todos eles, como Foe, são releituras de uma obra canônica sob
o prisma da descolonização: os dois primeiros retomam Coração das
trevas, de Joseph Conrad; o terceiro, A tempestade, de Shakespeare. O
protagonista de The River Between é Waiyaki, um jovem ambicioso que
fora enviado pelo pai a uma escola missionária onde pudesse adquirir os
conhecimentos necessários para se contrapor ao governo colonial e, com
isso, unificar as divisões entre duas aldeias do Quênia. Mostapha Sa’eed,
protagonista de Season of Migration to the North, retorna a sua aldeia no
Sudão, depois de anos de estudo na Grã-Bretanha, e lá se encontra com
o narrador, cujo nome jamais é revelado. Este também fora educado no
ocidente e fica intrigado em saber mais sobre o passado de Sa’eed depois
de ele recitar poesia em inglês fluente. A peça Une tempête, do caribenho
Césaire, segue de perto o enredo de A tempestade. Contudo, Césaire
dá uma atenção especial às personagens que habitavam a ilha antes da
chegada de Próspero e Miranda, isto é, Ariel e Caliban. Enquanto aquele é
retratado como “um solicito servidor de Próspero” (SAID, 2017, p. 334);
este é uma espécie de revolucionário, “que se livra de sua servidão atual
e de sua desfiguração física no processo de descobrir seu eu essencial,
pré-colonial” (SAID, 2017, p. 335). Mesmo que suas missões nem sempre
sejam bem sucedidas ou que suas condutas morais sejam reprováveis, os
protagonistas Waiyaki, Sa’eed e Caliban possuem uma história passível
de desenvolvimento que se manifesta, entre outros aspectos, por alguém
em pleno domínio da linguagem referencial e denotativa. Por contraste, o Sexta-Feira de Coetzee não só é mudo como
parece não ter sequer noção da sua condição de ex-escravo. É Barton
quem tenta conscientizá-lo. “Por que”, diz ela, “durante todos aqueles
anos sozinho com Cruso, você se submeteu ao domínio dele, quando
podia facilmente tê-lo matado, ou cegado e feito dele o seu escravo por
sua vez?” (COETZEE, 2016, p. 78). Mas aqui, com em todas as outras
interpolações da protagonista, ela fica sem resposta: “falar com você
é igual a falar com as paredes” (COETZEE, 2016, p. 78). Ao mesmo Aletria, Belo Horizonte, v. 31, n. 2, p. 3. Foe e o fracasso do sonho do poder total 67-89, 2021 85 tempo, a convivência de Barton com Sexta-Feira na Inglaterra não se
traduz em termos de senhor e escravo: “Sexta-Feira não era meu escravo,
mas de Cruso, e é um homem livre agora. Ele não pode nem mesmo ser
chamado de criado, tão ociosa é sua vida” (COETZEE, 2016, p. 70). As vezes em que Sexta-Feira se punha a dançar, produzindo “um som
cantarolante na garganta” (COETZEE, 2016, p. 84), sequer ouvia aos
chamados de Barton. Coetzee, com isso, oferece uma crítica à dominação
pela linguagem e se contrapõe ao argumento de que os oprimidos devam
desenvolver uma voz a partir dos mesmos códigos linguísticos herdados
do colono; ou, nas palavras de Spivak (1990, p. 18, tradução nossa), a
mensagem de Coetzee, talvez, resida na impossibilidade de “dar voz ao
nativo ‘no’ texto”.18 Segundo a hipótese que sigo neste artigo, são os
padrões narrativos tradicionais e a própria linguagem referencial que
estão em jogo.i Isso é o que parece sugerir o capítulo final do livro. Este breve
capítulo, de menos de cinco páginas, bastante conhecido pela sua
impenetrabilidade, pode ser interpretado como uma alegoria metaficcional
do leitor que dá sentido ao romance. Um narrador, não sabemos quem,
adentra uma sala escura (um convés, talvez) e se depara com os corpos
de Barton, seu capitão e Sexta-Feira. Os dois primeiros, ao que parece,
estão mortos. O mesmo poderia se deduzir de Sexta-Feira, não fosse pela
pulsação na garganta e pelo som débil e seco que seu corpo produz. Há,
com isso, indícios de que é precisamente a linguagem de Sexta-Feira que
se busca retratar neste capítulo. Sua linguagem certamente não pode ser
realista, tampouco uma na qual as palavras são signos de ideias. “Mas este
não é um lugar de palavras. Cada sílaba, assim que escapa, é capturada,
preenchida com água e dispersa. Este é um lugar onde corpos são seus
próprios sinais. É a morada de Sexta-Feira” (COETZEE, 2016, p. 142). O paralelo que V. S. Naipaul (1991) estabelece entre Colombo
e Crusoé, com o qual iniciei este artigo, está assentado na descoberta
heroica de um Novo Mundo que praticamente inexistia. Trata-se, claro,
de uma descoberta fantasiosa e é ela, segundo Naipaul (1991, p. 18 “[...] giving voice to the native ‘in’ the text”.
19 “It is a monologue; it is all in the mind”. 3. Foe e o fracasso do sonho do poder total 659,
tradução nossa), que justifica uma espécie de “monólogo; tudo se encontra
na mente”.19 Em um primeiro momento, como vimos, é a partir desse
monólogo que Crusoé passa a nomear e renomear as coisas, seu meio e Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 86 as pessoas com as quais se relaciona segundo o significado que melhor
lhe convir. Este é um dos sentidos do mito de Crusoé. Ao se voltar para
os próprios escritos de Defoe, Watt (1996) nos mostra o quanto o autor
tinha consciência de estar fabricando um mito, ou, em suas palavras,
“uma história emblemática”, cujo objetivo seria o “aprimoramento moral
e religioso” (DEFOE, 1704, apud WATT, 1996, p. 164, tradução nossa).20
Assim, o indivíduo, Crusoé, funcionaria como uma espécie de “emblema”
para outros indivíduos. Se tudo se encontra na mente, o mesmo vale para
a relação entre as palavras e as coisas. Defoe encontrou no Ensaio sobre
o entendimento humano de Locke seu fundamento filosófico, ainda que
conferisse uma extensão maior para a capacidade do indivíduo em regular
o significado das palavras do que seria aceita pelo filósofo. É gi
p
q
pi
É bastante conhecida a formulação de Watt de que um dos pilares
do mito de Crusoé é a ideia da dignidade do trabalho, que coincide
com o advento do capitalismo e que apela à solidão, representada
metonimicamente pela ilha deserta. Contudo, como procurei desenvolver
ao longo deste artigo, o mito não se materializaria sem a manipulação
dos signos verbais e seus ajustes tanto na consciência de Crusoé quanto
na relação de subordinação que ele estabelece com os demais habitantes
da ilha, sobretudo Sexta-Feira. Se, como vimos, a primeira impressão
do encontro de Crusoé com Sexta-Feira foi o prazer que aquele sentira
ao ouvir uma voz humana, o primeiro registro do selvagem vem
acompanhado do pronome possessivo “meu”. Prazer, dominação e
controle caminham lado a lado. Do mesmo modo, Crusoé trabalha não
apenas para suprir suas necessidades básicas ou para fugir do tédio, mas
também para usufruir dos prazeres oriundos de seu trabalho e isso mesmo
em coisas sem a menor utilidade prática, como seu cachimbo e outros
luxos e confortos que provém a si mesmo. Assim, seria um equívoco
identificar em Crusoé um asceta stricto senso e, nesse ponto, minha
interpretação diverge da de Watt (1996, p. 20 “[...] an emblematick history [...], of moral and religious improvement”.
21 “Even in language [...] Crusoe is a strict utilitarian”. 3. Foe e o fracasso do sonho do poder total 168, tradução nossa), para
quem o herói, “mesmo na linguagem, [...] é estritamente um utilitário”.21
Antes, há em Crusoé uma ânsia pelo convívio humano e, sobretudo, pela
voz humana. A natureza fabricada da sua linguagem e o estabelecimento
de sua colônia ficariam incompletos tivesse o náufrago permanecido
solitário na ilha durante todo o período. Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 87 Em sentido oposto ao do romance de Defoe (2012), em Foe,
os anos de confinamento do herói não resultaram em aprimoramento
tecnológico, acúmulo de bens materiais e ressignificação do espaço e
das relações de dominação por meio de uma reelaboração da linguagem. Assim, são significativas as escolhas de Coetzee (2016) pelo mudo
Sexta-Feira e pela escassez da linguagem na ilha de Cruso. Além disso, a
personagem e narradora de Foe, Susan Barton, não só fracassa em tramar
um enredo coerente para sua história de vida como fracassa, igualmente,
em instruir Sexta-Feira na linguagem verbal, escrita ou falada. Desse
modo, Foe parece minar a “combinação possível de palavras” [energy
of words], produzindo, assim, uma quebra tanto no mito de Crusoé – os
sonhos de ser o primeiro homem do mundo, de estar em controle do
mundo físico e do poder total – quanto nos modos tradicionais de se
contrapor à herança colonial e suas formas narrativas. Referências BONOMO, Daniel. Experimentum in Insula: Robinson Crusoé nas
origens do aborrecimento. Revista Literatura e Sociedade, São Paulo. v. 22, n. 25, p. 117-131, 2018. DOI: https://doi.org/10.11606/issn.2237-
1184.v0i25p117-131. COETZEE, John Maxwell. Foe. Tradução de José Rubens Siqueira. São
Paulo: Companhia das Letras, 2016. DEFOE, Daniel. Robinson Crusoé. Tradução de Sergio Flaksman,
Introdução e notas de John Richetti. São Paulo: Penguin; Companhia
das Letras, 2012. HILL, Christopher. O Mundo de Ponta Cabeça: ideias radicais durante
a Revolução Inglesa de 1640. Tradução de Renato Janine Ribeiro. São
Paulo: Companhia das Letras, 2002. JUNG, Sandro. The language(s) of hierarchy in Daniel Defoe’s Robinson
Crusoe. Nordic Journal of English Studies. Oslo, v. 2, n. 2, p. 265-277,
2003. DOI: https://doi.org/10.35360/njes.137. KEYMER, Thomas. Introduction. In: DEFOE, Daniel. Robinson
Crusoe. Edited with an Introduction by Thomas Keymer. Oxford:
Oxford University Press, 2008. DOI: https://doi.org/10.1093/
owc/9780199553976.001.0001. Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 88 LOCKE, John. Ensaio sobre o entendimento humano. Tradução de Pedro
Paulo Garrido Pimenta. São Paulo: Martins Fontes, 2012. MCKEON, Michael. The Origins of the English Novel 1600-1740. Baltimore: The John Hopkins University Press, 1987. MONTEIRO, Daniel. No limiar da visão: a poética do sublime em
Edmund Burke. São Paulo: LiberArs, 2018. NAIPAUL, Vidiadhar Surajprasad. Columbus and Crusoe. In: GROSS,
John. The Oxford Book of Essays. Oxford: Oxford University Press, 1991. p. 656-659. NOVAK, Maximillian E. Friday: or, the power of naming. In: RIVERO,
Albert J. Augustan Subjects: Essays in Honor of Martin C. Battestin. Newark: University of Delaware Press, 1997. p. 110-122. RICHETTI, John. Defoe as narrative innovator. In: RICHETTI, John. The Cambridge Companion to Daniel Defoe. Cambridge: Cambridge
University Press, 2008. p. 121-138. DOI: https://doi.org/10.1017/
CCOL9780521858403.008. RICHETTI, John. Introdução. In: DEFOE, Daniel. Robinson Crusoé. Tradução de Sergio Flaksman, Introdução e notas de John Richetti. São
Paulo: Penguin/Companhia das Letras, 2012. SAID, Edward W. Cultura e Imperialismo. Tradução de Denise Bottmann. São Paulo: Companhia de Bolso, 2017. SEIDEL, Michael. Robinson Crusoe: Varieties of Fictional Experience. In: RICHETTI, John. The Cambridge Companion to Daniel Defoe. Cambridge: Cambridge University Press, 2008. p. 182-199. DOI: https://
doi.org/10.1017/CCOL9780521858403.011. SHAKESPEARE, William. A tempestade. Tradução de Barbara
Heliodora. Rio de Janeiro: Editora Nova Fronteira, 2011. SPIVAK, Gayatri Chakravorty. The Theory on the Margin: Coetzee’s
Foe Reading Defoe’s Crusoe/Roxana. English in Africa. Makhanda, v. 17, n. 2, p. 1-23, 1990. STEELE, Richard. Alexander Selkirk, The Englishman. New York:
Brooklyn College, 2013. Disponível em: http://academic.brooklyn.cuny. edu/english/melani/novel_18c/defoe/selkirk.html. Referências Acesso em: 1 nov. 2020. Aletria, Belo Horizonte, v. 31, n. 2, p. 67-89, 2021 89 WATT, Ian. A ascensão do romance: estudos sobre Defoe, Richardson e
Fielding. Tradução de Hildegard Feist. 2 ed. São Paulo: Companhia das
Letras, 2007. WATT, Ian. Myths of Modern Individualism: Faust, Don Quixote, Don
Juan, Robinson Crusoe. Cambridge: Cambridge University Press, 1996. DOI: https://doi.org/10.1017/CBO9780511549236. WEBER, Max. A ética protestante e o espírito do capitalismo. Tradução
de José Marcos Mariani de Macedo. São Paulo: Companhia das Letras,
2012. WATT, Ian. A ascensão do romance: estudos sobre Defoe, Richardson e
Fielding. Tradução de Hildegard Feist. 2 ed. São Paulo: Companhia das
Letras, 2007. WATT, Ian. A ascensão do romance: estudos sobre Defoe, Richardson e
Fielding. Tradução de Hildegard Feist. 2 ed. São Paulo: Companhia das
Letras, 2007. WATT, Ian. A ascensão do romance: estudos sobre Defoe, Richardson e
Fielding. Tradução de Hildegard Feist. 2 ed. São Paulo: Companhia das
Letras, 2007. WATT, Ian. Myths of Modern Individualism: Faust, Don Quixote, Don
Juan, Robinson Crusoe. Cambridge: Cambridge University Press, 1996. DOI: https://doi.org/10.1017/CBO9780511549236. WEBER, Max. A ética protestante e o espírito do capitalismo. Tradução
de José Marcos Mariani de Macedo. São Paulo: Companhia das Letras,
2012. Recebido em: 11 de novembro de 2020. Aprovado em: 31 de maio de 2021. WEBER, Max. A ética protestante e o espírito do capitalismo. Tradução
de José Marcos Mariani de Macedo. São Paulo: Companhia das Letras,
2012.
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Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs
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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. Pe´ter L. Erdős☯¤*, Tama´s Ro´bert Mezei☯, Istva´n Miklo´s☯, Da´niel Solte´sz☯
Alfre´d Re´nyi Institute of Mathematics, Hungarian Academy of Sciences, Budapest, Hungary ☯These authors contributed equally to this work. ¤ Current address: Rea´ltanoda u 13-15, Budapest, H-1053, Hungary
* erdos.peter@renyi.mta.hu ☯These authors contributed equally to this work. ¤ Current address: Rea´ltanoda u 13-15, Budapest, H-1053, Hungary
* erdos.peter@renyi.mta.hu a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 Data Availability Statement: All relevant data are
within the paper. Funding: The authors of this paper were supported
in part by the National Research, Development and
Innovation Office | NK- FIH grant K 116769 and KH
126853. IM was supported in part by the National
Research, Development and Innovation Office |
NKFIH grant SNN 116095. DS was supported in
part by the National Research, Development and
Innovation Office | NKFIH grants K 108947 and K
120706. The WEB page of the founder is: https://
www.nkh.gov.hu/en/web/english/. The funders had Abstract Since 1997 a considerable effort has been spent on the study of the swap (switch) Markov
chains on graphic degree sequences. All of these results assume some kind of regularity
in the corresponding degree sequences. Recently, Greenhill and Sfragara published a
breakthrough paper about irregular normal and directed degree sequences for which
rapid mixing of the swap Markov chain is proved. In this paper we present two groups of
results. An example from the first group is the following theorem: let ~d be a directed degree
sequence on n vertices. Denote by Δ the maximum value among all in- and out-degrees
and denote by j~Ej the number of edges in the realization. Assume furthermore that
D <
1ffiffi
2
p
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
j~Ej 4
q
. Then the swap Markov chain on the realizations of ~d is rapidly mixing. This
result is a slight improvement on one of the results of Greenhill and Sfragara. An example
from the second group is the following: let d be a bipartite degree sequence on the vertex
set U ] V, and let 0 < c1 c2 < |U| and 0 < d1 d2 < |V| be integers, where c1 d(v) c2:
8v 2 V and d1 d(u) d2: 8u 2 U. Furthermore assume that (c2 −c1 −1)(d2 −d1 −1) < max
{c1(|V| −d2), d1(|U| −c2)}. Then the swap Markov chain on the realizations of d is rapidly mix-
ing. A straightforward application of this latter result shows that when a random bipartite or
directed graph is generated under the Erdős—Re´nyi G(n, p) model with mild assumptions
on n and p then the degree sequence of the generated graph has, with high probability, a
rapidly mixing swap Markov chain on its realizations. Since 1997 a considerable effort has been spent on the study of the swap (switch) Markov
chains on graphic degree sequences. All of these results assume some kind of regularity
in the corresponding degree sequences. Recently, Greenhill and Sfragara published a
breakthrough paper about irregular normal and directed degree sequences for which
rapid mixing of the swap Markov chain is proved. In this paper we present two groups of OPEN ACCESS Citation: Erdős PL, Mezei TR, Miklo´s I, Solte´sz D
(2018) Efficiently sampling the realizations of
bounded, irregular degree sequences of bipartite
and directed graphs. PLoS ONE 13(8): e0201995. https://doi.org/10.1371/journal.pone.0201995 results. An example from the first group is the following theorem: let ~d be a directed degree
sequence on n vertices. Denote by Δ the maximum value among all in- and out-degrees
and denote by j~Ej the number of edges in the realization. Assume furthermore that Editor: Arndt von Haeseler, Max F Perutz
Laboratories GmbH, AUSTRIA
Received: May 16, 2018
Accepted: July 25, 2018
Published: August 13, 2018
Copyright: © 2018 Erdo˝s 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: Arndt von Haeseler, Max F Perutz
Laboratories GmbH, AUSTRIA
Received: May 16, 2018
Accepted: July 25, 2018
Published: August 13, 2018 Editor: Arndt von Haeseler, Max F Perutz
Laboratories GmbH, AUSTRIA
Received: May 16, 2018
Accepted: July 25, 2018
Published: August 13, 2018 Editor: Arndt von Haeseler, Max F Perutz
Laboratories GmbH, AUSTRIA D <
1ffiffi
2
p
j~Ej 4
q
. Then the swap Markov chain on the realizations of ~d is rapidly mixing. This
result is a slight improvement on one of the results of Greenhill and Sfragara. An example
from the second group is the following: let d be a bipartite degree sequence on the vertex
set U ] V, and let 0 < c1 c2 < |U| and 0 < d1 d2 < |V| be integers, where c1 d(v) c2:
8v 2 V and d1 d(u) d2: 8u 2 U. Furthermore assume that (c2 −c1 −1)(d2 −d1 −1) < max
{c1(|V| −d2), d1(|U| −c2)}. Then the swap Markov chain on the realizations of d is rapidly mix-
ing. A straightforward application of this latter result shows that when a random bipartite or
directed graph is generated under the Erdős—Re´nyi G(n, p) model with mild assumptions
on n and p then the degree sequence of the generated graph has, with high probability, a
rapidly mixing swap Markov chain on its realizations. Copyright: © 2018 Erdo˝s et al. RESEARCH ARTICLE Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs The swap operation exchanges two disjoint edges ac and bd in the realization G with ad
and bc if the resulting configuration G0 is again a simple graph (we denote this operation by ac,
bd ) ad, bc). (For details see the next section). It is a well-known fact that the set of all possible
realizations of a graphic degree sequence is connected under this operation. (See, for example,
Petersen [2] or Havel [3] and Hakimi [4]). An analogous result applies for the swap operation
defined on bipartite graphs. (See, for example, Gale [5]). Here we have to be careful, as not
every edge exchange is eligible: on a bipartite graph we must ensure that vertices a and d
belong to different vertex classes. 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. Competing interests: The authors have declared
that no competing interests exist. In the literature, the name switch operation is also used, however, in our approach this lat-
ter is an operation on integer matrices slightly generalizing the swap operation. (See the sec-
tions on the analysis of the swap sequences). The situation is more complicated in case of directed degree sequences. In this case for
every vertex the number of incoming edges (in-degree) and the number of outgoing edges
(out-degree) is given in the degree bi-sequence. Here the ac, bd ) ad, bc type exchange pre-
serves the degree bi-sequence if both before and after the swap operation a and b are tails of
the directed edges. However, imagine that our graph ~G is a directed triangle C3
! while ~H is
the oppositely directed C3
. Both graphs have the same degree bi-sequence d = ((1, 1, 1); (1, 1,
1)). It is clear that there is only one way to transform the first one into the second one: if we
exchange three edges and three non-edges in ~G. We will call this operation a triple swap and
the previously defined “classical” one as a double swap. Kleitman and Wang proved in 1973
([6]) that any two realizations of a given graphic degree bi-sequence can be transformed into
each other using these two operations. The same fact was re-discovered in 2010 (see [7]). Introduction An important problem in network science is to algorithmically construct typical instances of
networks with predefined properties, often expressed as graph measures. In particular, special
attention has been devoted to sampling simple graphs (in our paper only graphs without paral-
lel edges and loops are considered) with a given degree sequence. In 1997 Kannan, Tetali, and
Vempala ([1]) proposed the use of the so-called switch Markov chain approach, which had
already been used in statistics. We call this the swap Markov chain approach. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 1 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs operations. (B) is due to Miklo´s, Erdős, and Soukup ([10]). Half-regularity means that in one
class the degrees are the same (i.e., regular), while in the other class the only restrictions are
those imposed by graphicality. (C) is due to Erdős, Miklo´s, and Toroczkai ([11]). Here almost-
half-regular means that for any pair of vertices on one side we have |d(v1) −d(v2)| 1. (D) was
proved by Erdős, Kiss, Miklo´s, and Soukup ([12]). This model will be introduced in detail and
its intrinsic connection with directed graphs will be fully explained in the section starting with
Lemma 12. Papers [9] and [12] are using slightly different Markov chains on regular directed
degree sequences, therefore (D) does not supersede (A). Finally, (E) was proved recently by
Greenhill and Sfragara ([13]). Their result has vastly extended the set of (normal and directed)
degree sequences for which the rapid mixing of the Markov chain is known (e.g., power-law
density-bounded normal degree sequences with parameter γ > 2.5). The papers [14] and [15]
fully characterize those degree bi-sequences where the set of all realizations is irreducible
under the double swap operation. In this paper we improve on the result of Greenhill and Sfragara on directed degree
sequences, and we prove the bipartite analogue of their degree sequence result for simple
graphs. We achieve this by applying our technique described in [12]. In addition, we further
extend the set of bipartite and directed degree sequences with rapidly mixing Markov chain
processes, using a condition on minimum and maximum degrees. Let d be a bipartite degree sequence on the underlying set U ] V. (So the underlying set is
equal to the disjoint union of the two classes). Theorem 2. Let d be a bipartite degree sequence on U and V as classes, let |E| be half of the
sum of the degrees, and let Δ = max d. If 2 D 1ffiffiffi
2
p
ffiffiffiffiffiffi
jEj
p
;
ð1Þ ð1Þ then the swap Markov chain on the realizations of d is rapidly mixing. then the swap Markov chain on the realizations of d is rapidly mixing. The following result describes another wide range of bipartite degree sequences with rapidly
mixing swap Markov chain. Theorem 3. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 The swap Markov chains corresponding to the most common graph models are irreducible,
aperiodic, reversible (obey detailed balance), have symmetric transition matrices, and thus
have uniform global stationary distributions. In their paper [1], Kannan, Tetali and Vempala conjectured that all these Markov chains
are rapidly mixing. The first rigorous proof in this topic is due to Cooper, Dyer and Greenhill
for regular graphs ([8]). Now, twenty years after the KTV conjecture, we are still far, probably
very far, from proving it in its full generality. However, many partial results have been proved;
those which play some role in this paper are summarized in the following theorem: Theorem 1. The swap Markov chain mixes rapidly for the following degree sequences: (A). d is a regular directed degree sequence. (B). d is a half-regular bipartite degree sequence. (C). d belongs to an almost-half-regular bipartite graph. (D). d is an almost-half-regular bipartite degree sequence, where every realization must avoid a
fixed (partial) matching. (D). d is an almost-half-regular bipartite degree sequence, where every realization must avoid a
fixed (partial) matching. (E). d is a directed degree sequence with 2 d max 1
4
ffiffiffiffiffi
M
p
, where M is the sum of the in-
degrees (or out-degrees), and where the set of all realizations under study is irreducible
under the double swap operation. There exist similar results on (normal) degree sequences as well, but their proofs are not
fully “compatible” with the proof of the above results (except case D). To our knowledge there
does not exist a fully developed proof machinery which is applicable for all three cases, that is,
for normal, bipartite, and directed degree sequences. The result (A) was proved by Greenhill ([9]). In the proof it is assumed that the set of all
realizations of the regular directed degree bi-sequence is irreducible under the double swap PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 2 / 20 Let 0 < c1 c2 < |U| = n and 0 < d1 d2 < |V| = m be integer parameters and
assume that d satisfies the following properties: Theorem 3. Let 0 < c1 c2 < |U| = n and 0 < d1 d2 < |V| = m be integer parameters and
assume that d satisfies the following properties: c1 dðvÞ c2;
8v 2 V
d1 dðuÞ d2;
8u 2 U:
ð2Þ c1 dðvÞ c2;
d1 dðuÞ d2; ð2Þ Furthermore, assume that ðc2 c1 1Þ ðd2 d1 1Þ maxfc1ðm d2Þ; d1ðn c2Þg
ð3Þ ð3Þ holds. Then the swap Markov chain on the realizations of d is rapidly mixing. We conjecture that a very similar result should apply to the case of normal degree
sequences. Our next two results are about directed degree sequences ~d on the n element vertex set X. The first one improves the constant 1
4 in the result of Greenhill and Sfragara to 1ffiffi
2
p . Moreover,
because both double and triple swaps are allowed, the irreducibility condition can be omitted
from the theorem. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 3 / 20 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs Theorem 4. Let ~d be a directed degree sequence on the n element V as its vertex set. Let j~Ej be
half of the sum of the degrees, and let Δ = max{max dout, max din}. If D < 1ffiffiffi
2
p
ffiffiffiffiffiffiffiffiffiffiffiffiffiffi
j~Ej 4
q
;
ð4Þ ð4Þ then the swap Markov chain on the realizations of ~d is rapidly mixing. Lastly, we show that conditions similar to that of Theorem 3 guarantee rapid mixing of the
swap Markov chains on a wide class of directed degree sequences. Theorem 5. Let 0 < c1 c2 < n and 0 < d1 d2 < n be integer parameters and assume that
graphic degree bi-sequence ~d satisfies the following properties: Theorem 5. Let 0 < c1 c2 < n and 0 < d1 d2 < n be integer parameters and assume that
graphic degree bi-sequence ~d satisfies the following properties: c1 doutðvÞ c2;
8x 2 X;
d1 dinðvÞ d2;
8x 2 X:
ð5Þ ð5Þ Furthermore, assume that ðc2 c1Þ ðd2 d1Þ 2 þ maxfc1ðn d2 1Þ þ d1 þ c2;
d1ðn c2 1Þ þ c1 þ d2g n ð6Þ ðc2 c1Þ ðd2 d1Þ 2 þ maxfc1ðn d2 1Þ þ d1 þ c2;
d1ðn c2 1Þ þ c1 þ d2g n ð6Þ holds. Then the swap Markov chain, using double and triple swap operations, is rapidly mixing
on the realizations of ~d. The proofs of our results strongly support Greenhill’s observation about the existing argu-
ments ([16]): “In each known case, regularity (or half-regularity) was only required for one
lemma, which we will call the critical lemma. This is a counting lemma which is used to bound
the maximum load of the flow (see [8, Lemma 4], [9, Lemma 5.6], [10, Lemma 6.15])”—and
some newer examples—([16, Section 3], [12, Lemma 18], [13, Lemma 2.5 and Lemma 3.6]). The main task is to prove the critical lemmas (Lemma 11 and 12) for our new conditions (2–
6). To that end, we first list the fundamental details from [12]. We would like to mention that after submitting a preprint of an earlier version of this paper
on arXiv, Amanatidis and Kleer have contacted us, claiming to have proved that the degree
sequences studied here are also “strongly stable” (see [17]). The next 3 sections lay down the foundations of the swap Markov chain on bipartite and
directed degree sequences. These have been originally described in our own paper [12]. For
the sake of readability and convenience, we recall in more detail some results from paper [12],
as they are crucial in understanding the presented approach. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Definitions and useful facts Formally, there exists a sequence of realizations G = G0, . . ., Gi−1, Gi = H, such that for each
j = 0, . . ., i −1 there exists a swap operation which transforms Gj into Gj+1. We denote the
swap Markov chain as M ¼ ðG; PÞ where the transition matrix P is the following: In any realization with probability 1
2 we stay in the current state (i.e., the chain is lazy) and
with probability 1
2 we uniformly choose two-two vertices u1, u2;v1, v2 from classes U and V,
respectively. We perform the swap u1v1, u2v2 ) u1v2, u2v1 if u1v1, u2v2 2 E(G) and the resulting
graph G0 is simple. Otherwise we do not perform a move. The swap moving from G to G0 is
unique, therefore the jumping probability from G to G0 6¼ G is: ProbðG ! G0Þ :¼ PðG0jGÞ ¼
1
2
m
2
n
2
:
ð8Þ ð8Þ The transition probabilities are time- and edge-independent and are also symmetric. The
chain is lazy, therefore aperiodic. It is also reversible, and so its globally stable stationary distri-
bution is uniform. Now we turn our attention to the notions and notation to describe Theorems 4 and 5. Liter-
ally these theorems are about directed graphs, however, we will use the machinery developed
in the paper [12], turning these statements into theorems about bipartite graphs with some
restriction on which edges can be used in the realizations. Let ~G be a simple directed graph (parallel edges and loops are forbidden, but oppositely
directed edges between two vertices are allowed) with vertex set Xð~GÞ ¼ fx1; x2; . . . ; xng and
edge set Eð~GÞ. For every vertex xi 2 X we associate two numbers: the in-degree and the out-
degree of xi. These numbers form the directed degree bi-sequence D. We transform the directed graph ~G into the following bipartite representation: let
Bð~GÞ ¼ ðU; V; EÞ be a bipartite graph where each class consists of one copy of every vertex
from Xð~GÞ. The edges adjacent to a vertex ux in class U represent the out-edges from x,
while the edges adjacent to a vertex vx in class V represent the in-edges to x (so a directed
edge xy corresponds the edge uxvy). Definitions and useful facts In this section, we recall some well-known definitions and results, furthermore we define our
swap Markov chains for the bipartite degree sequences and for the directed degree sequences. In this section, we recall some well-known definitions and results, furthermore we define our
swap Markov chains for the bipartite degree sequences and for the directed degree sequences. Let G be a simple bipartite graph on U ] V, where U = {u1, . . ., un} and V = {v1, . . ., vm}, and swap Markov chains for the bipartite degree sequences and for the directed degree sequences. Let G be a simple bipartite graph on U ] V, where U = {u1, . . ., un} and V = {v1, . . ., vm}, and
let its bipartite degree sequence be g
g
Let G be a simple bipartite graph on U ] V, where U = {u1, . . ., un} and V = {v1, . . ., vm}, and
let its bipartite degree sequence be dðGÞ ¼ ðdðUÞ; dðVÞÞ ¼
ðdðu1Þ; . . . ; dðunÞÞ; ðdðv1Þ; . . . ; dðvmÞÞ
:
ð7Þ ð7Þ For a ac, bd ) ad, bc swap operation to be valid it is not enough that ac, bd 2 E(G) and ad,
bc =2 E(G), we also need that ad can be an edge in some realization. In other words, we need
that a and d are in different vertex classes. We will use the name chord for any vertex pair u, v
where uv can be an edge in a realization, even if we do not know or do not care whether it is an
edge or a non-edge in the current realization. We can reformulate the definition of the swap
operation: it can be done if ac, bd 2 E(G) and ad, bc are chords. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 4 / 20 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs Now denote by G the set of all possible realizations of the graphic bipartite degree sequence
(d(U), d(V)). Consider two different realizations, G and H, of this bipartite degree sequence. As we already mentioned in the introduction, it is a well-known fact that the first realization
can be transformed into the second one (and vice versa) with a sequence of swap operations. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Definitions and useful facts If a vertex has zero in- (respectively out-) degree in ~G,
then we delete the corresponding vertex from Bð~GÞ. (Actually, this representation is an old
trick used by Gale [5], but one can find it already in [2]). The directed degree bi-sequence D
gives rise to a bipartite degree sequence. Here we make good use of the notion of chords: since there are no loops in our directed
graph, there cannot be any (ux, vx) edge in its bipartite representation—these vertex pairs are
non-chords. It is easy to see that these forbidden edges form a forbidden (partial) matching F
in the bipartite graph Bð~GÞ, or in more general terms, in B(D). To make it easier to remember
the nature of restriction, we will denote this restricted bipartite degree sequence with ~d. We consider all realizations Gð~dÞ which avoid the non-chords from F. Now it is easy to see
that the bipartite graphs in Gð~dÞ are in one-to-one correspondence with the possible realiza-
tions of the directed degree bi-sequence. Consider now again our example about two oppositely oriented triangles, C3
! and C3
. Con-
sider the bipartite representations BðC3
!Þ and BðC3
Þ, and take their symmetric difference r. It contains exactly one alternating cycle (the edges come alternately from BðC3
!Þ and BðC3
Þ),
s.t. each vertex pair of distance 3 along the cycle in r forms a non-chord. Therefore, in this 5 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs alternating cycle a “classical” swap cannot be performed. To address this issue, we introduce
a new swap operation: we exchange all edges coming from BðC3
!Þ with all edges coming from
BðC3
Þ in one operation. The corresponding operation for directed graphs is exactly the triple
swap operation. In general: if the current symmetric difference r contains a length-6 alternating cycle C6
such that all opposite vertex pairs form non-chords, then we allow performing the correspond-
ing C6-swap. In this notation, the original swap should properly be called a C4-swap (for obvi-
ous reasons), but for the sake of simplicity we only write swap instead of C4-swap. By the
constraints posed by the forbidden partial matching, only a subset of all bipartite swaps can
be performed. Definitions and useful facts These swaps together with the possible C6-swaps we just defined are called the
F-compatible swaps or F-swaps for short. Lemma 6 ([18], [12]). The set GðBðDÞÞ ¼ Gð~dÞ of all realizations is irreducible under
F-swaps. We are ready to define our swap Markov chain ~
M ¼ ðGð~dÞ; PÞ for the restricted bipartite
degree sequence ~d. The transition (probability) matrix P of the Markov chain is defined as follows: let the cur-
rent realization be G. Then 1. with probability 1/2 we stay in the current state, so our Markov chain is lazy; 1. with probability 1/2 we stay in the current state, so our Markov chain is lazy; 2. with probability 1/4 we uniformly choose two-two vertices u1, u2;v1, v2 from classes U and
V respectively and perform the swap if it is possible; 2. with probability 1/4 we uniformly choose two-two vertices u1, u2;v1, v2 from classes U and
V respectively and perform the swap if it is possible; 3. finally, with probability 1/4 we choose three-three vertices from U and V and check whether
they form three pairs of forbidden chords. If this is the case, then we perform a C6-swap if it
is possible. 3. finally, with probability 1/4 we choose three-three vertices from U and V and check whether
they form three pairs of forbidden chords. If this is the case, then we perform a C6-swap if it
is possible. The swap moving from G to G0 is unique, therefore the probability of this transformation
(the jumping probability from G to G0 6¼ G) is: ProbðG !b G0Þ :¼ PðG0jGÞ ¼ 1
4
1
jUj
2
jVj
2
;
ð9Þ
ProbðG !c G0Þ :¼ PðG0jGÞ ¼ 1
4
1
jUj
3
jVj
3
:
ð10Þ ProbðG !b G0Þ :¼ PðG0jGÞ ¼ 1
4
1
jUj
2
jVj
2
;
ð9Þ ð9Þ and and ProbðG !c G0Þ :¼ PðG0jGÞ ¼ 1
4
1
jUj
3
jVj
3
:
ð10Þ ð10Þ (These probabilities reflect the fact that G0 should be derived from G by a C4-swap or by a
C6-swap). The probability of transforming G to G0 (or vice versa) is time-independent and
symmetric. Therefore, P is a symmetric matrix, where the entries in the main diagonal are
non-zero, but (possibly) distinct values. Definitions and useful facts Our Markov chain is irreducible (by Lemma 6), and
it is clearly aperiodic, since it is lazy. Therefore, as it is well-known, the Markov process ~
M is
reversible with the uniform distribution as the globally stable stationary distribution. The general properties of the swap Markov chain on bipartite
degree sequences The proofs of our theorems closely follow the proof of Theorem 10 in [12], which, in turn, is
based on the proof method developed in [10]. Suppose ~d is a directed degree sequence and
d is the degree sequence of bipartite representations corresponding to the realizations of ~d. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 6 / 20 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs As we saw earlier the sets of all realizations GðdÞ and Gð~dÞ are slightly different: while
Gð~dÞ GðdÞ but there are realizations in GðdÞ that contain edges which are forbidden in
the realizations in Gð~dÞ: However, the following reasoning from [12] applies to both bipar-
tite and directed degree sequences. Therefore the notation G is used to refer either of the
two realization sets. As we saw earlier the sets of all realizations GðdÞ and Gð~dÞ are slightly different: while
Gð~dÞ GðdÞ but there are realizations in GðdÞ that contain edges which are forbidden in
the realizations in Gð~dÞ: However, the following reasoning from [12] applies to both bipar-
tite and directed degree sequences. Therefore the notation G is used to refer either of the
two realization sets. Consider two realizations X; Y 2 G, and take the symmetric difference r = E(X)ΔE(Y). Now
for each vertex in the bipartite graph (U, V; r) the number of incident X-edges (= E(X)\E(Y))
and the number of the incident Y-edges are equal. Therefore r can be decomposed into alter-
nating circuits and later into alternating cycles. The way the decomposition is performed is
described in detail in Section 5 of the paper [10]. Here we just summarize the highlights: p p
j
g
g
First, we decompose the symmetric difference r into alternating circuits in all possible
ways. In each case we get an ordered sequence W1, W2, . . ., Wκ of circuits. Each circuit is
endorsed with a fixed cyclic order. Now we fix one circuit decomposition. Each circuit Wi in the ordered decomposition has a
unique alternating cycle decomposition: Wi ¼ Ci
1; Ci
2; . . . ; Ci
ki. (This unique decomposition is
a quite delicate point and was discussed in detail in Section 5.2 of the paper [10]). The ordered circuit decomposition of r together with the ordered cycle decompositions of
all circuits provide a well-defined ordered cycle decomposition C1, . . The general properties of the swap Markov chain on bipartite
degree sequences ., Cl of r. This decompo-
sition does not depend on any swap operations, only on the symmetric difference of realiza-
tions X and Y. This ordered cycle decomposition singles out l −1 different realizations H1, . . ., Hl−1 from
G with the following property: for each j = 0, . . ., l −1 we have E(Hj)ΔE(Hj+1) = Cj+1 if we
apply the notation H0 = X and Hl = Y. This means that EðHiÞ ¼ EðXÞ 4
[
i0i
EðCi0Þ
! : It remains to design a unique canonical path from X to Y determined by the circuit decom-
position, which uses the realizations Hj as milestones along the path. In other words, for each
pair Hj, Hj+1 we should design a swap sequence which turns Hj into Hj+1. Here we slightly abuse the general naming conventions: in the original canonical path
method for any pair X; Y 2 G exactly one X ! Y path is defined. In Sinclair’s multicommod-
ity flow method ([19]) a (usually large) set of paths is defined, equipped with a probability dis-
tribution. In our presentation we use the expression canonical path to denote these paths,
differentiating the paths in G and the paths in some realizations. So, the canonical path under construction is a sequence X ¼ G0; . . . ; Gi; . . . ; Gm ¼ Y of realizations, where each Gi can be derived from Gi−1 with exactly one swap operation, and
there exists an increasing subscript subsequence 0 = n0 < n1 < n2 < < nℓ= m, such that we
have Gnk ¼ Hk for every 0 k ℓ. of realizations, where each Gi can be derived from Gi−1 with exactly one swap operation, and
there exists an increasing subscript subsequence 0 = n0 < n1 < n2 < < nℓ= m, such that we
have Gnk ¼ Hk for every 0 k ℓ. The construction of swap sequences between consecutive
milestones It is 2 if the edge is missing from Z but exists in both X and Y. It is 1 if the edge exists in all
three graphs (X, Y, Z) or it is there only in one of X and Y but not in Z. Finally, it is 0 if the
edge is missing from all three graphs, or the edge exists in exactly one of X and Y and in Z. (Therefore, if an edge exists in exactly one of X and Y then the corresponding chord in bM is
always 0 or 1). It is easy to see that the row and column sums of bMðX þ Y ZÞ are the same (Therefore, if an edge exists in exactly one of X and Y then the corresponding chord in bM is
always 0 or 1). It is easy to see that the row and column sums of bMðX þ Y ZÞ are the same
as the row and column sums in MX (or MY, or MZ). Now we are ready to determine the F-swap sequence between G and G0 and this is the
point where realizations from GðdÞ and Gð~dÞ start behave slightly differently. From now on
we will work with realizations from Gð~dÞ but we will point out those turning points where
there are real differences. The first such difference is that in the case of a directed realization
G 2 Gð~dÞ there may be a vertex vi along the cycle C s.t. u1vi is a non-chord, while for a simple
bipartite realization G 2 GðdÞ this does not happen. We determine the F-swap sequence between G and G0 from Gð~dÞ through an iterative algo-
rithm. In the first iteration we check, step by step, the positions (u1, v2), (u1, v3), . . ., (u1, vℓ)
and take the smallest j for which (u1, vi) is an actual edge in G. Since (u1, vℓ) is an edge in G,
such an i always exists. A typical configuration is shown in Fig 1. We call the chord u1vi the start-chord of the current sub-process and u1v1 is the end-
chord. We will sweep the alternating chords along the cycle. The vertex u1 will be the corner-
stone of this operation. This process works from the start-chord u1vi, viui (non-edge), uivi−1
(an edge) toward the end-chord v1u1 (non-edge)—switching their status in twos and fours. The construction of swap sequences between consecutive
milestones Next we define the canonical path corresponding to the cycle Ci. The procedure described here
is slightly different from the one in [12], since the excluded edge set F in [12] is slightly larger
than the one used here. 7 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs For convenience, we will use the names G; G0 2 G instead of Hi and Hi+1. These two graphs
have almost the same edge set: ðEðGÞ n ðCi \ EðXÞÞÞ [ ðCi \ EðYÞÞ ¼
EðG0Þ
ðEðG0Þ n ðCi \ EðYÞÞÞ [ ðCi \ EðXÞÞ ¼
EðGÞ: We refer to the elements of Ci \ E(X) as X-edges, while the rest of the edges of Ci are Y-edge
We denote the cycle Ci by C, which has 2ℓedges and its vertices are u1, v1, u2, v2, . . ., uℓ, vℓ. Finally, w.l.o.g. we may assume that the chord u1v1 is a Y-edge (and, of course, vℓu1 is an X-
edge). We will build our canonical path from G towards G0. At any particular step, the last con-
structed realization is denoted by Z. (At the beginning of the process we have Z = G). We are
looking for the next realization, denoted by Z0. We will control the canonical path system with
an auxiliary structure, originally introduced by Kannan, Tetali and Vempala in [1]: The matrix MG denotes the adjacency matrix of the bipartite realization G where the rows
and columns are indexed by the vertices of U and V, respectively, with the slight alteration that
a position corresponding to a forbidden edge (a non-chord) is indicated with a . There is a
natural correspondence between the entries of matrices on U × V and the chords of G. Our
auxiliary structure is the matrix bMðX þ Y ZÞ ¼ MX þ MY MZ: Summation does not change the positions with a . Since the non- entries of a bipartite
adjacency matrix are 0 or 1, the possible entries of bM are , −1, 0, 1, 2. An entry is if it corre-
sponds to a forbidden edge, and it is −1 if the edge is missing from both X and Y but it exists in
Z. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 The construction of swap sequences between consecutive
milestones We
check positions u1vi−1, u1vi−2 (all are non-edges) and choose the first chord among them,
which we call the current-chord. (Since u1 belongs to at most one non-chord we never have to
check more than two positions to find a chord). which we call the current-chord. (Since u1 belongs to at most one non-chord we never have to
check more than two positions to find a chord). 8 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs Fig 1. Sweeping a cycle. https://doi.org/10.1371/journal.pone.0201995.g001 Fig 1. Sweeping a cycle. https://doi.org/10.1371/journal.pone.0201995.g001 https://doi.org/10.1371/journal.pone.0201995.g001 Case 1: As we just explained the typical situation is that the current-chord is the “next” one,
so when we start this is typically u1vi−1. Assume that this is a chord. Then we can proceed with
the swap operation vi−1ui, viu1 ) u1vi−1, uivi. We just produced the first “new” realization in
our sequence, this is G0
1. For the next swap operation this will be our new current realization. This operation will be called a single-step. In a realization Z we call a chord bad if its state in Z (being edge or non-edge) is different
from its state in G, or equivalently, different from its state in G0, since G and G0 differ only on
the chords along the cycle C (recall that in our nomenclature a chord is a pair of vertices which
may form an edge). After the previous swap, we have two bad chords in G0
1, namely u1vi−1 and
viu1. Consider now the auxiliary matrix bMðX þ Y ZÞ (here Z ¼ G0
1). As we saw earlier, any
chord not contained in C has the same state in X, Y and Z. Accordingly, the corresponding
matrix value is 0 or 1 in bM. We call a position bad in bM if this value is −1 or 2. (A bad position
in bM always corresponds to a bad chord). Since we switch the start-chord into a non-edge, it
may become 2 in bM (in case the start-chord is an edge in both X and Y). Furthermore, the cur-
rent-chord turned into an edge. If it is a non-edge in both X and Y then its corresponding
value in bM becomes −1. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 The construction of swap sequences between consecutive
milestones After this step, we have at most two bad positions in the matrix, at
most one with 2-value and at most one with −1-value. Finishing our swap operation, the previ-
ous current-chord becomes the new start-chord, so it is the edge u1, vi−1. Case 2: If the position below the start-chord (this is now u1vi−2) is a non-chord, then we
cannot produce the previous swap. Then the non-edge u1vi−3 is the current-chord. For sake of
simplicity we assume that i −3 = 2 so we are in Fig 1. (That is, i −1 = 4). Consider now the PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 9 / 20 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs alternating C6 cycle: u1, v2, u3, v3, u4, v4. It has altogether three vertex pairs which may be used
to perform an F-swap operation. We know already that u1v3 is a non-chord. If neither v2u4
nor u3 v4 are chords, then this alternating cycle provides an F-compatible circular C6-swap. Again, we found the valid swap v2u3, v3u4, v4u1 ) u1v2, u3v3, u4v4. After that we again have 2
bad chords, namely u1v2 and v4u1, and together we have at most two bad positions in the new
bMðX þ Y ZÞ, with at most one 2-value and at most one −1-value. Finally, if one position, say v2u4, is a chord then we can process this C6 with two swap oper-
ations. If this chord is, say, an actual edge, then we swap v2u4, v4u1 ) u1v2, u4v4. After this
we can take care of the v2, u3, v3, u4 cycle. Along this sequence we never create more than 3
bad chords: the first swap makes chords v2u4, v4u1, and u1v2 bad ones, and the second cures
v2u4 but does not touch u1v2 and v4u1. So, along this swap sequence we have 3 bad chords, and
in the end we have only 2. On the other hand, if the chord v2u4 is not an edge, then we can
swap v2u3, v3u4 ) u3v3, u4v2, creating one bad edge, then by swapping the four cycle u1, v2, u4,
v4 we cure v2u4 but we switch u1v2 and v4u1 into bad chords. We finished our double-step
along the cycle. In a double-step we create at most three bad chords. The construction of swap sequences between consecutive
milestones When the first swap uses three chords
along the cycle then we may have at most one bad chord (with bM-value 0 or −1) and then the
next swap switches back the chord into its original status, and makes two new bad chords
(with at most one 2-value and one −1-value). When the first swap uses only one chord from
the cycle, then it creates three bad chords (changing two chords into non-edges and one into
an edge), therefore it may create at most two 2-values and one −1-value. After the second
swap, there will be only two bad chords, with at most one 2-value, and at most one −1-value. When only the third position corresponds to a chord in our C6 then after the first swap we
may have two −1-values and one 2-value. However, after the next swap we will have at most
one of both types. After finishing our single- or double-step, the previous current-chord becomes the new
start-chord and we look for the new current-chord. Then we repeat our procedure. There is
one important point to be mentioned: along the step, the start-chord switches back into its
original status, so it will not be a bad chord anymore. So even if we face a double-step the num-
ber of bad chords will never be larger than three (together with the chord viu1 which is still in
the wrong state, so it is a bad chord), and we always have at most two 2-values and at most one
−1-value in bMðX þ Y ZÞ. −1-value in bMðX þ Y ZÞ. When our current-chord becomes v1u2 then the last step will switch back the last start-
chord into its correct state, and the last current-chord cannot be in a bad state. So, when we
finish our sweep from u1vi to v1u1, we will only have one bad chord (with a possible 2-value in
bM). This concludes the first iteration of our algorithm. For the next iteration, we seek a new start-chord between viu1 and vℓu1 and the chord viu1
becomes the new end-chord. We repeat our sweeping procedure until there are no more
unprocessed chords. Upon completion, we find a realization sequence from G to G0. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 The construction of swap sequences between consecutive
milestones Now we are ready to describe the following, highly technical theorem from [10] which is
required to show that the defined swap Markov-chains are rapidly mixing. Theorem 9 (Section 4 in [10]). If the designed canonical path system satisfies the three condi-
tions below, then the MCMC process is rapidly mixing. The conditions are: (Θ). For each i < l the constructed path Hi ¼ G0
0; G0
1; . . . ; G0
m0 ¼ Hiþ1 satisfies m0 c |Ci+1| for
a suitable constant c. (O). 8j there exists a realization Kj 2 VðGÞ s.t. d MX þ MY MG0
j; MKj
O2, where MG is
the bipartite adjacency matrix of G, d denotes the Hamming distance, and O2 is a small
constant. (O). 8j there exists a realization Kj 2 VðGÞ s.t. d MX þ MY MG0
j; MKj
O2, where MG is
the bipartite adjacency matrix of G, d denotes the Hamming distance, and O2 is a small
constant. (O). 8j there exists a realization Kj 2 VðGÞ s.t. d MX þ MY MG0
j; MKj
O2, where MG is
the bipartite adjacency matrix of G, d denotes the Hamming distance, and O2 is a small
constant. (O). 8j there exists a realization Kj 2 VðGÞ s.t. d MX þ MY MG0
j; MKj
O2, where MG is
the bipartite adjacency matrix of G, d denotes the Hamming distance, and O2 is a small
constant. (X). For each vertex G0
j in the path being traversed the following three objects together uniquely
determine the realizations X, Y and the path itself: • The auxiliary matrix MX þ MY MG0
j, • The auxiliary matrix MX þ MY MG0
j, • the symmetric difference r = E(X)4E(Y), • the symmetric difference r = E(X)4E(Y), • and a polynomial size parameter set B. • and a polynomial size parameter set B. • and a polynomial size parameter set B. The meaning of condition (X) is that these structures can be used to control certain features
of the canonical path system; namely, their numbers give a bound on the number of canonical
paths between any realization pair X, Y which traverses G0
j. The construction of swap sequences between consecutive
milestones If in the
first sweep we had a double-step, then such a step will never occur later, so altogether with the
(new) bad end-chord we never have more than three bad chords (corresponding to at most
two 2-values and at most one −1-value). However, if the double-step occurs sometime later, for example in the second sweep, then
we face to the following situation: if we perform a circular C6-swap, then all together we have
at most two 2-values and one −1-value. Thus, we may assume that there is a chord suitable for
a swap in our C6. If this chord is a non-edge, then the swap around it produces one bad chord,
and at most one bad position in bM. The only remaining case is when that chord is an edge. After the first swap there will be four bad chords, and there may be at most three 2-values and After the first swap there will be four bad chords, and there may be at most three 2-values and PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 10 / 20 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs at most one −1 value. However, after the next swap (finishing the double step) we annihilate
one of the 2-values, and after that swap there are at most two 2-values and at most one
−1-value along the entire swap sequence. When we finish our second sweep, then chord vi u1
will be switched back into its original state and it will not be bad anymore. Iteratively applying the algorithm, the entire cycle C is processed after at most ℓsweep
sequences. This finishes the construction of the required F-swap sequence (and the required
realization sequence). Meanwhile we also proved the following important observations: Lemma 7. For the Markov chain M, we always have at most two 2-values and at most one
−1-value in our auxiliary matrix bMðX þ Y ZÞ along our procedure. −1-value in our auxiliary matrix bMðX þ Y ZÞ along our procedure. ~ Lemma 8. For the Markov chain ~
M, each auxiliary matrix bMðX þ Y ZÞ occurring along
our procedure is at most swap-distance one from a matrix with at most three bad positions: with
at most two 2-values and with at most one −1-value in the same column. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 The analysis of the swap sequences between milestones in M In this section, we will analyze the undirected case. We introduce the new switch operation on
integer matrices: we fix the four corners of a submatrix, and we add 1 to two corners in a diag-
onal, and add −1 to the corners on the other diagonal. This operation clearly does not change
the column and row sums of the matrix. (We will use this operation on adjacency matrices or
on auxiliary matrices of realizations). For example, if we consider the adjacency matrix MG of
a realization of d and make a valid swap operation, then this is equivalent to a switch in this
matrix. The next statement is trivial but very useful: Claim 10. If two matrices have switch-distance 1, then their Hamming distance is 4. Conse-
quently, if the switch-distance is c then the Hamming distance is bounded by 4c. The next lemma shows that property (O) holds for the auxiliary matrices along the
swap sequence from G toward G0 for degree sequences corresponding to Theorem 3 and
Theorem 2. Lemma 11. For any realization Z along the constructed swap sequence from G to G0 in GðdÞ
there exists a realization K = K(Z) such that dð bMðX þ Y ZÞ; MKÞ 16: Proof. The swap sequence transforming G to G0 only touches chords induced by VðCÞ. Therefore the row and column sums in A[Z] are the same as that of A[G] so the cornerstone Proof. The swap sequence transforming G to G0 only touches chords induced by VðCÞ. Proof. The swap sequence transforming G to G0 only touches chords induced by VðCÞ. Therefore, the row and column sums in A[Z] are the same as that of A[G], so the cornerstone
has the minimum row sum in A[Z] as well. Any entries of 2’s and −1’s in bM are in the row of u1, moreover, they are contained in A[Z]. Suppose bMu1;vj ¼ 2. The sum of entries of A[Z] in the column vj is < jU \ VðCÞj ¼ jV \ VðCÞj,
therefore 9 uk 2 U \ VðCÞ such that bMuk;vj ¼ 0. Since the sum of the entries in row u1 is mini-
mum among the rows of A[Z], there must 9vl 2 V \ VðCÞ such that bMuk;vl > bMu1;vl. Obviously,
bMuk;vl < 2, so bMu1;vl 2 f0; 1g. The analysis of the swap sequences between milestones in M The switch operation u1vj, ukvl ) u1vl, ukvj (decrease the
entries of the matrix by one at positions u1 vj and uk vl, and increase the entries at positions u1vl
and ukvj by one) in bM (and in A[Z]) eliminates the entry of 2 at u1vj, and creates an entry of 1 at
both u1vj and ukvj. In the column vl three scenarios are possible: either the entry −1 and a 0
exchange their positions, or a 0 and a 1 exchange their positions; finally, it is also possible that
the −1 and a 1 both become 0. By repeating the previous argument, we may eliminate one more entry 2, if necessary, from
A[Z]. (Recall that at the beginning we had at most two 2s in bM). Although it is possible that
the entry −1 is not in the u1-row anymore, it does not cause any hardship. Let bM0 be the matrix
we get after performing these at most two switches that eliminate the 2’s. Each entry of bM 0 is a
0 or a 1, except at most one −1 entry. The proof now diverges into two cases corresponding to Theorem 2 and Theorem 3,
respectively. The construction of swap sequences between consecutive
milestones Condition (O) implies that the space of auxiliary matrices is larger than VðGÞ by a multipli-
cative factor of at most ðnmÞ
2O2. To use this theorem we have to show that the defined swap sequences between Hi and Hi+1,
using the cornerstone u1 chosen in (F), satisfy conditions (Θ), (O), and (X) of Theorem 9. The
first one is easy to see, since we can process any cycle of length 2ℓin ℓ−1 swaps. Therefore,
we may choose c = 1 in (Θ). Condition (X) holds for the same reason as it holds in paper [12]. Thus only condition (O) remains to be checked. Until this very moment the choice of the cornerstone vertex u1 was arbitrary. Before we
turn to the analysis of the swap sequences, we choose which particular vertex of the cycle C will
serve as its cornerstone. Let the submatrix A contain those positions from any adjacency or any auxiliary matrix
which correspond to the positions ui vj defined by the vertices from C. Furthermore, denote by
A[Z] the submatrix of bMðX þ Y ZÞ spanned by the vertices of C. Then: PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 11 / 20 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs (F). Let u1 be a vertex which has the lowest row sum in the submatrix A[Hi] = A[G]. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 of d. Otherwise, observe that of d. Otherwise, observe that
ðu; vÞ
u 2 U n fu0; u1g; v 2 V n fv0; v1g; bM0
u;v ¼ bM0
u1;v ¼ bM0
u;v1 ¼ 1
ðdðv1Þ 2Þ ðD 1Þ þ ðdðu1Þ 2Þ ðD 1Þ
2ðD 1ÞðD 2Þ: The number of entries of 1 in bM0 that are incident on the same row as u0 or u1, on the same
column as v0 or v1, or in the above counted set is at most 2ðD þ 1Þ þ 2ðD 1Þ 1 þ 2ðD 1ÞðD 2Þ 2D2 2D þ 3 < jEj: The last inequality follows from Equality 1. Therefore there exists u2 2 U, v2 2 V such that
{u0, v0, u1, v1, u2, v2} is a set of 6 vertices where bM0
u2;v2 ¼ 1 and bM0
u2;v1 ¼ bM0
u1;v2 ¼ 0. Switching
along the six positions determined by the cyclically successive pairs, we get an adjacency
matrix corresponding to a realization of d. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Case of Theorem 2: Sequence d satisfies Eq 1 Suppose that bM0
u0;v0 ¼ 1. Since both u0 and v0 are at least 1, there 9v1 2 V and 9u1 2 U such
that bM 0
u0;v1 ¼ bM0
u1;v0 ¼ 1. If bM 0
u1;v1 ¼ 0, there is a switch which transforms bM0 into a realization 12 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs . Otherwise, observe that Case of Theorem 3: Sequence d satisfies Eqs 2 and 3 bM 0 is shown; each of the entries in the regions marked with 0/1 may be 0 or 1. Case of Theorem 3: Sequence d satisfies Eqs 2 and 3 From now on we will consider the entire matrix bM0 and not only A. Suppose that bM 0
u0;v0 ¼ 1. Let U0 ¼ fu 2 U j bM0
u;v0 ¼ 1g and V0 ¼ fv 2 V j bM0
u0;v ¼ 1g. If 9(u, v) 2 U0 × V0 such that
bM0
u;v ¼ 0, then switch operation u0v, uv0 ) u0v0, uv transforms bM 0 into an adjacency matrix. Suppose from now on, that 8(u, v)2U0 × V0 we have bM0
u;v ¼ 1. Let Suppose from now on, that 8(u, v)2U0 × V0 we have bM0
u;v ¼ 1. Let U00 ¼ fu 2 U j 9v 2 V0 :
bM0
u;v ¼ 0g;
V00 ¼ fv 2 V j 9u 2 U0 :
bM0
u;v ¼ 0g: Clearly, U@ \ U0 = V@ \ V0 = ;. Suppose there 9(u2, v2) 2 U@ × V@ such that bM0
u2;v2 ¼ 1. By
definition, there 9(u1, v1) 2 U0 × V0 such that bM0
u2;v1 ¼ 0 and bM0
u1;v2 ¼ 0. Clearly, applying first
the switch operation u1, u2 and v1, v2, and then the operation u0, u1 and v0, v1 transforms bM 0
into an adjacency matrix. Lastly, suppose that 8(u, v)2U@ × V@ we have bM0
u;v ¼ 0. This case is shown in Fig 2. In addition to the zeroes in U@ × V@, bM0
u;v0 ¼ 0 for any u 2 U@. We have jU00j ðm d1Þ
ðu; vÞ 2 U00 V j bM 0
u;v ¼ 0
¼
¼ jU00 V00j þ jU00j þ
ðu; vÞ 2 U00 ðV n V00 n fv0gÞj bM0
u;v ¼ 0
:
ð11Þ ð11Þ The right-hand side can be estimated from below as follows. Since the row and column
sums of bM0 are the same as that of MX, we have jU0j c1 bM0
u0;v0 ¼ c1 þ 1;
and
jV0j d1 bM 0
u0;v0 ¼ d1 þ 1: 13 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs Fig 2 bM 0 is shown; each of the entries in the regions marked with 0/1 may be 0 or 1 Fig 2. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Further simplifying: jU00j ðjV00j þ 1Þðc2 c1 1Þ þ ðc1 þ 1 c2Þm þ ðd1 þ 1Þn ðc1 þ 1Þðd1 þ 1Þ: Since we may suppose that c2 c1 + 2, we can substitute |V@| m −d2 and |U@| n −c1 −2
into the inequality, yielding Since we may suppose that c2 c1 + 2, we can substitute |V@| m −d2 and |U@| n −c1 −2
into the inequality, yielding ðc2 c1 1Þðd2 d1 1Þ d1ðn c2Þ þ 1: Symmetrically, a similar derivation gives ðc2 c1 1Þðd2 d1 1Þ c1ðn d2Þ þ 1: The last two inequalities clearly contradict the assumptions of this claim. In summary, in every case there exist at most 4 switches which transform bM into a 0 −1
matrix, which is a matrix with suitable row- and column sums, therefore it is the adjacency
matrix of a realization K of the degree sequence d. Fig 2. bM 0 is shown; each of the entries in the regions marked with 0/1 may be 0 or 1. https://doi.org/10.1371/journal.pone.0201995.g002 For any v 2 V0 and u 2 U\U@, we have bM0
u;v ¼ 1. Also, for any u 2 U0 and v 2 V\V@, we have
bM0
u;v ¼ 1. Therefore bM0
u;v ¼ 1. Therefore n c1 2 jU n U0 n fu0gj jU00j n c2;
m d1 2 jV n V0 n fu0gj jV00j m d2: Clearly, if c2 c1 + 1 or d2 d1 + 1 (i.e., G is almost half-regular), we already have a contra-
diction. We also have
ðu; vÞ 2 U00 ðV n V00 n fv0gÞj bM0
u;v ¼ 0
ðn c2Þðm jV00j 1Þ jU n U0 n U00j jV n V0 n V00 n fv0gj
ðn c2Þðm jV00j 1Þ ðn c1 1 jU00jÞ ðm d1 2 jV00jÞ:
ð12Þ ð12Þ PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018
14 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 14 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs Combining Eqs 11 and 12, ombining Eqs 11 and 12,
jU00j ðm d1Þ ðn c2Þ ðm jV00j 1Þ þ jU00jðm d1 1Þþ
þ ðjV00j þ 1Þ ðn c1 1Þ ðn c1 1Þ ðm d1 1Þ: The analysis of the swap sequences between milestones in ~
M Now we turn to discussing the directed case. As in the previous section, condition (O) is the
only remaining assumption of Theorem 9 which does not immediately follow from the con-
struction of swap sequences between consecutive milestones. Lemma 12. For any realization Z along the constructed swap sequence from G to G0 there
exists a realization K = K(Z) such that d
bMðX þ Y ZÞ; MK
20: Proof. As described by Lemma 8, it is possible that realization Z is derived by an F-swap
which is a first C4-swap to resolve an alternating C6 cycle along the sweep. It may introduce an
extra 2-value and/or a −1-value into the auxiliary structure. But Lemma 8 also shows that the
next C4 swap will revert these extra bad positions. Therefore let ZS denote the realization Z itself
if this extra swap is not needed, or the new realization if it is needed. Then bMðX þ Y ZSÞ has
at most two entries of 2 and at most one entry of −1. Now we have to show that there is a reali-
zation K such that d
bMðX þ Y ZSÞ; MK
16: As before we will use the shorthand bMðX þ Y ZSÞ ¼ bM: As before we will use the shorthand bMðX þ Y ZSÞ ¼ bM: If for all vl 2 V \ VðCÞ either bM uk;vl bMu1;vl or bMuk;vl ¼ or bMu1;vl ¼ holds: since the
sum of the entries in row u1 is minimum among the rows of A[Z], this is only possible if
there exist vl0, vl@ 2 V \ V(C) such that bMu1;vl0 ¼ , bMuk;vl0 ¼ 1, bMu1;vl00 ¼ 1, bMuk;vl00 ¼ ,
and for vl 2 V \ VðCÞ n fvj; vl0; vl00g we have bMuk;vl ¼ bM u1;vl. This is shown on Fig 3. If the second case applies, the first case must hold if we replace k by k0; if not, column vl@
would contain two , a contradiction. If the second case applies, the first case must hold if we replace k by k0; if not, column vl@
would contain two , a contradiction. By repeating the previous argument, we may eliminate one more entry 2, if necessary, from
A[Z] (and bM). (Recall that at the beginning we had at most two 2’s in bM). Although it is possi-
ble that the entry −1 is not in the u1-row anymore, it does not cause any hardship. Let bM0 be
the matrix we get after performing these at most two switches that eliminate the 2’s. Each entry
of bM0 is a 0 or a 1, except at most one −1 entry. The proof now diverges into two cases corresponding to Theorem 4 and Theorem 5,
respectively. As before we will use the shorthand bMðX þ Y ZSÞ ¼ bM: The swap sequence transforming the bipartite representation G to G0 (also, the previous extra
swap) only touches chords induced by VðCÞ. Therefore, the row and column sums in A[Z] are
the same as that of A[G], so the cornerstone has the minimum row sum in A[Z] as well. Any entries of 2’s and −1’s in bM are in the row of u1, moreover, they are contained in A[Z]. Suppose bMu1;vj ¼ 2. The column of vj in A[Z] contains at least one zero, therefore there exist
two vertices uk; uk0 2 U \ VðCÞ such that bMuk;vj ¼ 0 and bM uk0 ;vj ¼ 0, even if there is a in the
column of vj. We have two cases. 15 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs 1. There 9vl 2 V \ VðCÞ such that bMuk;vl > bMu1;vl: obviously, bMuk;vl < 2, so bMu1;vl 2 f0; 1g. The switch operation u1vj, ukvl ) u1vl, ukvj (decrease the entries of the matrix by one at
positions u1vj and ukvl, and increase the entries at positions u1vl and ukvj by one) in bM
(and in A[Z]) eliminates the entry of 2 at u1vj, and creates an entry of 1 at both u1vj and
ukvj. In the column vl three scenarios are possible: either the entry −1 and a 0 exchange their
positions, or a 0 and a 1 exchange their positions; finally, it is also possible that the −1 and a
1 both become 0. 2. If for all vl 2 V \ VðCÞ either bM uk;vl bMu1;vl or bMuk;vl ¼ or bMu1;vl ¼ holds: since the
sum of the entries in row u1 is minimum among the rows of A[Z], this is only possible if
there exist vl0, vl@ 2 V \ V(C) such that bMu1;vl0 ¼ , bMuk;vl0 ¼ 1, bMu1;vl00 ¼ 1, bMuk;vl00 ¼ ,
and for vl 2 V \ VðCÞ n fvj; vl0; vl00g we have bMuk;vl ¼ bM u1;vl. This is shown on Fig 3. 2. https://doi.org/10.1371/journal.pone.0201995.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Case of Theorem 4: Sequence d satisfies Eq 4 From now on we will consider the entire matrix bM0 and not only A. Suppose that bM 0
u0;v0 ¼ 1. The degrees of u0 and v0 are at least one, and so there are at least two entries of 1’s in the row Fig 3. bM is shown for Case 2 of the proof of Lemma 12. https://doi.org/10.1371/journal.pone.0201995.g003
.1371/journal.pone.0201995
August 13, 2018
16 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 16 / 20 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs and column of u0 and v0, respectively. Therefore there exists u1 2 U and v1 2 V such that they
are not copies of the same original vertex and bM0
u0;v1 ¼ bM0
u1;v0 ¼ 1. If bM0
u1;v1 ¼ 0, there is a
switch which transforms bM0 into a bipartite realization of ~d. Otherwise bM0
u1;v1 ¼ 1, so observe
that
ðu; vÞju 2 U n fu0; u1g; v 2 V n fv0; v1g; bM0
u;v ¼ 1;
bM0
u1;v ¼ 0 or bM0
u;v1 ¼ 0
ðdinðv1Þ 2Þ ðD 1Þ þ ðdoutðu1Þ 2Þ ðD 1Þ
2ðD 1ÞðD 2Þ: The number of entries of 1 in bM0 that are incident on either the rows of u0 or u1, on the col-
umn of v0 or v1, or on a position corresponding to an element of the above counted set is at
most 2ðD þ 1Þ þ 2ðD 1Þ þ 2ðD 1ÞðD 2Þ 2D2 2D þ 4: This implies that there exist u2 2 U and v2 2 V such that bM0
u2;v2 ¼ 1, bM 0
u1;v2 6¼ 1, and
bM0
u2;v1 6¼ 1. Because Equality 4 claims j~Ej is strictly larger than the right hand side by at least
2Δ, we may also assume that bM 0
u1;v2 6¼ , and bM0
u2;v1 6¼ . Therefore there exists u2 2 U, v2 2 V
such that {u0, v0, u1, v1, u2, v2} is a set of 6 vertices where bM0
u2;v2 ¼ 1 and bM 0
u2;v1 ¼ bM0
u1;v2 ¼ 0. Switching along the six positions determined by the cyclically successive pairs, we get an adja-
cency matrix corresponding to a realization of d. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Case of Theorem 5: Sequence d satisfies Eqs 5 and 6 Combining these inequalities, we get jU00j ðn d1Þ ðjU00 V00j jV00jÞ þ jU00jþ
þðn c2Þðn jV00j 1Þ jU n U0 n U00j jV n V0 n V00 n fv0gj: A few lines of computation similar to those in the previous section give A few lines of computation similar to those in the previous section give ðc2 c1Þðd2 d1Þ d1ðn c2 1Þ þ 3 þ c1 þ d2 n: Symmetrically, we also have Symmetrically, we also have ðc2 c1Þðd2 d1Þ c1ðn d2 1Þ þ 3 þ d1 þ c2 n: The last two inequalities clearly contradict the assumptions of this claim. In summary, in every case there exist at most 4 switches which transform bM into a 0 −1
matrix, which is a matrix with suitable row- and column sums, therefore it is the adjacency
matrix of a realization K of the degree sequence ~d. Case of Theorem 5: Sequence d satisfies Eqs 5 and 6 Since the row and column sums of bM0 are the same as that of MX, we have jU0j c1 bM0
u0;v0 ¼ c1 þ 1;
and
jV0j d1 bM 0
u0;v0 ¼ d1 þ 1: jU0j c1 bM0
u0;v0 ¼ c1 þ 1;
and
jV0j d1 bM 0
u0;v0 ¼ d1 þ 1: Also, n c1 2 jU n U0 n fu0gj jU00j n c2 1;
n d1 2 jV n V0 n fu0gj jV00j n d2 1: Clearly, if c2 = c1 or d2 = d1 (i.e., G is half-regular), we already have a contradiction. Each
column in V \ V@ may contain at most one , therefore
ðu; vÞ 2 U00 ðV n V00 n fv0gÞj bM 0
u;v 2 f0; g
ðn c2Þðn jV00j 1Þ jU n U0 n U00j jV n V0 n V00 n fv0gj: Moreover, bM0
u;v0 2 f0; g for any u 2 U@. Combining these inequalities, we get
jU00j ðn d1Þ ðjU00 V00j jV00jÞ þ jU00jþ
þðn c2Þðn jV00j 1Þ jU n U0 n U00j jV n V0 n V00 n fv0gj: Moreover, bM0
u;v0 2 f0; g for any u 2 U@. Combining these inequalities, we get
jU00j ðn d1Þ ðjU00 V00j jV00jÞ þ jU00jþ
þðn c2Þðn jV00j 1Þ jU n U0 n U00j jV n V0 n V00 n fv0gj: Moreover, bM0
u;v0 2 f0; g for any u 2 U@. Case of Theorem 5: Sequence d satisfies Eqs 5 and 6 From now on we will consider the entire matrix bM0 and not only A. Suppose that bM 0
u0;v0 ¼ 1. Let U0 ¼ fu 2 U j bM0
u;v0 ¼ 1g and V0 ¼ fv 2 V j bM0
u0;v ¼ 1g. If 9(u, v)2U0 × V0 such that
bM0
u;v ¼ 0, then switch operation u0v, uv0 ) u0v0, uv transforms bM0 into an adjacency matrix. Suppose from now on, that 8(u, v)2U0 × V0 we have bM0
u;v 2 f1; g. Let U00 ¼ fu 2 U j 9v 2 V0 :
bM0
u;v ¼ 0g;
V00 ¼ fv 2 V j 9u 2 U0 :
bM0
u;v ¼ 0g: Clearly, U@ \ U0 = V@ \ V0 = ;. Suppose bM0½U00; V00 contains more than |U@| entries of
1’s. A simple pigeon-hole principle argument implies that there 9(u2, v2) 2 U@ × V@ and
9(u1, v1) 2 U0 × V0 such that bM0
u2;v2 ¼ 1, bM 0
u2;v1 ¼ 0, bM0
u1;v2 ¼ 0, and M0
u1;v1 ¼ 1. Clearly,
applying first the switch operation u1, u2 and v1, v2, and then the operation u0, u1 and v0, v1
transforms bM0 into an adjacency matrix. Lastly, suppose that bM0½U00; V00 contains at most |U@| entries of 1’s. We have jU00j ðn d1Þ jU00j
ðu; vÞ 2 U00 V j bM 0
u;v ¼ 0
ðjU00 V00j jU00jÞ þ
ðu; vÞ 2 U00 ðV n V00Þj bM0
u;v 2 f0; g
jU00j; since bM0½U00; V contains exactly |U@| of . The right-hand side can be estimated from below as since bM0½U00; V contains exactly |U@| of . The right-hand side can be estimated from below as PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 17 / 20 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs follows. Since the row and column sums of bM0 are the same as that of MX, we have follows. PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 p
For completeness sake, we also state the respective theorem for directed random graphs. For completeness sake, we also state the respective theorem for directed random grap Corollary 14. If ~D is a directed Erdős-Rényi random graph on n vertices with out-edge proba-
bility p(n), such that 3
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
logn þ 1
2 log2
n
v
u
u
t
þ 2ffiffiffin
p
pðnÞ 1 3
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
logn þ 1
2 log2
n
v
u
u
t
2ffiffiffin
p ; then the swap Markov chain is rapidly mixing on the directed degree sequence of ~D with probabil-
ity at least 1 2
n. Comparing the applicability of the Theorems Next we discuss the applicability of our results. Theorem 2 (and the result of Greenhill and
Sfragara) is not applicable, when the average degree d > n
2. In case of Theorem 3 there is no
such region: for example if all degrees are between n
3 þ 1 and 2n
3 1, then inequality d > n
2 for
the average degree is possible. It is also easy to see that Theorem 3 applies to all half-regular
(consequently for all regular) bipartite degree sequences. However, if the degrees are evenly distributed between 1 and n
4, then Theorem 2 applies
while Theorem 3 does not. Therefore these results have different validity regions and they are
independent from each other. Generally speaking Theorem 2 and the Greenhill and Sfragara results are better applicable
to degree sequences developed under some scale-free random dynamics (with γ > 2.5), while
Theorem 3 is better fitted to degree sequences developed under the Erdős—Re´nyi model. For directed degree sequences similar analysis applies. Acknowledgments The authors of this paper were supported in part by the National Research, Development and
Innovation Office—NKFIH grant K 116769 and KH 126853. IM was supported in part by the
National Research, Development and Innovation Office—NKFIH grant SNN 116095. DS was
supported in part by the National Research, Development and Innovation Office—NKFIH
grants K 108947 and K 120706. A preliminary version of this paper can be found as arXiv
1712.01709v2. Erdős-Re´nyi random graphs The following statement is a straightforward, easy consequence of Theorem 3. Corollary 13. If G is a bipartite Erdős-Rényi random graph on vertex classes of size n and m,
with edge probability p(n, m), such that 3
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
logm þ 1
2 log2
n
v
u
u
t
pðn; mÞ 1 3
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
logn þ 1
2 log2
m
v
u
u
t
; then the swap Markov chain is rapidly mixing on the bipartite degree sequence of G with proba-
bility at least 1 1
n 1
m. (The roles of m and n can be interchanged). Proof. Let p = p(n, m), εc ¼ 1
3 pn and εd ¼ 1
3 ð1 pÞm. Also, let c1 = pn −εc, c2 = pn+ εc, d1 =
pm −εd, d2 = pm+ εd. Eq 3 holds, we only need to check that 4εcεd ðpn εcÞ ðm pm εdÞ: PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 18 / 20 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs Moreover, by Hoeffding’s inequality, Moreover, by Hoeffding’s inequality, Pr ðEquation 2 does not holdÞ
Pr ð9v 2 V jdðvÞ pnj > εcÞ þ Pr ð9u 2 U jdðuÞ pmj > εdÞ
m 2e 2p2n=9 þ n 2e 2ð1 pÞ2m=9 1
m þ 1
n ; which proves the statement. which proves the statement. p
For completeness sake, we also state the respective theorem for directed random graphs. References 1. Kannan R., Tetali P., Vempala S.: Simple Markov-chain algorithms for generating bipartite graphs and
tournaments, Extended abstract, in Proc. SODA’97 (1997), 193–200. ISBN:0-89871-390-0 2. Petersen J.: Die Theorie der regularen Graphen, Acta Math. 15 (1891), 193–220. https://doi.org/10. 1007/BF02392606 3. Havel V.: A remark on the existence of finite graphs. (in Czech), Časopis Pěst. Mat. 80 (1955), 477–
480. 4. Hakimi S.L.: On the realizability of a set of integers as degrees of the vertices of a simple graph. J. SIAM
Appl. Math. 10 (1962), 496–506. https://doi.org/10.1137/0110037 5. Gale D.: A theorem on flows in networks, Pacific J. Math. 7(2) (1957), 1073–1082. https://doi.org/10. 2140/pjm.1957.7.1073 6. Kleitman D.J., Wang D.L.: Algorithms for constructing graphs and digraphs with given valences and fac-
tors, Discrete Math. 6 (1973), 79–88. https://doi.org/10.1016/0012-365X(73)90037-X 7. Erdős P.L., Miklo´s I., Toroczkai Z.: A simple Havel-Hakimi type algorithm to realize graphical degree
sequences of directed graphs, Elec. J. Combinatorics 17(1) (2010), R66 (10 pp) 8. Cooper C., Dyer M., Greenhill C.: Sampling regular graphs and a peer-to-peer network, Comb. Prob. Comp. 16(4) (2007), 557–593. https://doi.org/10.1017/S0963548306007978 9. Greenhill C.: A polynomial bound on the mixing time of a Markov chain for sampling regular directed
graphs, Elec. J. Combinatorics 18 (2011), #P234. 10. Miklo´s I., Erdős P.L., Soukup L.: Towards random uniform sampling of bipartite graphs with given
degree sequence, Electronic J. Combinatorics 20(1) (2013), #P16, 1–49. 11. Erdős P.L., Miklo´s I., Toroczkai Z.: A decomposition based proof for fast mixing of a Markov chain over
balanced realizations of a joint degree matrix, SIAM J. Disc. Math. 29(1) (2015), pp. 481–499. https://
doi.org/10.1137/130929874 12. Erdős P.L., Kiss Z.S., Miklo´s I., Soukup L.: Approximate Counting of Graphical Realizations, PLOS
ONE (2015), pp 20. #e0131300. 13. Greenhill C., Sfragara M.: The switch Markov chain for sampling irregular graphs, Theoretical Comp. Sci 719 (2018), 1–20. https://doi.org/10.1016/j.tcs.2017.11.010 14. Berger A., Mu¨ller–Hannemann M.: Uniform sampling of digraphs with a fixed degree sequence, in
Graph Theoretic Concepts in Computer Science, LNCS 6410 Springer, Berlin, (2010), 220–231. 15. LaMar, M.D.: Directed 3-Cycle Anchored Digraphs And Their Application In The Uniform Sampling Of
Realizations From A Fixed Degree Sequence, in ACM & IEEE & SCS Proc. of 2011 Winter Simulation
Conference (Eds. S. Jain, R.R. Creasey et. al). (2011), 1–12. 16. Greenhill, C.: The switch Markov chain for sampling irregular graphs, in Proc. 26th ACM-SIAM Sympo-
sium on Discrete Algorithms, New York-Philadelphia (2015), pp. 1564–1572. 17. Author Contributions Writing – original draft: Pe´ter L. Erdős, Tama´s Ro´bert Mezei, Istva´n Miklo´s, Da´niel Solte´sz. Writing – review & editing: Pe´ter L. Erdős, Tama´s Ro´bert Mezei, Istva´n Miklo´s, Da´niel
Solte´sz. Writing – original draft: Pe´ter L. Erdős, Tama´s Ro´bert Mezei, Istva´n Miklo´s, Da´niel Solte´sz. 19 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 Efficiently sampling the realizations of bounded, irregular degree sequences of bipartite and directed graphs PLOS ONE | https://doi.org/10.1371/journal.pone.0201995
August 13, 2018 References Amanatidis, G., Kleer, P.: Rapid Mixing of the Switch Markov Chain for Strongly Stable Degree
Sequences, arXiv preprint 1803.01338 (2018), 1–21. 18. Erdős P.L., Kira´ly Z., Miklo´s I.: On graphical degree sequences and realizations, Combinatorics, Proba-
bility and Computing (2013), 1–22. 19. Sinclair A.: Improved bounds for mixing rates of Markov chains and multicommodity flow, Combin. Pro-
bab. Comput. 1 (1992), 351–370. https://doi.org/10.1017/S0963548300000390 20 / 20
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https://amb-express.springeropen.com/counter/pdf/10.1186/s13568-019-0809-2
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English
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An extreme halophilic xylanase from camel rumen metagenome with elevated catalytic activity in high salt concentrations
|
AMB express
| 2,019
|
cc-by
| 9,756
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© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. Abstract An extreme halophilic xylanase, designated as XylCMS, was characterized by cloning and expression of the encoding
gene from a camel rumen metagenome. XylCMS proved to be a GH11 xylanase with high identity to a hypotheti-
cal glycosyl hydrolase from Ruminococcus flavefaciens. XylCMS with a molecular weight of about 47 kDa showed
maximum activity at pH 6 and 55 °C. The enzyme activity was significantly stimulated by NaCl in 1–5 M concentra-
tions. Interestingly, the optimum temperature was not influenced by NaCl but the Kcat of the enzyme was enhanced
by 2.7-folds at 37 °C and 1.2-folds at 55 °C. The Km value was decreased with NaCl by 4.3-folds at 37 °C and 3.7-folds
at 55 °C resulting in a significant increase in catalytic efficiency (Kcat/Km) by 11.5-folds at 37 °C and 4.4-folds at 55 °C. Thermodynamic analysis indicated that the activation energy (Ea) and enthalpy (∆H) of the reaction were decreased
with NaCl by 2.4 and threefold, respectively. From the observations and the results of fluorescence spectroscopy, it
was concluded that NaCl at high concentrations improves both the flexibility and substrate affinity of XylCMS that are
crucial for catalytic activity by influencing substrate binding, product release and the energy barriers of the reaction. XylCMS as an extreme halophilic xylanase with stimulated activity in artificial seawater and low water activity condi-
tions has potentials for application in industrial biotechnology. Keywords: Xylanase, Halophilic, Extreme, Plant biomass feedstock for sustainable production of green fuels. However, the recalcitrant structure of plant biomass is
a major drawback in many industrial applications. Vari-
ous physical, chemical, and enzymatic treatments have
been developed for conversion of plant biomass into
soluble fermentable sugars. Given the costs, safety, and
environmental issues, enzymatic treatment has proved
to be superior to other treatments. In general, enzymes
with extremophilic characteristics are of special interest
for industrial applications where harsh conditions such
as high temperature, acidic or alkaline pH, and salt con-
centrations are required. Therefore, attempts have been
underway to discover or develop novel xylanases with
desired characteristics to meet the diversity of applica-
tions. In addition to the culture-dependent methods,
metagenomics has been used as a powerful and culture-
independent approach to mine various environments, Ghadikolaei et al. AMB Expr (2019) 9:86
https://doi.org/10.1186/s13568-019-0809-2 Ghadikolaei et al. AMB Expr (2019) 9:86
https://doi.org/10.1186/s13568-019-0809-2 Open Access An extreme halophilic xylanase from camel
rumen metagenome with elevated catalytic
activity in high salt concentrations Kamran Khalili Ghadikolaei, Elham Dasi Sangachini, Vasimeh Vahdatirad, Kambiz Akbari Noghabi
and Hossein Shahbani Zahiri* Kamran Khalili Ghadikolaei, Elham Dasi Sangachini, Vasimeh Vahdatirad, Kambiz Akbari Noghabi
and Hossein Shahbani Zahiri* *Correspondence: shahbani@nigeb.ac.ir
Department of Energy and Environmental Biotechnology, National
Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Introduction Xylanase is an important enzyme in the degradation
of plant biomass by hydrolysing xylan as a main con-
stituent of the plant cell wall. Xylan is composed of
xylose monomers connected by β-1,4-glycoside linkages
accounting for 30–35% of hardwoods, 15–30% of grami-
naceous plants, and 7–12% of gymnosperms. Xylanase
has application in various industries where plant bio-
mass hydrolysis is required for the production of human
food, animal feed, paper, pulp, and biofuels (Adigüzel
and Tunçer 2016; Kaur et al. 2016; Kumar et al. 2016;
Yegin et al. 2018). Plant biomass as the most abundant
renewable organic matter is regarded as a promising *Correspondence: shahbani@nigeb.ac.ir
Department of Energy and Environmental Biotechnology, National
Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran Chemicals and strainsh The chemicals were generally purchased from Sigma-
Aldrich (St. Louis, USA). Tryptone and yeast extract
were from Merck (Darmstadt, Germany). Restriction
enzymes, DNA marker, T4 DNA ligase, and DNA poly-
merase were purchased from Thermo Fisher Scientific
(Waltham, USA). Protein marker was purchased from
SinaClon (Tehran-Iran). The pET26b vector and E. coli
strains including DH5α and BL21(DE3) were from Nova-
gen (Madison, USA). The kits for PCR product purifica-
tion and plasmid extraction were from GeneAll (Seoul,
Korea). The Ni–NTA protein purification resin was pur-
chased from Qiagen (Hilden, Germany). Ghadikolaei et al. AMB Expr (2019) 9:86 Page 2 of 11 such as the rumen of herbivores, for novel xylanases
(Duan et al. 2009; Ghadikolaei et al. 2017, 2018; Hess
et al. 2011; Xing et al. 2012). The metagenome obtained
from environmental samples is a huge source of valuable
genes from culturable and uncultured microorganisms. As an extremozyme, halophilic xylanase can be suitable
for applications in low water activity conditions such
as food industries and biorefineries. It is estimated that
1.9–5.9 m3 water is required for the production of 1 m3
biofuel from plant biomass (Fang et al. 2015). Therefore,
the global scarcity of freshwater would be a drawback for
plant biorefineries. In contrast, seawater is amply avail-
able on earth. Seawater has been shown to be an efficient
solvent for hydrothermal pretreatment of plant biomass
as well (Fang et al. 2015; Ren et al. 2016). In this study,
extreme halophilic xylanase, designated as XylCMS, was
discovered by cloning and recombinant expression of
the encoding gene from a camel rumen metagenome, in
Escherichia coli. The extreme halophilic xylanase not only
was tolerant of salt but also its activity was stimulated in
high salt concentrations. The mechanism of the salt stim-
ulation was investigated using biochemical and biophysi-
cal methods. The halophilic xylanase may be interesting
due to its potential application in high salt conditions. and virtual cloning were performed using SnapGene soft-
ware (GSL Biotech, http://snapgene.com). Determination of xylanase activity Xylanase activity was measured by quantification of
reducing sugars released as a function of the enzyme
reaction (Miller 1959). Activity assays were conducted
in a reaction mixture composed of 90 µl of citrate buffer
(pH 5) containing 1 mg/ml oat-spelt xylan and 10 µl of
purified enzyme solution. All reactions were conducted
for 10 min before being stopped by the addition of 3 vol-
umes of DNS (3,5-dinitrosalicylic acid) reagent and heat-
ing in boiling water for 10 min. The light absorbance was
measured at 540 nm to calculate the concentration of
reducing sugars by a standard curve. One unit of enzyme
activity was defined as the amount of enzyme required to
produce one micromole of reducing sugars per minute
under the assay conditions. Recombinant protein production and purificationh Recombinant protein production and purification
The xylanase-encoding gene (xyl-CMS) was obtained
from a camel rumen metagenome which was available
from a previous study (Gharechahi et al. 2015). The gene
was isolated by PCR (polymerase chain reaction) using
a pair of primers as forward (5′-AAGAATTCCAAGAA
TGGCATTTTAAAGAAAC-3′) and reverse (5′-AACTC
GAGTTCGCCCTCAGCGC-3′) containing EcoRI and
XhoI restriction sites, respectively. The PCR product
was electrophoresed on a 1% agarose gel and purified by
GeneAll kit. The gene was digested by EcoRI and XhoI
and inserted using T4 DNA ligase into pET26b previ-
ously linearized by the same restriction enzymes. The
resulting plasmid, pETxyl-CMS, was cloned in E. coli
DH5α through chemical transformation and cultivation
of the transformants on LB (Luria–Bertani) medium
containing 50 µg/ml kanamycin. The recombinant plas-
mid was purified from the transformed E. coli DH5α cells
using a GeneAll plasmid isolation kit and verified for
authenticity by sequencing. For gene expression, pETxyl-
CMS was used to transform E. coli BL21(DE3) and the
transformants were grown on the LB/kanamycin medium
in 37 °C, 200 rpm shaking conditions. The growing cells
at an optical density (600 nm) of 0.8 were induced by
0.3 mM IPTG (Isopropyl β-d-1-thiogalactopyranoside)
and then incubated for 24 h further in 25 °C, 120 rpm
shaking conditions. The recombinant enzyme, Xyl-CMS,
was isolated from periplasmic space and purified by NI–
NTA (nickel nitrilotriacetic acid) resin using standard
protocols from Qiagen. The purity and concentration of
the enzyme were analyzed by SDS-PAGE (sodium dode-
cyl sulfate polyacrylamide gel electrophoresis) and Brad-
ford method, respectively. Substrate specificity and kineticshi The substrate specificity of XylCMS was studied by activ-
ity assays conducted under standard conditions with
different substrates including carboxymethyl cellulose
(CMC), starch, and oat-spelt xylan (OSX) at 1% con-
centration. The Michaelis constant (Km) and maximum
velocity (Vmax) of the enzyme for OSX hydrolysis at 37 °C
and 55 °C were obtained by activity assays conducted
with varying concentrations of the substrate in the range
of 2–50 mg/ml in citrate buffer (pH 6). The effect of NaCl
on the kinetic parameters was examined in two levels
including 4 M and 3 M as optima for assays conducted at
37 °C and 55 °C, respectively. The scatter plot of enzyme
activity versus substrate concentration was used to fit the
data to the Michaelis equation by SigmaPlot software. Bioinformatic analysesh The homology analysis was performed by Blast program
at the NCBI website (https://blast.ncbi.nlm.nih.gov/
Blast.cgi). The sequence alignment was conducted using
Clustal Omega at EMBL-EBI (https://www.ebi.ac.uk/
Tools/msa/clustalo/). The structural analyses were per-
formed using SignalP-5.0 Server (http://www.cbs.dtu.dk/
services/SignalP/) and the NCBI conserved domain data-
base (CDD) (https://www.ncbi.nlm.nih.gov/Structure/
cdd/wrpsb.cgi). The prediction of the enzyme structure
was performed using RaptorX web server (http://rapto
rx.uchicago.edu/). The primer design, sequence analysis, Ghadikolaei et al. AMB Expr (2019) 9:86 Ghadikolaei et al. AMB Expr (2019) 9:86 Page 3 of 11 Effect of pH, temperature, and NaCl on enzyme activityhf Effect of pH, temperature, and NaCl on enzyme activity
The effect of pH on enzyme activity was analyzed in dif-
ferent pH buffers including citrate buffer (pH 3–6), phos-
phate buffer (pH 6–8), and glycine–NaOH (pH 8–10) at
37 °C under standard conditions. The effect of tempera-
ture on enzyme activity was studied in citrate buffer (pH
5) at various temperatures in the range of 10–65 °C. The
thermodynamic parameters were calculated using the
Eyring’s absolute rate equation derived from the transi-
tion state theory (Eyring and Stearn 1939). The impact of
high salt concentrations on the enzyme activity was stud-
ied by activity assays conducted at optimum pH and tem-
perature in the presence of various NaCl concentrations
(1–5 M). The activity of XylCMS was also studied in arti-
ficial seawater composed of NaCl, 26.29 g/l; KCl, 0.74 g/l;
CaCl2, 0.99 g/l; MgCl2·6H2O, 6.09 g/l; MgSO4·7H2O,
3.94 g/l. The thermal stability of XylCMS was studied by
determination of the residual activity in 10-min intervals
during a 50-min incubation in citrate buffer (pH 5) at
50 °C, 55 °C, and 60 °C. BCM =
I() × ()
I()
, where I(λ) is the fluorescence intensity at wavelength λ.hil l
The first derivative of barycentric fluorescence plotted
versus temperature was used to investigate the alterations
in the melting pattern of XylCMS under the influence of
OSX and NaCl. The melting temperature (Tm) was esti-
mated using the following relation: Tm = maxdBCM
dT
(T), where max is the local maximum and dBCM/dT(T) is
the first derivative of barycentric fluorescence as a func-
tion of temperature. GenBank accession numberh The genes encoding for XylCMS was submitted to Gen-
Bank with the accession number of MG595703.1. Thin layer chromatography (TLC) p
y
g
p y
The SDS-PAGE was conducted using a 12% polyacryla-
mide gel according to Laemmli method (Laemmli 1970). The native-PAGE was performed in a similar way except
that SDS and β-mercaptoethanol were removed from
the protocols for running buffer, polyacrylamide gel,
and loading buffer preparation. In the preparation of
the native polyacrylamide gel, 1% oat-spelt xylan was
included and the electrophoresis was conducted at 4 °C
for 2 h. In the end, the gel was washed with distilled
water and incubated in citrate buffer (pH 5) at 37 °C for
1 h. Finally, the native gel was stained in 0.1% Congo
Red solution for 30 min and then destained in 1 M NaCl
solution. The products of oat-spelt xylan hydrolysis by XylCMS
were analyzed by TLC. For this purpose, an enzyme reac-
tion was conducted under standard conditions and, sub-
sequently, 3 µl of the reaction solution was loaded on a
TLC plate (silica gel 60 F254). The plate was allowed to
dry at room temperature and then developed in a TLC
tank using a mixture of 2-butanol, acetic acid, and water
(2:1:1) as a mobile phase. In the end, the plate was dried
at room temperature, sprayed with a mixture of ethanol
and sulfuric acid (9:1) and baked at 110 °C for 10 min. Fluorescence spectroscopyhf The effects of temperature, substrate, and NaCl on the
enzyme conformation was studied by fluorescence spec-
troscopy using a Cary–Eclipse spectrofluorometer (Var-
ian, Australia). For this purpose, the purified enzyme
alone and also in the presence of NaCl, OSX or both
was heated from 10 to 80 °C and the fluorescence emis-
sions in the range of 300–500 nm were recorded at 10 °C
intervals upon excitation at 280 nm. The spectra were
corrected for the buffer contribution and the barycentric
mean fluorescence (BCM) was calculated for each spec-
trum using the following equation: Enzyme activity in artificial seawater In order to investigate the potential application of Xyl-
CMS for catalysis in seawater, the enzyme activity was
assayed in artificial seawater as the reaction medium. Fig-
ure 4d shows that XylCMS not only was functional in the
artificial seawater but also its activity was stimulated by
27% at 55 °C and 77% at 37 °C as compared with corre-
sponding salt-free controls. Effects of pH and temperature on the enzyme activityhf Effects of pH and temperature on the enzyme activity
The effect of pH on the activity of XylCMS was studied
in the range of pH 3–10. The results showed that the
enzyme was active at all tested pH conditions with more
than 80% activity over the pH range of 5 to 9, and at
least 11% activity at other pH conditions (Fig. 3a)., The
maximum activity of XylCMS was obtained at pH 6 in
citrate buffer that was taken as the optimum pH condi-
tion for further activity assays. The profile of enzyme
activity in the temperature range of 10–65 °C showed
that the optimum temperature for XylCMS was 55 °C. The temperature profile of enzyme activity in 3 M NaCl
revealed that the optimum temperature of XylCMS was
not influenced by NaCl but the relative activity was sig-
nificantly enhanced at temperatures below the optimum. The enzyme activity in 3 M NaCl was less temperature
dependent and reached above 60% at a temperature
below 20 °C while a similar activity in the absence of salt
could be obtained at above 40 °C (Fig. 3b). Bioinformatic analysis A homology search using BlastP program at the NCBI
database revealed that XylCMS exhibited 99% identity
with a hypothetical protein of glycosyl hydrolase fam-
ily 11 from Ruminococcus flavefaciens. However, the
identity of XylCMS with already characterized xyla-
nases was quite low, exhibiting most identity with XynA
from Ruminococcus albus (59%), Xylanase 1 from a Ghadikolaei et al. AMB Expr (2019) 9:86 Page 4 of 11 Ghadikolaei et al. AMB Expr (2019) 9:86 Ruminococcus sp. (46%), and XynB from Ruminococcus
flavefaciens (44%). The structural analyses of XylCMS
revealed the presence of a lipoprotein signal peptide
(Sec/SPII), a xylanase domain, belonging to family 11 of
glycoside hydrolases, and a carbohydrate binding module
of family 4_9 (Fig. 1a). The homology modeling of Xyl-
CMS using RaptorX revealed a typical β-jelly roll struc-
ture for the xylanase domain and a β-sandwich structure
for the CBM domain (Fig. 1b). The multiple sequence
alignment of XylCMS with some other family 11 xyla-
nases was used to identify conserved residues including
two catalytic glutamic acids that are involved in substrate
binding and catalysis (Fig. 1c). Effects of NaCl on the enzyme activity and thermal stability
The effect of NaCl (1–5 M) on the activity of XylCMS was
studied in citrate buffer (pH 6) at 55 °C as the optimum
pH and temperature for the enzyme activity. The results
showed that the activity of XylCMS was remarkably stim-
ulated by NaCl in all tested concentrations. The highest
activity with 46% enhancement as compared with con-
trol was obtained by 3 M NaCl. Interestingly, even at 5 M
NaCl, the enzyme showed 31% higher activity compared
to the NaCl-free control condition (Fig. 4a). The effect of
NaCl on the enzyme activity was also assayed at 37 °C as
the body temperature of camels. The results showed that
the salt stimulation was even more striking at 37 °C giv-
ing rise to 2.4, 2.8, and 2.7 times enhancement of activity,
respectively, in 3 M, 4 M, and 5 M NaCl concentrations
as compared to the salt-free control condition at the
same temperature (Fig. 4a). Therefore, the optimum
NaCl concentration for maximum stimulation of the
enzyme activity assayed at 55 °C and 37 °C was 3 M and
4 M, respectively. As Fig. 4a shows, the salt stimulation at
37 °C can enhance the enzyme activity to higher than the
obtainable activity at 55 °C as the optimum temperature.h Heterologous protein expression and purificationh Heterologous protein expression and purification
The gene encoding XylCMS was cloned in pET-26b(+) in
frame with the vector’s pelb sequence at N-terminus and
the His-tag coding sequence at C-terminus. The result-
ing plasmid (pET-xylCMS) was successfully expressed
in E. coli BL21(DE3) and the recombinant XylCMS was
purified using Ni–NTA resin from the periplasmic cell
fraction. The purity of the enzyme was confirmed by
SDS-PAGE showing a single band of about 47 kDa which
corresponded with the calculated molecular mass of the
recombinant XylCMS (Fig. 2a). The zymography using
a native-PAGE gel containing 1% OSX revealed that the
purified enzyme was functionally active. The in-gel diges-
tion of OSX was discernible as a yellowish area in a red
background after staining with Congo Red (Fig. 2b). The
products of OSX hydrolysis with XylCMS were analyzed
by TLC. The result showed that XylCMS produced xylo-
tetraose as the only hydrolysis product of OSX (Fig. 2c). The thermal stability of XylCMS in NaCl-free condi-
tion as well as in 3 M NaCl was analyzed by determina-
tion of the residual activity of the enzyme in 10-minute
intervals during a 50-min incubation at 50 °C, 55 °C, and
60 °C (Fig. 4b, c). The results showed that in the absence
of NaCl, the enzyme with less than 8% loss of activity
was almost stable at 50 °C and 55 °C. However, at 60 °C,
its activity began to drop severely after 10 min and the
enzyme completely inactivated during 40 min incuba-
tion. The analysis of thermostability in the presence of
NaCl showed that the salt had a destabilizing effect on
XylCMS. As a result, the enzyme activity began to decline
after 10 min incubation at 55 °C and plunged to 47% dur-
ing 50 min. Likewise, the enzyme in 3 M NaCl was sig-
nificantly less stable at 60 °C with 83% loss of activity in
10 min and total inactivation in 20 min of incubation. Fluorescence spectroscopyh The impact of NaCl on the structure of XylCMS was
investigated by fluorescence spectroscopy. For this
purpose, the intrinsic fluorescence of the enzyme was Ghadikolaei et al. AMB Expr (2019) 9:86 Page 5 of 11 Ghadikolaei et al. AMB Expr b
c
a
Structural analysis of XylCMS. a Conserved domains including a family 11 glycoside hydrolase and a carbohydrate binding module of family
ere identified by NCBI’s conserved domain database. b The predicted tertiary structure of XylCMS confirming the modular structure of the
me. c Multiple sequence alignment of XylCMS with a few family 11 xylanases using Clustal Omega program. The identical and conserved
es are shown by stars and dots, respectively. The two glutamic acid catalytic residues are shown by arrowheads. The sequences are defined by
ccession numbers at GenBank b
a b c
Fig. 1 Structural analysis of XylCMS. a Conserved domains including a family 11 glycoside hydrolase and a carbohydrate binding module of family
4_9 were identified by NCBI’s conserved domain database. b The predicted tertiary structure of XylCMS confirming the modular structure of the
enzyme. c Multiple sequence alignment of XylCMS with a few family 11 xylanases using Clustal Omega program. The identical and conserved
residues are shown by stars and dots, respectively. The two glutamic acid catalytic residues are shown by arrowheads. The sequences are defined by
their accession numbers at GenBank c Fig. 1 Structural analysis of XylCMS. a Conserved domains including a family 11 glycoside hydrolase and a carbohydrate binding module of family
4_9 were identified by NCBI’s conserved domain database. b The predicted tertiary structure of XylCMS confirming the modular structure of the
enzyme. c Multiple sequence alignment of XylCMS with a few family 11 xylanases using Clustal Omega program. The identical and conserved
residues are shown by stars and dots, respectively. The two glutamic acid catalytic residues are shown by arrowheads. The sequences are defined by
their accession numbers at GenBank Fig. 1 Structural analysis of XylCMS. a Conserved domains including a family 11 glycoside hydrolase and a carbohydrate binding module of family
4_9 were identified by NCBI’s conserved domain database. b The predicted tertiary structure of XylCMS confirming the modular structure of the
enzyme. c Multiple sequence alignment of XylCMS with a few family 11 xylanases using Clustal Omega program. The identical and conserved
residues are shown by stars and dots, respectively. Fluorescence spectroscopyh 5). The conformational changes of XylCMS
as a function of temperature was investigated by the
plot of dBCM/dT (nm/ °C) versus T (°C). The results
showed that the melting profile of the enzyme was
remarkably influenced by OSX. The melting tempera-
ture of XylCMS was estimated to be about 28 °C but in
the presence of OSX, it was raised to 56 °C. In contrast,
the melting profile of XylCMS was not much altered by
3 M NaCl and the melting temperature of the enzyme
remained at about 28 °C in the presence of the salt. Interestingly, NaCl also counteracted the effects of OSX
so that the melting temperature of XylCMS in the pres-
ence of both NaCl and OSX was still retained at 28 °C
(Fig. 6). Temperature (°C) analyzed alone and also in the presence of NaCl, OSX or
both at various temperatures in the range of 10–80 °C. The results showed that the fluorescence intensity of
XylCMS declined progressively with the increase of
temperature and the intensity curve changed from a
peak to plateau. In the presence of OSX as a substrate,
the intrinsic fluorescence of the enzyme was severely
quenched irrespective of temperature. However, NaCl
(3 M) was shown to improve the fluorescence intensity
of XylCMS at all tested temperatures and even in the
presence of OSX, compared with the NaCl-free condi-
tions (Fig. 5). The conformational changes of XylCMS
as a function of temperature was investigated by the
plot of dBCM/dT (nm/ °C) versus T (°C). The results
showed that the melting profile of the enzyme was
remarkably influenced by OSX. The melting tempera-
ture of XylCMS was estimated to be about 28 °C but in
the presence of OSX, it was raised to 56 °C. In contrast,
the melting profile of XylCMS was not much altered by
3 M NaCl and the melting temperature of the enzyme
remained at about 28 °C in the presence of the salt. Interestingly, NaCl also counteracted the effects of OSX
so that the melting temperature of XylCMS in the pres-
ence of both NaCl and OSX was still retained at 28 °C
(Fig. 6). Fig. 3 a Effect of pH on the activity of XylCMS. Activity assays were
conducted in various pH buffers including citrate buffer for pH
3–6, phosphate buffer for pH 6–8, and glycine–NaOH buffer for pH
8–10 at 37 °C. Fluorescence spectroscopyh The two glutamic acid catalytic residues are shown by arrowheads. The sequences are defined by
their accession numbers at GenBank Fig. 1 Structural analysis of XylCMS. a Conserved domains including a family 11 glycoside hydrolase and a carbohydrate binding module of family
4_9 were identified by NCBI’s conserved domain database. b The predicted tertiary structure of XylCMS confirming the modular structure of the
enzyme. c Multiple sequence alignment of XylCMS with a few family 11 xylanases using Clustal Omega program. The identical and conserved
residues are shown by stars and dots, respectively. The two glutamic acid catalytic residues are shown by arrowheads. The sequences are defined by
their accession numbers at GenBank Ghadikolaei et al. AMB Expr (2019) 9:86 Ghadikolaei et al. AMB Expr Page 6 of 11 pH
3
4
5
6
7
8
9
10
Relative activity (%)
0
20
40
60
80
100
120
citrate buffer
phosphate buffer
glycin-NaOH buffer
Temperature (°C)
10
15 20
25
30 35 40 45
50
55 60
65
Relative activity (%)
0
20
40
60
80
100
120
salt-free standard
3 M NaCl
a
b
Fig. 3 a Effect of pH on the activity of XylCMS. Activity assays were
d
d
b ff
l d
b ff
f 100 KDa
75 KDa
63 KDa
48 KDa
35 KDa
25 KDa
1
2
4
G1
G2
G3
G4
G5
2
3
1
a
b
c
Fig. 2 Analysis of purity, functional activity, and mode of action of
XylCMS. a SDS-PAGE for purity analysis: Lane 1, protein molecular
weight marker; Lane 2, purified XylCMS; b Zymogram prepared by
Congo Red staining of a native PAGE gel indicating the functional
activity of the purified enzyme as an unstrained area; c TLC analysis
of OSX hydrolysis by XylCMS. Lane 1, enzyme-treated preparation;
lane 2, control without enzyme; lane 3, standard molecular marker
composed of mono-, di-, tri-, tetra-, and penta-glucose; lane 4,
enzyme-treated preparation. Fluorescence spectroscopyh The enzyme activities are shown as relative (%) to
the highest value obtained at pH 6. b Effect of temperature on the
activity of XylCMS. Activity assays were performed in citrate buffer
(pH 6) at various temperatures under salt-free standard and 3 M NaCl
conditions. The enzyme activities are shown as relative (%) to the
highest values obtained at 55 °C Fluorescence spectroscopyh The arrows indicate the hydrolysis
products of OSX 100 KDa
75 KDa
63 KDa
48 KDa
35 KDa
25 KDa
1
2
a 4
G1
G2
G3
G4
G5
2
3
1
c a c pH
3
4
5
6
7
8
9
10
Relative activity (%)
0
20
40
60
80
100
120
citrate buffer
phosphate buffer
glycin-NaOH buffer
a b Temperature (°C)
10
15 20
25
30 35 40 45
50
55 60
65
Relative activity (%)
0
20
40
60
80
100
120
salt-free standard
3 M NaCl
b Fig. 2 Analysis of purity, functional activity, and mode of action of
XylCMS. a SDS-PAGE for purity analysis: Lane 1, protein molecular
weight marker; Lane 2, purified XylCMS; b Zymogram prepared by
Congo Red staining of a native PAGE gel indicating the functional
activity of the purified enzyme as an unstrained area; c TLC analysis
of OSX hydrolysis by XylCMS. Lane 1, enzyme-treated preparation;
lane 2, control without enzyme; lane 3, standard molecular marker
composed of mono-, di-, tri-, tetra-, and penta-glucose; lane 4,
enzyme-treated preparation. The arrows indicate the hydrolysis
products of OSX Fig. 2 Analysis of purity, functional activity, and mode of action of
XylCMS. a SDS-PAGE for purity analysis: Lane 1, protein molecular
weight marker; Lane 2, purified XylCMS; b Zymogram prepared by
Congo Red staining of a native PAGE gel indicating the functional
activity of the purified enzyme as an unstrained area; c TLC analysis
of OSX hydrolysis by XylCMS. Lane 1, enzyme-treated preparation;
lane 2, control without enzyme; lane 3, standard molecular marker
composed of mono-, di-, tri-, tetra-, and penta-glucose; lane 4,
enzyme-treated preparation. The arrows indicate the hydrolysis
products of OSX salt-free standard
3 M NaCl Relative activity (%) Temperature (°C)
10
15 20
25
30 35 40 45
50
55 60
65 analyzed alone and also in the presence of NaCl, OSX or
both at various temperatures in the range of 10–80 °C. The results showed that the fluorescence intensity of
XylCMS declined progressively with the increase of
temperature and the intensity curve changed from a
peak to plateau. In the presence of OSX as a substrate,
the intrinsic fluorescence of the enzyme was severely
quenched irrespective of temperature. However, NaCl
(3 M) was shown to improve the fluorescence intensity
of XylCMS at all tested temperatures and even in the
presence of OSX, compared with the NaCl-free condi-
tions (Fig. Substrate specificity and kineticshi The substrate specificity of XylCMS was studied using
CMC, starch, and OSX as different substrates (1% w/v). The results showed that the enzyme could only hydro-
lyze OSX and no activity was detected on other sub-
strates. Using activity assays with varying concentrations
of OSX, the kinetic parameters of XylCMS were studied
in the absence and presence of NaCl at both 37 °C and
55 °C (Table 1). The results showed that NaCl increased
the Kcat of the enzyme by 2.7-folds at 37 °C and 1.2-
folds at 55 °C. The Km values were decreased with NaCl
by 4.3-folds at 37 °C and 3.7-folds at 55 °C. The overall
outcome was a significant increase in catalytic efficiency
(Kcat/Km) by 11.5-folds at 37 °C and 4.4-folds at 55 °C. The Ghadikolaei et al. AMB Expr (2019) 9:86 Page 7 of 11 Ghadikolaei et al. AMB Expr NaCl (M)
0
1
2
3
4
5
Relative activity (%)
20
40
60
80
100
120
140
160
55°C
37°C
Time (min)
0
10
20
30
40
50
Residual activity (%)
0
20
40
60
80
100
120
50 °C
55 °C
60 °C
Time (min)
0
10
20
30
40
50
Residual activity (%)
0
20
40
60
80
100
120
Control
3M NaCl
Seawater
Relative activity (%)
20
40
60
80
100
120
140
160
55°C
37°C
a
b
d
c
Salt conditions
50 °C
55 °C
60 °C
Fig. 4 a Effect of NaCl on the activity of XylCMS. Activity assays were conducted in various NaCl concentrations in citrate buffer (pH 6) at 37 °C and
55 °C. The enzyme activities are shown as relative (%) to the enzyme activity in salt-free condition at 55 °C. b Thermal stability of XylCMS during
50 min incubation at 50 °C, 55 °C, and 60 °C in citrate buffer (pH 6). The residual activity was determined at 10 min intervals in citrate buffer (pH 6) at
55 °C and is shown as relative (%) to the enzyme activity before incubation. c Thermal stability of XylCMS under the influence of salt during 50 min
incubation at 50 °C, 55 °C, and 60 °C in citrate buffer (pH 6) containing 3 M NaCl. The residual activity was determined at 10 min intervals in citrate
buffer (pH 6) at 55 °C and is shown as relative (%) to the enzyme activity before incubation. Substrate specificity and kineticshi d The catalytic activity of XylCMS in artificial seawater
compared with that in the salt-free control condition and 3 M NaCl at 37 °C and 55 °C. The activities are presented as relative (%) to the enzyme
activity in the salt-free condition at 55 °C Time (min)
0
10
20
30
40
50
Residual activity (%)
0
20
40
60
80
100
120
50 °C
55 °C
60 °C
b NaCl (M)
0
1
2
3
4
5
Relative activity (%)
20
40
60
80
100
120
140
160
55°C
37°C
a b NaCl (M)
Time (min)
0
10
20
30
40
50
Residual activity (%)
0
20
40
60
80
100
120
c
50 °C
55 °C
60 °C Control
3M NaCl
Seawater
Relative activity (%)
20
40
60
80
100
120
140
160
55°C
37°C
d d Residual activity (%) Fig. 4 a Effect of NaCl on the activity of XylCMS. Activity assays were conducted in various NaCl concentrations in citrate buffer (pH 6) at 37 °C and
55 °C. The enzyme activities are shown as relative (%) to the enzyme activity in salt-free condition at 55 °C. b Thermal stability of XylCMS during
50 min incubation at 50 °C, 55 °C, and 60 °C in citrate buffer (pH 6). The residual activity was determined at 10 min intervals in citrate buffer (pH 6) at
55 °C and is shown as relative (%) to the enzyme activity before incubation. c Thermal stability of XylCMS under the influence of salt during 50 min
incubation at 50 °C, 55 °C, and 60 °C in citrate buffer (pH 6) containing 3 M NaCl. The residual activity was determined at 10 min intervals in citrate
buffer (pH 6) at 55 °C and is shown as relative (%) to the enzyme activity before incubation. d The catalytic activity of XylCMS in artificial seawater
compared with that in the salt-free control condition and 3 M NaCl at 37 °C and 55 °C. The activities are presented as relative (%) to the enzyme
activity in the salt-free condition at 55 °C energy (∆G) of the reaction decreased but the entropy
(∆S) increased in the presence of NaCl (Table 2). The ∆H,
in particular, was remarkably decreased more than three-
fold by NaCl (Table 2). Substrate specificity and kineticshi thermodynamic analysis of OSX hydrolysis by XylCMS
indicated that NaCl decreased the activation energy (Ea)
of the reaction by 2.4-folds from 16.94 (kJ/mol) to 7.16
(kJ/mol). Likewise, the enthalpy (∆H) and Gibbs free Ghadikolaei et al. AMB Expr (2019) 9:86 Page 8 of 11 Temperature (°C)
10
20
30
40
50
60
70
80
dBCM/dT (nm/°C)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
XylCMS
XylCMS+NaCl
XylCMS+OSX
XylCMS+NaCl+OSX
Fig. 6 Influence of NaCl and OSX on the melting profile of XylCMS
illustrated by plotting the first derivative of barycentric mean
fluorescence as a function of temperature. The peak maximum
indicates the melting temperature (Tm) of the enzyme under various
conditions Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
10°C
20°C
30°C
40°C
50°C
60°C
C
°
0
8
C
°
0
7
Fig. 5 Analysis of intrinsic fluorescence of XylCMS alone and under
the influence of 3 M NaCl and OSX at various temperatures in the
range of 10–80 °C. At 10 °C intervals, the fluorescence intensity of
the enzyme alone (blue line), with NaCl (red line), with OSX (green
line), and in the presence of both NaCl and OSX (magenta line)
was recorded in the range of 300–500 nm. Substrate specificity and kineticshi At 10 °C intervals, the fluorescence intensity of
the enzyme alone (blue line), with NaCl (red line), with OSX (green
line), and in the presence of both NaCl and OSX (magenta line)
was recorded in the range of 300–500 nm. At all temperatures, the
fluorescence intensity of the enzyme was quenched with OSX but
NaCl could improve the fluorescence activity of XylCMS both in the
absence and presence of OSX Fig. 5 Analysis of intrinsic fluorescence of XylCMS alone and under
the influence of 3 M NaCl and OSX at various temperatures in the
range of 10–80 °C. At 10 °C intervals, the fluorescence intensity of
the enzyme alone (blue line), with NaCl (red line), with OSX (green
line), and in the presence of both NaCl and OSX (magenta line)
was recorded in the range of 300–500 nm. At all temperatures, the
fluorescence intensity of the enzyme was quenched with OSX but
NaCl could improve the fluorescence activity of XylCMS both in the
absence and presence of OSX Substrate specificity and kineticshi 6 Influence of NaCl and OSX on the melting profile of XylCMS
illustrated by plotting the first derivative of barycentric mean
fluorescence as a function of temperature. The peak maximum
indicates the melting temperature (Tm) of the enzyme under various
conditions Wavelength (nm)
Wa elength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
60°C Wavelength (nm)
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
50°C Table 1 Influence of NaCl on kinetic parameters of oat-
spelt xylan hydrolysis by XylCMS at 37 °C and 55 °C
Conditions
Specific
activity (U/
mg)
Km (mg/ml)
Kcat (1/s)
Kcat/Km
37 °C (no NaCl)
345
24.4
214
8.77
37 °C (4 M NaCl)
740
5.7
579
101.6
55 °C (no NaCl)
1766
23.3
1383
59.4
55 °C (3 M NaCl)
2170
6.5
1700
261.5 Table 1 Influence of NaCl on kinetic parameters of oat-
spelt xylan hydrolysis by XylCMS at 37 °C and 55 °C Table 1 Influence of NaCl on kinetic parameters of oat-
spelt xylan hydrolysis by XylCMS at 37 °C and 55 °C Wavelength (nm) Wavelength (nm)
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
C
°
0
8 Wavelength (nm)
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
C
°
0
7 role in the decomposition of plant biomass. The abil-
ity of the bacterium to degrade plant polysaccharides is
owing to a variety of lignocellulolytic enzymes that are
secreted to the surroundings or anchor in the cell wall. Accordingly, a few xylanases have been characterized
from R. flavefaciens (Flint et al. 1989, 1993; Zhang et al. 1994; Zhang and Flint 1992). However, XylCMS is distin-
guished from other characterized xylanases of the bacte-
rium in that its activity is significantly stimulated by high
salt concentrations. To the best of our knowledge, Xyl-
CMS is the first halophilic xylanase characterized from
rumen. Even in comparison with other halophilic xyla-
nases, XylCMS is noticeable for a significantly enhanced
activity in a high NaCl concentration of 5 M (Table 3). Fig. 5 Analysis of intrinsic fluorescence of XylCMS alone and under
the influence of 3 M NaCl and OSX at various temperatures in the
range of 10–80 °C. Substrate specificity and kineticshi At all temperatures, the
fluorescence intensity of the enzyme was quenched with OSX but
NaCl could improve the fluorescence activity of XylCMS both in the
absence and presence of OSX Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
10°C
20°C
30°C
40°C
50°C
60°C
C
°
0
8
C
°
0
7
Fig. 5 Analysis of intrinsic fluorescence of XylCMS alone and under
the influence of 3 M NaCl and OSX at various temperatures in the
range of 10–80 °C. At 10 °C intervals, the fluorescence intensity of
the enzyme alone (blue line), with NaCl (red line), with OSX (green
line), and in the presence of both NaCl and OSX (magenta line)
was recorded in the range of 300–500 nm. At all temperatures, the
fluorescence intensity of the enzyme was quenched with OSX but
NaCl could improve the fluorescence activity of XylCMS both in the
absence and presence of OSX Temperature (°C)
10
20
30
40
50
60
70
80
dBCM/dT (nm/°C)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
XylCMS
XylCMS+NaCl
XylCMS+OSX
XylCMS+NaCl+OSX Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
40°C Wavelength (nm)
300 350 400 450 500
Intensity (a.u.)
0
200
400
600
800
1000
1200
1400
30°C Intensity (a.u.) Fig. 6 Influence of NaCl and OSX on the melting profile of XylCMS
illustrated by plotting the first derivative of barycentric mean
fluorescence as a function of temperature. The peak maximum
indicates the melting temperature (Tm) of the enzyme under various
conditions Fig. Discussion In this study, XylCMS was obtained from a camel rumen
metagenome and characterized as a novel extreme halo-
philic xylanase. The blast search indicated that the gene
encoding XylCMS should belong to R. flavefaciens due to
the high identity of the enzyme with a hypothetical glyco-
syl hydrolase of the bacterium. R. flavefaciens is a strictly
anaerobic coccus in the rumen that plays an important For instance, in the case of two extremely halotoler-
ant xylanases of a halophilic bacterium, the maximum
activity was obtained with 1 M salt but the enzyme
activity was inhibited in higher salt concentrations
(Wejse et al. 2003). In another study on a haloalkaline
xylanase from Bacillus pumilus GESF-1, the enzyme
activity was enhanced by 60% in the presence of 2.5% Ghadikolaei et al. AMB Expr (2019) 9:86 Ghadikolaei et al. AMB Expr Page 9 of 11 Table 2 Influence of NaCl (3 M) on thermodynamic parameters of oat-spelt xylan hydrolysis by XylCMS at 37 °C and 55 °C
Conditions
∆H (kj/mol)
∆G (kj/mol)
∆S (kj/mol)
∆G(E–S) (kj/mol)
∆G(E–T) (kj/mol)
37 °C
14.36
62.22
− 0.154
7.62
− 6.19
37 °C + NaCl
4.58
61.25
− 0.182
5.54
− 9.24
55 °C
14.21
60
− 0.14
8.39
− 12.2
55 °C + NaCl
4.43
59.78
− 0.18
7.36
− 13.48 Table 2 Influence of NaCl (3 M) on thermodynamic parameters of oat-spelt xylan hydrolysis by XylCMS at 37 °C and 55 °C
Conditions
∆H (kj/mol)
∆G (kj/mol)
∆S (kj/mol)
∆G(E–S) (kj/mol)
∆G(E–T) (kj/mol)
37 °C
14.36
62.22
− 0.154
7.62
− 6.19
37 °C + NaCl
4.58
61.25
− 0.182
5.54
− 9.24
55 °C
14.21
60
− 0.14
8.39
− 12.2
55 °C + NaCl
4.43
59.78
− 0.18
7.36
− 13.48 Table 2 Influence of NaCl (3 M) on thermodynamic parameters of oat-spelt xylan hydrolysis by X aCl (3 M) on thermodynamic parameters of oat-spelt xylan hydrolysis by XylCMS at 37 °C and 55 °C Table 3 Comparison of molecular weight, optimum pH, optimum temperature and activity at given NaCl concentrations
between XylCMS and other characterized rumen and halophilic xylanases
a Molecular weight
b Optimum pH
c Optimum temperature
d Salt concentration
e Not determined
Name
Source
Mol. weighta
(kDa)
Opt. pHb
Opt. temp.c (°C)
Activity (%), salt
con.d (M)
References
Xyl-CMS
Camel rumen
46
6
55
132, 5
This study
Xyn10N18
Bovine rumen
54.5
6.5
35
ND
Gong et al. Discussion (2013)
XyIn-SH1
Holstein cattle rumen
39.5
6.5
40
ND
(Cheng et al. 2012)
XynGR40
Goat rumen
52.4
6.5
30
ND
Wang et al. (2011)
Xyn-lxy
Hu sheep rumen
71.3
6
50
ND
Wang et al. (2015)
NDe
Bacillus pumilus
39.6
8
40
73, 2.6
Menon et al. (2010)
XynSL3
Alkalibacterium sp. 150
9
55
60, 3
Wang et al. (2017)
XynFCB
Thermoanaerobacterium sac-
charolyticum
50
6.4
63
67, 2.6
Hung et al. (2011)
XynA
Zunongwangia profunda
43.7
6.5
30
100, 5
Liu et al. (2014)
ND
Aureobasidium pullulans
21.6
4
30–50
100, 3.4
Yegin (2017)
ND
Chromohalobacter sp. 15
9
65
90, 5
Prakash et al. (2012)
XynRBM26
Massilia sp. RBM26
45
5.5
45
86, 5
Xu et al. (2016)
Excg1
Colletotrichum graminicola
20
5.5
65
50, 3
Carli et al. (2016) Table 3 Comparison of molecular weight, optimum pH, optimum temperature and activity at give
between XylCMS and other characterized rumen and halophilic xylanases f molecular weight, optimum pH, optimum temperature and activity at given NaCl concentrations
other characterized rumen and halophilic xylanases NaCl (less than 0.5 M) but inhibited in higher salt con-
centrations (Menon et al. 2010). The activity of halo-
tolerant xylanase from Massilia sp. RBM26 has been
reported to be stable in NaCl concentrations of up to
1.5 M but at higher salt concentrations the activity
decreased steadily to 85% in 5 M NaCl (Xu et al. 2016). The salt-stable alkaline xylanase of Alkalibacterium
sp. SL3 has been shown to lose activity progressively
with the increase of NaCl concentration in the range of
0.25–4.5 M (Wang et al. 2017). In contrast, XylCMS was
proved in the current study to be an extreme halophilic
xylanase with elevated activity in high NaCl concentra-
tions up to 5 M that is unprecedented. It was revealed
by fluorescence spectroscopy that XylCMS in 3 M NaCl
exhibits more fluorescence activity during thermal
denaturation. In other words, although the enzyme fol-
lows a general trend of thermal denaturation, its fluo-
rescence activity tends to be higher in the presence of salt. The analysis of barycentric fluorescence indi-
cated that the melting temperature of XylCMS was not
altered by NaCl but raised from 28 °C to about 56 °C
by OSX as substrate. It means that XylCMS gains more
stability by the formation of the enzyme–substrate (ES)
complex. References Adigüzel AO, Tunçer M (2016) Production, characterization and application of
a xylanase from Streptomyces sp. AOA40 in fruit juice and bakery indus-
tries. Food Biotechnol 30:189–218 Carli S, Meleiro LP, Rosa JC, Moraes LAB, Jorge JA, Masui DC, Furriel RP (2016)
A novel thermostable and halotolerant xylanase from Colletotrichum
graminicola. J Mol Catal B Enzym 133:508–517 Carli S, Meleiro LP, Rosa JC, Moraes LAB, Jorge JA, Masui DC, Furriel RP (2016)
A novel thermostable and halotolerant xylanase from Colletotrichum
graminicola. J Mol Catal B Enzym 133:508–517 Chen GQ, Jiang XR (2018) Next generation industrial biotechnology based on
extremophilic bacteria. Curr Opin Biotechnol 50:94–100 Cheng F, Sheng J, Dong R, Men Y, Gan L, Shen L (2012) Novel xylanase from a
holstein cattle rumen metagenomic library and its application in xylooli-
gosaccharide and ferulic acid production from wheat straw. J Agric Food
Chem 60:12516–12524 Duan CJ, Xian L, Zhao GC, Feng Y, Pang H, Bai XL, Tang JL, Ma QS, Feng JX
(2009) Isolation and partial characterization of novel genes encoding
acidic cellulases from metagenomes of buffalo rumens. J Appl Microbiol
107(1):245–256 Eyring H, Stearn AE (1939) The application of the theory of absolute reacton
rates to proteins. Chem Rev 24(2):253–270 Fang C, Thomsen MH, Brudecki GP, Cybulska I, Frankær CG, Bastidas-Oyanedel
JR, Schmidt JE (2015) Seawater as alternative to freshwater in pretreat-
ment of date palm residues for bioethanol production in coastal and/or
arid areas. Chemsuschem 8(22):3823–3831 Flint HJ, McPherson CA, Bisset J (1989) Molecular cloning of genes from Rumi-
nococcus flavefaciens encoding xylanase and beta (1–3, 1–4) glucanase
activities. J Appl Environ Microbiol 55(5):1230–1233 Authors’ contributions KKG designed and performed the experiments; EDS performed the experi-
ments, VV drafted the manuscript, KAN evaluated and discussed the results;
HSZ conceived this study, edited and revised the manuscript. All authors read
and approved the final manuscript. Competing interests Competing interests
The authors declare that they have no competing interests. The authors declare that they have no competing interests. y
y
For industrial applications, extremophilic enzymes
such as halophilic xylanases are more practical than
normal enzymes to cope with the harsh conditions that
are required or desired in different processes (Chen
and Jiang 2018). An important challenge of industrial
biotechnology is the high consumption of fresh water. A practical solution is the replacement of fresh water
with abundant seawater in water-intensive industries
such as lignocellulosic biorefineries. In this context, a
hlophilic xylanase has potential application for enzy-
matic decomposition of plant biomass in seawater. In
the other hand, plant biomass has a complex and tough
structure that resists enzymatic hydrolysis. It has been
shown that pretreatment with NaCl is a cost-effective,
green method that can significantly improve the effi-
ciency of enzymatic hydrolysis of plant biomass (Jiang
et al. 2015). Therefore, XylCMS as a halophilic xylanase
may be useful in future applications in the production
of commodity chemicals such as biofuels from plant
biomass. The halophilic xylanase has also potential
application in bakery, pulp and paper industries due to
the ability of the enzyme to function under low water
activity conditions. Received: 1 June 2019 Accepted: 6 June 2019 Received: 1 June 2019 Accepted: 6 June 2019 Discussion In contrast, NaCl seems to exert a destabiliz-
ing effect on the enzyme conformation in the ES com-
plex as was indicated by the restoration of the melting
temperature to about 28 °C. The effect of NaCl gives
rise to a more flexible structure of the enzyme that has
less thermal stability as compared with the salt-free
condition. The destabilizing effect of NaCl was also
corroborated by the experimental study of thermal
deactivation indicating that XylCMS deactivated more
rapidly in the presence of NaCl. The Ea and ∆H of Xyl-
CMS were substantially decreased in the presence of
NaCl indicating that the reaction has lowered energy
requirements and, therefore, is more efficient under the Ghadikolaei et al. AMB Expr (2019) 9:86 Ghadikolaei et al. AMB Expr (2019) 9:86 Page 10 of 11 Page 10 of 11 influence of salt. In addition, a lower ∆H means that the
reaction is less temperature dependent resulting in ele-
vated rates of catalytic activity at temperatures below
the optimum temperature. From the findings, it can be
speculated that XylCMS is tolerant of high salt concen-
trations because of its high intrinsic flexibility. How-
ever, in the presence of substrate, the enzyme becomes
less flexible possibly due to the formation of new inter-
and intramolecular interactions. It seems that NaCl by
eliminating the new interactions can restore the flex-
ibility of the enzyme. The enzyme flexibility is crucial
for catalytic activity by influencing substrate binding,
product release and the energy barriers of the cata-
lyzed reaction (Hammes-Schiffer and Benkovic 2006). On the other hand, the substrate affinity of XylCMS
improves in the presence of salt as was indicated by a
significant decrease in Km value. Therefore, it seems
that NaCl exerts three distinct effects on XylCMS: (1) a
delicate stabilizing effect in certain locations by which
the enzyme becomes partially structured as indicated
by its elevated intrinsic fluorescence intensity; (2) an
overall destabilizing effect that leads to more flexibility
but less thermal stability of the enzyme in the ES com-
plex as revealed by the barycentric fluorescence and the
activity-stability analysis; (3) improvement of the affin-
ity of the enzyme to substrate. influence of salt. In addition, a lower ∆H means that the
reaction is less temperature dependent resulting in ele-
vated rates of catalytic activity at temperatures below
the optimum temperature. Discussion From the findings, it can be
speculated that XylCMS is tolerant of high salt concen-
trations because of its high intrinsic flexibility. How-
ever, in the presence of substrate, the enzyme becomes
less flexible possibly due to the formation of new inter-
and intramolecular interactions. It seems that NaCl by
eliminating the new interactions can restore the flex-
ibility of the enzyme. The enzyme flexibility is crucial
for catalytic activity by influencing substrate binding,
product release and the energy barriers of the cata-
lyzed reaction (Hammes-Schiffer and Benkovic 2006). On the other hand, the substrate affinity of XylCMS
improves in the presence of salt as was indicated by a
significant decrease in Km value. Therefore, it seems
that NaCl exerts three distinct effects on XylCMS: (1) a
delicate stabilizing effect in certain locations by which
the enzyme becomes partially structured as indicated
by its elevated intrinsic fluorescence intensity; (2) an
overall destabilizing effect that leads to more flexibility
but less thermal stability of the enzyme in the ES com-
plex as revealed by the barycentric fluorescence and the
activity-stability analysis; (3) improvement of the affin-
ity of the enzyme to substrate. acid; E. coli: Escherichia coli; Ea: activation energy; EMBL-EBI: European
Molecular Biology Laboratory-European Bioinformatics Institute; GH11:
glycosyl hydrolase family 11; IPTG: isopropyl β-d-1-thiogalactopyranoside; Kcat:
catalytic constant; Km: Michaelis constant; LB: Luria–Bertani; ml: millilitre; NCBI:
National Center for Biotechnology Information; NI–NTA: nickel nitrilotriacetic
acid; OSX: oat-spelt xylan; PCR: polymerase chain reaction; R. flavefaciens:
Ruminococcus flavefaciens; SDS-PAGE: sodium dodecyl sulfate polyacrylamide
gel electrophoresis; TLC: thin layer chromatography; Tm: melting temperature;
µg: microgram. BCM: barycentric mean fluorescence; CDD: conserved domain database; CMC:
carboxymethyl cellulose; DNA: deoxyribonucleic acid; DNS: 3,5-dinitrosalicylic Availability of data and materials Availability of data and materials
Not applicable. Funding This work was supported by National Institute of Genetic Engineering and
Biotechnology (NIGEB) (Grant No. 680). Ethics approval and consent to participate
Not applicable. Ethics approval and consent to participate
Not applicable. Abbreviations
BCM b
i Flint HJ, Martin J, McPherson CA, Daniel AS, Zhang JX (1993) A bifunctional
enzyme, with separate xylanase and beta (1, 3-1, 4)-glucanase domains, BCM: barycentric mean fluorescence; CDD: conserved domain database; CMC:
carboxymethyl cellulose; DNA: deoxyribonucleic acid; DNS: 3,5-dinitrosalicylic Ghadikolaei et al. AMB Expr (2019) 9:86 Page 11 of 11 Page 11 of 11 encoded by the xynD gene of Ruminococcus flavefaciens. J Bacteriol
175(10):2943–2951 encoded by the xynD gene of Ruminococcus flavefaciens. J Bacteriol
175(10):2943–2951 xylanase from Chromohalobacter sp. TPSV 101. J Mol Catal B-Enzym
74(3–4):192–198 xylanase from Chromohalobacter sp. TPSV 101. J Mol Catal B-Enzym
74(3–4):192–198 Ren H, Zong MH, Wu H, Li N (2016) Utilization of seawater for the biorefinery of
lignocellulosic biomass: ionic liquid pretreatment, enzymatic hydrolysis,
and microbial lipid production. ACS Sustain Chem Eng 4(10):5659–5666 Ghadikolaei KK, Noghabi KA, Zahiri HS (2017) Development of a bifunc-
tional xylanase-cellulase chimera with enhanced activity on rice
and barley straws using a modular xylanase and an endoglucanase
procured from camel rumen metagenome. Appl Microbiol Biotechnol
101(18):6929–6939 Wang G, Luo H, Wang Y, Huang H, Shi P, Yang P, Meng K, Bai Y, Yao B (2011) A
novel cold-active xylanase gene from the environmental DNA of goat
rumen contents: direct cloning, expression and enzyme characterization. Bioresour Technol 102:3330–3336 Ghadikolaei KK, Gharechahi J, Haghbeen K, Noghabi KA, Salekdeh GH, Zahiri
HS (2018) A cold-adapted endoglucanase from camel rumen with high
catalytic activity at moderate and low temperatures: an anomaly of truly
cold-adapted evolution in a mesophilic environment. Extremophiles
22(2):315–326 Wang Q, Luo Y, He B, Jiang LS, Liu JX, Wang JK (2015) Characterization of a
novel xylanase gene from rumen content of Hu sheep. Appl Biochem
Biotechnol 177:1424–1436 Wang G, Wu J, Yan R, Lin J, Ye X (2017) A novel multi-domain high molecular,
salt-stable alkaline xylanase from Alkalibacterium sp. SL3. Front Microbiol
7:2120 Gharechahi J, Zahiri HS, Noghabi KA, Salekdeh GH (2015) In-depth diversity
analysis of the bacterial community resident in the camel rumen. Syst
Appl Microbiol 38(1):67–76 Gong X, Gruniniger RJ, Forster RJ, Teather RM, McAllister TA (2013) Biochemical
analysis of a highly specific, pH stable xylanase gene identified from a
bovine rumen-derived metagenomic library. Appl Microbiol Biotechnol
97:2423–2431 Wejse PL, Ingvorsen K, Mortensen KK (2003) Purification and characterisation
of two extremely halotolerant xylanases from a novel halophilic bacte-
rium. Abbreviations
BCM b
i Extremophiles 7:423–431 Xing MN, Zhang XZ, Huang H (2012) Application of metagenomic techniques
in mining enzymes from microbial communities for biofuel synthesis. Biotechnol Adv 30(4):920–929 Hammes-Schiffer S, Benkovic SJ (2006) Relating protein motion to catalysis. Annu Rev Biochem 75:519–541 Xu B, Dai L, Li J, Deng M, Miao H, Zhou J, Mu Y, Wu Q, Tang X, Yang Y, Ding J,
Han N, Huang Z (2016) Molecular and biochemical characterization of a
novel xylanase from Massilia sp. RBM26 Isolated from the Feces of Rhino-
pithecus bieti. J Microbiol Biotechnol 26(1):9–19 Hess M, Sczyrba A, Egan R, Kim T-W, Chokhawala H, Schroth G, Luo S, Clark DS,
Chen F, Zhang T (2011) Metagenomic discovery of biomass-degrading
genes and genomes from cow rumen. Science 331(6016):463–467 Hung K-S, Liu S-M, Tzou W-S, Lin F-P, Pan C-L, Fang T-Y, Sun K-H, Tang S-J (2011)
Characterization of a novel GH10 thermostable, halophilic xylanase from
the marine bacterium Thermoanaerobacterium saccharolyticum NTOU1. Process Biotechnol 46(6):1257–1263 Yegin S (2017) Single-step purification and characterization of an extreme
halophilic, ethanol tolerant and acidophilic xylanase from Aureobasidium
pullulans NRRL Y-2311-1 with application potential in the food industry. Food Chem 221:67–75 Jiang Z, Yi J, Li J, He T, Hu C (2015) Promoting effect of sodium chloride on
the solubilization and depolymerization of cellulose from raw biomass
materials in water. Chemsuschem 8(11):1901–1907 Yegin S, Altinel B, Tuluk K (2018) A novel extremophilic xylanase produced
on wheat bran from Aureobasidium pullulans NRRL Y-2311-1: effects on
dough rheology and bread quality. Food Hydrocoll 81:389–397 Kaur P, Bhardwaj NK, Sharma J (2016) Pretreatment with xylanase and its
significance in hemicellulose removal from mixed hardwood kraft pulp as
a process step for viscose. Carbohydr Polym 145:95–102 Zhang JX, Flint HJ (1992) A bifunctional xylanase encoded by the xynA gene
of the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 com-
prises two dissimilar domains linked by an asparagine/glutamine-rich
sequence. Mol Microbiol 6(8):1013–1023 Kumar V, Marin-Navarro J, Shukla P (2016) Thermostable microbial xylanases
for pulp and paper industries: trends, applications and further perspec-
tives. World J Microbiol Biotechnol 32:34–44 Zhang JX, Martin J, Flint HJ (1994) Identification of non-catalytic conserved
regions in xylanases encoded by the xynB and xynD genes of the cel-
lulolytic rumen anaerobe Ruminococcus flavefaciens. Mol Gen Genet
245(2):260–264 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Abbreviations
BCM b
i Nature 227(5259):680–685 Liu X, Huang Z, Zhang X, Shao Z, Liu Z (2014) Cloning, expression and charac-
terization of a novel cold-active and halophilic xylanase from Zunong-
wangia profunda. Extremophiles 18(2):441–450 Publisher’s Note Menon G, Mody K, Keshri J, Jha B (2010) Isolation, purification, and charac-
terization of haloalkaline xylanase from a marine Bacillus pumilus strain,
GESF-1. Biotechnol Bioprocess Eng 15:998–1005 Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reduc-
ing sugar. Anal Chem 31(3):426–428 Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reduc-
ing sugar. Anal Chem 31(3):426–428 Prakash B, Vidyasagar M, Jayalakshmi SK, Sreeramulu K (2012) Purification
and some properties of low-molecular-weight extreme halophilic Prakash B, Vidyasagar M, Jayalakshmi SK, Sreeramulu K (2012) Purification
and some properties of low-molecular-weight extreme halophilic
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TO WHAT EXTENT CAN THE GYNECOLOGIST PREVENT AND CURE INSANITY IN WOMEN?
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JAMA
| 1,907
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public-domain
| 8,731
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He reported again in two days com-
plaining of the other eye and on examination it was found that
both optic discs were swollen, almost obscure,
an evidence of
retrobulbar neuritis. The pains were so severe that even with
opiates he could not sleep. This continued for several days,
when the patient became
more comfortable. The second eye
had never become quite
so bad
as the other; vision equalled
15/50. The pains subsided and the evidences of engorgement
were passing away, but the optic neuritis still persisted. There
Mas no history of specific trouble or rheumatism. might
by
operations. 4. All insane or epileptic women should have their
pelvic organs most thoroughly examined at once and all
diseased conditions should be relieved as quickly as pos-
sible by whatever means are necessary. by
necessary. 5. If these things be true, how important for the gen-
eral practitioner, under whose care all of these cases first
come, to see that all pelvic disorders in women are prop-
erly treated and cured as soon after their discovery as
possible, in order to prevent these mental troubles. Fi
ll
i poss b e,
p e e t
G. Finally, how important for our insane women to
have the benefit of what the gynecologist can do at the
earliest possible period. poss b e period. In our text-books on gynecology you will look almost
in vain for any encouragement along this line of thought. Pryor, Davenport, Gilliam, Penrose, Garrigues, Byford,
Hirst, Ashton. and Dudley give it
no consideration. Kelley gives it but scant notice, and one is cautioned
against any operative procedures in women who are in-
clined to mental trouble. In his edition of
1901 McNaughton-Jones devotes
one brief chapter to it and says: p
y
The relation of disordered sexual functions to mental diseases
has hardly received the careful attention
on the part of the
profession generally that it most decidedly demands. Picque
has found as high a proportion of affections of the generative
organs among the insane as 88 per cent. He reports favorably
of the effects of operations on the insane in a fair proportion of
these
affected. 1. The relations of insanity and pelvic diseases in
women have not heretofore received the attention which
their importance deserves, nor which they will receive
in the near future. and irrigation. He has seen great benefit derived from its be-
ing carefully carried out; there is
no doubt, however, of the
liability of a return of the condition if operative measures are
not resorted
to. He has reported
one
case of
retrobulbar
neuritis of frontal sinus origin. Another
case is under treat-
ment at the present time and is making rapid progress, vision
having improved from 6/lx to 6/xii in five days time with an
enlargement of the visual field and reduction of size in an ab-
solute central
scotoma. Further study of the subject will
undoubtedly be productive of some radical changes in the now
generally accepted etiology of many eye diseases. 2. Not all insane women who have pelvic disease as a
factor can be cured by local treatment or operations, but
in the early stages such treatment
or operations, in
many cases, would have been curative or preventive. many cases,
preventive. 3. Cases not benefited by local and conservative opera-
tions might still be cured by more radical operations many
,
preventive. 3. Cases not benefited by local and conservative opera-
tions might still be cured by more radical operations. generally accepted etiology
many eye
Dr. S. L. Leobetter, Birmingham, Ala., referred to the ease
of a man who complained of pain in the frontal region and de-
feet of vision in one eye. He gave a history of
a severe cold,
but no suppuration in the nasal cavities. On examination he
found that the eye was apparently perfectly healthy, except for
a slight cloudiness in
the macular region;
vision equalled
20/100. The other eye was perfectly normal. The pains in-
creased for the next two days. He made
a diagnosis of pos-
terior ethmoidal and probably sphenoidal engorgement, ordered
a Turkish bath, laxatives,
a little alterative tonic, and light
diet. In a few days the patient was only able to count fingers
at two feet with that eye and complained of much more pain,
so that opium had to be given. He was put on diaphoretics,
pilocarpin and other things, and kept up the laxative treat-
ment and ordered rest. Read in the Section
on Obstetrics and Diseases of Women of
the
American
Medical
Association,
at
the Fifty-seventh
Annual
Session, June, 1906. W. O. HENRY, M.D.
OMAHA. On the other hand, Beed says : Downloaded From: http://jama.jamanetwork.com/ by a Carleton University User on 06/20/2015 REVIEW OF LITERATURE. The reader can readily see why it is unnecessary to discuss
the relationship between the pelvic disease and epilepsy, chorea
and other
nervous diseases. The truth
can all
be summed
up in a word. There is no relation. The same truth obtains
with regard to the insanities. The question of the relationship existing between pel-
vic diseases in
women and insanity is
one which has
occupied the attention of the profession many years,
more particularly since 1857, when Loiseau presented a
thesis
on the subject, "Sympathetic Insanity," which
caused considerable discussion, and also since the paper
by Hergt, in 1870, on the subject, "Women's Diseases
and Disturbances of the Mind."
Although from that
time to the present the subject has been discussed to
some extent in medical societies and through medical
journals,
even yet great difference of opinion exists
among gynecologists and alienists. When I read a paper
before the Nebraska State Medical Society in 1898 on
the subject, "Relations of Insanity and Pelvic Diseases
in Women," there was little sympathy expressed by the
members of the society and my conclusions then given,
which more extensive experience has served to confirm,
were not received with favor. Those important conclu-
sions were as follows: In 189G W. P. Mantón said: Sherwood Dunn
reviews
the
entire subject
and the experience of a number of psychologic and gynecologic
authorities and comes to the general conclusion that affections
of the pelvic viscera in women
are often associated with in-
sanity in the relation of
cause and effect; that such lesions
predispose to the occurrence of puerperal insanity; that they
impede convalescence from mental disorders and operative in-
terference should be advocated in those cases in which pro-
nounced affection of the uterus or adncxa is discovered. y
p
Dr. Brawley said that he had probed and irrigated the
sinuses only when he discovered pus in the middle meatus,
because probing the sinuses complicates the diagnosis. He laid
stress on the fact that many of these cases, seen early, do not
have pus, and probing may lay
one open to the criticism of
having caused the suppuration when it occurs. In 189G W. P. Mantón said: So far as we have been able to determine from repeated
and persistent cross-examination, it is safe to say that there
had
been periods
of confusion, suspicion
and
dread
for at
least
a year before the untoward conduct or overt acts of the
individual made the nature of his conduct manifest to his
friends. g
g
Insanity
is
an expression only of functional
and organic
disorder;
remove the disorder on which the insanity depends
and the return to mental soundness is secured. Charles P. Bancroft says: Insanity is not a specific entity; it is rather an ever vary-
ing symptom complex, corresponding to many diverse underly-
ing disease processes. Insanity may be said to be a prolonged
departure from the individual's normal
method of thinking,
feeling and acting, due to functional or organic disturbances
of some portion of the encephalon. J. W. Wherry,1 Dansville, N. Y., writes
a very in-
structive article in which, among other things, he says: p
p
The definition of Bondurant of the Alabama Insane
Hospital is : ,
g
g ,
The evidences of brain pathology, in abnormal intellectual
states, as in idiocy, imbecility, dementia and general paralysis
are at least sufficiently established to call for further investiga-
tion, but in the emotional insanities, I believe it is useless to
probe the brain longer for a secret it does not hold. p
The facts obtained
seem to justify the opinion that many
of the patients in whom insanity and nephritis co-exist are in-
sane because of the nephritis; that is, the insanity is one of the
mental symptoms of acute or chronic uremic intoxication. probe
longer
I
can
see
no good reason why there should be any doubt
about the origin of melancholia in visceral conditions rather
than in a disease of the brain. H. A. Tomlinson, Minnesota, says: y
The pathologic history of insanity is known to be vague
and indefinite and from the standpoint of histology, furnishes
very little information
to the student in
search of specific
morbid changes which will explain the clinical manifestations
of aberrant
cerebral functioning. This apparent
absence of
definite anatomic change is to be explained by the conditions
which give rise to insanity; namely, the precedence of physical
disease,
shock, overwork, mental strain, infection,
or
auto-
intoxication
from
some
source. In 189G W. P. Mantón said: Consequently,
the insanity
can not be said to be dependent
on the changes found to be
present in the neuron, but rather, the histologie change and the
insanity follows
the conditions generated by antecedent
so-
matic changes to which they are consecutive. Maudsley said : y
It is clear as day that temporary bodily conditions, however
they may have been brought about, will play their part and it
may be well that future researches will discover the
causes
of the characteristic features of some varieties of mental de-
rangements in the diathetie states and the actual bodily dis-
orders which are associated with them. There is the most per-
fect harmony, the most intimate connection
or sympathy be-
tween the different organs of the body as the expression of its
organic life. Unity of the organism beneath consciousness and
the brain is quite aware that the body has a liver and a stom-
ach and feels the effects of disorder in any one of the organs. Ferrier says :
l
bl
f
li
d Ferrier says : g
Kohlberger says : y
Healthy states of viscera produce pleasurable feelings and
morbid states of viscera produce painful
or depressing feel-
ings. Visceral derangements are frequently the cause and al-
ways the accompaniment of melancholic depressions. g
y
The source of mental diseases is not only in the brain but
in all the organs. Morrell says : ways
p
p
C. C. Hersman,2 alienist to the South Side Hospital,
Pittsburg, also read a paper before the Section, in 1899,
in which he said : Although the brain is always the seat of insanity, it is not
always the seat of its cause. From these opinions I think we may say that insanity
is that mental condition characterized by
a prolonged
change in a person's usual manner of thinking, acting
and feeling without apparent cause and yet, doubtless,
the result of some brain or other bodily disease. It is
a well-known fact that the ovaries and testicles have
at least three distinct actions; the first, generation; the
sec-
ond, their action through absorption
on the central
nervous
system, which give to
men and women.their physical, moral
and intellectual characteristics;
the third is
a special tonic
action which reinforces in
a special way the action of the
spinal cord and brain. In 189G W. P. Mantón said: We long ago came to the conclusion that the idea of restor-
ing the sick mind to health as the result of gynecologic inter-
ference should be abandoned. Yet, strange to say, he goes on to admit : g
y
I have never operated on an insane woman yet, no matter
to what extent dementia has gone, without some relief to the
mental condition and
a decided improvement in the personal
comfort of the patient. p
So far as the testimony of gynecologists goes, it seems
to be overwhelmingly against the possibility of doing
anything either to prevent or to cure these sad com-
plaints. Thurman, of the York Betreat, said :
I
d
b
f p
,
,
In round numbers, of ten persons attacked by insanity, five
recover and five die sooner or later during the attacks. Of the
five who recover, not more than two remain well the rest of
their lives; the other three sustain subsequent attacks during
which at least two of them die. Tomlinson says that of 937 admitted, 212 had
a he-
redity of insanity, while 2G6 had a heredity of cancer
and 170 had a heredity of phthisis, 123 had sexual ex-
citement or perversion, 233 had some menstrual disorder. 443 had leucorrhea, 408 had uterine displacement, 222
of these last had adhesions, 79 had disease of the ovaries,
337 had laceration of the cervix, 424 had laceration of
the perineum, 238 had cystocele, 229 had rectocele. Then
he says : If this be a generally accepted prognosis, how impor-
tant for us to determine just what the gynecologist can
do to prevent insanity before it occurs or cure it after it
occurs. Chapin's definition is: p
Insanity is that mental condition characterized by
a pro-
longed change in the usual
manner of thinking, acting and
feeling; the result of disease or mental degeneration. g
g
The Alienist and Neurologist gives this definition: says
People become insane. Why? Because of defective nervous
organization and not because of cardiac or renal disease. The
physical disease simply upsets the unstable nervous organiza-
tion. 1. Am. Jour. of Insanity, January, 1906.
2. "Uterine
Diseases
and Insanity," The Journal
A.
M.
A.,
vol. xxxiii, p. 709. Downloaded From: http://jama.jamanetwork.com/ by a Carleton University User on 06/20/2015 In 189G W. P. Mantón said: y
In the report of the New York State Commission of
Lunacy for the year ending Sept. 30, 1906, the follow-
ing were the causes assigned for insanity in 4,819 cases
admitted to the New York hospitals for the insane in
the preceding twelve months : p
In addition to this I am not sure, but we will find
eventually that there are nerve centers in the uterus,
and possibly also in the tubes, which have an important
influence on the mental equilibrium. He cites in his
paper two interesting cases from the insane department
of the St. Francis Hospital as follows : p
g
Moral Causes:
Adverse conditions, loss of friends, business
troubles, etc., 354; mental strain, overwork, worry, 384;
re-
ligious excitement, 65 ; love affairs, including seduction, 32 ;
fright and nervous shock, 95. p
A Jewess, married, very excited, was in the hospital for
a
few
months. She
also
suffered
from chronic endometritis,
was curetted and went home in three weeks, with no mental
symptoms. Again,
a Polish girl who had been in the hospital
for more than a year with no improvement mentally, on exam-
ination
was found to be suffering from chronic endometritis. She went home cured in two weeks after curetting the uterus. They have both remained well
a period of about five years. g
Physical Causes:
Alcoholism, 476; sexual excess, 24;
ven-
ereal diseases, 94; masturbation, 90; sunstroke, 50; accident
or injury, 87; pregnancy, 9; parturition and puerperium, 136;
lactation, 15; change of life, 100; fevers, 23; privation and
overwork, 80; epilepsy,
183;
other convulsive disorders, 5;
diseases of the skull and brain, 76; old age, 230; exophthalmic
goiter, 1; epidemic influenza, 40;
abuse of drugs, 44; loss of
special sense, 6; uremic poisoning, 2; other autoinfections, 1;
heredity, 357; congenital defects, 44; unascertained, 1,413. Of
the total number 1,069 had inherited predisposition. Downloaded From: http://jama.jamanetwork.com/ by a Carleton University User on 06/20/2015 Downloaded From: http://jama.jamanetwork.com/ by a Carleton University User on 06/20/201 While
in the majority of these patients there had been some mental
improvement before the operation was done, yet, in all of the
twenty the psychic improvement following the operation was
marked,
as also
was the steady progress toward mental
re-
covery. I
am aware we usually look to the brain when searching
for the
cause of insanity and many times, I fear, when
we
should look elsewhere. It is necessarily the immediate seat of
the disturbances, but the remote
cause may be very foreign
to that organ. Many times, if the uterine disturbances were
relieved, the insanity would be removed. I have seen, as al-
ready stated, in the treatment of insanities the result of uter-
ine disease, the local or surgical treatment of the trouble not
only
cure the uterine disease, but effectually
cure
the
con-
comitant
disease occurring in the brain, thus showing
the
mysterious
(?)
and
unaccountable
(?)
connection
between
them. The "Beference Handbook of the Medical Sciences,"
1902, in the very able and rather exhaustive article on
insanity, says, when speaking of the accessory causes : y,
y ,
p
g
y
It is noticeable that organic diseases of the uterus and its
appendages
are
seldom
accompanied
by
profound
nervous
storms. On the other hand, the leucorrheas, amenorrheas, pro-
fuse menses, vaginismus, chronic uterine catarrh, and
a host
of
other comparatively benign affections
are
now and then
followed by mental disturbances. Although it may be said, in
general, that the influence of the affections of the generative
apparatus as a cause of insanity in woman has been exagger-
ated, as an accessory factor it is often worthy of consideration. y
Thus we see that the general
consensus of opinion
now among alienists seems to be that various bodily dis-
eases may be the exciting cause of insanity, and hence
the importance of curing these diseases as a prerequisite
to restoring mental health. Then, too, I think, it is
generally conceded that there is a closer and more inti-
mate relationship between the brain and sexual organs
than other viscera,
so that any disturbance there will
more easily affect the mental condition than diseases of
any other organs and it is remarkable how large the per-
centage of pelvic troubles is among insane women and
to what extent these women are cured of their mental
troubles when the local pelvic irritation is removed. Downloaded From: http://jama.jamanetwork.com/ by a Carleton University User on 06/20/201 For
example, in 1895, Dr. Bohe, of Maryland, said: p ,
y
The number of my cases is too small (34) to allow me to draw
conclusions, but if anything of practical
value
can
be
de-
duced from them, it is that puerperal insanity, melancholia
and simple
mania offer the best
chances of
cure from
the
proper treatment of local
lesions in the pelvis. I
am
con-
vinced that earlier operation in appropriate cases would have
largely increased the proportion of recoveries. While I do not wish to criticise the author of this
paper, still I am confident that his small percentage of
cures resulted from the fact that he did not entirely re-
move all pelvic disturbance, or, in other words, his oper-
ative work was not radical enough. Of course, simple
justice will require us to admit the possibility of an-
other factor, and that is too long duration of the mental
disturbance before the pelvic trouble was looked after,
but the former seems to me far more likely, because the
doctor's point of view was not altogether right, for he
should have gone at his pelvic work with the primary ob-
ject of so thoroughly getting rid of the irritation there
as not only to cure the physical ailment, but to be confi-
dent that by perfect results here he would reap much
greater benefits, namely,
a restored mentality and, no
doubt, richer and better results would have crowned bis
labors. To illustrate what I mean, I have selected a few
cases from his report read at the sixty-first annual meet-
ing of the American Medico-Psychological Society at
San Antonio, April, 1905. g y
p
p
And yet of his 34 patients operated on, 14 were cured,
5 improved and none made worse mentally. p
y
Dr. A. T. Hobbs,
of the London (Ontario) Asylum,
with the gynecologist, Dr. Meek, carefully and system-
atically examined 220 women who were confined in the
asylum, and found 188 of them, or 85 per cent., suffer-
ing from some pelvic lesion. They proceeded to operate
on 173 of these, and their report is that 42 per cent, of
these recovered, 24 per cent, were improved, 32 per cent,
remained the same and 2 per cent. died. 3. New York Med. Jour., 1905. Downloaded From: http://jama.jamanetwork.com/ by a Carleton University User on 06/20/201 p
LeBoy Broun, of the Manhattan State Hospital, read
a very excellent article before the Brooklyn Medical As-
sociation in January of the present year, in which, after
citing freely from various hospital reports, he says: g
y
p
p
y
Thus it is seen that in all insane asylums
a large majority
of the women inmates suffer in some form from
a disease of
the pelvic organs. Downloaded From: http://jama.jamanetwork.com/ by a Carleton University User on 06/20/201 ing this course, fully three-fourths of the patients operated on
were sufferers from forms of mental disease recognized as un-
favorable and in whom little permanent progress can be made
in ameliorating their mental state. The unfortunates of this
class have as much claim for physical relief as those of more
amenable forms of mental disease. Two hundred and forty-
two patients have been given some form of gynecologic opera-
tion, which is a little less than 5 per cent, of the total number
of women in the hospital during the period covering the oper-
ations. Of those operated on there have been 62 abdominal sec-
tions, 51 operations for displaced uteri and 129 minor plastic
operations. As a result of all the operations done (242)
112
patients have been physically benefited in
a marked degree, j
107 have been noticeably improved, though not to such a great |
extent as in the previous number. Of the remaining patients
five died,
of
which
number
two
deaths
were
attributable
to
the operation
and
three to natural
causes. The
state-
ment made by some writers that operative measures for dis-
eased conditions at times aggravate the insanity of the patient
is not in accord with my experience. No instance of such an
occurrence exists
among patients I have operated
on. The
same
statement
is
made by
Mantón concerning
his
opera-
tions in the East Michigan Asylum covering
a period of 20
years and by Picque, who has been operating for
12 years
among the insane of Paris. The rare occurrence of true psy-
chosis following gynecologic operations
on
women
not
pre-
viously insane has been brought out by Rohe.3
He states that,
as
a result of communication with all the asylums of the
United States and Canada, he found that in the course of ten
years prior to 1893 only 25 patients had been registered in all
these asylums
as having
become
insane after gynecologic
operations. In studying the histories of the patients on whom
I have operated, I find that of the 242 patients, 138 still re-
main in the institution and
104 have been discharged. Of
those
discharged
43
are
recorded
as
recovered
mentally. Twenty of these 43 discharged as cured have had their men-
tal recovery materially hastened as a result of the physical im-
provement arising from the operation done
on them. AUTHOR'S CASES. My' own cases now comprise
a total of 28. Of this
number only one patient has been operated on too re-
cently to tell what the ultimate result will be. The others
have been operated on long enough to signify something
of the benefit they will receive. The first
ones
were
operated on nine, ten and eleven years ago and remain
well to the present time. Sixteen of the patients made a
perfect physical and mental recovery. One died soon after
the operation. All except this
one
were temporarily
benefited and none made worse. Two have since died
of the intercurrent troubles. Eight, although slightly
improved, I have not been able to trace and do not know
their condition. The one
so recently operated on and
very much better I can not yet report on, although the
improvement is marked, and apparently the recovery
will be perfect. g
y
Miss
G.,
age
40. Disease:
Chronic melancholia,
retro-
flexion of uterus, with fibroids. Duration:
Two years. Oper-
ation:
Myomectomy with Bissell's operation. Result:
Not
satisfactory; mental state unimproved. y;
p
In this case there is no question that there was some
ovarian trouble and that
a hysterectomy woufd have
given better results. y
p
In this case there is no question that there was some
ovarian trouble and that
a hysterectomy woufd have
given better results. g
Mrs. F., age 28. Disease:
Chronic melancholia, rétrover-
sion and ovarian cyst. Duration:
Three years. Operation:
Curettage anterior vaginal section, removal right ovarian cyst
and shortening of round ligaments. Result:
Operation satis-
factory; mental condition unimproved. y
p
There can be little doubt that there was left behind
sufficient difficulty to maintain the irritation previously
existing before the operation. y
There can be little doubt that there was left behind
sufficient difficulty to maintain the irritation previously
existing before the operation. p
Case 1.—Woman, aged 34, married ten years, never preg-
nant, had chronic endometritis with cystic ovaries. Removal of
the tubes and ovaries was done in 1895 and she has remained
well from that time until now. She had acute mania. g
p
Mrs. R., age 32. Disease:
Acute melancholia, rétroversion,
laceration of the cervix and perineum. Operation:
Curettage
hysterorrhaphy. Result:
Operation
was successful;
no im-
provement mentally. Case 2.—Woman, aged 34, mother of
one child, ten years
old. Patient
had
retroflexed
uterus,
lacerated
cervix
and
cystic ovaries. AUTHOR'S CASES. Supravaginal amputation of the uterus
and removal of the tubes and ovaries resulted in perfect phy-
sical and mental health. Case 7.—Woman, aged 40, had melancholia, large retro-
flexed uterus, prolapsed tubes and ovaries. Vaginal hyster-
ectomy resulted in immediate improvement, but after she
re-
turned home I lost sight of her and have not been able to lo-
cate her since. Mrs. H., age 35. Disease:
Paranoia with adherent retro-
version
and laceration
of cervix and perineum. Duration:
One year. Operation:
Uterus suspended, left tube and ovary
removed, excision of cervix and repair of perineum. Result:
Satisfactory but
no mental improvement. Mrs. H., age 35. Disease:
Paranoia with adherent retro-
version
and laceration
of cervix and perineum. Duration:
One year. Operation:
Uterus suspended, left tube and ovary
removed, excision of cervix and repair of perineum. Result:
Satisfactory but
no mental improvement. Case 8.—Woman, aged 42, mother of five children, suffered
from melancholia, chronic oöphoritis and cystic degeneration
of the ovaries. Removal of tubes and ovaries resulted in per-
fect mental and physical health. C
W Certainly these operations, while they removed part
of the pathology, left as much behind to maintain the
mental aberration and, therefore, were not adequate. Certainly these operations, while they removed part
of the pathology, left as much behind to maintain the
mental aberration and, therefore, were not adequate. p y
Case 9.—Woman, aged
30,
married
several
years,
never
pregnant, had retroflexed uterus, degeneration of the ovaries
and melancholia for several months. Vaginal hysterectomy re-
sulted in slight improvement, but she finally refused to take
food and died in the asylum. q
Mrs. M.,
age
25. Disease:
Acute
melancholia
of
two
months' duration, with double
adnexa
diseases. Operation:
Both adnexa were removed and hysterorrhaphy. Result:
Men-
tal condition unchanged. q
Mrs. M.,
age
25. Disease:
Acute
melancholia
of
two
months' duration, with double
adnexa
diseases. Operation:
Both adnexa were removed and hysterorrhaphy. Result:
Men-
tal condition unchanged. g
Miss F., age 36. Disease:
Chronic melancholia of eight
years' duration with endometritis
and rétroversion. Opera-
tion:
Curettage. Result:
Physical condition improved and
mental condition only slightly improved. g
Miss F., age 36. Disease:
Chronic melancholia of eight
years' duration with endometritis
and rétroversion. Opera-
tion:
Curettage. Result:
Physical condition improved and
mental condition only slightly improved. Case 10.—Woman, aged 50, mother of two children, had
passed the change of life. AUTHOR'S CASES. Cervix
was repaired, tubes and ovaries were
removed
and patient
made
a good
recovery mentally
and
physically. p
y
Here, too, the local irritation was not entirely re-
moved. Mrs. L.,
age
28. Disease:
Acute melancholia,
adherent
rétroversion with double pyosalpinx. Operation:
Excision of
cervix and perineorrhaphy with suspension of uterus. p y
y
Case
3.—Woman,
aged
42,
mother
of
several
children,
suffered from a large subinvoluted uterus with lacerated cer-
vix, retroflexion and prolapsed ovaries. She had melancholia
and dementia for
a year and
a half. Supravaginal amputa-
tion of the uterus, tubes and ovaries was made after which she
made a perfect physical and mental recovery. No wonder the patient did not recover, for there must
have remained an abnormal amount of pathology in the
pelvis. No wonder the patient did not recover, for there must
have remained an abnormal amount of pathology in the
pelvis. p
Mrs. S., age 34. Disease:
Chronic melancholia with retro-
flexion
and
laceration
of
cervix
and perineum. Duration:
Three and one-half years. Operation:
Hysterorrhaphy curet-
tage,
excision of
cervix
and perineorrhaphy. Result:
No
mental improvement. p
p y
y
Case 4.—Woman, aged 30, mother of one child, had melan-
cholia, subinvoluted uterus, degeneration of ovaries. Curette-
ment was done and tubes and ovaries removed. Patient made
good recovery from operation and was mentally improved, but
I have lost sight of her and can not now trace her. p
Mrs. L., age
39. Disease :
Chronic mania with multiple
fibromyomata and rétroversion. Duration :
Ten years. Oper-
ation:
Myomectomy
and hysterectomy. Result:
Operation
successful but no mental improvement. p
Mrs. L., age
39. Disease :
Chronic mania with multiple
fibromyomata and rétroversion. Duration :
Ten years. Oper-
ation:
Myomectomy
and hysterectomy. Result:
Operation
successful but no mental improvement. g
Case 5.—Woman, aged 35, mother of
two children,
had
retroflexion of uterus, chronic oöphoritis and melancholia. Re-
moval of tubes and ovaries resulted in perfect physical and
mental health. Here there can be no doubt that a supravaginal am-
putation, with probabde removal of tubes and ovaries,
would have given better results, and certainly if done
earlier the chances of recovery would have been in-
creased. Case 6.—Woman, aged 45, mother of two children, suffered
from melancholia, had
a large retroflexed uterus, prolapsed
tubes and
ovaries. ment, for here, whatever else may have been wrong, the
suspension of the uterus granted a new course of pelvic
irritation, being wholly irrational and unreasonable. ment, for here, whatever else may have been wrong, the
suspension of the uterus granted a new course of pelvic
irritation, being wholly irrational and unreasonable. ment, for here, whatever else may have been wrong, the
suspension of the uterus granted a new course of pelvic
irritation, being wholly irrational and unreasonable. SELECTIONS FROM LE ROT RROUN's
CASES. Mrs. S., age 39. General health:
Fair. Disease: Dementia,
paralytica
ovarian cyst and retroflexed
uterus. Operation:
Removal
of cyst, suspension
of
uterus. Result:
Physical
condition satisfactory;
no marked mental improvement. p
Insane patients, being as much subject to physical diseases
as their more fortunate sisters have equally as much right to
be relieved. Such is now recognized and the majority of the in-
stitutions have on their staff consulting surgeons who render
such relief as needed. Those unfortunates are now treated as
if they were not insane,
as far as their physical condition is
concerned. As stated,
no operations
have
been undertaken
with the direct object of influencing the mental status. The
physical status alone has been considered. As a result of adopt- Although the result of this operation was satisfactory
as far as the physical condition was concerned, we are
not surprised that there was no marked mental improve- Downloaded From: http://jama.jamanetwork.com/ by a Carleton University User on 06/20/2015 AUTHOR'S CASES. y
y
p y
Case 22.—Woman, aged 36, mother of two children, had
multiple uterine fibroid. Abdominal hysterectomy resulted in
physical and mental cure. p y
Case 23.—Young girl, 19.years of
age, with
retroflexed
uterus, prolapsed tubes and ovaries, developed melancholia with
mania. Venetral suspension was done but she made
no im-
provement and the girl is still in the asylum. p
y
I wish, then, to conclude my paper by insisting that
the gynecologist can prevent the occurrence of insanity
in many women with very unstable nervous organization
if he will, by treatment or operation, remove all pelvic
irritation; and, again, he may cure various forms of
insanity in women if such irritation is entirely removed,
and it is important not to substitute a lesser form of irri-
tation in getting rid of the more serious one. Conserva-
tive treatment and operations are all right, if they really
secure complete removal of all pelvic irritation; but to
get all the results we have a right to expect from gyne-
cology, and which we can undoubtedly secure in a very
large proportion of cases, we must wholly eradicate the
pelvic irritation by whatever means are necessary, no
matter how radical the work required. p
g
y
Case 24.—Woman, aged 38, mother of four children, had
retroflexed uterus with cystic ovaries and melancholia and was
bedfast for
several
months. Supravaginal amputation
and
removal of tubes and ovaries resulted in satisfactory physical
recovery and
some mental improvement, since which time I
have not been able to trace the case. C Case 25.—Married woman, aged 26, mother of two children,
developed puerperal mania after her last child was born and
after a thorough curettement for septic uterus and wiping-out
with carbolic acid she made
a rapid recovery both mentally
and physically. p y
y
Case
26.—Unmarried
woman,
aged
28,
with pronounced
melancholia was confined to her bed with intramural fibroid,
the removal of which resulted in
some physical and mental
improvement, but two years later she died without having
fully recovered her normal mentality. 27 p y
y
Case
26.—Unmarried
woman,
aged
28,
with pronounced
melancholia was confined to her bed with intramural fibroid,
the removal of which resulted in
some physical and mental
improvement, but two years later she died without having
fully recovered her normal mentality. 27 AUTHOR'S CASES. Granted that
a number of
cases of puerperal in-
sanity will recover without operation, I know positively, from
experience, that two things
have occurred and will
occur ;
namely, that good mental health is hastened by surgical inter-
ference and that
a number of patients would never have
re-
covered without surgical interference and that the recovery is
due entirely to the removal of serious diseases interfering with
the return to physical and through it to mental health. Les-
ions of the ovaries are, without doubt, causative factors, and
disease of the uterus too, in producing insanity, in
cases of
nervous heredity. cholia. Removal of tubes and ovaries resulted in physical
and mental health. cholia. Removal of tubes and ovaries resulted in physical
and mental health. Case 14.—Woman, aged 38, retroflexed adherent uterus and
prolapsed tubes and ovaries with melancholia and dementia,
had been confined to her bed for several months. Vaginal hys-
terectomy was done without benefit. The patient died two
weeks later. Case 14.—Woman, aged 38, retroflexed adherent uterus and
prolapsed tubes and ovaries with melancholia and dementia,
had been confined to her bed for several months. Vaginal hys-
terectomy was done without benefit. The patient died two
weeks later. Case 15.—Woman, aged 35, married several years, no chil-
dren, had cystic ovaries and melancholia. Removal of tubes
and ovaries resulted in physical and mental health. Case 15.—Woman, aged 35, married several years, no chil-
dren, had cystic ovaries and melancholia. Removal of tubes
and ovaries resulted in physical and mental health. p ys ca
Case 16.—Married woman, aged 35,
never pregnant, had
cystic degeneration of ovaries with melancholia. Removal of
tubes and ovaries resulted in physical improvement and some
mental improvement. I lost sight of her and have not been
able to locate her since. p ys ca
Case 16.—Married woman, aged 35,
never pregnant, had
cystic degeneration of ovaries with melancholia. Removal of
tubes and ovaries resulted in physical improvement and some
mental improvement. I lost sight of her and have not been
able to locate her since. Case 17.— (Details lost.)
C
18
W
d Case 17.— (Details lost.)
C
18
W
d Case 17.— (Details lost.)
C
18
W )
Case 18.—Woman, aged 42, mother of three children, de-
veloped puerperal mania with septic condition of the uterus. Supravaginal amputation with removal of tubes and ovaries
resulted in physical
and mental improvement. DISCUSSION. Db. W. P. Mantón, Detroit, Mich., said that his experience
with this subject extended over many years; that he was the
pioneer in this line and the first to take up systematic work
among insane
women. For seventeen years he has been con-
nected with two large state institutions in Michigan, and has
had to do also with two private hospitals for the insane,
so
that several thousand insane women have passed under his ob-
servation. One of the best recent articles on this subject is by
Dr. LeRoy Broun in a late number of the Journal of Medical
Sciences. Dr. Broun has had large opportunities for observa-
tion and arrives at the same conclusions as Dr. Mantón. He
took exception to the first item in Dr. Henry's abstract. When
he began work every asylum superintendent in the land, with
the exception of Dr. Henry M. Hurd, now of Johns Hopkins
University, but then superintendent of the Eastern Michigan
Asylum, was utterly opposed to any gynecologic interference
with insane women. If the alienist does not now know any-
thing about these conditions it is his own fault, said Dr. Mantón, because he and
a great many others have brought y
y
Case 27.—Young woman, aged 27, severe dysmenorrhea since
early girlhood with periodic attacks of mania and melancholia
and cystic degeneration of both ovaries. Removal of tubes and
ovaries has resulted in marked relief both mentally and physic-
ally, but the time is yet too short for a final report in this
case. S ) Case 28.—Mrs. B. (Dr. S.), aged 30, mother of two chil-
dren had retroflexed uterus and cystic ovaries which resulted
in melancholia. Abdominal hysterectomy was done, tubes and
ovaries being removed. She made
a good recovery and
was
mentally improved when she left the hospital, but I have been
unable to trace her since and do not know the final outcome. AUTHOR'S CASES. Uterine fibroid with calcareous de-
generation
was present. Vaginal hysterectomy was done and
resulted in physical and mental soundness. Here, with an endometritis and rétroversion probably
of eight or ten years' duration, there could not have been
a healthy condition of the ovaries and tubes. Neither
could the rétroversion be left alone and the pelvic irrita-
tion relieved. Hence, this ease did not have the relief
which might have been afforded by more thorough treat-
ment, and certainly we could have reasonably expected
more had the operation been done earlier. p y
Case 11.—Woman, aged 36, mother of two children, had bad
laceration of the perineum, subinvoluted and reflexed uterus
and prolapsed ovaries. Repair of lacerations and removal of
tubes and ovaries resulted in perfect cure, physical and mental. Case 12.—Woman, aged 30, married several years,
never
pregnant, had retroflexed uterus and periodical mania. After
vaginal hysterectomy she made physical and mental recovery. Case 13.—Woman, aged 27, married
several years,
never p y
Case 11.—Woman, aged 36, mother of two children, had bad
laceration of the perineum, subinvoluted and reflexed uterus
and prolapsed ovaries. Repair of lacerations and removal of
tubes and ovaries resulted in perfect cure, physical and mental. C p
and prolapsed ovaries. Repair of lacerations and removal of
tubes and ovaries resulted in perfect cure, physical and mental. Case 12.—Woman, aged 30, married several years,
never
pregnant, had retroflexed uterus and periodical mania. After
vaginal hysterectomy she made physical and mental recovery. C
13
W
d 2
i d p
, p y
Case 12.—Woman, aged 30, married several years,
never
pregnant, had retroflexed uterus and periodical mania. After
vaginal hysterectomy she made physical and mental recovery. g
y
y
p y
recovery. Case 13.—Woman, aged 27, married
several years,
never
pregnant, had cystic degeneration of the ovaries and meian- Downloaded From: http://jama.jamanetwork.com/ by a Carleton University User on 06/20/2015 Through reduction of physical well being and through reflex
irritation. The removal of disease, the restoration of dis-
placements, the extirpation of tumors and repair of injuries
will first restore the physical health and through it the men-
tal condition;
and, second, will
remove
a
source of reflex
irritation. Downloaded From: http://jama.jamanetwork.com/ by a Carleton University User on 06/20/2015 AUTHOR'S CASES. I have lost
sight of the case and do not know the ultimate result. C I would like to emphasize another point, and that is
that many insane women who have pelvic trouble do not
complain of it. While this also obtains in women who
are not insane, yet it is very much more apt to be the case
in women who are mentally unbalanced, as is attested
by those who have given the subject careful thought and
as shown by my own cases, and again I would like to
insist
on the point that sometimes there is serious
ovarian degeneration which is not discoverable by ordi-
nary pelvic examination. So that I am sure some of
these patients in asylums who are examined by the most
competent gynecologists have serious ovarian disease
which could only be detected by opening the abdominal
cavity and personally making a macroscopic inspection
of the ovary. So that it is not sufficient to say that ex-
amination has been made and no pelvic trouble found,
for, as every operator knows, a good many times when
the abdomen is opened and the ovary is carefully in-
spected serious disease is found,
even when not sus-
pected by bimanual examination. g
Case
19.—Woman, aged
52,
mother of
several children,
passed the
menopause six years,
had suffered from
hemor-
rhoids and retroflexed
uterus with ulcération of the cervix
and developed melancholia of severe type. Vaginal hysterec-
tomy and removal of hemorrhoids resulted in perfect physical
and'mental health. g
Case
19.—Woman, aged
52,
mother of
several children,
passed the
menopause six years,
had suffered from
hemor-
rhoids and retroflexed
uterus with ulcération of the cervix
and developed melancholia of severe type. Vaginal hysterec-
tomy and removal of hemorrhoids resulted in perfect physical
and'mental health. Case 20.—Woman, aged 23, single, had severe dysmenorrhea
and periodical attacks of mania, with melancholia and pro-
lapsed cystic right ovary. On removal of ovary patient im-
proved both physically and mentally. p
p y
y
y
Case 21.—Woman, aged 40, mother of four children, de-
veloped melancholia, after her last child
was born. There
was retroflexed uterus with prolapsed tubes and ovaries. Vag-
inal hysterectomy resulted in physical and mental health. y
y
p ys ca
Case 22.—Woman, aged 36, mother of two children, had
multiple uterine fibroid. Abdominal hysterectomy resulted in
physical and mental cure. PHILADELPHIA. The ques-
tion among alienists has been pretty thoroughly determined,
that back of it all is an unstable nervous organization, and the
question of attempting to cure insanity per se, by surgery, is
somewhat far-fetched. As Dr. Mantón said, the insane person
is entitled to the
same character of surgical relief that the
sane person expects. The surgical condition may be causative
in a sense in a great many cases. The possibility cf a patient
recovering her mental balance is greater if all physical suffer-
ing can be removed. If she has
a bleeding fibroid she has
a
better chance to recover her health if it is removed. The late
Dr. Carpenter
and
he had
examined between
100
and
200
patients. In the neighborhood of
84 per cent, of the total
number had some form of intrapelvic trouble. Of that number
the history of only about 21 per cent, showed that the abdom-
inal condition preceded the mental. He advocated surgery for
all surgical conditions, whether in sane or insane; but in the
absence of gross pathology insanity or other neuroses can not
be reasonably expected to yield to any form of surgery. The classification of endometritis should be purely anatomic. In other words, it should be divided into the acute and the
chronic forms. While acute endometritis involves all of the
component parts of the endometrium indifferently, the chronic
form may affect especially the glands or the stroma. Any
classification
of endometritis,
based
on etiologic factors,
is
faulty, because the etiology of chronic forms
can not always
be determined, and, therefore, the classification in many
cases
would be useless. There is also no relation in the chronic form
between the cause and the form of the anatomic change in the
endometrium. A classification based
on clinical symptoms,
as catarrhal,
purulent,
or hemorrhagic, is also bad, for such terms might
be easily applied to different stages of the same process, and
a classification based on symptoms leads to an almost endless
variety of forms. y
p
y
y
surgery. Dr. W. O. Henry, Omaha, said that he was glad to hear the
remarks of Drs. CONCLUSION-. Dr. A. T. Hobbs, now superintendent of
a private
sanitarium at Guelph, Ont., in a recent letter, sums up
his views as follows: Pelvic diseases interfere with mental health in two ways: Downloaded From: http://jama.jamanetwork.com/ by a Carleton University User on 06/20/2015 this matter up repeatedly. He
does not believe this to
be
true, however, for
as the result of work done there is to-day
hardly
an institution which has not a gynecologist connected
with it. While there
is unquestionably
a relationship
be-
tween the pelvic organs in woman and the brain, Dr. Mantón
does not believe that, except in possibly rare instances, disease
of the former gives rise to mental alienr.Jon. Whenever in-
sanity exists, however, and there is a local point of irritation
present in either the pelvis or the abdomen, the mental
con-
dition is often made worse, and by removing the irritation, no
matter how demented the woman may be, she will be made
more comfortable and is more easily taken care of. In a large
number of
cases he has never
seen
an instance in which the
mental.condition was not relieved to some extent as the result
of this kind of treatment. In his experience some 81 per cent. of insane women suffer from locai disorders; but if 100 women
who
are walking about in apparent perfect health
were
ex-
amined it will probably be found that
as large
a percentage
are suffering from some abdominal or pelvic disorder also. So
that the finding of this percentage of local disease among in-
sane
women really
means that
a large
number
of
women,
sane or insane, have this burden to carry. Operations under-
taken solely for the cure of mental disorder in insane women
are wrong; but every insane woman suffering from abdominal
or pelvic disease is as much entitled to relief from the somatic
condition as is her sane sister. The late Dr. Robe, who wrote
a good deal regaruing the curability of insanity in
women
through surgical means, appears to have been too sanguine,
and Dr. Mantón is not altogether certain about Dr. Robe's
cases. Another observer, Dr. Hobbs,
was assistant physician
at the London (Ont.) asylum under Dr. Buck, when he began
his investigations. Hobbs' results and Dr. Manton's do not
tally, and Dr. Mantón is left to conclude that Dr. Hobbs deals
with
a different class of
cases. Dr. Report
of the Committee
on Nomenclature of Endometritis to
the Section on Obstetrics and Diseases of Women at the Fifty-sev-
enth Annual
Session of the American Medical Association, June,
1906. PHILADELPHIA. The word endometrium
should be used to refer to the mucous
membrane which lines the body of the uterus. The mucosa of
the cervical canal is anatomically and physiologically a differ-
ent structure, and although it may properly
be called endo-
metrium, usage more or less restricts that term to the mucosa
of the body of the uterus. y
The endometrium is intimately connected with the muscula-
ture of the uterus and its circulation is greatly influenced by
changes which occur in the blood vessels of the myometrium
and by the tonicity of the uterine muscle. The endometrium
is continuous with the lining membrane of the tubes and with
the epithelial layer of the cervix, therefore, it follows that the
endometrium is influenced by all inflammatory affections, dis-
placements,
new
growths
and
congestions
of
the
genital
organs. g
By
some it is held that the term endometritis
(inflamma-
tion of the endometrium)
should
be applied only to those
lesions
of
the
endometrium
which
show histologically
the
products of an active reaction of the tissues to
an irritant. According to this view, endometritis is always the result of in-
fection with a micro-organism. By others it is held that there
are causes of inflammation other than bacterial; and that a
long continued inflammatory process, produced either by bac-
terial infection, or by mechanical or other forms of irritation,
may produce hyperplastic
or atrophie changes in the endo-
metrium, without any histologie evidence of
an acute inflam-
matory reaction. y
Such
a distinction results from
a difference in the concep-
tion of what constitutes an inflammation and has little prac-
tical importance. Thus, what is considered glandular hyper-
plasia of the endometrium by the adherents to the first view is
called glandular endometritis by the adherents to the second. It is our belief that it will lead to less confusion of terms to
regard
"glandular hypertrophy"
of
the
endometrium
and
"atrophy" of the endometrium as the end results of a chronic
endometritis, or as Siinger has aptly stated it, the residuum
of an inflammation. g
g
p
y
Dr. F. F. Lawrence, Columbus, 0., said that this subject
was discussed in
a symposium
in Denver, when Dr. Joseph
Price was chairman; also in Atlantic City in 1900. CONCLUSION-. Buck became very
en-
thusiastic regarding the operative cure of insanity. THE
NOMENCLATURE
OF
ENDOMETRITIS. ERNEST
F. TUCKER,
M.D.,
PORTLAND,
ORE.,
HENRY
O. MARCY, M.D., BOSTON, JOHN G. CLARK, M.D., Downloaded From: http://jama.jamanetwork.com/ by a Carleton University User on 06/20/2015 PHILADELPHIA. Mantón and Lawrence, but that his contention
is that these pelvic diseases in
women of unstable
nervous
organization
are the particular thing that
often upsets the
mind, and the curing of these things before they have upset
the mind will, in such cases, prevent the breakdown. So long
as
no
one
can tell what insanity really is,
he thinks that
practical experience in
cases that
are cured counts for
more
than anything else. When he has operated on
cases and five
or ten years later the patients are still well, that counts for
more than all the theory
on earth. Of 28
eases he had
16
patients go home well. This is of real practical value. He
does not claim that every insane woman can be cured by opera-
tion, but many can, and every insane woman should be given
the benefit of all possible aid which
can be afforded by the
removal of all pelvic irritation. And finally, by curing these
pelvic irritations in women of unstable nervous organizations,
before insanity occurs, its development :^ay often be wholly
prevented. The age of the individual also should be given no place in
the nomenclature. Senile endometritis conveys
no pathologic
meaning;
the terms juvenile
or adolescent endometritis may
be employed just as properly. In conformity with inflammatory lesions elsewhere, we may
speak of acute and chronic endometritis. The chronic form
may further be divided into glandular and interstitial, when
the glands
on the
one hand or the stroma
on the other
are
especially involved. Pathologists may subdivide the chronic
forms in order to indicate anatomic peculiarities, but this is
not necessary for the clinician. Endometritis, except in the acute form, rarely exists -alone. It is usually complicated by inflammatory lesions of the cer-
vix or of the pelvic viscera; lacerations of the cervix or of the Downloaded From: http://jama.jamanetwork.com/ by a Carleton University User on 06/20/2015
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